Construction of infectious clone of HBV adefovir-resistant strain RT A181T and its expression in Huh7 cells
10.3969/j.issn.1673-4130.2016.23.010
- VernacularTitle:乙型肝炎病毒阿德福韦酯耐药株（RT A181T）感染性克隆的构建及在 H uh7细胞中的表达
- Author:
Jianhua ZHAO
;
Renfei LU
- Keywords:
hepatitis B virus;
adefovir;
drug resistance;
infectious clone
- From:
International Journal of Laboratory Medicine
2016;37(23):3266-3268
- CountryChina
- Language:Chinese
-
Abstract:
Objective To rapidly construct hepatitis B virus(HBV)adefovir(ADV)‐resistant strain(RT A181T)infectious clone by using SOE‐PCR ,to observe the expression of recombinant plasmids in Huh7 cells and to establish the in vitro cell model of HBV ADV‐resistant strain(RT A181T) .Methods The conservative primers were designed ,the full‐length HBV genome was amplified from serum in the patients with ADV‐resistant chronic hepatitis B .Then ,the 1 .3‐fold genome‐length infectious clone pHBV1 .3 was constructed by using the SOE‐PCR technique ,which was transfected into human hepatoma cells Huh7 cell line .ELISA ,Western Blot and Real‐time PCR were adopted to detect infectious cloning replication and expression ,meanwhile the antiviral drugs lamivu‐dine(LAM ) and ADV were used to verify the infectious clone copy and inhibition of expression .Results The HBV ADV‐resistant infectious cloning plasmids pHBV1 .3 was successfully constructed .This plasmid could be effectively replicated ,transcripted and ex‐pressed in Huh7 cell lines .LAM could effectively inhibit the replication and expression of the infectious clone ,while ADV could not inhibit the replication and expression of the infectious clone .Conclusion The constructed infectious clone plasmids pHBV1 .3(RT A181T ) of HBV ADV‐resistant strain can efficiently replicate and express protein in vitro ,its transfected cells can be used in the study of HBV replication mechanism and antiviral study .
