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目的构建丙肝病毒(HCV)核心基因克隆载体PMD18T-HCV/core,为进一步研究将HCV核心蛋白的基因调节功能应用于构建可调控性表达载体治疗病毒性肝炎做好前期基础。方法将含HCV全长基因的pHCV质粒于大肠杆菌DH5α内扩增,提取pHCV质粒,从pHCV质粒中PCR扩增出HCV核心基因片段并将其插入PMD18T克隆载体得到PMD18T-HCV/core克隆载体,将PMD18T-HCV/core克隆载体转化DH5α,筛选阳性克隆,抽提重组质粒并进行PCR及酶切鉴定,再行序列分析。结果经PCR获得582bp含限制性内切酶位点的阳性产物,T载体克隆后,PCR、酶切鉴定及序列分析证实,克隆片段与GeneBank中该基因的序列同源性为97%。结论该实验成功构建了含HCV核心蛋白基因序列的T载体克隆,提示该克隆是用作亚克隆和抑制肝炎病毒研究的理想克隆。
Objective To construct HCV core gene cloning vector PMD18T-HCV / core for further study of HCV core protein gene regulatory function in the construction of a regulable expression vector for the treatment of viral hepatitis. Methods The pHCV plasmid containing the full-length HCV gene was amplified in E. coli DH5α, and the pHCV plasmid was extracted. The HCV core gene fragment was amplified by PCR from plasmid pHCV and inserted into PMD18T cloning vector to obtain PMD18T-HCV / core cloning vector. PMD18T-HCV / core cloning vector was transformed into DH5α, positive clones were screened, recombinant plasmids were extracted and identified by PCR and restriction enzyme digestion. Results A 582bp fragment containing the restriction enzyme site was obtained by PCR. The cloned T vector was identified by PCR, restriction enzyme digestion and sequence analysis. The sequence identity of the cloned fragment to GeneBank was 97%. Conclusion This experiment successfully constructed a T vector clone containing the HCV core protein gene sequence, suggesting that this clone is an ideal clone for subcloning and suppression of hepatitis virus.