摘要
目的 配合HBV载体的研究 ,为HBV载体提供包装。方法 HBV全基因经删除包装信号ε区后 ,插入到潮霉素抗性pMEP4载体 ,转染HepG2 细胞系 ,用潮霉素筛选形成细胞克隆。检测表达HBsAg及HBcAg较多者作为HBV包装细胞系 ,进一步转染稳定表达复制缺损型HBV的质粒 ,PCR方法观察上清液中病毒的形成。结果 包装细胞系高表达HBsAg和HBcAg ;携带重组HBV的G4 18抗性重组逆转录病毒感染潮霉素抗性包装细胞系后 ,经两种抗生素同时筛选 ,在细胞培养上清液中能检出突变型HBV ,未检出野生型HBV。结论 删除了包装信号的HBV次全基因 ,失去了形成完整HBV颗粒的能力 ,但能为复制缺损型HBV提供包装所需的结构蛋白。
Objective To cooperate with the study of HBV vector,hygromycin-resistant packaging cell line was developed that allows encapsidation of plasmids into HBV particles. Methods Free of packaging signal,HBV genome was inserted into plasmid pMEP4,which expresses the HBV structural proteins including core,pol and preS/S proteins. HepG 2 cell lines were employed to transfect with the construct. Hygromycin selection was done at a concentration of 150 μg/ml in the culture medium. The hygromycin-resistant clones with the best expressions of HBsAg and HBcAg were theoretically considered as packaging cell line and propagated under the same conditions. It was infected with recombinant retrovirus vector and then selected with G418 and hygromycin in the culture medium. The existence of recombinant HBV virion in the culture medium was examined by PCR. Results Hygromycin-resistant HBV packaging cell line was generated,which harbored an HBV mutant whose packaging signal had been deleted. Expressions of HBsAg and HBcAg were detectable. Infected with recombinant retrovirus pRV-CP,the hygromycin-resistant packaging cell line was found to secrete mutant HBV particles and no wild-type HBV was detectable in the culture medium. Conclusion After the packaging signal was deleted and transfected into HepG 2 cell lines,the partial HBV genome lost its ability to form wild-type HBV,but conserves cis-action providing structural proteins for the packaging of the replication-defective HBV.
出处
《中华实验和临床病毒学杂志》
CAS
CSCD
北大核心
2004年第1期28-30,共3页
Chinese Journal of Experimental and Clinical Virology
基金
国家自然科学基金 (3 0 170 85 4)
全军"十五"课题(0 1MA0 10 )
