低拷贝HBV病毒全长DNA的制备和体外复制水平的鉴定

Low copy number amplification of full-length hepatitis B virus gene and identification of in-vitro replication level in serum

摘要:

目的：从乙型肝炎病毒（hepatitis B virus，HBV）低拷贝的血清样本中提取病毒DNA并通过PCR扩增得到全长DNA片段。将全长片段经过环化处理后转染HuH7细胞，体外评价病毒的复制水平。方法：设计含有BspQⅠ酶切位点的HBV全长扩增引物和分段扩增引物，选择HBV低拷贝的临床病人血清样本，经抽提纯化后的病毒DNA作为模板，用巢式PCR结合分段扩增的方法扩增得到HBV全长DNA，PCR产物经BspQⅠ酶切后自连环化，将环化的HBV DNA转染HuH 7细胞，经5 d细胞培养，提取细胞中的病毒复制中间体，通过Southern blot分析病毒复制水平。结果：通过PCR扩增得到5个HBV全长DNA，经过酶切连接全长基因后转染HuH7细胞，Southern blot检测均有复制表达。结论：本实验成功构建能够有复制表达的HBV环化全长，为研究血清中低拷贝HBV病毒的不同病人复制水平与DNA序列关系打下基础。

Abstract:

Objective：To evaluate the replication of virus by extracting virus DNA from low copy number of hepatitis B virus （HBV） serum samples，doing PCR amplification to acquire full-length HBV DNA fragments and transfecting HuH7 cells with full-length HBV DNA fragments after treatment. Methods：Primers for fragment and full-length amplification containing BspQⅠ restrictive en－zyme cutting site were designed and HBV DNA from patients was used as templates. Full-length HBV was acquired by nested PCR combined with sectional amplification. Closed circular DNA was got through restrictive enzyme digestion and T4 DNA ligase ligation. HBV replication level was analyzed by Southern blot after HuH 7 cells being transfected with closed circular HBV DNA. Results：Five complete HBV genomes were successfully obtained using the new PCR method. After digestion，ligation and tansfection experiments，Southern blot results all supported replication expression. Conclusions：This method lays a foundation for investigating the correlation of different HBV replication levels and DNA variation in patients with low copy number of virus.