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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 the CK-1 gene of Trichinella spiralis and hepatocarcinoma cell and identify it. Methods The CK-1 gene was amplified by PCR using Trichinella spiralis cDNA as template, and cloned into the prokaryotic expression vector pET-28a(+) to construct the recombinant expression plasmid pET-28a-CK-1 and transform competent Escherichia coli (E). BL21 (DE3) and IPTG were induced for 3, 4, and 5 h, respectively, and the expressed product was analyzed by 12% SDS-PAGE. Mice were immunized with purified Trichinella muscle larvae to prepare mouse anti-Trichinella muscle larva positive serum. Mouse anti-H7402 whole cell protein positive serum was prepared by immunizing mice with H7402 cells. Mouse anti-CK- was prepared by immunizing mice with recombinant CK-1 protein. 1 protein positive serum, respectively, as its primary antibody, Western blot identification. Results Recombinant expression plasmid pET-28a(+)-CK-1 was proved to be constructed correctly by double restriction enzyme digestion and sequencing. The optimal induction time of IPTG was 4 h. The expressed recombinant CK-1 protein existed in the form of inclusion bodies, and the expression level accounted for 37.5% of total bacterial protein; recombinant CK-1 protein can be recognized by mouse anti-Trichinella muscle larva positive serum and mouse anti-H7402 whole cell protein positive serum. Mouse anti-CK-1 protein positive serum can recognize Trichinella spiralis muscle larval protein and H7402 whole cell protein showed specific bands at a relative molecular weight of 38 500, indicating that the recombinant CK-1 protein is a cross antigen and has a good reactogenicity. Conclusion The prokaryotic expression of Trichinella spiralis CK-1 gene lays a foundation for further study on the anti-tumor effect of Trichinella spiralis.