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

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、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1279nt（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。关键词：丙型肝炎病毒；包膜蛋白；序列分析；准种；插入突变

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

This paper investigated the envelope protein E1/E2 quasispecies genetic characterization of 4 HCV positive sera(Genotype 1b:274,366,383;Genotype 2a:283) in China.Nucleotide acid was extracted and glycoprotein E1/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 E1/E2 coding genes,and some vital functional regions of E1/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 interruptionat 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.