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目的研究拉米夫定治疗前后慢性乙型肝炎患者体内乙型肝炎病毒P基因区的变异情况。方法从5例慢性乙型肝炎患者拉米夫定治疗前和治疗12个月的血清标本中,扩增目的片段,阳性结果双酶切后克隆至JM105感受态细胞。每份标本随机挑取20个阳性克隆,以错配聚合酶链反应一限制性片段长度多态性分析法检测YMDD基因序列,出现变异者进行双向测序。结果 5例慢性乙型肝炎患者在拉米夫定治疗12个月后,其中2例HBV DNA为阴性;2例HBV DNA转阴后又转阳,测序结果提示出现M552I变异;1例HBV DNA始终阳性,和治疗前一样存在D553G的变异。结论 D553G变异可能是慢性乙型肝炎患者拉米夫定治疗无效的原因之一;拉米夫定治疗后出现的乙型肝炎病毒基因组P 区YMDD变异是抗病毒药物诱导的结果。
Objective To study the variation of P gene region of hepatitis B virus in patients with chronic hepatitis B before and after lamivudine treatment. Methods The target fragment was amplified from 5 serum samples of 5 patients with chronic hepatitis B before lamivudine treatment and 12 months of treatment. The positive results were double digested and cloned into JM105 competent cells. 20 positive clones were randomly selected from each specimen, and the YMDD gene sequence was detected by mismatched PCR-restriction fragment length polymorphism (PCR-RFLP). Mutants were sequenced bidirectionally. Results In 5 patients with chronic hepatitis B, HBV DNA was negative in 2 of 12 patients after lamivudine treatment. Two patients with HBV DNA turned negative after switching negative. The result of sequencing showed M552I mutation and HBV DNA in one patient Positive, as before treatment, there is variation of D553G. Conclusion D553G mutation may be one of the reasons for the failure of lamivudine treatment in patients with chronic hepatitis B. YMDD mutation in HBV P region of hepatitis B virus after lamivudine treatment is the result of antiviral drug induction.