庚型肝炎病毒基因组全长cDNA的拼接及克隆

Splicing and cloning of the full-length genomic cDNA of GB virus C/hepatitis G virus

目的：构建ＧＢ病毒Ｃ／庚型肝炎病毒（ＧＢＶ－Ｃ／ＨＧＶ）基因组全长ｃＤＮＡ克隆。方法：以覆盖ＧＢＶ－Ｃ／ＨＧＶ分离株ＨＧＶ－Ｉｗ全长基因组且互相重叠的５个基因片段Ｉｗ５，Ｉｗｑ２，Ｉｗｈ６，Ｉｗ３和Ｉｗ３为起始材料，采用重叠延伸ＰＣＲ拼接和连接酶连接的方法，构建ＧＢＶ－Ｃ／ＨＧＶ基因组全长ｃＤＮＡ克隆。结果：ＧＢＶ－Ｃ／ＨＧＶ基因组全长ｃＤＮＡ克隆的酶切图谱与预期一致；核苷酸序列分析显示，克隆的ＧＢＶ－Ｃ／ＨＧＶ基因组全长ｃＤＮＡ的核苷酸及推测的氨基酸序列与原ＨＧＶ－Ｉｗ序列一致。结论：ＧＢＶ－Ｃ／ＨＧＶ基因组全长ｃＤＮＡ的克隆已经构建成功。该克隆的构建，为深入研究ＧＢＶ－Ｃ／ＨＧＶ的致病性及致病机制、复制及转录和翻译机制。

Objective: To construct GBVC/HGV full-length genomic cDNA clone. Methods: On the basis of 5 cDNA fragments termed in order from 5 end to 3 end of the genome: Iw5,Iwq2,Iwh6,Iw3 and Iw3 of a GBVC/HGV isolate HGVIw,the GBVC/HGV fulllength genomic cDNA clone was constructed using overlap extension PCR and ligation methods. Results: By digesting with the predicted endonucleases,the physical map of GBVC/HGV fulllength genomic cDNA clone was identical with the expected results. The determination of nucleotide sequence showed that the nucleotide and amino acid sequences of GBVC/HGV fulllength genomic cDNA was the same as original isolate HGVIw. Conclusion: GBVC/HGV full-length genomic cDNA clone has been constructed successfully,and it will play an important role in further studing on the virus pathogenicity and pathogenesis,replication,transcription and translation machanism,virus morphology and morphogenesis.