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目的构建1.3倍乙型肝炎病毒(HBV)全基因的真核表达载体pcDNA3.1(+)-HBV1.3,并观察其在Bewo细胞中的表达。方法以质粒pMD18T-HBV上的HBV DNA序列为模板,构建1.3倍HBV全基因序列,将其插入到真核表达载体pcDNA3.1(+),用酶切、PCR及测序鉴定,并把该载体转染Bewo细胞,以Western免疫印迹、微粒子酶免疫分析法(MEIA)和荧光定量PCR法分别检测胞内和上清中HBsAg、HBeAg蛋白的表达及HBV DNA水平。结果通过酶切、PCR及测序鉴定,成功构建1.3倍HBV全基因表达载体,该载体可以在Bewo细胞系中表达和分泌HBsAg与HBeAg,并可检测到高水平的HBV DNA。结论成功构建了1.3倍HBV全基因真核表达载体pcDNA3.1(+)-HBV1.3,为研究胞内HBV宫内感染奠定了基础。
Objective To construct an eukaryotic expression vector pcDNA3.1 (+) - HBV1.3 of 1.3 times of hepatitis B virus (HBV) gene and observe its expression in Bewo cells. Methods HBV DNA sequence of plasmid pMD18T-HBV was used as a template to construct a 1.3-fold full-length HBV genome sequence and inserted into the eukaryotic expression vector pcDNA3.1 (+). The vector was identified by restriction enzyme digestion, PCR and sequencing. The vector The expression of HBsAg and HBeAg protein and HBV DNA in intracellular and supernatant were detected by Western blotting, microparticle enzyme immunoassay (MEIA) and fluorescence quantitative PCR respectively. Results The full-length 1.3-fold HBV gene expression vector was successfully constructed by restriction enzyme digestion, PCR and sequencing. The vector can express and secrete HBsAg and HBeAg in Bewo cell line and detect high level of HBV DNA. Conclusion 1.3-fold full-length HBV eukaryotic expression vector pcDNA3.1 (+) - HBV1.3 was successfully constructed, which laid the foundation for the study of intrauterine HBV intrauterine infection.