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HBVDNA为阴性,2例HBVDNA转阴后又转阳,测序结果提示出现M5521变异;1例HBVDNA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 variation features of the hepatitis B virus polymerase gene in chronic hepatitis B patients before and after lamivudine treatmeat. Methods From the serum samples of five CHB patients both before and after 12 months' lamivudine treatment, HBV polymcrasc gent was amplified and positive DNA fragments were cloned into JM105. Twenty positive clones of every sample were checked with mismatched polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and YMDD variants were sequenced. Results Among five patients after 12 months lamivudine treatment, M552I mutations in two patients with HBV DNA rebounding and D553G mutation in one non-responder were detected except in two patients with negative HBV DNA. Conclusions D553G mutation is probably one of the reasons which caused non-responsiveness to the lamivudine treatment. The mutations of YMDD motif that occurred after lamivudine treatment are caused by the induction of the drug.