应用单链构象多态性和异源双链分析研究拉米夫定耐药变异

Application of SSCP/HDA in mutation of lamivudine resistance

目的 应用单链构象多态性/异源双链分析技术研究慢性乙型肝炎患者拉米夫定治疗前后乙肝病毒准种组成的改变。方法 聚合酶链反应扩增6例患者治疗前后共12份血清标本中目的片段并逐一分析。结果 拉米夫定治疗前患者体内乙肝病毒聚合酶基因序列准种组成较复杂,准种数在7-14(平均9.8),均高于治疗后准种数4-8(平均5.7),t=3.98,P<0.05。6例患者治疗前的准种分布中有一种或两种优势准种,但比例较低(33.3％-81.80％);治疗后显著升高(78.80％-90.9％),t=3.24,P<0.05。选择优势克隆测序,6例患者经拉米夫定治疗后2例在酪氨酸-蛋氨酸-天冬氨酸-天冬氨酸(YMDD)序列出现M550V/L528M,3例为M5501变异,1例无变异。结论 采用单链构象多态性/异源双链分析技术对人体内病毒作整体性的研究,可避免其他研究方法的片面性,为发现新的变异点提供机会,进一步解释拉米夫定的耐药机制。

Objective To study changes of quasispecies composing hepatitis B virus in pre- and post-therapy of lamivudine in chrocnic hepatitis B patients by SSCP/HDA. Methods 12 samples of 6 patients were analysis after PCR. Results Pre-therapy quasispecies composing was more complicated than post-therapy. The number of quasispecies was 7-14 and 4 - 8, respectively ( t = 3. 98, P < 0. 05 ). 6 patients had one or two predominant quasispecies before therapy, but percentage was lower than post-therapy samples, that was 33. 3%-81. 8% and 78. 8%-90. 9% , respectively (t =3. 24,P <0. 05). By sequence analysis of predominant quasispecies, it was found that 2 patients with M550V/L528M, 3 patients with M550I and 1 patient with wild type after lamivudine therapy. Additionally, there were individual mutations which had no obvious current Conclusion To study virus in body by SSCP/HDA could avoid some methods'unilateralism , find new mutations and explain the mutation mechanism of lamivudine.