人源化抗肝癌scFv融合RC-RNase在大肠埃希菌中的表达及对人肝癌细胞SMMC-7721的体外杀伤作用

目的 :在大肠埃希菌中可溶性表达新型重组人源化抗肝癌单链抗体融合RC RNase(scFv RC RNase) ,观察其对体外培养的人肝癌细胞SMMC 772 1的杀伤作用。方法 :将原核表达质粒Tih scFv RC RNase以大肠埃希菌E coliBL2 1(DE3 )为宿主菌 ,用异丙基硫代 -β -D -半乳糖苷 (IPTG )诱导表达。工程菌经超声碎菌、离心后 ,上清液用Ni NTAAgarose亲合层析法进行纯化。四甲基噻唑蓝 (MTT)法检测纯化蛋白对体外培养的人肝癌细胞SMMC 772 1的体外杀伤作用。结果 :IPTG诱导 5h后 ,SDS PAGE显示抗肝癌scFv RC RNase在大肠埃希菌E coliBL2 1(DE3 )中主要以可溶性的形式表达 ,表达量占菌体总蛋白量的 10 5 %。MTT法提示 ,纯化后的表达产物对体外培养的人肝癌细胞SMMC 772 1具有一定的杀伤作用 ,其半数致死量 (LD50 )为 0 0 0 7g/L。结论 :抗肝癌scFv RC RNase融合蛋白有望成为一种具有一定临床应用潜力的抗肝癌导向治疗药物。

Expression of humanized anti-HCC scFv fusing to RC-RNase in E.coli and its killing effect on human hepatocellular carcinoma cell SMMC-7721 in vitro

OBJECTIVES:To express solubly new recombinant humanized anti hepatocellular carcinoma (HCC) scFv fusing to RC RNase (scFv RC RNase) in E.coli and to observe its killing effect on HCC cell SMMC 7721 in vitro .METHODS:Prokaryotic expression vetor containing scFv RC RNase was induced to be expressed by IPTG in E.coli BL21(DE3).The E.coli was lysed by ultrasonication. Supernatant of the lysate was purified by Ni NTA Agarose affinity chromatography.MTT method was used to detect the killing effect of scFv RC RNase fusion proteins in vitro .RESULTS:After induction with IPTG for 5 h,a new anticipated band appeared on SDS PAGE gel.The expressed product existed in soluble form,and was amount to 10 5% of total bacterium protein.After being purified, target protein killed the HCC cell SMMC 7721 in vitro with the LD 50 0 007 g/L.CONCLUSIONS:The humanized anti HCC scFv RC RNase fusion protein has been successfully solubly expressed in E.coli (DE3) with killing effect on HCC cell, and may be a potential anti HCC targeting agent.