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目的:构建绿脓杆菌外毒素PE38KDEL融合全人源抗c-Met单链抗体原核表达载体,并对其产物进行变性、复性和纯化,检测其对肝癌细胞系的毒性作用。方法:以从人源天然Fab抗体库中筛选出的AM2-26克隆作为模板扩增出人源c-Met单链基因,采用基因工程原理,将扩增出的c-Met单链基因和PE38KDEL基因克隆入pBAD/GⅢA表达载体中,重组克隆载体经酶切及DNA序列测定证实两片段连接正确;转化大肠杆菌Top10F′,左旋阿拉伯糖诱导表达,采用镍离子螯合层析法纯化变性的包涵体样品,并用连续梯度透析的方法对纯化后的包涵体进行复性;采用流式细胞术鉴定复性产物与靶细胞的结合活性,MTT法检测c-Met/PE38KDEL免疫毒素对肝癌细胞的体外杀伤活性。结果:成功构建了免疫毒素表达载体pBAD/GⅢA/c-Met/PE38KDEL;诱导产物主要以包涵体形式存在;复性后的免疫毒素c-Met/PE38KDEL具有与靶细胞特异性结合的活性并且对SMMC7721细胞系具有较明显的杀伤作用。结论:全人源抗c-Met单链抗体与PE38KDEL重组免疫毒素的成功表达纯化及对人肝癌细胞系的试验结果,为进一步肝癌的导向治疗提供依据。
OBJECTIVE: To construct a prokaryotic expression vector for PE38KDEL fusion of human Pseudomonas exotoxin PE38KDEL and to denature, renaturation and purify the product, and to examine its toxicity on hepatocellular carcinoma cell lines. Methods: The human c-Met single-stranded gene was amplified from the AM2-26 clone screened from the human natural Fab antibody library. The c-Met single-stranded gene and PE38KDEL The gene was cloned into pBAD / GⅢA expression vector. The recombinant cloning vector was confirmed by restriction enzyme digestion and DNA sequence analysis. The two fragments were ligated correctly. The recombinant plasmid was transformed into E.coli Top10F ’and induced by L-arabinose. Nickel ion chelate chromatography The purified inclusion bodies were refolded by continuous gradient dialysis. The binding activity of the refolded product to the target cells was identified by flow cytometry. The immunotoxin of c-Met / PE38KDEL was detected by MTT assay in vitro Killing activity. Results: The immunotoxin expression vector pBAD / GⅢA / c-Met / PE38KDEL was constructed successfully. The induced products mainly existed as inclusion bodies. The refolded immunotoxin c-Met / PE38KDEL had specific binding activity with target cells SMMC7721 cell line has a more obvious killing effect. Conclusion: The successful expression and purification of the whole human anti-c-Met scFv and PE38KDEL recombinant immunotoxin and the experimental results on human hepatocellular carcinoma cell lines provide the basis for the further treatment of liver cancer.