旋毛虫与肝癌细胞相关基因CK-1的原核表达及鉴定

Prokaryotic expression and identification of liver cancer cells-associated CK-1 gene of Trichinella spiralis

目的原核表达旋毛虫与肝癌细胞相关基因CK-1,并进行鉴定。方法以旋毛虫cDNA为模板,PCR扩增CK-1基因,克隆至原核表达载体pET-28a(+)中,构建重组表达质粒pET-28a-CK-1,转化感受态大肠埃希菌(E.coli)BL21(DE3),IPTG分别诱导表达3、4、5 h,表达产物进行12%SDS-PAGE分析。以纯化的旋毛虫肌幼虫免疫小鼠制备鼠抗旋毛虫肌幼虫阳性血清,以H7402细胞免疫小鼠制备鼠抗H7402全细胞蛋白阳性血清,以重组CK-1蛋白免疫小鼠制备鼠抗CK-1蛋白阳性血清,分别以其作为一抗,进行Western blot鉴定。结果重组表达质粒pET-28a(+)-CK-1经双酶切及测序鉴定证明构建正确;IPTG最佳诱导时间为4 h,表达的重组CK-1蛋白以包涵体形式存在,表达量占菌体总蛋白的37.5%;重组CK-1蛋白可被鼠抗旋毛虫肌幼虫阳性血清和鼠抗H7402全细胞蛋白阳性血清识别,鼠抗CK-1蛋白阳性血清可识别旋毛虫肌幼虫蛋白和H7402全细胞蛋白,在相对分子质量38 500处均可见特异性条带,表明重组CK-1蛋白是一个交叉抗原,且具有良好的反应原性。结论原核表达了旋毛虫CK-1基因,为进一步研究旋毛虫的抗肿瘤效应奠定了基础。

Objective To express and identify liver cancer cells-associated CK-1 gene of Trichinella spiralis in prokaryotic cells. Methods CK-1 gene was amplified by PCR using the cDNA of T. spiralis as a template and cloned into prokaryotic expression vector pET-28a （＋）. The constructed recombinant plasmid pET-28a-CK-1 was transformed to E. coli BL21 （DE3） which was induced with IPTG for 3, 4 and 5 h respectively. The expressed product was analyzed by 12% SDS-PAGE. Mouse antisera were prepared by immunizing mice with purified T. spiralis muscle larvae, H7402 cells and recombinant CK- 1 protein, and used as the first antibody for Western blot respectively. Results Restriction analysis and sequencing proved that recombinant plasmid pET-28a （＋）-CK-1 was constructed correctly. The optimal time for induction with IPTG was 4 h. The expressed recombinant CK-1 protein, in a form of inclusion body, contained 37. 5% of total somatic protein. Recombinant CK-1 protein was recognized by positive mouse sera against T. spiralis muscle larvae and against whole H7402 cells, with specific bands with relative molecular mass of 38 500, which was proved as a cross antigen with good reactogenicity. Conclusion The CK-1 of T. spiralis was successfully expressed in prokaryotic cells, which laid a foundation of further study on the anti-tumor effect of T. spiralis.