人源化抗肝癌单链抗体与牛蛙核糖核酸酶融合基因的构建及表达

Preparation of a fusion protein of Rana catesbeiana ribonuclease and the humanized scFv against hepatocellular carcinoma

利用RT-PCR的方法从牛蛙肝脏中克隆牛蛙核糖核酸酶(RC-RNase)基因并进行序列测定;将人源化抗肝癌单链抗体scFv基因与RC-RNase基因相连接,制备scFv-RC-RNase融合基因表达质粒,转化大肠杆菌BL21(DE3),用IPTG诱导进行表达。以SDS-PAGE和免疫印迹法对表达产物进行分析鉴定。序列分析表明,扩增出的RC-RNase基因片断大小约为405bp。经SDS-PAGE和免疫印迹分析显示,scFv-RC-RNase融合基因表达质粒在大肠杆菌中的诱导表达产物出现相对分子质量约为38000的一条新生蛋白带,与预期结果相符。融合蛋白表达量占菌体总蛋白量的18.5%,主要以包涵体形式存在。表明成功地构建了抗肝癌scFv-RC-RNase融合基因,并在大肠杆菌中获得有效表达,为进一步进行肝癌的导向治疗研究奠定了基础。

To prepare fusion gene of the humanized scFv against hepatocellular carcinoma (HCC)and RC-RNase for a new immunotoxin in E.coli.RC-RNase gene was cloned by RT-PCR from the liver of Rana catesbeiana and identi-fied by nucleotide sequencing.An expression plasmid of the fusion gene scFv-RC-RNase was constructed by linking anti-HCC scFv with RC-RNase.Then the plasmid was introduced into E.coli BL2(DE3).Anti-HCC scFv-RC-RNase expression was induced by IPTG.SDS-PAGE and Western blotting analyzed the expression product.The sequencing data indicated that the RC-RNase amplification was as long as405bp.After induction,anti-HCC scFv-RC-RNase was expressed in E.coli.A new protein band was observed at the position of Mr38000on SDS-PAGE gel,account-ing for18.5%of total amount of bacterial proteins.The expressed product was found with the inclusion bodies.Western blotting further identified the target protein.The fusion gene of anti-HCC scFv and RC-RNase was suc-cessfully constructed and expressed in E.coli,the fusion protein may ultimately be used in targeting therapy of HCC.