论文部分内容阅读
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HCV 1b型是山东省部分地区主要的基因型
Objective To understand the genotype and gene variation of hepatitis C virus (HCV) isolates in Shandong Province. Methods Sixty-four serum samples of HCV-positive in clinical tests were screened by UBIHCVEIA 4.0 diagnostic kit in Germany, 54 cases were positive. Among them, 28 of them were randomly selected. The 399 bp HCV NS5 gene fragment was amplified by RT nested PCR. As a result, 12 bands showed specific bands. Subsequently, the 12 NS5 region fragments were directly subjected to T vector cloning, and 10 cloned NS5 region gene fragments were successfully sequenced by the Sanger method. The resulting 10 sequences were compared for homology with all HCV isolates in GenBank. Results All 10 genotypes of HCV isolates in Shandong Province belonged to HCV 1b genotype. Analysis of the obtained 10 NS5 fragments showed that all nucleotide changes were caused by substitutions and no insertions and deletions were found. Most of the mutations belonged to synonymous mutations, accounting for the total number of mutations Of the 74%. The G D D motif of RNA-dependent RNA polymerase and all cysteines are completely conserved. Conclusion This study demonstrates that HCV 1b is the major genotype in some parts of Shandong Province