乙型肝炎病毒前S基因区缺失突变发生机制的探讨

Putative Mechanism of HBV Pre-S Gene Mutation

检测慢性乙型肝炎病毒(HBV)携带者和患者外周血内HBV前S区基因缺失突变的分子结构特点,探讨其发生机理。用聚合酶链反应方法从慢性乙肝患者和携带者血清中扩增出前S区基因片段,克隆、测序,分析缺失发生的结构特点,从而推测这些前S区基因缺失突变的产生机制。从262例慢性乙肝患者和103例无症状HBV携带者体内扩增出前S区片段,共在30例患者和携带者中检测出多种前S区基因缺失突变,主要集中于前S1区的3′端和前S2区的5′端。其中有9例患者和携带者体内存在完全一样的nt3019～nt3201183bp的缺失突变,该缺失突变符合真核细胞mRNA剪接机制,在此位置上各基因型的序列高度保守。同时有另外两种缺失突变,即nt3019～nt3147129bp缺失、nt3019～nt310991bp缺失也符合该剪接机制。有23种缺失突变部分于重复序列之间,符合逆转录过程中的模板转换机制所导致的缺失。根据前基因组RNA预测出二级结构,仅部分缺失突变在RNA二级结构中对应于局部的结构。此结果表明:HBV在外界因素mRNA的剪接机制和内在因素聚合酶蛋白的功能特点的共同作用下,产生各种突变,不同的机制将导致不同类型的缺失突变。除真核细胞mRNA剪接机制外,逆转录过程中的模板转换是主要机制之一。

This paper was to study the molecular character of HBV preS mutation in chronic hepatitis B patients and chronic HBV carriers and accordingly to discuss its possible mechanismHBV DNA extracted from sera of chronic hepatitis B patients and HBV carriers were subjected to polymerase chain reaction,and then cloning and sequencingThe mutational structural characters were analyzed,the possible mechanisms were put forwardThe PreS gene segment was amplified from sera of 262 hepatitis B patients and 103 asymptomatic carriers,different kinds of mutations were found in 30 casesThe deletions were mainly located in 3′end of PreS1 and 5′end of PreS2Among these mutations,inframe 183bp deletion(nt3019nt3201)was found in 9 cases,this mutation coincided with mRNA splicing mechanism in mammalian cell and was highly conserved in all genotypes129bp deletion(nt3019nt3147)and 91bp deletion(nt3019nt3109)were also detected and also coincided with mRNA splicing mechanismTwentythree different kinds of deletions were found among repeat sequences,which coincided with template switch mechanism during reverse transcriptionThe secondary structures of pregenomic RNAs were predicted,only part of deleted sequences correlated to local RNA secondary structureWhen HBV is subjected to mutation under both the external factor,mRNA splicing mechanism,and the internal factor,the function of HBV polymerase,different mutations can be producedBesides mRNA splicing mechanism in mammalian cell,template switch mechanism during reverse transcription is one of the major mechanism for deletion mutation PreS nt3019nt3201, 183bp deletion may be genotypic nonspecific,but its high occurrence may result from selection pressure