乙型肝炎病毒全基因组研究的新方法

A novel method to study the full length genome of hepatitis B virus

目的为从全基因水平研究乙型肝炎病毒变异与致病性的关系，建立血清标本经聚合酶链反应（ＰＣＲ）加酶切方法扩增及克隆ＨＢＶ全基因组的新方法。方法设计了含ＳｐｅⅠ，ＳａｌⅠ及ＳａｐⅠ酶切点的引物，分别扩增１．１５ｋｂ和２．０５ｋｂ的单链及双链ＤＮＡ区。经酶切及连接后获得ＨＢＶ全基因克隆。结果用新建立的方法直接从少量血清中克隆了ＨＢＶ全基因，转染ＨｅｐＧ２细胞可表达抗原并有胞内复制。

Objective To study the association between pathogenicity and variants of hepatitis B virus(HBV) by full length HBV genome, a novel method of amplification of the whole genome of HBV strains from serum samples of patients by PCR, followed by enzyme digestion and cloning was established. Methods Two sets of primers were designed: one with SpeⅠ restriction site, the other with restriction sites of SalⅠ and SapⅠ. PCR products were amplified separately from the single stranded and the double stranded regions of the genome, and the two PCR products obtained were 1.15 kb and 2.05kb. After cleavage and ligation, the full length HBV genome was used to transfect HepG2 cell line. Results Expression of viral antigens and intracellular replication of HBV were observed with the full length HBV genome by the novel method. Conclusion This novel method could serve to study the structure and function of HBV genome from viral strains of clinical samples.