心脏基因工程研究:柯萨奇B2病毒云南分离株VP1基因的分子特征

Heart genetic engineering: molecular characteristics of VP1 gene of coxsackievirus B2 Yunnan strain

目的:探讨CVB2云南分离株编码主要中和抗原的衣壳蛋白VP1基因的分子特征,以期为中国CVB2感染致病的分子基础、基因疫苗的研制和心脏基因工程实施提供参考依据。方法:采用逆转录PCR技术扩增CVB2云南分离株VP1基因,经分子克隆获得pMD18-T-CVB2VP1并测序,应用生物信息学进行其序列、同源性分析、预测蛋白质二级结构,并建立进化树。结果:CVB2云南分离株VP1基因全长约846bp,无起始密码及终止密码,与Ohio-1株(GenBank登录号:AF081312)核苷酸、氨基酸序列同源性最高为98%。BC环的βB区与βC区之间缺失6个腺嘌呤,其编码2个构成无规卷曲结构的赖氨酸。CVB的共同抗原表位区中RIYFKP-KHVKA高度保守,为云南分离株及其他参考毒株的共同序列,变异主要在CVB2云南株的251～252位,W→Y,V→I。结论:CVB2云南分离株与Ohio-1株同源性最高,亲缘关系最近,以主要中和抗原表位区存在缬氨酸缺失,CVB共同抗原表位区存在具有规律性的变异及保守序列等为其主要分子特征。

AIM:To investigate the molecular characteristics of the capsid protein VP1 gene of coxsackievirus B2(CVB2) Yunnan strain main neutralizing antigen,so as to provide references for the molecular basis of CVB2 infection induced disease,prepare of gene vaccine and the implement of heart genetic engineering. METHODS:The VP1 gene of CVB2 Yunnan strain was amplified with reverse transcriptase polymerase chain reaction(RT PCR),and pMD18 T CVB2VP1 was constructed by molecular cloning and sequenced.Bioinformatics was employed to analyze the nucleotide and amino acid sequence and phylogeny,and predict the secondary structure of protein, and cladogram was established as well. RESULTS:The complete VP1 gene of CVB2 Yunnan strain had a total length of 846bp and without initiator codon and terminator codon.The highest similarity of VP1 gene with Ohio 1 strain(GenBank logging number:AF081312) nucleotide and amino acid sequence was 98% .There were six adenines deletion coding for two valines which constructed random coil structure between β B and β C region in the BC loop of Yunnan strain,where located the major neutralizing immunogenic epitope.RIYFKPKHVKA sequence among the common antigenic determinants of CVB was highly conserved in all CVB2 strain, variations were observed mainly at 251 to 252 sites of CVB2 Yunnan strain,W→ Y, V→ I. CONCLUSION:CVB2 Yunnan strain shows the highest similarity to Ohio 1 strain, and they are closely related,which is characterized by two valines deletion in the major neutralizing immunogenic epitope and regular variation with a highly conserved sequence in the common antigenic determinants of CVB.