逆转录病毒载体介导的HBs Ag抗原表达及其稳定性研究  

Retroviral vector-mediated HBsAg expression and it's stability

作　　者：周智[1] 张海红[2] 卢建溪[2] 姚集鲁[2] 邓练贤[2] 

机构地区：[1]重庆医科大学附二院传染科,400010 [2]中山医科大学中山三院传染病科

出　　处：《中华实验和临床病毒学杂志》2002年第3期236-238,共3页Chinese Journal of Experimental and Clinical Virology

基　　金：国家自然科学基金青年基金项目 (3 0 10 0 15 8) ;第 2 6届中国博士后基金〔1999〕17号

摘　　要：目的　探讨逆转录病毒载体介导的HBsAg抗原的表达及其稳定性。方法　将HBVS基因插入逆转录病毒载体pLXSN中 ,构建成重组逆转录病毒载体。用电穿孔法转染PA317细胞 ,包装成假病毒颗粒 ,在不同温度下冻存。于不同时间用假病毒颗粒感染HepG2 、NIH3T3及 2 93细胞 ,用RT PCR及ELISA法检测HBsAg表达 ;以感染后G4 18抗性克隆形成数确定假病毒颗粒的活力。结果　在各个时间段HBsAg表达量均差异无显著性。在 - 2 0℃冻存时 ,6个月后克隆数即下降过半 ,12个月后仅形成少数克隆 ,2 4个月后无克隆形成 ;在 - 4 0℃冻存时 12个月后HepG2 、NIH3T3、2 93形成的克隆数分别为 12 1、332和 89,2 4个月后分别为 4 2、137和 4 3,与冻存前比较差异有显著性 ;- 70℃冻存时 ,2 4个月后克隆数分别为 15 9、4 6 3和 112 ,与冻存前比较差异无显著性。结论　HBsAg重组逆转录病毒颗粒在 - 70℃保存 2年后 ,病毒活力未受明显影响 ,HBsAg表达量亦无明显改变。Objective To explore use of retroviral vector in gene therapy of hepatitis B. Methods The recombinant vector pLXSN HBs was constructed by inserting HBV S gene into pLXSN. The pseudovirus, which was produced from PA317 after transferring with pLXSN HBs by electroporation, were frozen at different temperature. The activities of the pseudovirus to infect eukaryotic cells and express antigen were determined by comparing the numbers of G418 resistant clones and assaying HBsAg in the supernatant of the cells with ELISA after infection HepG 2, NIH3T3 and 293 cells. Results It was hard to find changes in HBsAg amount at different intervals and different temperatures. G418 resistant clones, however, were variable. When frozen at -20℃, the numbers of clones were half less than that of the beginning after 6 months, few clones were formed after 12 months, and no clone was found after 24 months. When frozen at -40℃, the numbers of clones were 121, 332 and 89, 42, 137 and 43 for HepG 2, NIH3T3 and 293 cell lines at 12 and 24 months, respectively. When frozen at -70℃, the numbers of clones were 159,463 and 112 for HepG 2, NIH3T3 and 293 cell lines at 24 months, respectively. There was no statistical difference compared to that of zero months. Conclusion The activity of the peseudovirus to infect eukaryotic cells and expressed antigen was not changed after 2 years frozen at -70℃.

关 键 词：逆转录病毒载体 HBsAg抗原 表达 稳定性 乙型肝炎 

分 类 号：R392[医药卫生—免疫学]