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

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Author:
No author available
Journal Title:
JOURNAL OF THE FOURTH MILITARY MEDICAL UNIVERSITY
Issue:
7
DOI:
10.3321/j.issn:1000-2790.1999.07.004
Key Word:
肿瘤坏死因子;融合表达;单域抗体;谷胱甘肽巯基转移酶

Abstract: 目的:RT-PCR法获得鼠抗人肿瘤坏死因子-α(hTNF-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pGE6VH和pGE6VL,利用大肠杆菌系统表达VH和VL单域抗体蛋白. 方法:将RT-PCR法获得的E6VH和E6VL基因分别克隆入融合表达载体pGEX4T-1中,使它们受控于Ptac启动子, 转化大肠杆菌DH5α,经异丙基-β-D-硫代半乳糖苷(IPTG)诱导,100 g/L SDS-PAGE检测表达产物. 结果:SDS-PAGE显示E6VH表达产物分子质量约为38 ku,E6VL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(GST)-VH融合蛋白占菌体总蛋白的45%,GST-VL 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GST-VH和GST-VL融合蛋白的显色条带. 结论:在大肠杆菌DH5α中成功地表达了hTNF-α的单域抗体基因.

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