湖北地区119例 HCV 1b 型 NS3丝氨酸蛋白酶区的序列变异研究

摘要

目的：分析丙型肝炎病毒(hepatitis C virus, HCV)1b 型非结构蛋白3(non-structure protein 3, NS3)丝氨酸蛋白酶区序列的变异规律及影响意义。方法使用基因型别特异性引物,通过巢式 PCR 的方法扩增119例慢性 HCV 1b 型患者血清中 HCV NS3区。对 PCR 产物进行测序后获得 NS3区核苷酸及氨基酸序列。分析119例 HCV 1b 型 NS3区序列的同源性及种系进化,分析 NS3丝氨酸蛋白酶区的变异情况及重要功能位点的突变情况。结果湖北地区 HCV 1b 型与 HCV 1b 型标准株及亚洲地区序列同源性较高,核苷酸序列同源性为85.94％及87.68％,氨基酸序列同源性可达96.83％及92.39％,而与欧美地区1b 型同源性较低,核苷酸序列同源性均为85.57％,氨基酸序列同源性分别为92.17％及93.38％。进化树分析提示湖北地区序列与中国其他地区序列亲缘性较接近,而与日本、东南亚地区及欧美地区的1b 型亲缘性较远。 NS3丝氨酸蛋白酶区185个氨基酸内有34个位点有氨基酸突变,但其重要的功能区包括催化酶底物特异性结合位点以及 Zn2﹢结合位点在所有序列中均高度保守,无一例发生突变。结论对湖北地区 HCV 1b型 NS3丝氨酸蛋白酶区的变异规律及生物学意义的研究进一步丰富和完善了对 HCV 1b 型基因组变异情况的认识。为了解 HCV1b 型在中国地区的进化过程提供一定理论基础。%Objective To analyze the sequence diversity of hepatitis C virus 1b NS3 serine protease domain in Chinese patients and study its influence on viral function and virus replica-tion. Methods Genotype-specific primers were designed according to the published sequences from database. Nested-PCR was used to amplify the NS3 region of 119 patients. PCR product was se-quenced. Homology analysis and phylogenetic tree were calculated by using the software. The muta-tion rate and variation of crucial functional amino acid were analyzed. Results Sequences in pa-tients from Hubei province of China shared a higher homology with HCV 1b standard strain and Asi-an sequences than the European and American sequences. The nucleotide and amino acid homology to 1b standard strain and Asian sequences ranged from 85. 94% to 87. 68% and 96. 83% to 92. 39% , respectively, and the nucleotide and amino acid homology to European and American se-quences was 85. 57% , and 92. 17% to 93. 38% , respectively. Phylogenetic analysis showed that the sequence from Hubei Province has a closer genetic distance to other published Chinese se-quences than sequences from Japan, Southeast Asia, Europe and America. There were 34 amino acid sites that had mutations within the 185 amino acids of serine protease region, but its important functional sites including the binding site for substrate of the catalytic enzyme and the Zn2 ﹢ binding sites were highly conserved in all sequences. Conclusion The analysis of the sequence diversity of HCV 1b NS3 serine protease domain and the biological significance enriches the understanding of variation and mutation of HCV 1b genome, and offers important molecular biology information for the study of HCV evolution in China.