我国丁型肝炎病毒全基因克隆及序列分析

Cloning and sequence analysis of full-length cDNA of hepatitis D virus in China

为构建我国丁型肝炎病毒(HDV)全基因克隆,分别从3份HDV阳性血清中提取病毒RNA,通过反转录-聚合酶链反应(RT-PCR)分段扩增,获得4个相互重叠的DNA片段,将PCR产物测序,利用DNAStar软件拼接,获得HDV全基因组序列;设计引物,用重叠PCR扩增全长HDV片段并连接至克隆载体,构建全基因克隆。结果成功克隆出3株1 675bp的HDV全长基因组。经与GenBank标准序列比对,3株HDV均为基因Ⅰb型,核酸序列同源性达98%以上,与我国Ⅰ型X77627的同源性均达98%以上,与其他基因Ⅰ型的同源性均高于84%。与基因Ⅱ型和Ⅲ型的同源性分别低于77%和66%。本研究构建了3株具有我国代表性的HDV全长基因cDNA克隆,为进一步开展HDV分子生物学研究提供了基础。

The present paper aims to clone and analyze the sequence of full-length cDNA of hepatitis D virus （HDV） in China. HDV RNA was extracted from three HDV seropositive samples, cDNA was obtained by reverse transcription-polymerase chain reaction （RT-PCR）. Four overlapped fragments were amplified by nested PCR, and the products were sequenced. The full-length sequences were joined by DNAStar. The full- length cDNA of HDV was amplified by overlapping PCR using designed primers and connected to the cloning T vector. Three strains of HDV were successfully amplified and cloned. The three HDV strains were all genotype lb. The nucleic acid sequence homology of the three HDV strains was over 98%. They had over 98% sequence homology to genotype Ⅰ Chinese strain X77627, and 〉84% sequence homology to other genotype Ⅰ strains. They had no more than 77% and 66% sequence homology to genotype Ⅱand genotype Ⅲrespectively. In conclusion, we cloned full-length cDNA of three strains of HDV, which could be helpful for the further study of HDV molecular biology.