中国大陆新发现丙型肝炎病毒3′非编码区终止密码子变异

A novel gene mutation of stop coden in 3′untranlated region of hepatitis C virus from China

目的获得全长中国大陆1b型丙型肝炎病毒(HCV)3′非编码区(3′UTR)cDNA,并分析一级结构的变异,为进一步研究其在HCV复制、翻译中的调控机制和开发新的抗HCV药物奠定基础。方法利用逆转录套式聚合酶链反应(RT-PCR)限制性内切酶长度多态性分析(RFLP)初步筛选出1例1b型HCV感染者,采用半套式RT-PCR法扩增出约400bp的cDNA片段,克隆测序。结果获得的全长1b型HCV3′端序列,由高变区、Poly(u)区、Poly(u/c)区及98碱基区4部分组成;首次发现终止密码子突变由TGA突变为TGG,可导致NS5B的翻译不能及时终止。结论半套式RT-PCR法可有效获得病毒基因组的全长末端序列;首次报道终止密码子突变导致NS5B区延长,3′UTR缩短。该发现对了解HCV的复制和翻译机制可能有一定的理论和实践意义。

Objective To obtain full-length cDNA of hepatitis C virus （HCV） 3′untranslated region （3′UTR） in a Chinese patient, and analysis its primary structure. Methods By reverse transcription-nested polymerase chain reaction （RT-PCR） and restriction fragment length polymorphism（RFLP） assay, A patient infected genotype 1 b HCV was identified. The fragment of the eDNA of 3 untranlated region was amplified using semi-nested RT-PCR, then subjected to cloning and sequencing. The acquired sequence was compared with a prototype lb sequence HCV-J3 from genebank. Results The full-length eDNA of lb genotype HCV 3′ UTR was obtained. The 3′UTR consists of four elements： the 5′region, the poly（U）, poly（U/C） and 98-base region. Compared with HCV-J3, A substitution is identified at the stop coden, but the G9374A substitution did not alter the stop coden to tryptophan. Conclusion The seminested RT-PCR is efficient to obtain full-length of 3′untranslated region. The stop coden substitutions here first reported at the 3′ UTR may be of help for further study the HCV gene replication and translation.