乙型肝炎病毒聚合酶基因重组腺病毒表达系统的构建和初步鉴定

Construction and identification of the recombinant adenoviral expressing system with hepatitis B virus reverse polymerase gene

目的构建乙型肝炎病毒聚合酶基因(RT/RNaseH区段)腺病毒表达系统并初步鉴定其转录表达。方法采用PCR方法从pUC19-1.24HBV质粒中扩增RT/RNaseH区段,将该片段与pCMV-Tag2B载体连接构建腺病毒穿梭载体,将其在Bj5183细菌中重组,获得的重组质粒转染到Ad293细胞中包装,待细胞裂解后收集含病毒的上清并感染Huh7细胞,48h后通过RT-PCR鉴定目的基因转录。结果成功扩增HBVRT区并插入pC-MV-Tag2B载体,成功获得腺病毒重组质粒并在AD293中包装成病毒颗粒,病毒颗粒感染Huh7细胞后RT-PCR可以在Huh7细胞中检测到HBVRT区段mRNA转录。结论成功构建乙型肝炎病毒聚合酶基因RT/RNaseH区段的腺病毒表达系统,该系统能在Huh7细胞中转录,但逆转录蛋白的表达仍待进一步检测。

Objective To construct the adenoviral expressing system of hepatitis B virus reverse polymerase gene （RT/RnaseH domain） and identify the transcription of this system in Huh7 cells. Methods HBV/P（RT/RNAaseH） gene fragment was amplified from pUC19 - 1.24HBV and subsequently inserted into pCMV - Tag2 vector, pCMV - Tag,2 - RT/RNAaseH vector was recombined after transformed into Bj5183 bacterium. The recombinant plasmids extracted from Bj5183 bacterium was transfected into Ad293 cells and encapsidated into adenovirus particles. The adenovirus in the supernatant of Ad293 cells was collected and used to infect Huh7 cells. After 48h, the transcription of HBV/P gene （ RT/ RnaseH domain） was detected by reverse -transcript PCR. Results HBV/P（RT/RNAaseH） gene fragment was successfully amplified and pCMV - Tag2 - RT/RNAaseH vector was successfully constructed. The recombinant plasmid was transfected into AD293 cells. The supernatant containing adenovirus could infect Huh7 cells and the transcription of HBV/ P gene can be detected in the cells. Conclusion The aderloviral expressing system of hepatitis B virus reverse polymerase gene （RT/RnaseH domain） was successfully constructed and they can transcribe the targeted gene in Huh7 cells but the expression of RT/RNAaseH protein needs further determination.