丙型肝炎病毒核心蛋白基因克隆载体的构建

Construction of Cloning Plasmid Containing Core Gene of HCV

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 the PMD18T vector containing the core gene of the hepatitis C virus（HCV）,preparing for the research on the therapy for virus hepatitis with expression regulation vector which involves the application of the modulating mechanism of HCV core protein.Methods After expanding plasmid pHCV（containing the whole genome of HCV） through E.coli DH5α,the core gene is amplified by PCR from pHCV.Then the core segment is inserted into the clone plasmid PMD18T.Plasmid PMD18T-HCV/core is transformed into DH5α and the positive clones were screened out and the recombinant plasmids were isolated from such clones.Finally,the recombinants were identified by PCR,restriction endonucleases analysis and sequencing.Results PCR assay showed that a DNA fragment of 582bp could be amplified from the recombinant plasmid.Restriction endonucleases digestion confirmed the target fragment had been inserted into PMD18T successfully.The sequence results verified that the sequence of the target fragment had high homology（97%） with the core gene sequence of HCV in GeneBank. Conclusion The T vector clone with core gene of HCV has been constructed successfully and it is effective for the study on subcloning and the inhibition of hepatitis virus.