携HBx基因逆转录病毒载体的构建

Construction of a Retroviral Vector Containing HBx Gene

【目的】构建携HBx基因的逆转录病毒载体 ,为研究HBx基因与肝癌生物学行为间的关系提供基础。【方法】采用PCR技术从HBV全基因组中扩增HBx基因 ;将HBx基因亚克隆至质粒pLNSX ,构建质粒 pLNSHBx ,磷酸钙 DNA共沉淀法将重组体导入包装细胞系PA317,检测培养上清病毒滴度。【结果】应用PCR技术成功地从HBV基因组中克隆出全长HBx基因 ,并经序列分析证实 ;建立了产病毒细胞株PA317/HBx ,检测其培养上清病毒滴度为 8.9× 10 4 CFU ,PCR证实重组病毒中含有HBx基因。【结论】成功构建了携HBx基因的较高滴度逆转录病毒载体。

Objective Establish a retroviral vector carrying HBx gene for investigation of the effects of HBx gene on the biological behavior of hepatocellular carcinoma. Methods HBx gene was amplified from genomic DNA of HBV by PCR technique, and subcloned to plasmid pLNSX to construct recombinant plasmid pLNSHBx. Applying the calcium phosphate-DNA co-precipitation technique, the pLNSHBx was transferred into PA317 packaging cell line. Culture supernatant of these cells was collected for titration of the recombinant virus. Results HBx gene was cloned from HBV genome by PCR successfully, and proved by sequence determination. A vector producing cell line PA317/HBx was established. The titres of the recombinant virus in the culture supernatant were 8.9×10 4 CFU. An HBx gene integration was detected by PCR in the recombinant retroviral vectors. Conclusions A retrovirus vector containing HBx gene was successfully constructed.