乙肝病毒X基因慢病毒载体的构建和鉴定

Construction and Identification of a Recombinant Lentiviral Vector Expressing Hepatitis B Virus X Protein

目的构建乙型肝炎病毒(HBV)X蛋白的重组慢病毒载体pRRLsincPPT-hPGK-X-IRES-eGFP-WPRE(pRRL-X),并感染正常组织来源的人肝细胞HL-7702,以验证其在体外的感染效率。方法采用PCR技术扩增出HBV X基因的DNA片段,用IN-Fusion技术构建表达载体pRRL-X并鉴定,jet-PEI转染试剂与三质粒共转染293T细胞后浓缩、计算慢病毒颗粒的滴度;用慢病毒液感染正常组织来源的人肝细胞HL-7702,120 h后初步观察慢病毒颗粒的感染情况。Real-time PCR和Western blot法检测细胞HBV X基因的表达。结果成功构建HBV X基因的慢病毒表达载体质粒pRRL-X。经293T细胞包装后,用慢病毒感染正常组织来源的人肝细胞HL-7702,120 h后被感染的细胞内可见强弱不等的荧光。结论成功构建HBV X慢病毒表达载体。

Objective To validate the transfection efficiency of the recombinant HBV X-containing lentiviral vector（pRRL-X） driven by the hPGK promoter in the HL-7702 cell line.Methods The lentiviral vector（pRRL） was reconstructed by inserting the lentiviral vectors with the HBV X gene fragment,which was isolated from pcDNA3-X vector.It was analyzed with PCR,restriction and protein were existed in cells infected with the lentivirus pRRL-X.The recombination lentiviral particles were produced by the packaging 293T cell by using the jet-PEI method,the culture supernatant was harvested and the titration of them was calculated by the limiting dilution.The HL-7702 cells were transduced by the lentiviral particles,after 120h,the GFP expression was observed under the fluorescence microscope.The expression level of HBV X in cells was examined by the Real-time PCR and Western blot.Results 120 h after pRRL-X lentivirus infection of HL7702 cells,various intensities of florescence can be seen in the cell.The expression of HBVX gene in recombinant HL7702 was confirmed in the way of Real-time PCR and Western blot.Conclusion Recombinant Lentiviral vectors expressing Hepatitis B virus X Gene were successfully constructed.