我国主要乙型肝炎病毒流行株中C抗原18～27位细胞毒性T淋巴细胞表位特点分析

目的 探讨我国HBV感染者中人类白细胞抗原(HLA)-A*0201限制性特异性细胞毒性T淋巴细胞(CTL)表位HBcAg(18～27)V/I变异体的病毒学背景特点.方法 应用生物信息学方法分析GenBank中30条不同HBV基因型的病毒株HBcAg(18～27)区段的序列特点.设计错配引物,构建针对HBV B、C基因型HBcAg(18～27)V/I的PCR限制性片段长度多态性分析(RFLP)筛选方法.以PCR-RFLP和测序方法检测来自我国8省区160份HBV基因型B、C标本的HBcAg(18～27)V/I基因特点.用表位预测网站分析不同HBcAg(18～27)特异性CTL表位基因变异体与HLA-A*0201分子结合力的差异.结果 成功构建了针对HBV基因B、C型HBcAg(18～27)V/I的PCR RFLP筛选方法;在调查的HBV B、C基因型血清中发现HBcAg(18～27)V的阳性率仅为1.88%(3/160);生物信息学分析显示HBcAg(18～27)I与HLA-A*0201分子的结合力明显低于HBcAg(18～27)V(HLA Ligand评分123与156,SYFPEITHI评分22与24).结论 我国流行的主要HBV流行株中HBcAg(18～27)的最后一位氨基酸主要表现为异亮氨酸,而非缬氨酸;在进行细胞免疫学研究时,以HBcAg(18～27)V作为参照需要充分考虑这一病毒学背景.

Characteristics of HBcAg(18-27) CTL epitopes of the main epidemic HBV strains in China

OBJECTIVE: To study the characteristics of the virology background of HLA-A2 restricted HBcAg(18-27) epitope mutations in HBV infected patients in China. METHOD: 30 HBV sequences with different genotypes from Genbank were analyzed by bioinformatics and the mismatched primers were designed for constructing a PCR-RFLP method to screen HBcAg(18-27)V/I in China. The distributions of HBcAg(18-27)V/I of 160 samples with HBV genotype B/C infection from 8 areas in China were screened and analyzed by PCR-RFLP and sequencing. The affinity of HBcAg(18-27)V/I to HLA-A0201 was analyzed through referencing the bioinformatics websites. RESULTS: We successfully constructed a PCR-RFLP method for screening HBcAg(18-27)V/I from genotype B/C, and only 3 samples with HBcAg(18-27)V sequence were found in the 160 samples (3/160, 1.88%). The affinity of HBcAg(18-27)I to HLA-A 0201 was lower than the one of HBcAg(18-27)V through bioinformatic analysis (HLA ligand score was 123 vs 156, and the SYFPEITHI score was 22 vs 24). CONCLUSION: The last amino acid of most HBcAg(18-27) sequences of epidemic HBV strains in China is isoleucine, and not valine. Therefore HBcAg(18-27) sequence background in different HBV genotypes should be thoroughly considered when using it as a reference or control in immunological research about HBV.