CAJ | 학술논문

目的就拉米夫定治疗慢性乙型肝炎(CHB)1a的疗效、HBV DNA P基因变异特点及编码的蛋白质的二级结构加以研究.方法提取HBV DNA后,经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.结果按照ALT水平分组,各组间促进ALT正常化没有明显差异.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,还发现了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 1a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.
목적취랍미부정치료만성을형간염(CHB)1a적료효、HBV DNA P기인변이특점급편마적단백질적이급결구가이연구.방법 제취HBV DNA후,경반소식취합매련반응(PCR)확증,PCR산물자동측서.총결랍미부정적료효、P기인변이출현적시간,용SAS연건분석영향료효적인소.용DNA STAR연건적CLUSTAL V방법대HBV DNA P기인편단적핵감산화안기산차이、변이류형진행분석.용DNAclub、DNAsis 연건분석출현P기인변이전후소편마적단백질적이급결구적차이.결과 안조ALT수평분조,각조간촉진ALT정상화몰유명현차이.재치료후6개월혈청표본중미발현YMDD변이,재치료후12개월혈청표본중발현2례YMDD변이.재대조조중발현료1례L528M변이;재치료후12개월발현료2례YVDD변이,환발현료2례지유L528M적변이.재발생변이후,YMDD적전각결구변위절첩,L528M적전각결구몰변.결론 1a적랍미부정치료가이유효억제HBV DNA적복제.YMDD변이발생재랍미부정치료6개월지후,L528M변이가단독재체내존재,미접수항병독치료적만성HBV휴대자가발생L528M변이.발생YMDD변이적P기인편마적단백질이급결구유전각변위절첩.L528M변이불영향단백질이급결구.
[Objective] To research the outcome of one-year lamivudine therapy and the characters of HBV DNA polymerase gene mutations. [Methods] First extraction of HBV DNA from serum samples, then hemi-nested PCR, purification, sequencing reaction, precipitation and automatic sequencing. Summarize the outcome of lamivudine therapy and observe the time of YMDD mutation development, then analysis the influence factors of outcome by SAS software. Analysis the difference of amino acid of part P gene of HBV DNA, and draw the classification of mutation by CLUSTAL V method of DNA STAR software. Analysis the difference of protein secondary structure of HBV DNA P gene encoded before and after mutation by DNAclub and DNAsis software. [Results] There were not different in improving ALT normalization in four individual groups. After 12 months of therapy, the rate of HBeAg loss increased linearly with ALT level of baseline increasing, the rate was 72.41%; but the rate of HBeAg seroconversion decreased linearly with ALT level of baseline increasing, the rate was 13.79%. Two YMDD mutations had been detected from serum specimens of 12 months after therapy. One L528M single mutation was detected in control group.Two YVDD mutations were accompanied with L528M mutation. YMDD faced to 70-73AA, L528M faced to 47AA.The secondary structure of 47AA and 70-73AA protein was the turn when no mutation. After developing mutation,the turn of 70-73AA changed to the sheet, but the turn of 47AA did not change. [Conclusion] One-year lamivudine therapy can inhibit HBV DNA replication in patients with chronic hepatitis B, and improve ALT normalization.And the rate of HBeAg loss was 72.41%, the rate of HBeAg seroconversion was 13.79%. The ALT level of pretherapy was not enough to predict HBeAg seroconversion alone. YMDD mutations induced by lamivudine therapy usually develop after 6 months, and the YMDD mutant rate in patients with lamivudine therapy was 6.06% one-year.YVDD mutantion usually accompanied with L528M, L528M can exist in vivo alone, and L528M mutation can develop in asymptomatic carriers who did not accept antiviral therapy. Protein secondary structure of HBV DNA P gene encoded changed after P gene mutation, and the turn changed to the sheet. L528M mutation did not affect protein secondary structure.