四份中国HCV阳性血清中包膜蛋白E1/E2准种基因的序列分析及新型插入突变的发现

Quasispecies Sequence Analyses of Envelope Protein E1/E2 Coding Genes from four Chinese HCV Patients and Identification of a Novel Insertion Mutation of HCV

为分析四份中国丙型肝炎病毒(HCV)阳性血清中包膜蛋白E1/E2基因的准种特征。本研究对从4份中国HCV阳性血清(1b亚型:274、366和383;2a亚型:283)中提取的HCV核酸,采用逆转录-聚合酶反应扩增编码全长E1/E2蛋白(191～764aa)的基因片段,随机挑取多个克隆测序。根据E1/E2基因核苷酸的序列与其他相关序列(来自于GenBank)构建亲缘性关系进化树,进行核苷酸与氨基酸同源性分析并对重要的基因位点进行分析。共获得阳性克隆序列43个(274株10个,283株12个,366株13个,383株8个),发现高变区HVR1、HVR2的基因异质性高,而其他抗体中和表位及跨膜区I、II及N末端糖基化位点相对保守。并首次发现在HCV 2a亚型(283血清)中多个准种序列存在1 279nt(E1区,313aa)处单碱基插入优势基因突变,导致HCV包膜蛋白编码突变与中断(E2区,398aa)。本研究对中国HCV代表株包膜蛋白E1/E2编码基因的准种多样性及一种新型插入突变进行了描述,可为进一步研究HCV免疫逃避与慢性化机制提供重要信息。

This paper investigated the envelope protein El/E2 quasispecies genetic characterization of 4 HCV positive sera （Genotype lb ： 274,366,383 ; Genotype 2a： 283） in China. Nucleotide acid was extracted and glycoprotein El/E2 （191-764aa） coding genes were obtained by RT-PCR, positive clones were randomly selected for sequencing. The phylogenetic relationships and the homology of nucleotide and amino acid were analyzed based on El/E2 coding genes, and some vital functional regions of El/E2 were characterized. A total of 43 sequences （274 ： 10; 283 ： 12; 366 ： 13; 383 ： 8） were obtained showing high genetic heterogeneity in HVR1 and HVR2 regions, while sequences of the neutralizing epitopes, transmembrane domain I, II and N-terminal ectodomain were comparatively conservative. Single base（C）insertion mutation at nt1279 （ E1 region, aa313） ,resulting in a mutated E1 coding protein （beginning at aa 313） and interrup- tionat N terminus （aa 398） of HVR1 region of E2, was dominant quasispecies sequence（11/12） found in serum 283 . This is the first report on E1/E2 quasispecies in Chinese HCV patients and this novel pattern of insertion mutation provides important information for further study on HCV pathogenesis and immune evasion.