人源抗TNF—α抗体Fab基因的单链化及其表达和纯化

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－linkerVL的结构将人源抗TNF－a的VH，VL基因拼接成单链抗体（ScFv)基因，并将构建好的ScFv基因插入表达载体pQE30，转染E.coli M15,以IPTG诱导表达，Ni-NTA树脂纯化表达产物，构建的ScFv基因长723bp, 列分析表明，该序列拼接正确，SDS－PAGE显示，用重组的pQE30转化的M15菌经诱导后，有相对分子质量（M）约为52000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TNFa的ScFv基因，并在E.coli DH5a中表达和纯化的该基因的产物。

Construction andExpression of the Anti-TNF-

To 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 Mr 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.