☆如何阅读质粒图谱

如何阅读质粒图谱最近由于实验需要,需要查阅载体图谱,到园子里搜罗一番,发现虽然有人问载体图谱阅读的问题,也有前辈回答,但都不详细,借自己也在琢磨这个问题的机会,将我学到的东西整理一下,于大家分享。

载体主要有病毒和非病毒两大类,其中质粒DNA是一种新的非病毒转基因载体。

一、一个合格质粒的组成要素#复制起始位点Oril 即控制复制起始的位点。

原核生物DNA分子中只有一个复制起始点。

而真核生物DNA分子有多个复制起始位点。

＃抗生素抗性基因可以便于加以检测，如Amp+l ,Kan+＃多克隆位点MCS 克隆携带外源基因片段l＃P/E 启动子/增强子l＃Termsl 终止信号＃加poly（A）信号l 可以起到稳定mRNA作用二、如何阅读质粒图谱第一步：首先看Ori的位置，了解质粒的类型（原核/真核/穿梭质粒）第二步：再看筛选标记，如抗性，决定使用什么筛选标记。

（1）Ampr 水解β－内酰胺环，解除氨苄的毒性。

（2）tetr 可以阻止四环素进入细胞。

（3）camr 生成氯霉素羟乙酰基衍生物，使之失去毒性。

（4）neor（kanr）氨基糖苷磷酸转移酶使G418（卡那霉素衍生物）失活（5）hygr 使潮霉素β失活。

第三步：看多克隆位点（MCS）。

它具有多个限制酶的单一切点。

便于外源基因的插入。

如果在这些位点外有外源基因的插入，会导致某种标志基因的失活，而便于筛选。

决定能不能放目的基因以及如何放置目的基因。

第四步：再看外源DNA插入片段大小。

质粒一般只能容纳小于10Kb的外源DNA片段。

一般来说，外源DNA片段越长，越难插入，越不稳定，转化效率越低。

第五步：是否含有表达系统元件，即启动子－核糖体结合位点－克隆位点－转录终止信号。

这是用来区别克隆载体与表达载体。

克隆载体中加入一些与表达调控有关的元件即成为表达载体。

选用那种载体，还是要以实验目的为准绳。

启动子－核糖体结合位点－克隆位点－转录终止信号＃启动子－促进DNA转录的DNA顺序，这个DNA区域常在基因或操纵子编码顺序的上游，是DNA分子上可以与RNApol特异性结合并使之开始转录的部位，但启动子本身不被转录。

＃增强子/沉默子－为真核基因组（包括真核病毒基因组）中的一种具有增强邻近基因转录过程的调控顺序。

其作用与增强子所在的位置或方向无关。

即在所调控基因上游或下游均可发挥作用。

/沉默子－负增强子，负调控序列。

＃核糖体结合位点/起始密码/SD序列（Rbs/AGU/SDs）：mRNA有核糖体的两个结合位点，对于原核而言是AUG（起始密码）和SD序列。

l＃转录终止顺序（终止子）/翻译终止密码子：结构基因的最后一个外显子中有一个AATAAA的保守序列，此位点down－stream有一段GT或T富丰区，这2部分共同构成poly（A）加尾信号。

回答有人之前提出的一个问题：为什么质粒图谱上有的箭头顺时针有的箭头逆时针？那其实是代表两条DNA 链，即质粒是环状双链DNA，它的启动子等在其中一条链上，而它的抗性基因在另一条链上.三、介绍一下关于载体的知识（虽然课本上都有写）1. 什么是载体即要把一个有用的基因（目的基因——研究或应用基因）通过基因工程手段送到生物细胞（受体细胞），需要运载工具（交通工具）携带外源基因进入受体细胞，这种运载工具就叫做载体（vector）。

P.S.基因工程所用的vector实际上是DNA分子，是用来携带目的基因片段进入受体细胞的DNA2. 载体的分类＃按功能分成：（1）克隆载体都有一个松弛的复制子，能带动外源基因，在宿主细胞中复制扩增。

它是用来克隆和扩增DNA片段（基因）的载体。

（所以有时实验时扩增效率低下，要注意是不是使用的严谨型载体）（2）表达载体具有克隆载体的基本元件（ori,Ampr,Mcs等）还具有转录/翻译所必需的DNA 顺序的载体。

＃按进入受体细胞类型分：（1）原核载体（2）真核载体（3）穿梭载体（sbuttle vector）指在两种宿主生物体内复制的载体分子，因而可以运载目的基因（穿梭往返两种生物之间）. P.S. 穿梭质粒含原核和真核生物2个复制子，以确保两类细胞中都能扩增。

3. 基因工程载体的3个特点：（一）都能独立自主的复制：载体DNA分子中有一段不影响它们扩增的非必需区域，如MCS，插在其中的外源DNA片段，能被动的跟着载体一起复制/扩增，就像载体的正常成分一样。

（二）都能便利的加以检测：如载体的药物抗性基因，多是抗生素抗性基因，将受体细胞放在含有该抗生素培养板上培养生长时，只有携带这些抗性基因的载体分子的受体细胞才能存活。

（三）都能容易进入宿主细胞中去，也易从宿主细胞中分离纯化出来。

4. 载体的选择和制备：选择载体主要依据构建的目的，同时要考虑载体中应有合适的限制酶切位点。

如果构建的目的是要表达一个特定的基因，则要选择合适的表达载体。

载体选择主要考虑下述3点：【1】构建DNA重组体的目的，克隆扩增/表达，选择合适的克隆载体/表达载体。

【2】.载体的类型：－－－（1）克隆载体的克隆能力－据克隆片段大小（大选大，小选小）。

如<10kb选质粒。

－－－（2）表达载体据受体细胞类型－原核/真核/穿梭，E.coli/哺乳类细胞表达载体。

－－－（3）对原核表达载体应该注意3点：－－－－－－①选择合适的启动子及相应的受体菌；－－－－－－②用于表达真核蛋白质时注意克服4个困难和阅读框错位；－－－－－－③表达天然蛋白质或融合蛋白作为相应载体的参考。

【3】载体MCS中的酶切位点数与组成方向因载体不同而异，适应目的基因与载体易于链接，不产生阅读框架错位。

选用质粒（最常用）做载体的4点要求：①选分子量小的质粒，即小载体（1－1.5kb）→不易损坏，在细菌里面拷贝数也多（也有大载体）；②一般使用松弛型质粒在细菌里扩增不受约束，一般10个以上的拷贝，而严谨型质粒<10个。

③必需具备一个以上的酶切位点，有选择的余地；④必需有易检测的标记，多是抗生素的抗性基因，不特指多位Ampr（试一试）。

无论选用哪种载体，首先都要获得载体分子，然后采用适当的限制酶将载体DNA进行切割，获得分子，以便于与目的基因片段进行连接。

质粒载体质粒（plasmid）是细菌或细胞染色质以外的，能自主复制的，与细菌或细胞共生的遗传成分。

其特点如下：①是染色质外的双链共价闭合环形DNA（covalently closed circuar DNA,cccDNA），可自然形成超螺旋结构，不同质粒大小在2-300kb之间，＜15kb的小质粒比较容易分离纯化，＞15kb的大质粒则不易提取。

②能自主复制，是能独立复制的复制子（autonomous replicon）。

一般质粒DNA复制的质粒可随宿主细胞分裂而传给后代。

按质粒复制的调控及其拷贝数可分两类：严紧控制（stringent control）型质粒的复制常与宿主的繁殖偶联，拷贝数较少，每个细胞中只有1个到十几个拷贝；另一类是松弛控制（relaxed control）型质粒，其复制宿主不偶联，每个细胞中有几十到几百个拷贝。

每个质粒DNA上都有复制的起点，只有ori能被宿主细胞复制蛋白质识别的质粒才能在该种细胞中复制，不同质粒复制控制状况主要与复制起点的序列结构相关。

有的质粒的可以整合到宿主细胞染色质DNA中，随宿主DNA复制，称为附加体，例如细菌的性质粒就是一种附加体，它可以质粒形式存在，也能整合入细菌的DNA，又能从细菌染色质DNA上切下来。

F因子携带基因编码的蛋白质能使两个细菌间形成纤毛状细管连接的接合（conjugation），通过这细管遗传物质可在两个细菌间传递。

③质粒对宿主生存并不是必需的。

这点不同于线粒体，线粒体DNA也是环状双链分子，也有独立复制的调控，但线粒体的功能是细胞生存所必需的。

线粒体是细胞的一部分，质粒也往往有其表型，其表现不是宿主生存所必需的，但也不妨碍宿主的生存。

某些质粒携带的基因功能有利于宿主细胞的特定条件下生存，例如，细菌中许多天然的质粒带有抗药性基因，如编码合成能分解破坏四环素、氯霉素、氨芐表霉素等的酶基因，这种质粒称为抗药性质粒，又称R质粒，带有R质粒的细菌就能在相应的抗生素存在生存繁殖。

所以质粒对宿主不是寄生的，而是共生的。

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Seamless Cloning.酵母表达载体Gateway® pYES-DEST52 V ectorPichiaPink™Secreted Protein Vector KitPichiaPink™Secretion Signal SetPichiaPink™Vector KitpAO815pFLD1 Vector KitpGAPZ A, B, & CpGAPZαA, B, & CpPIC3.5KpPIC6 A, B, & CpPIC6αA, B, & CpPIC9KpPICZ A, B, & CpPICZαA, B, & CpTEF1⁄ZeopYES2pYES2⁄CTpYES2⁄NT A, B, & CpYES2.1 TOPO® TA Expression KitpYES3⁄CT基因酷质粒图谱/bbs/forum-38-1.html，收藏了将近800种质粒的图谱及相关信息特向大家推荐，介绍及使用方法见：/bbs/thread-417-1-1.html质粒图谱信息一.九种表达载体Pllp-OmpA, pllp-STII, pMBP-P, pMBP-C,pET-GST, pET-Trx, pET-His, pET-CKS, pET-DsbA二.克隆载体pTZ19R DNApUC57 DNAPMD18TPQE30pUC18pUC19pTrcHisApTrxFuspRSET-ApRSET-BpVAX1PBR322pbv220pBluescript II KS (+)L4440pCAMBIA-1301pMAL-p2XpGD926三.PET系列表达载体Protein Expression » Prokaryotic Expression » pET Dsb Fusion Systems 39b and 40bProtein Expression » Prokaryotic Expression » pET Expression System 33b Protein Expression » Prokaryotic Expression » pET Expression Systems Protein Express ion » Prokaryotic Expression » pET Expression Systems plus Competent CellsProtein Expression » Prokaryotic Expression » pET GST Fusion Systems 41 and 42Protein Expression » Prokaryotic Expression » pET NusA Fusion Systems 43.1 and 44Protein Expressio n » Prokaryotic Expression » pET Vector DNAProtein Purification » Purification Systems » Strep•Tactin Resins and Purification Kits四.PGEX系列表达载体T EcoR pGEX-1 I/BAPpGEX-2TpGEX-2TKpGEX-3XpGEX-4T-1pGEX-4T-2pGEX-4T-3pGEX-5X-1pGEX-5X-2pGEX-5X-3pGEX-6P-1pGEX-6P-2pGEX-6P-3五.PTYB systemPTYB1PTYB2PTYB11PTYB12六.真核表达载体pCDNA3.1(-)pCDNA3.1(+)pPICZ alpha ApGAPZαAPYES2.0pBI121pEGFP-N1pEGFP-C1pPIC9KpPIC3.5K答学生问：克隆载体与表达载体FROM：红泥小火炉问题：基因工程中有克隆载体和表达载体，克隆载体可以在受体菌中大量复制，表达载体用于表达目的蛋白，那么实际应用中，我们的最终目的是要得到目的蛋白，克隆载体不能完成表达，有何用呢？还是说利用克隆载体实现目的基因的大量复制后，再将其转移到表达载体中实现表达？它是否有何缺陷不能整合克隆载体的功能？构建兼有克隆和表达双重功能的载体有何困难？回答：1.克隆的目的比较单一，就是将你感兴趣的DNA片段，重组进入载体，然后于宿主细胞中大量繁殖，主要用于各种文库的建立，比如人类基因组计划；同时由于载体所能容纳的目的片段的长度是有限的，而克隆载体没有表达所需的各种片段，所以可以容纳更长的目的片段，即可以克隆足够长的基因，效率更高。