Adr亚型乙型肝炎病毒转染细胞模型的构建

Construction of cell models transfected with HBV adr subtype

摘要:
目的建立乙型肝炎病毒(HBV)复制状态的细胞模型。

方法利用分子亚克隆技术,将9600bp的adr亚型HBV全基因克隆至pcDNA3的EcoRI和HindIII位点构建pcDNA3-3HBV,通过脂质体介导的方法转染HepG2细胞,G418筛选。

结果成功构建了质粒pcDNA3-3HBV,稳定转染后培养上清,ELISA检测结果显示,HBsAg、HBeAg阳性,且PCR检测前S/S基因阳性。PCR证实转染细胞中有HBV cccDNA存在,RT-PCR证实HBV S基因mRNA的表达。

结论重组质粒pcDNA3-3HBV能在HepG2细胞中表达、转录、复制。

Abstract:
Objective To establish HBV replication cell models.

Methods Using molecular subclone technique,pcDNA3-3 HBV was constructed by insertion of 9 600 bp fragment of HBV adr subtype genome into the EcoRⅠ and HindⅢ sites of pcDNA3.Hep G2 was transfected with pcDNA3-3 HBV by using lipofectamine transfection reagent and screened with antibiotic G418.

Results The plasmid pcDNA3-3 HBV was constructed successfully.After stable transfection,HBsAg and HBeAg were positive in the supernatant which tested by ELISA.S gene and pre-S gene were also positive which tested by PCR.cccDNA was found in the cells by PCR,and HBV pre2S1 mRNA was measured by RT-PCR.

Conclusion pcDNA3-3 HBV can be expressed,transcribed and replicated in Hep G2 cells.