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1675 bp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 Ib .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 II and genotype III respectively .In conclusion ,we cloned full‐length cDNA of three strains of HDV ,which could be helpful for the further study of HDV molecular biology .