在西安地区HBeAg阴性患者中检出乙型肝炎病毒前C区基因突变株

Detection of pre-C region mutants in hepatitis B virus from HBeAg-negative patients in Xi'an area of China

作者用HBeAg阴性、抗HBe阳性患者血清提取HBV DNA作为模板,用前C区引物进行聚合酶链反应(PCR),选择3份PCR扩增阳性产物,用EcoRI酶切获得长为312 bp的基因片段。其克隆至PUC18质粒中,转化大肠杆菌JM109,提取质粒,经PCR扩增及酶切鉴定后,用双脱氧末端标记法进行基因序列测定。结果发现,在2例患者HBV DNA克隆中存在有前C区基因突变,即1896位的鸟嘌吟为腺嘌吟所替代,因此产生1个终止性的密码子TAG。本研究发现在我国西安地区存在HBV前C区突变株的感染,对进一步研究乙型肝炎发病机理具有重要意义。

HBV DNA extracted from the sera of HBeAg-negative and anti-HBe positive patients was amplified in polymerase chain reaction (PCR) with pre-C region primer. Positive products of PCR from 3 patients were chosen and digested with EcoR I. After purified from 1% low melting point agarose gel, we obtained the resulting fragment of 312 base pairs (bp) representing nt 1653 to 1964 and containing the pre-C region (nt 1814 to 1900); then they were cloned into plasmid PUC18 and transformated into E. coli JM109. Gene sequence was determined by didoxy chain termination method. The results showed that the gene mutation in 1896 site of HBV pre-C region was found in the clones of two patients. In that place, Guanine was replaced by Adenine. It, therefer, converted Trp-28 (TGG) into a stop codon (TAG) inhibiting the synthesis and secretion of HBeAg. This is the first report about the infection of mutants in HBV pre-C region detected in Xi'an area of China and it would play an important role in studying the pathogenesis of hepatitis B.