拉米夫定对乙型肝炎病毒DNA复制及基因变异的影响

目的:研究拉米夫定治疗慢性乙型肝炎(CHB)的疗效、乙型肝炎病毒(HBV)DNA P基因变异特点及编码蛋白质的二级结构.方法:提取HBV DNA后,半巢式聚合酶链反应(PCR)扩增,PCR产物自动测序.总结拉米夫定的疗效、P基因变异出现的时间,用SAS软件分析影响疗效的因素.用DNA STAR软件的CLUSTAL V方法对HBV DNA P基因片段的核苷酸和氨基酸差异、变异类型进行分析.用DNAclub,DNAsis软件分析出现P基因变异前后所编码的蛋白质的二级结构的差异.结果:在治疗后6个月血清标本中未发现YMDD变异,在治疗后12个月血清标本中发现2例YMDD变异.在对照组中发现了1例L528M变异;在治疗后12个月发现了2例YVDD变异,2例只有L528M的变异.在发生变异后,YMDD的转角结构变为折叠,L528M的转角结构没变.结论:1年的拉米夫定治疗可以有效抑制HBV DNA的复制.YMDD变异发生在拉米夫定治疗6个月之后,L528M变异可单独在体内存在,未接受抗病毒治疗的慢性HBV携带者可发生L528M变异.发生YMDD变异的P基因编码的蛋白质二级结构由转角变为折叠.L528M变异不影响蛋白质二级结构.

Effects of lamivudine on DNA duplication and genetic mutation of hepatitis B

Objective: To study the outcome of one-year lamivudine therapy and the characters of HBV DNA polymerase gene mutation.Methods: We extracted HBV DNA from serum samples,then performed hemi-nested PCR,purification,sequencing reaction,precipitation,and automatic sequencing.The outcome of lamivudine therapy was summarized,and the developing time of YMDD mutation was observed.The factors affecting outcome were analyzed by using SAS software.The difference of amino acid of part P gene in HBV DNA was analyzed, and the mutation was classified by using CLUSTAL V method with DNA STAR software.The difference of secondary structure of protein encoded by HBV DNA P gene before and after mutation was analyzed by using DNAclub and DNAsis software.Results: No YMDD mutation was found in serum specimen 6 months after the therapy.Two YMDD mutations had been detected in serum specimen 12 months after therapy.One L528M single mutation was detected in the control group.Two YVDD mutations were accompanied with L528M mutation.After the mutation,the turn of secondary structure of YMDD protein changed to the sheet,but the turn of L528M did not change.Conclusion: One-year lamivudine therapy can inhibit HBV DNA replication in patients with chronic hepatitis B and improve the normalization of alanine aminotransferase.Mutations of YMDD induced by lamivudine therapy usually develop 6 months after therapy.The YMDD mutant rate in patients receiving lamivudine therapy was 6.06% 1 year later.YVDD mutation is usually accompanied with L528M.Mutation of L528M can exist in vivo alone and can develop in asymptomatic carriers without antiviral therapy.Secondary structure of protein encoded by HBV DNA P gene changes after P gene mutation,and the turn changes to the sheet.L528M mutation has no effect on secondary structure of protein.