中文摘要：

目的构建抗肿瘤坏死因子-α（Tumornecrosisfactor-d，TNF．仅）单链抗体（Singlechainfragmentvariable，scFv）的原核高效表达体系，并优化表达条件。方法在引物中添加编码6×His的核苷酸片段，使目的蛋白C．末端带有6xHis标签。从质粒pCANTAB5E-seF中PCR扩增scFv基因，连接至pBV220载体，连接产物分别转化至E．coliBL2l（DE3）和DH5ct中，42％诱导表达，并对诱导时间、培养基pH值和类型进行优化，Westernblot分析表达产物的反应原性。结果PCR扩增出的scFv基因片段长度约770bp，且序列正确；重组表达质粒pBV220．scFv经酶切鉴定证明构建正确；在EcoliBL2I（DE3）中可获得表达，在E．coliDH5c~中不表达；表达的重组scFv相对分子质量约为28000，以包涵体形式存在于胞浆中，表达量约占菌体总蛋白的15％，且可与抗His．Tag抗体发生特异性反应；工程菌的最佳诱导表达条件为：以EcoliBL21（DE3）为宿主菌，在pH7．4的LB培养基中，42℃诱导5h。结论将scFv的表达载体更换为pBV220，可提高scFv的表达量，在优化表达条件下，scFv的表达水平进一步提高。

英文摘要：

Objective To construct a high prokaryotic expression system for single chain fragment variable （scFv） against tumor necrosis factor-or （TNF-ct） and optimize the condition for expression. Methods The nucleotide fragment encoding 6 x His was added into primers in order to produce 6 x His Tag at C-terminus of target protein. The seFv gene was amplified from plasmid pCANTAB 5E-seFv by PCR and inserted into vector pBV220. The constructed recombinant plasmids were transformed to E. coli BL21 （DE3） and DH5ct and induced at 42~C. The temperature for induction as well as pH value and kind of medium were optimized, and the expressed product was analyzed for reactogenicity by Western blot. Results The scFv fragment with correct sequence, at a length of about 770 bp, was amplified by PCR. Restriction analysis proved that recombinant plasmid pBV220-scFv was constructed correctly and expressed in E. coli BL21 （DE3） but not in E. coli DH5o~. The expressed recombinant scFv, with a relative molecular mass of about 28 000, existed in a form of inclusion boody in cytoplasma, contained about 15% of total somatic protein, and showed specific reaction with anti-His-Tag antibody. The condition for expression was optimized as induction of E. coli BL21 （DE3） in LB medium （pH 7. 4） at 42℃ for 5 h. Conclusion The expression level of scFv was increased by substitution of expression vector pCANTAB 5E with pBV220, and further increased by induction under optimal condition.