噬菌体展示技术构建重组人TNF_单链抗体库

上海交通大学学报(医学版)

Vol .26No .2Feb .2006

Journal of Shanghai J iaotong University (Medical Science )

【作者简介】柴蔚然(1973-),女,河北任丘人,博士生

【文章编号】　0258-5898(2006)02-0155-04

·基础研究·

噬菌体展示技术构建重组人TNF 2

α单链抗体库柴蔚然1,　杨　涛2,　胡晓年3,　牛　勃

2

(1上海交通大学基础医学院生物化学与分子生物学教研室,上海　200025;2山西医科大学生物化学与分子生物学教研室;

3

中国协和医科大学基础研究所)

【摘　要】目的应用噬菌体展示技术构建抗重组人肿瘤坏死因子-α(rhT NF 2α)的单链抗体库。方法利用噬菌体表面展示技术、绕过杂交瘤技术,提取经免疫BALB /c 小鼠淋巴细胞总RNA,用相应引物扩增小鼠免疫球蛋白VH 基因和VL 基因,构建

VH 、VL 抗体库。由L inker 2p ri m er m ix 经重叠延伸反应将重链可变区基因与轻链可变区基因拼接成单链抗体。再用RS p ri m er

m ix 以单链抗体为模板进行第二次聚合酶链反应,从而获得全套抗rhT NF 2

α的单链抗体基因。以Sfi Ⅰ/N ot Ⅰ,位点定向插入pCANT AB 25E 噬菌粒载体,转化大肠杆菌TG1后,经M13K O7辅助噬菌体拯救,构建成全套抗rhT NF 2

α单链抗体表面展示文库。结果成功构建了rhT NF 2α全套单链抗体噬菌体表面展示文库。经M13K O7超感染后,噬斑计数滴度为2.1×1011

pfu 。结论

rhT NF 2

α全套单链抗体噬菌体表面展示文库的构建,为研究rhT NF 2α抗体结合位点,了解其结构与功能的关系以及研究用于临床免疫治疗的新型抗体打下了基础。

【关键词】肿瘤坏死因子-α;　单链抗体;　噬菌体展示技术【中图分类号】Q78 【文献标识码】A

Con structi on of Phage D ispl ay L i brary of S i n gle 2Cha i n Fv An ti bod i es Aga i n st

Recom b i n an t Human TNF 2

αCHA IW e i 2ran 1,YANG Tao 2,HU X i a o 2n i a n 3,N I U Bo

2

(1

D epa rt m en t of B ioche m istry and M olecu larB iology,B asicM edical College,Shanghai J iaotong U niversity,Shanghai 200025,China;

2

D epart m ent of B ioche m istry and M olecular B iology,ShanxiM edica l U niversity;

3

Institution of B asic

M edical Science,Peking U nion M edica l College )

Abstract:　O b jective To construct a phage dis p lay library of single 2chain antibodies against recombinant human

tumor necr osis fact or 2

α(rhT NF 2α).　M ethods By the technique of phage dis p lay,the heavy 2chain and kappa light 2chain variable regi on genes (VH and VL )were asse mbled int o single 2chain antibodies (ScFv )with overlap extensi on reacti on by L inker 2p ri m er m ix .The ScFv were cl oned int o pCANT AB 25E phage m id and intr oduced int o

E .coli TG 1.The phage m id 2containing bacterial col onies were infected with M 13K O 7,then the t otal surface dis p lay

library of anti 2rhT NF 2

αwas established .　R esu lts The phage antibody library was constructed .The titre of p laque was 2.1×1011

pfu after superinfecti on with M 13K O 7.　C onclusion ScFv fusi on p r otein was dis p layed on the sur 2

face of recombinant phage successfully .It is the basis of studying the recep t or binding site of rhT NF 2αand the rela 2ti onshi p bet w een its structure and functi on as well as the neutralizati on characteristic of monocl onal antibody .

Key words:　tu mor necr osis fact or 2

α;　single 2chain antibody;　phage dis p lay technique 肿瘤坏死因子-α(tu mor necr osis fact or 2

α,T NF 2α)是机体内主要的炎症因子之一,在机体的多种病理生理过程中起着非常重要的作用

[1]

。许多实验表

明,内毒素对机体的多种病理和生理作用,主要是由

T NF 2

α介导的。由于T NF 2α大量释放而致的炎症、损伤、感染性休克等,往往症状凶险,难以治疗,典型的例子是恶病质、败血症休克和炎症性肠病[2]

。而且在艾滋病

[3]

、移植物抗宿主病

[4]

、脑型疟疾

[5]

、类

风湿性关节炎[6]

、再生障碍性贫血、骨髓增生异常综

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