论文部分内容阅读
目的:克隆人细胞角蛋白8(CK8)基因cDNA并在大肠杆菌中表达CK8蛋白产物。方法:运用DNA重组技术,设计CK8基因特异引物,采用反转录聚合酶链反应(RT-PCR)技术从人肝细胞癌7721细胞中扩增CK8编码区cDNA,将其插入克隆载体pMD18-T simple vector中,获得重组质粒pMD18-CK8,转化感受态大肠杆菌DH5α。继而采用PCR技术从重组质粒pMD18-CK8中扩增出约1500bp目的片段,再次级克隆到原核表达质粒pET-28a(+)载体中,获得pET-28a-CK8重组原核表达质粒;经酶切、PCR及测序鉴定后转化入BL21(DE3)菌株,在IPTG诱导下进行融合蛋白的表达。采用SDS-PAGE电泳及Western blot检测CK8蛋白表达水平。结果:成功建立了人CK8基因cDNA重组体的无性繁殖系。②在原核细胞中成功地表达出人CK8蛋白,该蛋白具有很好的免疫原性。结论:运用原核细胞基因工程技术成功构建和表达了人细胞角蛋白8(CK8)基因,为进一步研究CK8的基因型及探讨该基因编码的CK8蛋白的性质和生物学活性创造条件。
Objective: To clone human cytokeratin 8 (CK8) cDNA and express CK8 protein in E. coli. Methods: CK8 gene-specific primers were designed by using DNA recombination technique. CDNA of CK8 coding region was amplified from human hepatocellular carcinoma cell line 7721 by reverse transcription-polymerase chain reaction (RT-PCR) and cloned into the cloning vector pMD18-T simple vector, the recombinant plasmid pMD18-CK8 was obtained and transformed into competent E. coli DH5α. Then about 1500bp fragment was amplified by PCR from the recombinant plasmid pMD18-CK8 and cloned into the prokaryotic expression plasmid pET-28a (+) vector again to obtain the recombinant prokaryotic expression plasmid pET-28a-CK8. After identification by PCR and sequencing, the strain was transformed into BL21 (DE3) strain and the expression of the fusion protein was induced under the induction of IPTG. The protein expression of CK8 was detected by SDS-PAGE and Western blot. Results: Clone line of cDNA recombinant of human CK8 gene was successfully established. ② in human prokaryotic cells successfully expressed human CK8 protein, the protein has good immunogenicity. CONCLUSION: The human cytokeratin 8 (CK8) gene was successfully constructed and expressed by using prokaryotic gene engineering technology, which provided conditions for further research on the genotype of CK8 and its biological properties and biological properties.