探索用PGenesil-1(Pg)构建的靶向乙型肝炎病毒表面抗原(HBsAg)基因的shRNA表达载体PGenesil-1-HBs(简称Pgs),对体外培养HepG2.2.15细胞中的HBV基因及其抗原表达的抑制作用.设计、合成靶向HBV S区的3对DNA序列,分别插入PGenesil-1中构建3个siRNA表达载体Pgs1、Pgs2、Pgs3,经限制性内切酶,DNA序列测定等技术鉴定确认.筛选并确定最佳细胞接种量及重组质粒转染量后,分别或按不同组合转染HepG-2.2.15细胞,48 h后采用半定量RT-PCR检测HBVsmRNA转录水平,免疫细胞化学技术检测HBsAg表达水平,MEIA分别检测细胞裂解液和培养上清中HBsAg和HBeAg的表达水平.结果表明,HBV真核表达载体Pgs1、Pgs2、Pgs3均能不同程度地抑制HepG2.2.15细胞中的HBsAg、HBeAg合成和HBs-mRNA转录.成功构建的HBV真核表达载体Pgs1、Pgs2、Pgs3,其中PgS3能显著抑制HBsAg表达(P＜0.01).多种表达载体联合对抗原表达的抑制作用效率不同.%It was to construct the shRNA expressing vectors targeting HBsAg gene of HBV, to probe their inhibitation on the HBV antigen expression. Three couple of DNA sequence targeting the HBV S gene was synthesized and cloned into PGenesil-1 plasmid vector,to construct Pgsl ,Pgs2,Pgs3 shRNA expressing vectors,which was confirmed by restriction enzyme digestion,PCR and sequencing. Then, the vectors were respectively or grouping differently transfected into HepG-2. 2. 15 cells. Transcription level of HBs mRNA was tested by means of semiquantitative RT-PCR,and expressional level of HBsAg in HepG-2. 2. 15 cells was tested by ICC. The observed results made sure the Pgsl ,Pgs2,Pgs3 shRNA expression vectors constructed could all inhibit HBsAg expression in the HepG-2. 2.15 in vitro. Thus,the Pgs3 shRNA expression vectors constructed successfully could significantly inhibit HBsAg expression compareed with control group(P <0.01 ) ,while inhibition of combination shRNA expression vector on expression of HBV antigen were not different.
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