HBx基因重组逆转录病毒载体的构建及表达

目的:构建携带HBx基因的重组逆转录病毒载体,为进一步研究HBx基因在HBV相关性HCC(原发性肝癌)发病机制中的作用以及建立肝癌动物模型奠定基础.方法:根据基因库中已公布的HBx序列设计引物,采用PCR技术从HBx腺病毒质粒中扩增出HBx基因.将HBx基因克隆到逆转录病毒载体pSEB-HUS质粒中.酶切、PCR及测序鉴定后经脂质体介导转染L02细胞,Western Blotting检测HBx蛋白的表达.结果:PCR、酶切及测序鉴定证实目的基因正确克隆至逆转录病毒质粒pSEB-HUS中,目的基因序列与GENE BANK报道一致,转染L02细胞后荧光显微镜可观察到GFP的表达.Western Blotting检测可见HBx蛋白的表达.结论:成功构建HBx重组逆转录病毒载体.

Construction and expression of Retroviral Vector containing HBx gene

Objective:To construct the retroviral vector containing HBx gene so as to investigate the effects of HBx gene in the pathogenesis of HBV-dependent hepatic cell carcinoma(HCC)and further construct animal models of hepatic cell carcinoma.Methods:HBx gene was amplified from the HBx adenovirus Plasmid by polymerase chain reaction(PCR)technique whose primer was designed according to the announced sequences of HBx gene in gene bank,then cut by two endonucleases and directionally cloned into the pseb-hus vector and analyzed by gene sequencing analyzer,the correct constructed vectors were transfected into L02 cell line.At last,HBx protein was detected by Western Blotting.Results:Through PCR,endonuclease cutting and gene sequencing,the target gene was verified into be correctly cloned to retroviral plasmid pSEB-HUS and the high expression of green fluorescence protein(GFP)in L02 cell line was observed under fluorescent microscope.The expression of HBx protein was also observed by Western Blotting.Conclusion:Recombinant retroviral vector expressing HBx protein was successfully constructed.