山东省丙型肝炎病毒分离株NS5区核苷酸序列分析  

Nucleotide sequence analysis of HCV NS5 region in Shandong province

作　　者：王芹[1] 张文卿[2] 于红[2] 王笑峰[2] 张健 邵济钧[2] 

机构地区：[1]青岛大学医学院附属医院检验科,266003 [2]青岛大学医学院分子病毒学实验室 [3]山东省省立医院检验科

出　　处：《中华传染病杂志》2001年第6期363-366,共4页Chinese Journal of Infectious Diseases

基　　金：受山东省科委科研基金资助 [( 96)第1 2 2 672 2 ]

摘　　要：目的　了解山东省丙型肝炎病毒 (HCV)分离株的基因型及其基因的变异情况。方法　应用德国UBIHCVEIA 4 .0诊断试剂盒筛选山东省部分地区 64例临床检验为抗 HCV阳性的血清标本 ,有 54例阳性。随意抽取其中 2 8例 ,应用逆转录套式 聚合酶链反应 (RT nested PCR)扩增3 1 9bp的HCVNS5区基因片段 ,结果 1 2例出现特异性条带。随后将这 1 2个NS5区片段直接进行T载体克隆 ,并用Sanger法对克隆成功的 1 0个NS5区基因片段进行序列测定。将所得到的 1 0个序列与GenBank中所有的HCV分离株进行同源性比较。结果　 1 0例山东省部分地区HCV分离株的基因型均属于HCV 1b型。对获得的 1 0个NS5区片段进行变异性分析发现 ,所有的核苷酸变化都是由于替代作用引起的 ,没有碱基的插入和缺失 ;大部分的突变都属于同义突变 ,占突变总数的 74 %。RNA 依赖性RNA聚合酶的G D D基序和所有的半胱氨酸都完全保守。结论　本研究证明了HCVObjective To study the genotypes and the variations of HCV isolates in some areas of Shandong province.Methods The kit of AKZO UBI EIA 4.0 assay for antibody to hepatitis C virus(HCV)was used to screen 64 clinical anti HCV Ab positive serum samples, and 54 samples of which gave positive results. With RT nested PCR that utilized highly efficient primers,a product of 319bp in the NS5 region was amplified from 12 of 28 samples.These 12 PCR products were directly cloned using TA cloning kit, and 10 independent clones were isolated and sequenced by dideoxynucleotide chain termination reaction. Ressults Every sequencing analysis followed by comparison with reported sequences in the GenBank revealed that 10 sequences were all classified as HCV type 1b. All nucleotides changes in NS5 region were due to substitution without insertion and deletion,and most of the substitution (74%)occurred in the third base of a codon; The motif of G D D and all cysteines were conserved. Conclusion The result has demonstrated that HCV 1b is predominant in some areas of Shandong province.

关 键 词：肝炎病毒 丙型 基因型 NS5区 序列 

分 类 号：R373.21[医药卫生—病原生物学]