人源抗TNF-α抗体Fab基因的单链化及其表达和纯化  

Construction and Expression of the Anti-TNF-α ScFv Gene

作　　者：袁斌[1] 白洁[2] 张广育[3] 刘一平[1] 何君[1] 王慧[1] 黄策[1] 

机构地区：[1]军事医学科学院微生物流行病研究所,中国北京100071 [2]白求恩医科大学,中国吉林长春130021 [3]首都医科大学微生物教研室,中国北京100054

出　　处：《生命科学研究》2001年第1期56-59,共4页Life Science Research

基　　金：国家自然科学基金资助项目!(396 70 0 43)

摘　　要：构建人源抗 TNF-α单链抗体基因 ,并尝试其在 E.coli中的表达和纯化 .采用人工接头 ,按 VH-linker-VL的结构将人源抗 TNF-α的 VH、VL基因拼接成单链抗体 ( Sc Fv)基因 ;并将构建好的 Sc Fv基因插入表达载体 p QE30 ;转染 E.coli M1 5,以 IPTG诱导表达 ,并用 Ni-NTA树脂纯化表达产物 .构建的 Sc Fv基因长 72 3bp,序列分析表明 ,该序列拼接正确 ;SDS-PAGE显示 ,用重组的 p QE30转化的 M1 5菌经诱导后 ,有相对分子质量 ( Mr)约为 52 0 0 0的外源蛋白表达 ,Ni-NTA树脂纯化的表达产物纯度大于 90 % .因此 ,成功地构建了人源抗 TNFα的 Sc Fv基因 ,并在 E.coliTo construct an anti TNFαsingle chain Fv gene and to express and purify it in E.coli . The VH and VL genes of anti TNFαmAb were connected into ScFv gene by a flexible linker and the ScFv gene fragments were inserted into expression vector pQE30. The recombinant plasmid were transfected into E.coli M15 and the expression of target protein was induced with IPTG. The expressed protein was purified using Ni NTA resin. The results showed that the length of ScFv gene fragments was 723 bp and the sequence was correct. SDS PAGE indicated that the recombinant plasmid pQE30 expressed a new protein band with a M r about 52 000 with the purity of the product >90%. Thus, the ScFv gene was sucessfully constructed and highly expressed in E.coli , and purified recombinant protein was obtained.

关 键 词：单链抗体 人源抗体 肿瘤坏死因子 基因表达 纯化 

分 类 号：R392.2[医药卫生—免疫学] R392－33[医药卫生—基础医学]