慢性乙型肝炎患者体内乙型肝炎病毒DNA序列异质性及准种特点的研究

The study on heterogeneity of hepatitis B virus DNA

目的 以乙型肝炎病毒 (HBV)基因异质性来探讨HBV准种群在慢性感染者中的存在状态。方法 应用聚合酶链反应 (PCR)法 ,自 18例慢性患者血清中分别扩增前C/C基因、P基因逆转录酶区 (RT)、HBV全S区、X基因和全基因组序列 ,克隆入pGEMTeasy质粒 ,挑选 81克隆进行DNA测序以确定病毒的变异程度。结果 除外大段缺失突变的克隆 ,来源于同一患者前C/C区、RT区、全S区、X区和全基因组不同克隆之间的碱基序列同源性分别为 98 0 %～ 99 1%、98 7%～ 99 3%、97 5 %～ 10 0 %、93 0 %～ 98 2 %和 96 6 %～ 97 5 %。前C区A83点突变、C抗原内部缺失较为常见 ,缺失突变、终止替换突变、短序列的插入或缺失突变造成的移框突变在各个区域中均可检出 ,X基因 (核心蛋白启动子区 ,CP)内部缺失突变不仅导致X蛋白羧基端的多样性 ,而且调控HBeAg的表达。编码截短型表面抗原中蛋白和缺陷病毒基因亦在患者外周血中。结论 多区域测序结果提示HBV慢性患者体内HBV呈现准种群共存 ,X区 (CP区 )内存在热点缺失突变区 ,CP区的变异可能影响前C蛋白的表达 ,这些变异可能与患者不良预后有关。

Objective To investigate the HBV quasispecies groups in the patients with chronic HBV infection. Methods Specific primers ware synthesized according to HBV strain found in China, the preC/C gene, reverse transcriptase region, whole S region, X gene and whole genome ware amplified by PCR method from the serum of 18 patients with chronic HBV infection, and then the PCR products were subcloned into pGEM Teasy vectors. Positive clones with target sequences were selected out for sequencing. Sequence comparison of the selected clones ware made to find the difference. Results The homology between clones from one patient of preC/C gene, reverse transcriptase of polymerase region, whole S region, X gene and whole genome are 98 0%～99 1%?98 7%～99 3%?97 5%～100%?93 0%～98 2% and 96 6%～97 5%, respectively. There was a high frequency A83 substitution and core antigen internal deletion (CID) in preC/C region. Substitution, deletion and frame shift by insertion or deletion of short sequence were found in 4 open reading frames. Deletion in X gene (Core promoter, CP) will not only result in the polymorphism of X protein at the carboxyl end, but also regulate the expression of HBeAg. Coding sequence of truncated middle surface antigen and defective HBV genome could also be detected in this study. Conclusion There are HBV quasispecies groups in patients with chronic HBV infection. Hot deletion region in X region (CP) will influence the prognosis of the HBV infection. Individually characterized substitutions in amino acid sequence of viral protein is worthy of further study.