鼠抗人TNF-α单域抗体基因在大肠杆菌中的融合表达

Expression of single domain antibody fragments of anti human TNF α antibody in E.coli as fusions with GST

目的： Ｒ Ｔ Ｐ Ｃ Ｒ 法获得鼠抗人肿瘤坏死因子α（ｈ Ｔ Ｎ Ｆα）单克隆抗体 Ｅ６ 轻、重链可变区基因序列，分别构建融合表达载体 ｐ Ｇ Ｅ６ Ｖ Ｈ 和ｐ Ｇ Ｅ６ Ｖ Ｌ，利用大肠杆菌系统表达 Ｖ Ｈ和 Ｖ Ｌ单域抗体蛋白． 方法：将 Ｒ Ｔ Ｐ Ｃ Ｒ 法获得的 Ｅ６ Ｖ Ｈ 和 Ｅ６ Ｖ Ｌ基因分别克隆入融合表达载体 ｐ Ｇ Ｅ Ｘ４ Ｔ１ 中，使它们受控于 Ｐｔａｃ 启动子， 转化大肠杆菌 Ｄ Ｈ５α，经异丙基β Ｄ硫代半乳糖苷（ Ｉ Ｐ Ｔ Ｇ）诱导，１００ ｇ／ Ｌ Ｓ Ｄ Ｓ Ｐ Ａ Ｇ Ｅ检测表达产物．结果： Ｓ Ｄ Ｓ Ｐ Ａ Ｇ Ｅ显示 Ｅ６ Ｖ Ｈ 表达产物分子质量约为 ３８ ｋｕ， Ｅ６ Ｖ Ｌ表达产物分子质量约为 ３７ ｋｕ，与预期结果相符． 光密度扫描结果表明，谷胱甘肽巯基转移酶（ Ｇ Ｓ Ｔ） Ｖ Ｈ 融合蛋白占菌体总蛋白的 ４５％ ， Ｇ Ｓ Ｔ Ｖ Ｌ 融合蛋白占菌体总蛋白的３６％ ， Ｗｅｓｔｅｒｎｂｌｏｔ 证实在相应分子质量处，有 Ｇ Ｓ Ｔ Ｖ Ｈ 和 Ｇ Ｓ Ｔ Ｖ Ｌ融合蛋白的显色条带． 结论：在大肠杆菌 Ｄ Ｈ５α中成功地表达了ｈ Ｔ Ｎ Ｆα的单域抗体基因．

AIM: To express the light and heavy chain variable region genes of anti TNFα antibody in E.coli using a fusion protein expression vector. METHODS: The light and heavy chain variable region genes, amplified from a marine anti human TNF α Hybridoma cell line E6 by RT PCR, were inserted into a fusion protein expression vector pGEX4T 1 respectively, in which the foreign gene was controlled by Ptac promoter. The recombinant plasmids pGE6VH and pGE6VL were transformed into E.coli DH5α and induced with IPTG. 10% SDS PAGE and Western blot identified the expression products. RESULTS: After induction, 38 ku (E6VH) and 37 ku(E6VL) protein bands appeared on 10% SDS PAGE gel and Western blotting filter paper. CONCLUSION: The single domain antibody of E6VH and E6VL has been successfully expressed in E.coli DH5α.