丙型肝炎病毒结构区基因变异的研究

Study of genetic variation of the structural region of hepatitis C virus

目的 探讨丙型肝炎病毒 (HCV)结构区变异在HCV感染慢性化中的作用。方法 采用逆转录 聚合酶链反应 (RT PCR)扩增HCV的C、E1及E2 /NS1区核苷酸片断 ( 1kb) ,扩增产物进行克隆 ,以单链构象多态性 (SSCP) /异质性双体分析 (HDA)方法对每份血清中 30个克隆的C/E1和E2 /NS1区准种 (Quasispecies )进行优化筛选 ,分析HCV急性感染者和持续性感染者血清准种复杂性。结果 HCV的C/E1区和E2 /NS1区扩增片段形成的SSCP条带间差异明显 ,但E2区形成的异质性双体与同源双体之间差距更显著 ,准确地反映结构区准种的复杂性 ;HCV持续性感染者比急性感染者血清的E2区准种复杂性明显增加。

Objective To study the role of the structural gene variation of hepatitis C virus(HCV) in the chronicity of HCV infection. Methods Serum samples from four HCV infection acquired during six month follow up and four patients with chronic hepatitis C type were obtained. A 1 kb fragment containing the 3' half of core ,completed E1 and 5' half of E2/NS1 was amplified by nested reverse transcription polymerase chain reaction (RT PCR). For each,30 cloned cDNAs were examined by a method that combined heteroduplex analysis (HAD) and a single stranded conformational polymorphisim (SSCP)assay to optimize selection of clones with unique gel shift patterns (clonotypes )as a measure of the quasispecies complexity. Results There was obvious difference between the SSCP bands of cloned cDNAs representing core E1 and E2 region,but more distinct difference between heteroduplex and the homoduplex of clones spanning E2 region which accurately reflected the degree of heterogeneity. Quasispecies complexity was higher in those with persistent viremia than those with acute viremia. Conclusion HCV persistence is associated with complex quasispecies in viral structural region.