SLA 1
云计算服务等级协议SLA[V1
云计算服务等级协议SLA[V11. 引言本云计算服务等级协议（Service Level Agreement，简称SLA）旨在明确云计算服务提供商（以下简称"供应商"）与客户之间的服务级别和责任。
2. 服务范围本SLA适用于供应商所提供的云计算服务，包括但不限于计算资源的分配和管理、存储空间的提供、数据备份和恢复等。
3. 服务等级供应商将努力保障以下服务等级指标的达成：- 可用性：系统每月至少可用99.9%的时间。
- 响应时间：供应商将在故障报告收到后的1小时内作出响应，并在24小时内提供解决方案或更新。
- 数据备份与恢复：供应商将每日进行数据备份，并保证在数据恢复请求发起后的24小时内完成恢复。
4. 服务维护供应商将定期进行系统维护，并提前至少48小时通知客户。
在维护期间，供应商将尽力减少可能对客户造成的影响，并在维护结束后恢复正常服务。
5. 数据安全供应商将采取合理的技术和管理措施，以确保客户数据的安全性和隐私性。
供应商将进行定期的安全性评估和风险分析，并在发现风险时及时采取措施进行修复和预防。
6. 服务变更供应商在进行重大服务变更时，将提前至少2周通知客户，并尽可能提供过渡方案以最大程度减少客户的影响。
7. 违约责任如果供应商未能达到本SLA所规定的服务等级指标，客户将有权根据具体情况要求赔偿、合同解除或其他补救措施。
8. 有效期本SLA自发布之日起生效，有效期为一年，到期后可根据双方协商决定是否续约。
9. 其他条款本SLA除上述规定外，还应符合相关法律法规的要求，在遵守法律法规的前提下，供应商和客户可根据实际情况协商修改、补充或解释本SLA的具体条款。
SLA(服务等级协议)(2024两篇)
SLA(服务等级协议)（二）SLA（服务等级协议）（二）一. 引言服务等级协议（SLA）是一种约定，用于明确服务提供商和客户之间的责任和义务，确保提供的服务能够满足客户的期望和需求。
本文是SLA（服务等级协议）系列的第二部分，将继续探讨如何制定一个有效的SLA，以及如何监控和调整SLA以确保其持续有效性。
二. 制定一个有效的SLA1. 清晰的目标和指标在制定SLA时，首先需要明确确定服务的目标和指标。
这些目标和指标应该具体、可衡量和可达成。
例如，如果是一个网络服务提供商，目标可以是确保网络的可用性达到99.9％，指标可以是测量网络的平均故障时间。
2. 可接受的服务水平明确定义客户可以接受的服务水平是制定有效SLA的关键。
这应该基于客户的业务需求和优先级。
不同的客户可能有不同的可接受水平，因此需要根据他们的需求进行个性化的设置。
3. 定期沟通SLA应该在服务提供商和客户之间进行定期的沟通和回顾。
这有助于确保双方对SLA的理解和满意度，并提供机会进行任何必要的调整和改进。
定期沟通还可以帮助建立互信和合作关系，以实现更强的合作效果。
4. 风险管理在制定SLA时，必须考虑潜在的风险和不可控因素。
这些风险可能包括自然灾害、供应商故障、网络攻击等。
在SLA中应明确如何应对这些风险，并制定相应的应急计划。
三. 监控和调整SLA1. 监控指标一个有效的SLA应该有具体的监控指标，以评估服务提供商是否符合SLA的要求。
这些指标可以包括服务的可用性、故障时间、响应时间等。
监控指标应该根据SLA的要求进行定期的测试和评估。
2. 持续改进监控SLA是为了发现潜在的问题和改进的机会。
服务提供商应该根据监测结果进行持续改进，以提高服务质量和满足客户的需求。
客户的反馈也是宝贵的资源，应该被积极地参考和整合。
3. 合同条款如果发现SLA需要调整或修订，应遵守合同条款并与客户进行协商。
变更可能需要在SLA中进行全面的审查和修改，以确保新的要求能够得到满足。
SLA资料1
3.4 The same sequence for SLA?To what degree might this proposed developmental sequence of syntactic acquisition apply in SLA?SLA is different from L1A in numerous respects, particularly with regard to:i mature conceptual development :a in child language acquisition knowledge of the world and knowledge of language are developing simultaneously whereas adult SLA builds upon pre-existing conceptual knowledge;b adult learners have sophisticated formal operational means of thinking and can treat language as an object of explicit learning, that is, of conscious problem-solving and deduction, to a much greater degree than can children (N. Ellis, 1994a);ii language input: the typical L1 pattern of acquisition results from naturalistic exposure in situations where caregivers naturally scaffold development (Tomasello and Brooks, 1999), whereas classroom environments for second or foreign language teaching can distort the patterns of exposure, of function, of medium, and of social interaction (N. Ellis and Laporte,1997);iii transfer from L1: adult SLA builds on pre-existing L1 knowledge (MacWhinney, 1992; Odlin, this volume), and, thus, for example, whereas a young child has lexically specific patterns and only later develops knowledge of abstract syntactic categories which guide more creative combinations and insertions into the slots of frames, adults have already acquired knowledge of these categories and their lexical membership for L1, and this knowledge may guide creative combination in their L2 interlanguage to variously good and bad effects. Nevertheless, unless there is evidence to the contrary, it is a reasonable default expectation that naturalistic SLA develops in broadly the same fashion as does L1 – from formulae, through low-scope patterns, to constructions – and that this development similarly reflects the influences of type and token frequencies in the input. (But see Doughty, this volume, for a discussion of how L1 and L2 processing procedures differ.) There are lamentably few longitudinal acquisition data for SLA that are of sufficient detail to allow the charting of construction growth. Filling this lacuna and performing analyses of SLA which parallel those for L1A described in section 3.2 is an important research priority. But the available evidence does provide support for the assumption that constructions grow from formulae through low-scope patterns to more abstract schema. For a general summary, there are normative descriptions of stages of L2 proficiency that were drawn up in as atheoretical a way as possible by the American Council on the Teaching of Foreign Languages (ACTFL) (Higgs, 1984). These Oral Proficiency Guidelines include the following descriptions of novice and intermediate levels that emphasize the contributions of patterns and formulae to the development of later creativity:Novice Low: Oral production consists of isolated words and perhaps a few high frequency phrases … Novice High: Able to satisfy partially the requirements of basic communicative exchanges by relying heavily on learned utterances but occasionally expanding these through simple recombinations of their elements … Intermediate: The intermediate level is characterized by an ability to create with the language bycombining and recombining learned elements, though primarily in a reactive mode.(ACTFL, 1986, p. 18)Thus, the ACTFL repeatedly stresses the constructive potential of collocations and chunks oflanguage. This is impressive because the ACTFL guidelines were simply trying to describe SLA as objectively as possible – there was no initial theoretical focus on formulae – yet nonetheless the role of formulae became readily apparent in the acquisition process.There are several relevant case studies of child SLA. Wong Fillmore (1976) presented the first extensive longitudinal study that focused on formulaic language in L2 acquisition. Her subject, Nora, acquired and overused a few formulaic expressions of a new structural type during one period, and then amassed a variety of similar forms during the next. Previously unanalyzed chunks became the foundations for creative construction (see also Vihman's, 1982, analyses of her young son Virve's SLA). Such observations of the formulaic beginnings of child L2 acquisition closely parallel those of Pine and Lieven for L1.There are a few studies which focus on these processes in classroom-based SLA. R. Ellis (1984) described how three classroom learners acquired formulae which allowed them to meet their basic communicative needs in an ESL classroom, and how the particular formulae they acquired reflected input frequency – they were those which more often occurred in the social and organizational contexts that arose in the classroom environment. Weinert (1994) showed how English learners’ early production of complex target-like German foreign language negation patterns came through the memorization of complex forms in confined linguistic contexts, and that some of these forms were used as a basis for extension of patterns. Myles, Hooper, and Mitchell (1998; Myles, Mitchell, and Hooper, 1999) describe the first two years of development of interroga-tives in a classroom of anglophone French L2 beginners, longitudinally tracking the breakdown of formulaic chunks such as comment ťappelles-tu? (what’ s your name?), commentt'appelle-t-il? (what’ s his name?), and ou habites-tu? (where do you live?), in particular the creative construction of new interrogatives by recombination of their parts, and the ways in which formulae fed the constructive process. Bolander (1989) analyzed the role of chunks in the acquisition of inversion in Swedish by Polish, Finnish, and Spanish immigrants enrolled in a4-month intensive course in Swedish. In Swedish, the inversion of subject-verb after asentence-initial non-subject is an obligatory rule. Bolander identified the majority of the inversion cases in her data as being of a chunk-like nature with a stereotyped reading such as det kan man saga (that can one say) and det tycker jag (so think I). Inversion in these sort of clauses is also frequent when the object is omitted as in kan man saga (can one say) and tycker jag (think I), and this pattern was also well integrated in the interlanguage of most of these learners. Bolander showed that the high accuracy on these stereotyped initial-object clauses generalized to produce a higher rate of correctness on clauses with non-stereotyped initial objects than was usual for other types of inversion clause in her data, and took this as evidence that creative language was developing out of familiar formulae.Although there are many reviews which discuss the important role of formula use in SLA (e.g., Hakuta, 1974; Nattinger and DeCarrico, 1992; Towell and Hawkins, 1994; Weinert, 1995; Wray, 1992), there is clearly further need for larger-sampled SLA corpora which will allow detailed analysis of acquisition sequences. De Cock (1998) presents analyses of corpora of language-learner productions using automatic recurrent sequence extractions. These show that second language learners use formulae at least as much as native speakers and at times at significantly higher rates. There is much promise of such computer-based learner corpus studies (Granger, 1998), providing that sufficient care is taken to gather the necessarily intensive longitudinal learner data. There is also need to test the predictions of usage-based theoriesregarding the influences of type frequency and token frequency as they apply in SLA.2.1 Construct definition: interpretations about L2 acquisitionIn order to define acquisition as a construct for measurement purposes, the particular interpretations to be made about L2 acquisition must first be sought within existing SLA theories. Table 21.1 summarizes some of the essential features (interpretive as well as evidentiary) for three main theoretical approaches to SLA.Generative SLA views language as a symbolic system, autonomous from cognition, and too complex to be acquired by training or through inductive or deductive learning from the input. Since it adheres to the tenets of first language nativism, generative SLA research aims at elucidating empirically whether learners can have indirect, partial, full, or no access to the principles of Universal Grammar in the process of acquiring an L2, and it prioritizes interpretations about linguistic competence, not language performance (Gregg, 1990; Schwartz, 1993; White, 1991). Further, this epistemological approach to L2 acquisition focuses on constructs which describe and explain the origins of linguistic mental representations (the “competence problem” central in a property theory) and does not concern itself so much with interpreting how such representations unfold or become available to the learner in a predictable route (the “developmental problem” central in a transition theory) (see Gregg, 1996).Therefore, generative SLA research confines itself to formal descriptions of interim learner grammars (i.e., syntax) as reflected in a learner's tacit ability to judge ungrammaticality in the L2, because it assumes that the goal of SLA as a theory is to explain how learners can acquire a full mental representation of many of the complexities of the L2, and why they cannot acquire all aspects of an L2 syntax (and precisely which aspects learners may fail to acquire). Generative linguistic studies of SLA are likely to rely almost exclusively on the outcomes of grammaticality judgment tasks of various kinds, where acquired means nativelike levels of rejection of illegal exemplars of the target grammar.Interactionist SLA, on the other hand, is based on functionalist views of language as a symbolic system that develops from communicative needs (Tomlin, 1990; Tomasello, 1998a). Language is believed to be a complex faculty that is acquired by the learner through engagement with the environment, through inductive and/or deductive learning from input, and in a constructive process (in the Piagetian sense) constrained by general cognition (see Long, 1996; Richards and Gallaway, 1994). Hence, language acquisition is thought of as a gradual process of activeform/function mapping, and the traditional dichotomy between competence and performance is not maintained; instead, language learning is inextricably related to language use in that performance is viewed as driving competence (Hymes, 1972; see papers in G. Brown, Malmkjaer,and Williams, 1996; and discussion in McNamara, 1996, ch. 3). Interactionist epistemologies,drawing on functionalist linguistic theories, such as variationist sociolinguistics (Preston, 1989), functional grammar (Givon,1979), and discourse analysis (Sinclair and Coulthard, 1975), focus not so much on the origin and description of linguistic representation as on the “developmental problem” (e.g., Pienemann, 1998).Not only, therefore, do interactionist SLA theories need to describe and explain learner transitional grammars, but their interpretations must also invoke non-linguistic (i.e., cognitive and environmental) constructs thought to be crucial in accounting for how learning of an L2 takes place on a predictable route and with differential ultimate success. Interactionist SLA researchers maintain that acquisition of L2 forms cannot be demonstrated until such forms are productively used in a variety of contexts in spontaneous performance; a multiplicity of performance data is therefore required to produce a complete picture of language development. In addition, this type of theory argues that incremental, non-linear changes (not necessarily target-oriented improvements) in patterns of language use can be taken as indications that gradual learning is taking place (e.g., Mellow, Reeder, and Forster, 1996).Consequently, interactionist studies (at least logically ought to) draw on measures of implicit and explicit memory for L2 forms (i.e., recognition tasks where acquired means detected or noticed), measures of explicit knowledge of rules (i.e., metalinguistic verbalization tasks, where acquired means understood with awareness), and measures of the use of L2 forms in spontaneous, meaningdriven discourse (i.e., comprehension and production tasks involving sentence-level and, preferably, text-level performance, where ability for use is demonstrated). In sum, under interactionist approaches to SLA, acquired may mean a number of gradual and non-linear changes in the linguistic (and, in some theories, metalinguistic) behavior that characterize the developmental course of L2 acquisition, based on construct interpretations such as: (i) a form has “emerged,” has been “detected,”“noticed,” “attempted,” or “restructured”; (ii) a learner is “aware” of a form or a form-related pattern; and/or (iii) a learner is “able to use a form appropriately and fluently.”Finally, emergentism provides a combined functional and neurobiological approach to language acquisition that views grammar as a complex, rule-like, but not rule-governed system arising from the interaction of very simple learning mechanisms in the organism (the architecture of the human brain) with the environment (massive exposure to input). Emergentist theories of L2 acquisition seek to explain the frequency and regularity of linguistic input to which the learner must be exposed in order for the processing system (i.e., the brain) to develop a functional set of weights (i.e., degree of interconnectivity among nodes) that will match patterns underlying that input (Sokolik, 1990).Speeded, accurate production of output that matches the input provides evidence that such functional sets of weights in the neural networks have been established on the basis of simple learning algorithms and exposure to positive input alone (N. Ellis, 1998). Consequently, emergentistconnectionist studies typically employ computer modeling experiments and trials with human subjects under laboratory conditions, with interpretations based on reaction-time decision tasks involving carefully controlled input (e.g., N. Ellis and Schmidt, 1997). Acquired, for emergentists, means fast, accurate, and effortless performance attained along attested learning curves that reflect non-linear, exemplar-driven learning.Obviously, each of the preceding theoretical approaches to SLA defines acquisition in unique ways and calls for particular construct interpretations to be made on the basis of measurement data.Indeed, what counts as acquisition is so dependent on the theoretical premises of the research domain that the same measurement data may be interpreted as evidence of acquisition or the lack thereof, depending on the theoretical approach adopted. A good illustration of this point can be found in a well-known study by Trahey and White (1993). Measurement outcomes from this study showed that young francophone learners in intensive ESL programs in Quebec, after a two-week regime of exposure to English input flooded with adverbs, accepted more cases ofSubject-Adverb- Verb-Object sentences (ungrammatical in the L1 but grammatical in English) than they had accepted before. However, positive evidence alone (i.e., exposure to only correct SAVO exemplars in the flooded input) did not cause these learners to rejectSubject-Verb-Adverb-Object sentences (grammatical in the L1 and ungrammatical in English). From the generativist perspective of the authors, these measurement observations were interpreted to show that acquisition had not occurred, because there was no evidence of parameter resetting, which would require simultaneous acceptance of SAVO and rejection of *SVAO. However, arguments from interactionist SLA, including developmental accounts of L2 learning (e.g., Meisel, Clahsen, and Pienemann, 1981; Mellow et al.,1996) and claims about the role of attention and awareness in L2 learning (e.g., Schmidt, 1993, 1994; Tomlin and Villa, 1994), would call for an alternative interpretation of the same data as evidence for incipient acquisition of adverb placement in L2 English. In fact, in studies of implicit and incidental instructional conditions (i.e., external interventions that do not orient learners to learning with intention; see Schmidt, 1993) researchers have repeatedly found evidence for acquisition in small post-instructional increases in recognition of or preference for the targeted form (a behavior typically observed in input flood treatments, as in Trahey and White, 1993) and/or in increased, albeit initially unsuccessful, attempts to produce the targeted form (a behavior typically observed in typographical input treatments; see Alanen, 1995; Jourdenais et al., 1995).To summarize, what counts as acquisition (theoretically defined), as well as the utility of viewing L2 acquisition in particular ways, may be disputed by researchers from differing paradigms. However, such disagreements themselves bear witness to the fact that construct definitions are available. Given theoretical construct definitions, additional conceptual bases for measurement may be evaluated. Therefore, we turn now to an examination of the evidence required for making interpretations about acquisition and the measurement tasks used to provide such evidence.。
软件开发服务等级协议SLA[V1
软件开发服务等级协议SLA[V1 ---协议编号：SLA[V1.0]生效日期：[日期]1. 引言本协议旨在明确软件开发服务提供商（以下简称“服务提供商”）与客户之间的服务等级协议。
本协议规定了服务提供商在软件开发服务方面的责任和义务，以及客户可以期望的服务水平。
2. 定义在本协议中，以下术语的定义如下：- 服务提供商：指负责提供软件开发服务的公司或个人。
- 客户：指与服务提供商签订本协议并购买软件开发服务的公司或个人。
- 服务等级协议（SLA）：指服务提供商和客户之间达成的关于服务水平的约定。
3. 服务范围服务提供商将根据客户的需求，提供以下软件开发服务：- 软件需求分析- 系统设计与架构- 编码与开发- 软件测试与质量保证- 上线部署与发布- 技术支持与维护4. 服务等级目标服务提供商将努力满足以下服务等级目标：- 响应时间：服务提供商将在接收到客户的请求后的【指定时间】内进行回应。
- 项目交付：服务提供商将根据双方约定的时间表按时交付软件开发项目。
- 质量标准：服务提供商将确保软件开发服务符合行业标准和最佳实践，并提供高质量的软件解决方案。
5. 服务支持- 技术支持：服务提供商将通过电话、电子邮件或在线聊天等方式为客户提供技术支持，解答相关问题和提供解决方案。
- 故障处理：服务提供商将在发现软件故障或问题时，立即采取行动解决，并提供相应的故障跟踪和排除过程。
6. 服务计费- 费用结构：软件开发服务的计费方式以和客户签订的合同为准。
- 付款方式：客户需按照合同约定的付款方式和时间节点支付软件开发服务费用。
7. 不可抗力双方因不可抗力事件（如自然灾害、战争、政府行为等）而无法履行本协议下的任何义务时，应及时通知对方，并根据具体情况商议必要的解决方案。
8. 争议解决本协议的任何争议应通过友好协商解决，若协商无果，可以由双方约定的仲裁机构进行仲裁。
9. 协议变更任何对本协议的修改或变更应经双方以书面方式达成一致，并由双方的授权代表签署。
能力服务sla标准
能力服务sla标准在当今竞争激烈的市场环境下，企业需要不断提升自身的竞争力，而服务水平的提升是其中至关重要的一环。
为了确保客户能够获得高质量的服务，许多企业都会制定能力服务SLA标准，以明确自身的服务承诺和责任。
本文将针对能力服务SLA标准展开讨论，旨在帮助企业更好地理解和制定相关标准。
首先，能力服务SLA标准是指企业为了保证服务质量和客户满意度而设定的一系列指标和承诺。
这些指标通常涵盖了服务响应时间、问题解决时间、服务可用性、服务级别协议等内容。
通过明确这些标准，企业可以更好地管理和监控自身的服务水平，从而提升客户体验和忠诚度。
其次，制定能力服务SLA标准需要考虑到企业自身的实际情况和客户需求。
不同的行业和企业可能面临的挑战和要求各不相同，因此在制定SLA标准时需要充分考虑到这些因素。
同时，还需要与客户充分沟通，了解他们的期望和需求，以便制定出更加切实可行的标准。
在制定能力服务SLA标准时，企业还需要充分考虑到自身的能力和资源情况。
制定过高的标准可能导致企业难以实现，从而影响客户满意度；而过低的标准则可能无法满足客户的需求，影响企业的声誉和竞争力。
因此，制定能力服务SLA标准需要在客户需求、企业实际情况和市场竞争力之间取得平衡。
除了制定能力服务SLA标准，企业还需要不断监测和评估这些标准的执行情况。
只有通过不断地跟踪和分析，企业才能及时发现问题并进行改进，从而不断提升服务水平。
同时，企业还需要与客户进行及时的沟通和反馈，以了解他们对服务的满意度和建议，从而进一步完善能力服务SLA标准。
总之，能力服务SLA标准对于企业提升服务质量和客户满意度具有重要意义。
通过制定明确的标准，并不断监测和改进，企业可以更好地满足客户需求，提升竞争力，实现可持续发展。
因此，制定能力服务SLA标准是每个企业都应该重视和努力实践的重要环节。
SLA_1_余达
immediate cocial context, but used for future travel or other crosscultural situations, or studied as a crurricular requiement or elective in school, but with no immediate or practical application
postgraduate schools(英语教学法方向)
Why SLA?
laying the foundation for your further academic research
being curious about SLA phenomenon
Q1: How do people manage to learn an L2?
"native language"?
Are they interchangeable?
A case study
My cousin was born in Korea and the very first language
she learned was obviously Korean; however, her whole family moved to the US when she was 7 and she grew up in New Jersey, USA. (She has been living in the US for nearly 20 years.) So she now speaks English much better than Korean. The language she speaks best, most comfortably and confidently is English. Is her first language still Korean? What about her native language? Her Korean became so rusty and she sounds like a brain-damaged person whenever she speaks Korean. Can we still call her a native speaker of Korean?
如何使用网络流量监控软件实现网络SLA管理(一)
如何使用网络流量监控软件实现网络SLA管理随着互联网的普及和网络应用的广泛应用，网络服务品质的保障变得越来越重要。
作为网络管理人员，我们需要确保网络服务水平协议（Service Level Agreement，SLA）的达成。
在这个过程中，使用网络流量监控软件是非常必要的。
一、什么是网络流量监控软件网络流量监控软件是一种用于实时监测和分析网络流量的工具。
它可以对网络流量进行实时抓包，并提供相关统计数据，帮助我们了解网络的负载情况、带宽利用率、延迟等重要参数。
常用的网络流量监控软件包括Wireshark、PRTG、SolarWinds等。
二、网络SLA管理的重要性网络SLA管理是确保网络服务质量的关键。
通过网络流量监控软件，我们可以实时监测网络的状态，快速发现和解决网络问题，从而保证网络服务的可用性、性能和安全性。
1. 提升网络服务的可用性网络服务的可用性是SLA管理的重要指标之一。
通过网络流量监控软件，我们可以对网络设备、链路等进行实时监测，及时发现故障和异常，快速采取措施修复，避免网络中断给用户带来的影响。
2. 优化网络服务的性能网络服务的性能是SLA管理的另一个关键指标。
网络流量监控软件可以分析网络的负载情况、带宽利用率、延迟等参数，帮助我们优化网络架构和资源分配，提升网络的响应速度和吞吐量，提高用户体验。
3. 加强网络服务的安全性网络服务的安全性是SLA管理的重要方面之一。
网络流量监控软件可以监测网络流量中的异常行为和安全事件，及时发现潜在的安全威胁，从而采取相应的安全措施，确保网络的安全性和数据的保密性。
三、使用网络流量监控软件实现网络SLA管理的关键步骤1. 定义关键指标和阈值在使用网络流量监控软件之前，我们需要明确网络SLA管理的关键指标和阈值。
例如，可用性要求、网络延迟要求、带宽利用率要求等。
根据这些指标和阈值，我们可以在监控软件中设置相应的警报条件，以便及时发现和响应异常情况。
2. 配置监控设备和接口网络流量监控软件需要与网络设备进行连接，以抓取网络流量数据。
performance_sla_1
© 2008 Sombers Associates, Inc., and W. H. Highleyman
1
Part 3 answers the SLA configuration question for servers with exponential service times. It provides charts and an Excel spread sheet to determine the probability that the response will not exceed a specified value. Part 4 introduces the Gamma distribution and shows with charts and a spread sheet how to use it to answer the SLA configuration question for servers with arbitrary response times. Part 5 concludes with an example of determining the probability/maximum response time curve for a complex series of servers with differing service time distributions.
L rTs
(2)
© 2008 Sombers Associates, Inc., and W. H. Highleyman
2
That is, if transactions are arriving at an average rate of one per second, and if the average service time for a transaction is 0.5 seconds, the server is busy 50% of the time. It is carrying a load of 0.5. When a transaction arrives to be serviced, it will find W transactions waiting in front of it. In addition, with probability L, there will be a transaction in the process of being serviced (remember that L is the probability that the server is busy – that is, it is currently servicing a transaction). If a transaction is currently being serviced, then only kTs time is left to complete its servicing. The newly arrived transaction must wait while the current transaction is completed (kTs seconds L of the time) and then for the W transactions in front of it to be serviced (WTs seconds). Thus, it must wait for a time Tq (the queuing delay) of
1-SLA-1
Second Language Acquisition: An Overview
I. What is Second Language Acquisition (SLA)? 1. Current status of SLA An independent discipline Multidisciplinary in nature Significant development in the past 40-45 years Far from a complete theoretical framework
I. What is Second Language Acquisition (SLA)? 3. The Definition of SLA:
the process learning another language after the learning of the native language competence: a language user’s underlying knowledge of language performance: the use of the grammar in the comprehension and production of language
N. Chomsky (1928- )
How to characterize learner’s L2 competence? inference of L2 competence via L2 learners’ performance
Second Language Acquisition: An Overview
to become permanently established in the interlanguage of a second language learner in a form that is deviant from the TL norm and that continues to appear in performance regardless of further exposure to the target language
SLA(1)
(3)Acquisition and Learning
The Acquisition-Learning distinction is the most fundamental of all the hypotheses in Krashen's theory. According to Krashen there are two independent systems of second language performance: 'the acquired system' and 'the learned system'. The 'acquired system' or 'acquisition' is the product of a subconscious process, which is very similar to the process children undergo when they acquire their first language. It requires meaningful interaction in the target language - natural communication - in which speakers are concentrated on the communicative act. Yet the 'learned system' or 'learning' is the product of formal instruction and it comprises a conscious process, which results in conscious knowledge 'about' the language, for example knowledge of grammar rules. Krashen (1981) uses the term „acquisition‟ to refer to the spontaneous and incidental process of rule internalization that results from natural language use, where the learner‟s attention is focused on meaning rather than form. (Ellis, 1994:692) Krashen (1982) uses the term „learning‟ to refer to the development of conscious knowledge of an L2 through formal study. It means that same as explicit knowledge (Ellis, 1994: 712). Stephen Krashen makes a distinction between „Acquisition‟ and „Learning‟.
移动集团客户网络分级服务SLA规范范本
移动集团客户网络分级服务SLA规范范本第一章总则为了规范移动集团客户网络分级服务，确保客户网络的稳定运行和高质量服务，特制定本规范。
第二章服务级别协议1. 定义服务级别协议（SLA）：服务级别协议是指网络服务提供商（SP）与客户之间以合同形式约定的服务质量标准和责任分工。
2. SLA的适用范围：本SLA规范适用于移动集团客户网络分级服务。
3. 客户网络分类：(1) 一级网络客户：指移动集团的重要客户，其网络服务对企业业务运行至关重要。
(2) 二级网络客户：指一般的企业客户，其网络服务对企业业务运行具有一定的重要性。
(3) 三级网络客户：指小型企业客户或个人用户，其网络服务需求相对较低。
4. SLA服务内容：(1) 一级网络客户：提供24小时不间断的网络监控与维护服务，并保证在故障时能够及时响应并解决问题，将平均恢复时间控制在2小时以内。
(2) 二级网络客户：提供12小时不间断的网络监控与维护服务，并保证在故障时能够及时响应并解决问题，将平均恢复时间控制在4小时以内。
(3) 三级网络客户：提供8小时不间断的网络监控与维护服务，并保证在故障时能够及时响应并解决问题，将平均恢复时间控制在8小时以内。
第三章级别服务要求1. 响应时间：SP在下述情况下，需在规定的时间内响应客户的服务请求。
(1) 一级网络客户：30分钟内响应(2) 二级网络客户：1小时内响应(3) 三级网络客户：2小时内响应2. 故障恢复时间：SP在下述情况下，需在规定的时间内解决故障。
(1) 一级网络客户：2小时内恢复(2) 二级网络客户：4小时内恢复(3) 三级网络客户：8小时内恢复3. 进行例行维护：SP应提前通知客户进行例行维护，并选择非工作时间进行维护。
第四章服务支持1. 技术支持：SP应为客户提供网络故障的调查与解决、技术支持等服务。
2. 服务报告：SP应定期向客户提供网络性能、故障处理情况等相关报告。
3. 培训服务：SP应为客户提供相应的网络技术培训，帮助客户提升网络运维能力。
实验用荣昌猪SLA-1等位基因鉴定及分子遗传特征分析
畜牧兽医学报 2023,54(7):3064-3077A c t a V e t e r i n a r i a e t Z o o t e c h n i c a S i n i c ad o i :10.11843/j.i s s n .0366-6964.2023.07.037开放科学(资源服务)标识码(O S I D ):实验用荣昌猪S L A -1等位基因鉴定及分子遗传特征分析刘弘毅,罗霆宇,李昌文,于海波,路小野,陈洪岩,夏长友*,高彩霞*(中国农业科学院哈尔滨兽医研究所,动物疫病防控全国重点实验室,黑龙江省实验动物与比较医学重点实验室,国家禽类实验动物资源库,哈尔滨150069)摘 要:旨在明确实验用荣昌猪S L A 遗传背景,深入研究S L A -1分子相关的抗原递呈和免疫应答机制,本研究对27头荣昌猪采集抗凝血,分离外周血淋巴细胞,提取总R N A ,设计特异性引物对S L A -1基因进行R T -P C R 扩增㊁克隆和测序,对获得序列的分子特征进行分析㊂结果显示,荣昌猪中共获得11个S L A -1等位基因,其中,9个为新等位基因,全部获得G e n B a n k 登录号和I S A G S L A 命名委员会官方命名,S L A -1*24:01等位基因在该群体中频率最高㊂S L A -1基因编码区全长1086b p,存在127个核苷酸多态位点,非同义单核苷酸多态性位点数高于同义单核苷酸多态性位点数㊂核苷酸多样度为0.0449,单倍型多样度为1,平均核苷酸差异数为48.782,G+C 含量为64.9%㊂S L A -1基因编码的361个氨基酸中,有75个氨基酸变异位点;第2和第3外显子编码区多态性最高,且第2外显子区域的氨基酸变异程度高于第3外显子区,组成抗原肽结合槽6个口袋的33个关键氨基酸位点中,11个在人与荣昌猪之间高度保守,与β2-微球蛋白结合的19个关键氨基酸位点中,10个保持一致,C D 8分子与MH C 结合的关键氨基酸位点中,只有225(T h r /S e r )和228(T h r /M e t )位点不同㊂同源性和系统进化树分析表明,S L A -1*10:03和S L A -1*18:03分别与人H L A -A *02:01和H L A -A *11:01等位基因的同源性最高,荣昌猪与亚洲野猪㊁巴马小型猪和融水小型猪等亚洲猪种具有较近的亲缘关系㊂本研究成功获得了中国地方猪种荣昌猪S L A -1基因,发现其具有极为丰富的多态性,为揭示荣昌猪的S L A 遗传背景和开展异种移植研究奠定了遗传学基础㊂关键词:荣昌猪;S L A -1等位基因;鉴定;分子特征中图分类号:S 852.4 文献标志码:A 文章编号:0366-6964(2023)07-3064-14收稿日期:2022-11-17基金项目:国家重点研发计划项目(2021Y F F 0703000);国家自然科学基金(31872313);中央级公益性科研院所基本科研业务费专项(1610302022018);兽医生物技术国家重点实验室课题(S K L V B P 202120;S K L V B P 202101)作者简介:刘弘毅(1997-),男,湖南常德人,硕士生,主要从事实验动物免疫遗传学研究,E -m a i l :m 175********@163.c o m*通信作者:夏长友,主要从事实验动物学研究,E -m a i l :x i a c h a n g y o u @c a a s .c n ;高彩霞,主要从事实验动物免疫遗传学研究,E -m a i l :ga o c a i x i a @c a a s .c nI d e n t i f i c a t i o n o f S L A -1A l l e l e s a n d A n a l ys i s o f M o l e c u l a r G e n e t i c C h a r a c t e r i s t i c s i n R o n g c h a n g P i gs L I U H o n g y i ,L U O T i n g y u ,L I C h a n g w e n ,Y U H a i b o ,L U X i a o y e ,C H E N H o n g ya n ,X I A C h a n g yo u *,G A O C a i x i a *(S t a t e K e y L a b o r a t o r y f o r A n i m a l D i s e a s e C o n t r o l a n d P r e v e n t i o n ,H e i l o n g j i a n g P r o v i n c i a l K e y L a b o r a t o r y o f L a b o r a t o r y A n i m a l a n d C o m p a r a t i v e M e d i c i n e ,N a t i o n a l P o u l t r y L a b o r a t o r yA n i m a l R e s o u r c e C e n t e r ,H a r b i n V e t e r i n a r y R e s e a r c h I n s t i t u t e ,C h i n e s e A c a d e m y o fA gr i c u l t u r a l S c i e n c e s ,H a r b i n 150069,C h i n a )A b s t r a c t :T h e s t u d y a i m e d t o c l a r i f y t h e S L A g e n e t i c b a c k g r o u n d o f e x p e r i m e n t a l R o n g c h a n g p i gs a n d f u r t h e r s t u d y S L A -1m o l e c u l e -r e l a t e d a n t i g e n p r e s e n t a t i o n a n d i mm u n e r e s po n s e m e c h a n i s m.7期刘弘毅等:实验用荣昌猪S L A-1等位基因鉴定及分子遗传特征分析I n t h i s s t u d y,t h e a n t i c o a g u l a n t b l o o d s w e r e c o l l e c t e d f r o m27R o n g c h a n g p i g s,a n d p e r i p h e r a l b l o o d l y m p h o c y t e s w e r e i s o l a t e d.T o t a l R N A w e r e e x t r a c t e d a n d S L A-1g e n e s w e r e a m p l i f i e d u s i n g s p e c i f i c p r i m e r s w i t h R T-P C R m e t h o d,c l o n e d a n d s e q u e n c e d.M o l e c u l a r g e n e t i c c h a r a c t e-r i s t i c s o f t h e o b t a i n e d s e q u e n c e s w e r e f u r t h e r a n a l y z e d.T h e r e s u l t s s h o w e d t h a t a t o t a l o f11 S L A-1a l l e l e s w e r e o b t a i n e d i n R o n g c h a n g p i g s,o f w h i c h n i n e w e r e n o v e l a l l e l e s.A l l a l l e l e s o b-t a i n e d G e n B a n k a c c e s s i o n n u m b e r s a n d o f f i c i a l n a m e s w e r e a s s i g n e d b y t h e I S A G S L A N o m e n c l a-t u r e C o mm i t t e e.T h e f r e q u e n c y o f S L A-1*24:01a l l e l e w a s t h e h i g h e s t i n t h i s p o p u l a t i o n.T h e f u l l-l e n g t h o f t h e c o d i n g r e g i o n o f S L A-1g e n e w a s1086b p,w i t h127n u c l e o t i d e p o l y m o r p h i c s i t e s.T h e n u m b e r o f n o n-s y n o n y m o u s S N P s w a s h i g h e r t h a n t h a t o f s y n o n y m o u s S N P s.T h e n u-c l e o t i d e d i v e r s i t y w a s0.0449,t h e h a p l o t y p e d i v e r s i t y w a s1,t h e a v e r a g e n u c l e o t i d e d i f f e r e n c e n u m b e r w a s48.782,a n d t h e G+C c o n t e n t w a s64.9%.T h e r e w e r e75a m i n o a c i d v a r i a t i o n s i t e s a m o n g361a m i n o a c i d s e n c o d e d b y S L A-1g e n e.T h e c o d i n g r e g i o n o f e x o n2a n d e x o n3h a d t h e h i g h e s t p o l y m o r p h i s m,a n d t h e d e g r e e o f a m i n o a c i d v a r i a t i o n i n e x o n2r e g i o n w a s h i g h e r t h a n t h a t i n e x o n3r e g i o n.A m o n g t h e33k e y a m i n o a c i d s i t e s t h a t m a k e u p t h e s i x p o c k e t s o f t h e p e p-t i d e-b i n d i n g g r o o v e,11o f t h e m w e r e h i g h l y c o n s e r v e d b e t w e e n h u m a n a n d R o n g c h a n g p i g,a n d 10o f t h e19k e y a m i n o a c i d s i t e s w e r e c o n s i s t e n t w i t hβ2-m i c r o g l o b u l i n.O n l y t w o s i t e s,225 (T h r/S e r)a n d228(T h r/M e t),w e r e d i f f e r e n t a m o n g t h e k e y a m i n o a c i d s i t e s t h a t C D8m o l e-c u l e s b i n d t o MH C.T h e a n a l y s i s o f h o m o l o g y a n d p h y l o g e n e t i c t r e e s h o w e d t h a t S L A-1*10:03 a n d S L A-1*18:03h a d t h e h i g h e s t h o m o l o g y w i t h h u m a n H L A-A*02:01a n d H L A-A*11:01a l-l e l e s,r e s p e c t i v e l y.R o n g c h a n g p i g w a s c l o s e l y r e l a t e d t o A s i a n b o a r,B a m a m i n i a t u r e p i g,R o n g-s h u i m i n i a t u r e p i g a n d o t h e r A s i a n p i g b r e e d s.T h e S L A-1a l l e l e s o f C h i n e s e R o n g c h a n g p i g s w e r e s u c c e s s f u l l y i d e n t i f i e d,a n d i t w a s f o u n d t o h a v e h i g h l y r i c h p o l y m o r p h i s m.T h e r e s u l t s o f t h i s s t u d y p r o v i d e d a g e n e t i c f o u n d a t i o n f o r r e v e a l i n g t h e S L A g e n e t i c b a c k g r o u n d o f R o n g c h a n g p i g s a n d c o n d u c t i n g x e n o t r a n s p l a n t a t i o n r e s e a r c h.K e y w o r d s:R o n g c h a n g p i g;S L A-1a l l e l e;i d e n t i f i c a t i o n;m o l e c u l a r c h a r a c t e r i s t i c s*C o r r e s p o n d i n g a u t h o r s:X I A C h a n g y o u,E-m a i l:x i a c h a n g y o u@c a a s.c n;G A O C a i x i a,E-m a i l: g a o c a i x i a@c a a s.c n主要组织相容性复合体(m a j o r h i s t o c o m p a t i-b i l i t y c o m p l e x,MH C)是一组编码主要组织相容性抗原的基因群总称,最早在小鼠体内被发现[1-2]㊂MH C作为组织细胞的遗传标记分子,与动物的抗病性㊁抗原识别㊁递呈和免疫调节密切相关[3-5]㊂此外,MH C编码的高度多态的蛋白对器官移植成功与否具有关键作用[6-9]㊂猪MH C又称猪白细胞抗原(s w i n e l e u k o c y t e a n t i g e n,S L A),位于猪第7号染色体并横跨着丝点,是包含猪基因组中许多免疫相关基因的紧密连锁区域[10-11]㊂S L A基因包含高度多态的S L A I类㊁S L A I I类和相对保守的S L A Ⅲ类基因,高度多态的S L A-1基因是经典的S L A I 类基因,目前I P D-MH C(I mm u n o P o l y m o r p h i s m D a t a b a s e-MH C)数据库共收录了89个S L A-1基因(R e l e a s e3.9.0.107/2022b u i l d209)㊂S L A-1基因编码的S L A I类分子通过内源性抗原递呈,将胞内的抗原肽递呈到细胞表面,经C D8+T细胞识别并引起细胞免疫,对于机体的健康以及对疾病的抗病性具有重要作用㊂S L A-1包含8个外显子,第1外显子编码前导多肽链,第2~4外显子分别编码α1~α3胞外域,第5外显子编码跨膜结构域,第6~ 8外显子编码胞内域,其中,第2和第3外显子编码的α1和α2胞外域多态性最高,二者形成的抗原肽结合槽(p e p t i d e-b i n d i n g g r o o v e,P B G)决定了与之结合的T淋巴细胞表位的特异性[12]㊂有研究利用S L A-1分子体外构建S L A I类复合体研究与病毒T细胞表位结合的特异性,发现不同S L A-1分子结合T细胞表位的能力不同[13],这就提示携带不同S L A-1等位基因的猪对疫病的抵抗力不同㊂猪是世界范围内广泛培育的重要经济动物,是5603畜牧兽医学报54卷人们日常生活主要的动物蛋白来源,而且由于猪与人类在基因组㊁血液生理指标㊁解剖学结构和成分等方面高度相似,使其逐渐成为一种重要的实验动物,应用于临床器官移植㊁动物疾病模型构建和病原致病机制研究等领域[14-17]㊂荣昌猪是中国西南地区的本土猪种,由于其肉质好和抗逆性强成为中国主要的优良猪种之一,目前,已在各领域表现出重要的应用价值,如用于建立人类医学动物模型,M I T F-M基因突变形成的白化耳聋荣昌猪可以作为一种人类遗传性神经性听力缺陷的大型哺乳动物模型,在人工耳蜗应用以及研究中国耳聋遗传方面具有十分重要的意义[18-20]㊂同时荣昌猪具有较强的抗病性,已用于建立猪病原感染模型并研究自身免疫机制,为重大疫病防控提供支撑,同时也用于筛选独特的免疫相关分子,为抗病育种奠定基础㊂2019年,中国农业科学院哈尔滨兽医研究所从重庆市畜牧科学院生物工程研究所引入27头荣昌猪,屏障环境饲养繁育,主要用于培育标准化的实验猪种群,同时开展了疫病净化㊁疾病模型构建及应用研究㊂本研究就该荣昌猪种群S L A-1基因特征进行分析,旨在为标准化实验荣昌猪的培育和推广应用奠定基础,也为异种移植研究提供遗传学参考㊂1材料与方法1.1试验材料通过前腔静脉采集中国农业科学院哈尔滨兽医研究所保存的27头荣昌猪(:6,:21)的抗凝血5m L,置于E D T A抗凝管,用于分离外周血淋巴细胞,27头荣昌猪的编号如表1所示㊂1.2主要试剂猪外周血淋巴细胞分离液购自天津市灏洋生物制品科技有限公司,S i m p l y总R N A提取试剂盒购自B i o s h a r p公司,D N A胶回收试剂盒购自OM E-G A公司,O n e s t e p P C R试剂盒㊁感受态细胞大肠杆菌(E s c h e r i c h i a c o l i)D H5α和载体p M D18-T v e c t o r均购自宝生物工程(大连)有限公司㊂1.3外周血淋巴细胞的分离和R N A的提取根据猪外周血淋巴细胞分离液说明书,将猪外周血淋巴细胞分离,-80ħ保存㊂根据S i m p l y总R N A提取试剂盒说明书提取外周血淋巴细胞总R N A,利用超微量分光光度计(N a n o P h o t o m e t e r,德国)测量总R N A浓度,-80ħ保存㊂1.4引物设计与合成参考G e n B a n k中大白猪H01单倍型序列(登录号:A J251829),利用P r i m e r P r e m i e r5软件设计引物扩增S L A-1基因,扩增片段约为1537b p㊂S L A1-F:5'-C T C A G C T T C T C C C C A G A C C C C G A-G G C T G A G G A T C-3',S L A1-R:5'-G G A T T C T G G A A G G T T C T C A A T C C T T C C A T T T A T T T C C T C-3'㊂引物由吉林省库美生物科技有限公司合成㊂1.5S L A-1的扩增和测序利用O n e S t e p P C R试剂盒以及合成的引物对目的基因进行扩增㊂R T-P C R反应体系50μL:P r i-m e S c r i p t1S t e p E n z y m e M i x2μL,2ˑ1s t e p B u f f e r 25μL,上㊁下游引物(10p m o l㊃μL-1)各1.5μL,R N A 模板2μL(约300n g㊃μL-1),R N a s e F r e e H2O 18μL㊂P C R反应条件:50ħ反转录30m i n;94ħ预变性2m i n;94ħ变性30s,55ħ退火30s,72ħ延伸1m i n,35次循环;72ħ延伸8m i n㊂利用1%琼脂糖凝胶电泳对扩增产物进行鉴定,由吉林省库美生物科技有限公司直接测序㊂对于杂合子,利用胶回收试剂盒纯化目的片段,与p M D-18T载体在16ħ条件下连接2h,然后转化至大肠杆菌D H5α感受态细胞中,并利用含A m p抗性的L B平板进行筛选㊂鉴定阳性单克隆菌落后,送至吉林省库美生物科技有限公司测序㊂1.6序列比对及分析测序获得的原始序列数据通过C h r o m a s V e r-s i o n2.6.6进行人工编辑,校对电泳峰图与碱基对应关系㊂利用M E G A7.0软件对直接测序和克隆测序结果进行比对,根据不同猪个体中S L A-1基因序列差异确定等位基因序列㊂当同一个体克隆测序中至少3个单克隆的序列相同且该序列在不同个体的直接或克隆测序中多次出现,判定为一个等位基因,即一个等位基因的判定必须至少出现在2个不同的个体或来自2个独立的P C R反应㊂利用D n a S P5.10.01软件进行等位基因多态性分析,利用D N A S T A R L a s e r g e n e软件进行同源性分析,以人白细胞抗原(h u m a n l e u k o c y t e a n t i g e n,H L A)频率最高的H L A-A*02:01等位基因(G e n B a n k登录号:F N806801)为参照[21],利用M E G A7.0采用邻接法(N e i g h b o r-J o i n i n g,N-J)构建系统进化树㊂所有序列提交G e n B a n k获得登录号㊂将新等位基因提交国际动物遗传学学会(T h e I n t e r n a t i o n a l S o c i e-t y o f A n i m a l G e n e t i c s,I S A G)S L A命名委员会获得66037期刘弘毅等:实验用荣昌猪S L A -1等位基因鉴定及分子遗传特征分析官方命名㊂S L A 等位基因的命名采用H L A 命名系统,等位基因前两位数字用于指定具有相似D N A序列的等位基因组( 组 是基于系统发育分析和D N A 序列基序的识别);第3和第4位数字用于指定具有不同蛋白质序列的等位基因;第5和第6位数字用于指定因同义核苷酸替换而不同的等位基因;以冒号(:)作为区域分隔符㊂2 结 果2.1 R T -P C R 扩增与克隆R T -P C R 扩增获得1537b p 左右的目的条带(图1),清晰且单一,表明已成功扩增S L A -1基因㊂将27头猪的扩增产物全部进行直接测序,分析测序结果,发现4头猪测序结果为单峰(猪编号为3㊁9㊁13和27),且序列相同,表明是纯合子个体,对其中1头(3号猪)进行了克隆㊂其它23头猪均为套峰,表明为杂合子个体,将具有相同杂合套峰的个体进行分类后,分别对10个个体(猪编号为3㊁5㊁10㊁15㊁16㊁17㊁18㊁19㊁23和24)进行了克隆㊂所有克隆鉴定阳性单克隆菌落并测序(图2)㊂M.D L 2000D N A 分子标记;1.P C R 产物(猪编号2);2.P C R 产物(猪编号3);3.P C R 产物(猪编号5);4.P C R 产物(猪编号10);5.P C R 产物(猪编号14);6.P C R 产物(猪编号15);7.P C R 产物(猪编号17);8.P C R 产物(猪编号18)M.D L 2000D N A m a r k e r ;1.P C R p r o d u c t o f N o .2;2.P C R p r o d u c t o f N o .3;3.P C R p r o d u c t o f N o .5;4.P C Rpr o d u c t o f N o .10;5.P C R p r o d u c t o f N o .14;6.P C R pr o d u c t o f N o .15;7.P C R p r o d u c t o f N o .17;8.P C R pr o d u c t o f N o .18图1 S L A -1基因部分扩增结果F i g .1 P a r t i a l a m pl i f i c a t i o n r e s u l t s o f S L A -12.2 S L A -1等位基因序列分析分析27头荣昌猪S L A -1基因直接测序和克隆测序结果,根据等位基因的判定原则共鉴定出11个等位基因(表1),将其提交G e n B a n k 数据库和I P D -MH C 数据库,分别获得登录号和I S A G S L A 命名委员会的官方命名(表2)㊂与I P D -MH C 数据库中M.D L 2000D N A 分子标记;1~4.编号10的单克隆鉴定部分结果;5~8.编号14的单克隆鉴定结果M.D L 2000D N A m a r k e r ;1-4.P a r t i a l r e s u l t s o f n o .10m o n o c l o n a l i d e n t i f i c a t i o n ;5-8.P a r t i a l r e s u l t s o f n o .14m o n -o c l o n a l i d e n t i f i c a t i o n图2 部分阳性单克隆菌落P C R 扩增结果F i g.2 P a r t i a l r e s u l t s o f m o n o c l o n a l i d e n t i f i c a t i o n 已提交的89个S L A -1等位基因序列相比较,获得2个已知等位基因,S L A -1*11:03和S L A -1*15:03,分别存在于韩国地方猪种和中国融水小型猪㊁皖南花猪中㊂此外,发现9个新等位基因,命名为S L A -1*02:04㊁02:05㊁10:03㊁16:05㊁16:06㊁18:03㊁23:05㊁24:01和25:01㊂27头荣昌猪均至少包含1个S L A -1新等位基因,且10头荣昌猪中至少包含3个S L A -1等位基因,编号10的个体中含有4个不同的S L A -1等位基因,表明S L A -1是双拷贝基因㊂在该群体中,对被鉴定出在S L A -1基因座上具有两个以上等位基因的个体进行等位基因之间的两两组合分析,对在不同基因型个体中出现次数超过3次以上的组合确定为同一条染色体上的双拷贝等位基因,在编号分别为1㊁6㊁10㊁18㊁24㊁25的个体中不仅鉴定出S L A -1*23:05㊁S L A -1*11:03等位基因而且还有其他等位基因存在,因此在该群体中S L A -1*23:05与S L A -1*11:03,S L A -1*02:04与S L A -1*18:03之间存在连锁关系㊂连锁的等位基因S L A -1*23:05和S L A -1*11:03以及S L A -1*02:04和S L A -1*18:03分别用双拷贝等位基因A 和B表示,等位基因S L A -1*02:05㊁S L A -1*10:03㊁S L A -1*15:03㊁S L A -1*16:05㊁S L A -1*16:06㊁S L A -1*24:01㊁S L A -1*25:01分别用等位基因C ㊁D ㊁E ㊁F ㊁G ㊁H ㊁I 表示,该群体在S L A -1基因座上共获得11种基因型(表1)㊂群体的等位基因频率显示,频率最高的是等位基因H (S L A -1*24:01),频率为44.23%,其次是双拷贝等位基因A (S L A -1*23:05,S L A -1*11:03)和等位基因E (S L A -1*15:03),频率分别为19.23%㊁17.31%,H (S L A -1*24:01)是该荣昌猪群体中最流行的等位基因(图3)㊂7603畜 牧 兽 医 学 报54卷表1 荣昌猪S L A -1等位基因T a b l e 1 S L A -1a l l e l e o f R o n g c h a n g p i g荣昌猪编号N o .o f R o n g c h a n g性别S e xS L A -1等位基因A l l e l e s o f S L A -1S L A -1基因型1G e n o t y pe s of S L A -11123:05,11:03,24:01A H215:03,24:01E H324:01HH515:03,24:01E H623:05,11:03,15:03A E723:05,11:03,24:01A H823:05,11:03,24:01A H924:01HH 1023:05,11:03,02:04,18:03A B1115:03,24:01E H 1216:05,24:01F H 1324:01HH 1416:05,24:01F H 1515:03,16:06E G1616:05,24:01F H 1710:03,15:03,24:01-1823:05,11:03,02:05A C 1923:05,11:03,15:03A E2015:03,24:01E H 2116:05,24:01F H2310:03,24:01D H2423:05,11:03,25:01A I2523:05,11:03,16:05A F2615:03,24:01E H 2724:01HH 2010315:03,24:01E H2020323:05,11:03,24:01A H1.A~I 所表示的等位基因见表2;-.基因型无法判断1.T h e a l l e l e s r e p r e s e n t e d b y A t o I a r e m a r k e d i n t a b l e 2;-.G e n o t y pe c a n n o t b e d e t e r m i n e d 表2 荣昌猪S L A -1等位基因提交信息T a b l e 2 S u b m i s s i o n i n f o r m a t i o n f o r S L A -1a l l e l e i n R o n g c h a n g p i g 已鉴定等位基因I d e n t i f i e d a l l e l e等位基因A l l e l e G e n B a n k 登录号G e n B a n k a c c e s s i o n n u m b e rS L A -1*23:05,S L A -1*11:03A O N 859881,O P 249499S L A -1*02:04,S L A -1*18:03BO N 859887,O N 859888S L A -1*02:05C O N 859886S L A -1*10:03DO N 859883S L A -1*15:03E O P 249500S L A -1*16:05F O N 859884S L A -1*16:06G O N 859885S L A -1*24:01HO N 859880S L A -1*25:01IO N 85989086037期刘弘毅等:实验用荣昌猪S L A -1等位基因鉴定及分子遗传特征分析图3 S L A -1等位基因及频率分布F i g .3 T h e d i s t r i b u t i o n o f S L A -1a l l e l e s a n d f r e q u e n c y2.3 S L A -1等位基因多态性与二级结构分析11个S L A -1等位基因编码区(c o d i n g se -qu e n c e s ,C D S )D N A 多态性分析显示(图4),碱基变异(b a s e v a r i a t i o n ,S )主要集中在225~325b p 和500~575b p 两个区域,核苷酸多样度(n u c l e o t i d e d i v e r s i t y,P i )的变化趋势与碱基变异数的分布趋势基本保持一致,而且两者的峰值均处于第2和第3外显子区域内,表示该区域是S L A -1基因的高频变异位点区㊂在C D S 区内,整体的P i 为0.0449,单倍型多样度(h a p l o t y p e d i v e r s i t y,H d )为1,平均核苷酸差异数(a v e r a ge n u m b e r of n u c l e o t i d e d i f f e r e n c e s ,K )为48.782,G+C 含量为64.9%,在所编码的361个氨基酸中,产生了75个氨基酸变异位点(a m i n oa c i d v a r i ab l e s i t e s ,A V );在1~1086b p 的核苷酸位点中,涵盖了127个核苷酸多态位点,其中,包含简约信息位点85个,单一多态位点42个,此外,核苷酸多态位点包含18个同义单核苷酸多态性位点和109个非同义单核苷酸多态性位点㊂第2和第3外显子区域的多态性规律与整个C D S 区变异相似(表3)㊂图4 S L A -1等位基因碱基变异位置分布(A )和核苷酸多样度变化趋势(B )F i g .4 T h e d i s t r i b u t i o n o f b a s e v a r i a t i o n p o s i t i o n s (A )a n d n u c l e o t i d e d i v e r s i t y (B )o f S L A -1a l l e l e s e qu e n c e 由于S L A 提呈不同抗原,因此第2和第3外显子编码的α1和α2结构域构成的P B G 多态性较高,对荣昌猪S L A -1编码蛋白的α1和α2结构域的氨基酸序列变异性进行分析,结果显示,有14个氨基酸位点的W u -K a b a t 指数高于6.0,其中,第74个氨基酸指数最高,达到13.8(图5A )㊂第2外显子区域的氨基酸变异程度高于第3外显子,且变异程度最高的位点位于第2外显子区域㊂此外,由于S L A -1*04:01分子所编码的蛋白结构已经解析[22],共存在33个关键氨基酸组成P B G 的A~F 6个口袋,因此将获得的荣昌猪S L A -1的α1和α2结构域氨基酸序列与S L A -1*04:01进行比对分9603畜牧兽医学报54卷表3S L A-1等位基因序列多态性分析T a b l e3P o l y m o r p h i s m a n a l y s i s o f S L A-1a l l e l e s e q u e n c e s序列信息S e q u e n c e i n f o r m a t i o nS L A-1编码区S L A-1C D S r e g i o n第2和第3外显子E x o n2a n d e x o n3比对序列范围/b p A l i g n e d S e q.r e g i o n 1~108665~610核苷酸数N u m b e r o f n u c l e o t i d e 1086546核苷酸保守性位点数量N u m b e r o f n u c l e o t i d e i n v a r i a b l e s i t e s 959444核苷酸多态位点数量N u m b e r o f n u c l e o t i d e v a r i a b l e s i t e s 127102简约信息位点数量N u m b e r o f n u c l e o t i d e p a r s i m o n y i n f o r m a t i o n s i t e s 8565单一多态位点数量N u m b e r o f n u c l e o t i d e s i n g l e t o n v a r i a b l e s i t e s 4237同义单核苷酸多态性数量N u m b e r o f s y n o n y m o u s S N P(S i n g l e n u c l e o t i d e p o l y m o p h i s m)187非同义单核苷酸多态性数量N u m b e r o f n o s y n o n y m o u s S N P 10995氨基酸数量N u m b e r o f a m i n o a c i d s 361182氨基酸变异位点数量N u m b e r o f a m i n o a c i d s v a r i a b l e s i t e s 7561G+C含量G+C c o n t e n t 0.6490.653平均核苷酸差异数量A v e r a g e n u m b e r o f n u c l e o t i d e d i f f e r e n c e s 48.78239.145单倍型多样性H a p l o t y p e d i v e i t y 1.0001.000核苷酸多样性N u c l e o t i d e d i v e r s i t y0.04490.0717析,结果显示,182个氨基酸位点中,121个位点完全保守,而33个关键氨基酸位点中,11个氨基酸位点完全保守,分别为L e u5(L)㊁T y r7(Y)㊁M e t45(M)㊁T y r59(Y)㊁L e u81(L)㊁T y r84(Y)㊁T y r123(Y)㊁L y s146(K)㊁T y r159(Y)㊁L e u160(L)㊁T y r171(Y),其余22个位点均发生高频率突变(图5B)㊂比对H L A-A*02:01㊁H L A-A*11:01与荣昌猪S L A-1等位基因氨基酸序列,结果显示,在与β2-微球蛋白(β2-m i c r o g l o b u l i n,β2m)结合的19个氨基酸位点中,有10个氨基酸在人H L A-A与荣昌猪S L A-1等位基因中保持一致,分别是T y r7(Y)㊁P h e8(F)㊁V a l25(V)㊁T y r27(Y)㊁P h e33(F)㊁P h e36(F)㊁H i s93 (H)㊁L e u110(L)㊁G l y112(G)和G l n115(Q)㊂另外,比较人和荣昌猪C D8分子与MH C结合的7个关键氨基酸位点(223~229)[23],只有225(T/S)和228(T/M)两个位点存在差异,其它位点高度保守(表4)㊂二级结构分析发现,荣昌猪不同S L A-1等位基因显示出S L A-1基因的典型二级结构,α1和α2区主要为α-螺旋和β-折叠,α1区具有1个α-螺旋和4个β-折叠,α2区具有4个α-螺旋和4个β-折叠(图5B)㊂2.4S L A-1等位基因同源性分析荣昌猪不同S L A-1等位基因的核苷酸相似性为93.6%~99.4%,氨基酸相似性为86.5%~ 98.3%,其中,S L A-1*02:04和S L A-1*02:05同源性最高,核苷酸和氨基酸相似性分别为99.4%和98.3%,二者存在7个核苷酸变异和6个氨基酸变异㊂荣昌猪与I P D-MH C数据库中已提交的69个具有C D S全长序列的S L A-1等位基因相似性分析显示,核苷酸序列和氨基酸序列分别为92.6%~07037期刘弘毅等:实验用荣昌猪S L A -1等位基因鉴定及分子遗传特征分析▼.人白细胞抗原(H L A )与β2-微球蛋白(β2m )结合位点,*.S L A -1*04:01等位基因抗原肽结合槽关键位点▼.A m i n o a c i d p o s i t i o n b i n d i n g h u m a n l e u k o c y t e a n t i g e n (H L A )a n d β2-m i c r o g l o b u l i n (β2m ),*.K e y a m i n o a c i d p o s i t i o n o f P B G l o c a t e d i n S L A -1*04:01a l l e l e 图5 荣昌猪S L A -1等位基因第2和第3外显子区域氨基酸变异性(A )和氨基酸序列比对(B )F i g .5 T h e a m i n o a c i d v a r i a b i l i t y (A )a n d c o m p a r i s o n (B )o f e x o n 2a n d e x o n 3o f S L A -1a l l e l e s i n R o n g c h a n g p i g99.9%和85.9%~99.4%㊂荣昌猪S L A -1等位基因和人群中流行的H L A -A *02:01和H L A -A *11:01等位基因的核苷酸相似性为83.3%~86.8%,氨基酸相似性为70.3%~76.9%,其中,与H L A -A *02:01相似性最高的等位基因是S L A -1*10:03(核苷酸85.0%,氨基酸74.4%),与H L A -A *11:01相似性较高的等位基因是S L A -1*18:03(核苷酸86.8%,氨基酸76.9%)(表5)㊂2.5 S L A -1等位基因系统进化树分析从I P D -MH C 数据库中选取与荣昌猪S L A -1等位基因核苷酸相似性较高(93.3%~99.9%)的31个具有C D S 全长的S L A -1等位基因,与荣昌猪获得的11个S L A -1等位基因序列比对,以人群中H L A -A 频率最高的H L A -A *02:01和H L A -A*11:01等位基因为外群对照,利用邻接法构建系统进化树(图6)㊂结果显示,荣昌猪中9个新等位基因分布在不同聚类分支中,根据系统进化树,I S A GS L A 命名委员会对其进行了命名㊂S L A -1*02:04和S L A -1*02:05聚在02等位基因组分支中,S L A -1*02:05与S L A -1*02:02具有3个核苷酸差异,S L A -1*02:04与S L A -1*02:01具有4个核苷酸差异,与多种猪品种和猪细胞系亲缘关系较近,如:1703畜牧兽医学报54卷表4人和荣昌猪C D8分子与M H C结合的关键氨基酸氨基酸位点比较T a b l e4C o m p a r i s o n o f k e y a m i n o a c i d p o s i t i o n o f h u m a n a n d R o n g c h a n g p i g C D8m o l e c u l e s c o m b i n e w i t h M H C等位基因A l l e l e氨基酸位置A m i n o a c i d p o s i t i o n 223224225226227228229H L A-A*02:01A s p(D)G l n(Q)T h r(T)G l n(Q)A s p(D)T h r(T)G l u(E) H L A-A*11:01ӘӘӘӘӘӘӘS L A-1*02:04ӘӘS e r(S)ӘӘM e t(M)ӘS L A-1*02:05ӘӘS e r(S)ӘӘM e t(M)ӘS L A-1*10:03ӘӘS e r(S)ӘӘM e t(M)ӘS L A-1*11:03ӘӘS e r(S)ӘӘM e t(M)ӘS L A-1*15:03ӘӘS e r(S)ӘӘM e t(M)ӘS L A-1*16:05ӘӘS e r(S)ӘӘM e t(M)ӘS L A-1*16:06ӘӘS e r(S)ӘӘM e t(M)ӘS L A-1*18:03ӘӘS e r(S)ӘӘM e t(M)ӘS L A-1*23:05ӘӘS e r(S)ӘӘM e t(M)ӘS L A-1*24:01ӘӘS e r(S)ӘӘM e t(M)ӘS L A-1*25:01ӘӘS e r(S)ӘӘM e t(M)ӘӘ.与H L A-A*02ʒ01相同的氨基酸Ә.S a m e a m i n o a c i d w i t h H L A-A*02:01表5荣昌猪S L A-1和I P D-M H C数据库中提交的S L A-1以及人H L A-A流行等位基因的相似性比较T a b l e5C o m p a r i s o n o f n u c l e o t i d e a n d a m i n o a c i d h o m o l o g y a m o n g S L A-1a l l e l e s o f R o n g c h a n g p i g,S L A-1a l l e l e s s u b m i t t e d i nI P D-M H C d a t a b a s e a n d f r e q u e n t h u m a n H L A-A a l l e l e s%同源性H o m o l o g y荣昌猪S L A-1等位基因S L A-1a l l e l e s o fR o n g c h a n g p i gI P D-MH C数据库S L A-1等位基因S L A-1a l l e l e s o fI P D-MH C d a t a b a s eH L A-A*02:01等位基因H L A-A*02:01a l l e l eH L A-A*11:01等位基因H L A-A*11:01a l l e l e核苷酸相似性N u c l e o t i d e h o m o l o g y 93.6~99.492.6~99.983.3~85.085.1~86.8氨基酸相似性A m i n o a c i d h o m o l o g y86.5~98.385.9~99.770.3~74.472.8~76.9辛克莱小型猪㊁N I H小型猪和小型猪肾细胞系等㊂S L A-1*10:03等位基因与汉福德㊁大花白以及融水小型猪中获得的S L A-1*10:01等位基因聚于同一分支,与S L A-1*10:01具有5个核苷酸差异㊂S L A-1*16:05和S L A-1*16:06遗传距离较近,与S L A-1*16:03聚为一支,属于16等位基因组,特别是S L A-1*16:05与S L A-1*16:03仅有一个核苷酸差异,两个等位基因均与亚洲野猪的亲缘关系较高㊂S L A-1*18:03与S L A-1*18:01聚于同一分支㊂S L A-1*23:05独立聚为一支,表明有特定的肽结合基序,而且与S L A-1*11:10和S L A-1*23:01聚为一大类分支,表明与巴马小型猪亲缘关系较近㊂S L A-1*24:01与亚洲野猪㊁巴马小型猪中获得的S L A-1*19:01聚于一大支,表明荣昌猪和其亲缘关系较近㊂S L A-1*25:01独立分为一支,表明也具有独特的肽结合基序㊂此外,已知的S L A-1*11:03和S L A-1*15:03等位基因在韩国地方猪㊁中国融水小型猪和皖南花猪也存在,表明这些品种猪可能有相同血缘关系的祖先㊂3讨论实验猪在畜牧兽医生产实践中发挥重要作用,被广泛用于研究病原致病机制㊁T细胞表位识别和疫苗评估等,在这些研究中S L A对抗原的识别具有严格的MH C限制性,从而引起个体差异[24-27]㊂因此鉴定S L A基因可减少实验动物遗传背景差异对各项研究结果的影响㊂经典的S L A I类基因S L A-1编码的I类分子主要参与内源性抗原递呈,识别27037期刘弘毅等:实验用荣昌猪S L A -1等位基因鉴定及分子遗传特征分析һ.荣昌猪S L A -1等位基因һ.S L A -1a l l e l e s o f R o n g c h a n g p i g 图6 荣昌猪S L A -1等位基因系统发育树F i g .6 P h y l o g e n e t i c t r e e o f S L A -1a l l e l e f r o m R o n g c h a n g p i g3703畜牧兽医学报54卷抗原并诱导细胞免疫应答㊂S L A-1基因具有高度多态性,目前I P D-MH C数据库共收录了89个S L A-1等位基因(R e l e a s e3.9.0.107/2022b u i l d209),存在于不同的猪品种或猪细胞系㊂荣昌猪是我国优良的地方猪品种,已作为实验动物在动物医学模型㊁药物评价模型和异种器官供体等研究中广泛应用,但目前尚未对荣昌猪的S L A-1基因进行系统鉴定,本研究在荣昌猪S L A-1基因座上共获得11个等位基因,其中,9个为新等位基因,2个为已知等位基因,在现有群体数量有限的情况下S L A-1基因座表现出较高的多态性㊂荣昌猪S L A-1基因也发现了双拷贝现象㊂在之前的研究中,有学者发现S L A-1基因座双拷贝现象在韩国本地猪㊁S N U小型猪㊁N I H小型猪㊁巴克夏猪㊁约克夏猪㊁杜洛克猪㊁长白猪㊁梅山猪和巴马小型猪等众多猪品种中均存在,且部分双拷贝现象在某些S L A单倍型中持续存在[28-31]㊂一般认为,基因多样性或杂合度的增加有利于机体抵御不良环境,但无论是不同S L A基因座的等位基因杂合还是S L A-1基因座双拷贝杂合,在病原体和S L A等位基因之间的关系都是较为复杂的㊂首先,在同一个S L A基因座存在多个等位基因可能会增加其对不同抗原的结合能力,从而提高生存概率;其次,如果在群体中不存在对特定病原敏感的特定等位基因,即便杂合度很高,该种群对于病原的抵抗力依然较弱,而存在特定等位基因的纯合子群体却能够获得高的抵抗力,这可以从杂合子优势假说以及稀有等位基因优势假说得到解释[32-33]㊂S L A-1基因的第2和第3外显子所编码的抗原肽结合槽相较于其它区域具有非常高的多态性,这种现象在不同猪品种中都存在[34-36]㊂本研究对荣昌猪S L A-1等位基因的D N A多态性进行了分析,从核苷酸多态性位点数量占比和非同义核苷酸多态性位点数量的比例发现,第2和第3外显子区域都表现出绝对的优势㊂氨基酸变异位点数量以及氨基酸变异系数和位置与核苷酸多态性具有相似性,同样证明S L A-1基因的高度多态区域集中于第2和第3外显子㊂S L A I类分子主要通过递呈病原的抗原肽给C D8+T淋巴细胞识别[37],继而诱导细胞杀伤效应清除感染性细胞㊂抗原肽是通过结合S L A I类分子P B G中的6个口袋(A~F),然后与β2m形成复合物递呈在细胞表面供T细胞识别[38-39]㊂因此不同等位基因和递呈抗原肽的差异会对P B G的立体结构造成影响,利用已解析的S L A-1*04:01的三维结构,对荣昌猪S L A-1等位基因6个口袋的关键氨基酸进行比对,发现有较高的差异性,提示获得的等位基因可能展现出与S L A-1*04:01不同的三维结构㊂猪已被选定是最佳的异种移植供体,但猪-人异种器官/细胞移植主要障碍是人体对植入外源器官的免疫反应,其中S L A是与同种和异种免疫识别密切相关的一个重要分子标记基因,能被各种人类免疫细胞亚群直接识别[40]㊂因此异种移植成功与否的前提在于猪S L A与人H L A之间的适配性所导致的急性或慢性免疫排斥反应能否得到解决㊂比较荣昌猪S L A-1与β2m结合以及人和荣昌猪C D8分子与MH C结合的关键氨基酸位点,发现氨基酸位点保守性较高㊂将获得的S L A-1等位基因与人群体中频率较高的等位基因进行同源性分析,显示S L A-1*10:03和S L A-1*18:03相似性最高,预示着携带这两个等位基因的猪可能和人之间存在较高的适配性㊂目前,I P D-MH C数据库已收录了世界各类猪种以及猪细胞系的S L A-1等位基因,选取与荣昌猪相似性较高的S L A-1等位基因建立了系统进化树,发现荣昌猪种群中获得的S L A-1*15:03和S L A-1*11:03也分别存在于中国融水小型猪㊁皖南花猪以及韩国本地猪中,这些猪种起初都源自亚洲㊂获得的新S L A-1等位基因少部分与各类猪源细胞系亲缘性较高,大多数均与亚洲猪种亲缘性较高,因此可推测荣昌猪可能和这些猪种存在血缘相同的祖先㊂本研究获得的荣昌猪S L A-1等位基因遗传信息仅针对于饲养于屏障设施中这个小型封闭群,并不能代表全国整个荣昌猪品种的群体遗传信息,但是对标准化实验荣昌猪的培育和推广应用必不可少㊂此外在荣昌猪上获得的S L A-1基因也可用于深入研究病原的抗原递呈机制,筛选猪重要病原的S L A限制性T细胞表位,对开发安全有效的表位肽疫苗意义重大㊂4结论本研究通过对荣昌猪S L A-1基因C D S区域进行P C R扩增测序,成功获得了11个S L A-1等位基因,其中,9个为新发现等位基因,S L A-1*24:01是47037期刘弘毅等:实验用荣昌猪S L A-1等位基因鉴定及分子遗传特征分析该荣昌猪群体中最流行的等位基因㊂S L A-1基因多态性主要集中在第2和第3外显子区域,P B G中的33个关键氨基酸位点有11个位点完全保守;与β2m结合的19个关键氨基酸位点有10个位点在人与荣昌猪之间保持一致,C D8分子与MH C结合的关键氨基酸位点高度保守,只有225(T h r/S e r)和228(T h r/M e t)位点不同㊂同源性以及系统进化树表明,S L A-1*10:03和S L A-1*18:03分别与人H L A-A*02:01和H L A-A*11:01等位基因的同源性最高,荣昌猪与亚洲野猪㊁巴马小型猪和融水小型猪等亚洲猪种具有较近的亲缘关系㊂本研究结果为荣昌猪S L A遗传背景的研究以及抗原递呈过程中S L A与不同抗原肽的相互作用关系奠定基础㊂参考文献(R e f e r e n c e s):[1]马丽颖,岳秉飞.主要组织相容性复合体(MH C)研究进展[J].中国比较医学杂志,2005,15(3):182-185.MA L Y,Y U E B F.A d v a n c e s i n r e s e a r c h o n m a j o rh i s t o c o m p a t i b i l i t y c o m p l e x[J].C h i n e s e J o u r n a l o fC o m p a r a t i v e M e d i c i n e,2005,15(3):182-185.(i nC h i n e s e)[2]刘子展.贵州小型猪S L A经典Ⅰ类和Ⅱ类基因克隆和生物信息学分析及P C R-S S P分型方法的建立[D].合肥:安徽农业大学,2012.L I U Z Z.C l o n i n g a n d b i o i n f o r m a t i c s a n a l y s i s o fs w i n e l e u k o c y t e a n t i g e n s c l a s s I a n d c l a s s I I g e n e s i nG u i z h o u m i n i p i g s a n d e s t a b l i s h m e n t o f P C R-S S Pt y p i n g m e t h o d[D].H e f e i:A n h u i A g r i c u l t u r a lU n i v e r s i t y,2012.(i n C h i n e s e)[3] Y AMA G U C H I T,D I J K S T R A J M.M a j o rh i s t o c o m p a t i b i l i t y c o m p l e x(MH C)g e n e s a n d d i s e a s er e s i s t a n c e i n f i s h[J].C e l l s,2019,8(4):378. [4] C A R R I N G T O N M,B O N T R O P R E.E f f e c t s o fMH C c l a s s I o n H I V/S I V d i s e a s e i n p r i m a t e s[J].A I D S,2002,16:S105-S114.[5] R O C K K L,R E I T S E,N E E F J E S J.P r e s e n tY o u r s e l f!B y MH C c l a s s I a n d MH C c l a s s I Im o l e c u l e s[J].T r e n d s I mm u n o l,2016,37(11):724-737.[6] S E E T H A R A M A,T I R I V E E D H I V,M O H A N A K U M A RT.A l l o i mm u n i t y a n d a u t o i mm u n i t y i n c h r o n i cr e j e c t i o n[J].C u r r O p i n O r g a n T r a n s p l a n t,2010,15(4):531-536.[7] A F Z A L I B,L OM B A R D I G,L E C H L E R R I.P a t h w a y s o f m a j o r h i s t o c o m p a t i b i l i t y c o m p l e xa l l o r e c o g n i t i o n[J].C u r r O p i n O r g a n T r a n s p l a n t,2008,13(4):438-444.[8] W E I S S R A.X e n o t r a n s p l a n t a t i o n[J].B M J,1998,317(7163):931-934.[9] C O O P E R D K C,G A S T O N R,E C K HO F F D,e t a l.X e n o t r a n s p l a n t a t i o n-t h e c u r r e n t s t a t u s a n d p r o s p e c t s[J].B r M e d B u l l,2018,125(1):5-14.[10] C E L I S-G I R A L D O C T,B OHÓR Q U E Z M D,C AMA R G O M,e t a l.A c o m p a r a t i v e a n a l y s i s o fS L A-D R B1g e n e t i c d i v e r s i t y i n C o l o m b i a n(c r e o l e sa n d c o mm e r c i a l l i n e)a n d w o r l d w i d e s w i n ep o p u l a t i o n s[J].S c i R e p,2021,11(1):4340. [11]权金强,江新杰,李昌文,等.实验用S P F大白猪和长白猪S L A-1基因特征分析[J].农业生物技术学报,2017,25(5):770-780.Q U A N J Q,J I A N G X J,L I C W,e t a l.C h a r a c t e r i z a t i o n a n a l y s i s o f S L A-1g e n e f r o m S P Fy o r k s h i r e(S u s s c r o f a)a n d l a n d r a c e[J].J o u r n a l o fA g r i c u l t u r a lB i o t e c h n o l o g y,2017,25(5):770-780.(i n C h i n e s e)[12] HAMM E R S E,HO C S,A N D O A,e t a l.I m p o r t a n c e o f t h e m a j o r h i s t o c o m p a t i b i l i t y c o m p l e x(S w i n e l e u k o c y t e a n t i g e n)i n s w i n e h e a l t h a n db i o m e d ic a l r e s e a r c h[J].A n n u R e v A n i m B i o s c i,2020,8:171-198.[13] G A O C X,H E X W,Q U A N J Q,e t a l.S p e c i f i c i t yc h a r a c t e r i z a t i o n o f S L A c l a s s I m o l e c u l e s b i nd i n g t os w i n e-o r i g i n v i r a l c y t o t o x i c T l y m p h o c y t e e p i t o p ep e p t i d e s i n v i t r o[J].F r o n t M i c r o b i o l,2017,8:2524.[14] L U N N E Y J K.A d v a n c e s i n s w i n e b i o m e d i c a l m o d e lg e n o m i c s[J].I n t J B i o l S c i,2007,3(3):179-184.[15] S W I N D L E M M,MA K I N A,H E R R O N A J,e t a l.S w i n e a s m o d e l s i n b i o m e d i c a l r e s e a r c h a n dt o x i c o l o g y t e s t i n g[J].V e t P a t h o l,2012,49(2):344-356.[16] G E I S L E R K,KÜN Z E L J,G R U N D T N E R P,e t a l.T h e p e r f u s e d s w i n e u t e r u s m o d e l:l o n g-t e r m p e r f u s i o n[J].R e p r o d B i o l E n d o c r i n o l,2012,10:110.[17] R AM I R E Z-M E D I N A E,V U O N O E,R A I A,e t a l.D e l e t i o n o f E184L,a p u t a t i v e D I V A t a r g e t f r o m t h ep a n d e m i c s t r a i n o f a f r i c a n s w i n e f e v e r v i r u s,p r o d u c e sa r e d u c t i o n i n v i r u l e n c e a n d p r o t e c t i o n a g a i n s t v i r u l e n tc h a l l e n g e[J].J V i r o l,2022,96(1):e0141921.[18] C H E N W,Y I H J,Z HA N G L,e t a l.E s t a b l i s h i n gt h e s t a n d a r d m e t h o d o f c o c h l e a r i m p l a n t i nR o n g c h a n g p i g[J].A c t a O t o-L a r y n g o l,2017,1375703。
sla的组成部分和流程和注意事项
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SLA成型技术的应用(1)(精)
课程小结
光固化成型技术在生物医疗领域的应用 复杂外科手术的规划 头部、颌面修复 假体模具制作 组织工程 制作 光固化成型技术作为快速成型技术中发展最成熟的 技术 ，在生物医疗领域具有广阔的应用前景，正继 续向着工业化、产业化方向发展。
光固化成型的应用
1 2 3 4
概念原型制作 产品模型制造 快速模具制造 功能原件制造
5
难加工材料构件的制造
生物医疗-光固化成型非常有前景的一个应用领域。
光固化成型在生物医疗领域的应用
光固化成型在生物医疗领域的应用
SLA制造模型，以便策划头颅修补手术
光固化成型在生物医疗领域的应用
连体婴儿手术方案策划
光固化成型的应用
课程：激光3D打印技术 主讲教师：高尧
教学目标
了解光固化成型在生物医疗领域的应用现状
光固化成型
在应用较多的几种快速成型工艺方法中，光固 化成型由于具有成型过程自动化程度高、制作原 型表面质量好、尺寸精度高以及能够实现比较精 细的尺寸成型等特点，使之得到最为广泛的应用。
目前，已广泛应用于航空、汽车、电器、消费 品、文化创意以及生物医疗等行业。
概念原型制作产品模型制造产品模型制造难加工材料构件的制造sla制造模型以便策划头颅修补手术连体婴儿手术方案策划sla打印头颅模型sla制作的聚乳酸支架光固化前沿研究领域组织工程植入细胞并培养一天后的显微图此外利用sla技术还可以制造出包含多种复杂微结构的肝组织工程支架该支架系统可保证多种肝脏细胞的有序分布可为组织工程肝脏支架材料微观结构的模拟提供参考
sla工作原理(一)
sla工作原理(一)SLA工作什么是SLASLA，全称Service Level Agreement，即服务级别协议。
它是一种合同，用于规定服务提供商和客户之间的服务水平和对应的服务指标。
SLA可以帮助客户了解他们将得到什么样的服务，以及如何处理问题和投诉。
SLA工作原理SLA的工作原则是从业务功能的角度出发，定义每个功能的响应时间、同时在线用户数、故障时长、恢复时间等指标。
具体SLA的内容包括以下部分：•服务范围：包括服务的种类、规模、服务工作原理、具有相关的业务目标。
•响应时间：指服务提供商应该在哪个时间期限内完成客户的获取请求或解决问题的请求。
•可用率：指服务的运行时间与规定时间的比率，用于衡量服务是否可靠。
•故障处理：指故障的处理流程以及相关的响应时间，包括修改、测试和发布方面的内容。
•保密性：指服务提供商保护客户个人隐私的信誉和承诺。
•安全性：指因素，如防护、备份、灾难恢复、安全性控制，以及设施和设备的安全控制。
如果SLA的相关标准没有被满足，客户可以要求服务提供商对其进行赔偿，或提出有适当措施进行鼓励型奖励。
SLA工作的优点SLA不仅有助于服务提供商提高服务质量，而且还可以帮助客户了解服务提供商对客户的承诺并维护服务质量标准，进一步增强服务品牌的信誉。
此外，它还有以下优点：•帮助提供者进行业务规划和进程控制，进一步提高服务度量。
•降低成本，改善运维效率。
•使公司与客户之间的关系更为紧密和持久，促进业务增长。
SLA工作的局限性虽然SLA有许多优点，但也存在一些限制和局限性。
以下是主要局限性之一：•SLA的实施需要付出比较大的成本和努力，包括一些服务和工具的购买、额外的工作等。
•SLA常常会引起压力，因为它们对高质量的服务质量有着高要求。
•如果在SLA的实施中发现服务性能低于某些标准，客户可能不会被及时通知。
•SLA并不总能满足客户的本质要求，因为它往往只能强制执行与和岗位等方面的规定，但无法对事情本身产生保障作用。
sla一12vdc一sl一c的工作原理
sla一12vdc一sl一c的工作原理
sla-12vdc-sl-c的工作原理涉及到其作为一个继电器的功能。
继电器是一种电子控制器件，它具有控制系统（输入回路）和被控制系统（输出回路），通常应用于自动控制电路中，它实际上是用较小的电流去控制较大电流的一种“自动开关”。
具体来说，sla-12vdc-sl-c是一个常开常闭型的继电器，它的工作原理主要是通过输入信号的控制，来驱动其内部的触点开关，从而实现对输出电路的控制。
当输入信号发生变化时，继电器内部的触点开关会随之变化，从而改变输出电路的状态。
在sla-12vdc-sl-c中，其最大电源电压和最小电源电压的设定，是为了保证继电器在正常工作时的稳定性和可靠性。
同时，继电器的常开常闭功能，也是根据实际应用需求进行设计的。
SLA的定义、背景信息、名词解释及指标
引言概述SLA（Service Level Agreement），即服务级别协议，是一种合同协议，用于确保服务提供商向客户提供可接受的水平的服务。
本文将探讨SLA的定义、背景信息、名词解释和指标等方面内容，以帮助读者更好地了解SLA。
正文内容1. SLA的定义1.1 什么是SLASLA是一种协议，旨在明确服务提供商将为客户提供的服务水平。
它通常包括服务的范围、质量和响应时间等方面的具体要求。
1.2 SLA的重要性SLA的制定和实施可确保服务提供商遵守承诺，提供高质量的服务，同时也为客户提供了一种保障，确保能够获得满意的服务体验。
2. 背景信息2.1 SLA的发展历程SLA最早起源于互联网服务提供商（ISP），随着IT服务管理的发展，已广泛应用于各个行业。
SLA的发展历程反映了对服务水平的关注度的提高，旨在推动服务提供商提供更好的服务。
2.2 SLA的应用领域SLA广泛应用于各个行业，如云计算、电信、软件开发等，其目的是为了确保用户获得高质量的服务体验。
SLA可以用于服务供应商与客户之间的合作关系，也可以在组织内部的不同部门之间制定。
3. 名词解释3.1 服务水平协议（SLE）服务水平协议是指SLA中约定的具体服务水平要求，包括性能、可靠性、可用性等方面的指标和标准。
3.2 幸存时间（Mean Time to Repair，MTTR）幸存时间是指从服务出现故障到故障修复完成所需的平均时间。
较短的幸存时间意味着服务可以更快地恢复正常运行。
3.3 故障率（Failure Rate）故障率是指单位时间内发生故障的频率。
较低的故障率意味着服务更加可靠。
4. SLA的指标4.1 服务可用性（Service Availability）服务可用性是指服务在一定时间内处于可用状态的百分比。
例如，99.9%的服务可用性意味着服务每年只有0.1%的时间不可用。
4.2 响应时间（Response Time）响应时间是指服务提供商对客户请求做出响应的时间。
sla工作原理
sla工作原理SLA（Service Level Agreement）是指服务水平协议，它是一种合同形式的文件，用于明确服务提供商和客户之间的责任和义务。
SLA工作原理主要包括协商、制定、执行和监控四个阶段。
在协商阶段，服务提供商和客户就服务的各项指标进行洽谈和协商，明确双方的期望和需求。
这些指标通常包括服务可用性、响应时间、故障恢复时间等。
协商的过程中，双方需要充分沟通，确保SLA的内容能够满足客户的需求，并且符合服务提供商的能力。
制定阶段是根据双方协商的内容，将SLA具体化为一份合同文件。
在这个阶段，需要明确服务提供商的责任和义务，以及客户的权益和义务。
合同中还应该包括补偿措施、争议解决机制等内容，以保证双方在合作过程中的权益得到保障。
执行阶段是指服务提供商按照SLA的约定提供服务，客户按照合同要求享受服务。
服务提供商需要严格遵守SLA中约定的各项指标，确保服务的质量和稳定性。
同时，客户也需要履行合同中规定的义务，如按时支付费用等。
监控阶段是对服务水平进行监控和评估，以确保SLA的执行情况符合合同要求。
监控可以通过收集和分析服务数据、客户反馈等方式进行。
如果发现SLA规定的指标未能达到或超出了约定的范围，需要及时采取措施进行调整和改进，以保证服务质量。
SLA的工作原理可以简单总结为协商、制定、执行和监控四个阶段。
通过这一过程，服务提供商和客户能够明确双方的责任和义务，保证服务的质量和稳定性。
同时，SLA也为服务提供商和客户之间的合作提供了一种有效的管理和监督机制，有助于双方建立长期稳定的合作关系。
SLA作为一种合同形式的文件，在服务提供商和客户之间起到了重要的桥梁作用。
通过明确双方的责任和义务，SLA能够帮助双方建立起良好的合作关系，确保服务的质量和稳定性。
同时，SLA也为双方提供了一种有效的管理和监督机制，有助于解决合作过程中可能出现的问题和争议。
因此，了解SLA的工作原理对于服务提供商和客户来说都是至关重要的。
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2000, Linguistics and Second Language Acquisition, FLTRP)
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L2 Lesson by Zhangsirui
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• What Constitutes Knowledge of Language?
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• How is Knowledge of Language Acquired?
A second goal for linguistics is discovering how knowledge of language comes into being---how linguistic competence is acquired by the human mind. It is proposed to achieve this goal by describing how innate principles of the child’s mind create linguistic competence.
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• How is knowledge of languages put to use?
People who know two languages can decide how to use them according to where they are, what they are talking about, who they are talking to, and so on. Describing their language use means showing how knowledge of two or more languages is used by the same speaker psychologically and
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L2 Lesson by Zhangsirui
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Lecture 1
• We begin our lesson from “The goals of second language research”, based on Cook’s The Background to Current
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• The above questions need to be rephrased to take in knowledge of more than one language when we discuss second language acquisition. The goal of linguistics is not monolingual but becomes multilingual.
The prime goal of linguistics is to describe the language contents of the human mind: its task is to represent what native speakers know about language--their linguistic competence. Achieving this goal means producing a fully explicit representation of the speaker’s competence.
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kinds of knowledge. Grammar is conceptualized as a module and consists of a set of autonomous subsystems, each with its own criteria for well-formedness. This contrasts with cognitive theories that assume the mind is a single unitary system. Cognitive view considers the mechanisms to be general in nature. It sees the process of learning a language---whether a first or a second one---as essentially the same as any other kind of learning.
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• I-language tradition The I-language tradition in linguistics is concerned with mental reality and with knowledge, in short with representing the internal aspects of the mind; it is based linguistic competence. Observable behavior is only one way of getting into these non-observable aspects.
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• E-language tradition
The E-language tradition in linguistics is concerned with behavior, with social convention, and with language as an external social reality; hence its methodology is based on collecting large samples of spoken language data. The grammar of the language is derived by working out the “structure” or patterns in these data; a grammar is a collection of descriptive statements concerning the Elanguage.
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• Second language acquisition takes many forms and occurs in many situations; in particular second languages are not only picked up by learners in natural circumstances similar to first language acquisition but are also taught in classrooms. Hence there is often a tension between approaches to second language research that see ideas about language and about language acquisition as directly relevant and approaches that see the ways in which the mind acquires other types of knowledge as
201hangsirui
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• What constitutes knowledge of languages?
One brain, two systems? A person who speaks knows two grammars; two systems of language knowledge are present in the same mind. One goal of SLA research is to describe grammars of more than one language simultaneously existing in the same person.
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• How is Knowledge of language Put to Use?
That means to see how it relates to thinking, comprehension, and communication. This involves both the psychological process through which the mind understands and produces speech, and the processes through which speech is adapted to an actual moment of speaking in a particular context of situation.
sociologically.
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• Linguistics can be divided into external and internal language approaches, or, E-language and Ilanguage approaches. (Chomsky 1986) • When we make explanation of SLA, we consider external factors and internal factors. (Ellis 1994)