Qi Z T, Ren H, Zhu F L, Shao L, Pan W, Hu W J, He J W, Miao X H, Du P
Department of Microbiology, Second Military Medical University, Shanghai 200433, China.
Ross Gastroenterol Zh. 2001(2):46-56.
To construct a single cDNA clone with full-length genome of hepatitis G virus (HGV) could be transcribed and expressed in vitro.
The 5 initial HGV cDNA fragments of Iw5, Iwq2, Iwh6, Iw3 and Iw3 used in this study were amplified from serum of a Japanese non A-E hepatitis patient. These fragments overlapped and covered the entire genome from 5'-end to 3'-end of HGV cDNA. Overlap extension PCR and ligation methods were used with 12 primers for the construction of a full-length genomic HGV cDNA clone from the subgenomic fragments.
A single HGV cDNA clone (pHGVqz) was successfully constructed, physical mapping of the generated pHGVqz found identical to what we expected, and the sequence was deposited with the GenBank under the Accession number AF081782. The analysis of the full-length sequence, which was able to be in vitro transcribed and expressed, showed that this single clone contained 9373 nucleotides (encoding 2873 amino acids), and shared high homologies with other compared HGV isolates.
A full-length genomic HGV cDNA clone is generated for the first of the kind in this study, it could be expressed and transcripted. This single cDNA clone is expected to be of importance in the investigation on replication and pathogenicity of HGV.
构建一个能在体外转录和表达的庚型肝炎病毒（HGV）全长基因组的单一cDNA克隆。
本研究中使用的Iw5、Iwq2、Iwh6、Iw3和Iw3这5个初始HGV cDNA片段是从一名日本非甲 - 戊型肝炎患者的血清中扩增得到的。这些片段相互重叠，覆盖了HGV cDNA从5'端到3'端的整个基因组。使用重叠延伸PCR和连接方法，用12条引物从亚基因组片段构建全长基因组HGV cDNA克隆。
成功构建了一个单一的HGV cDNA克隆（pHGVqz），对产生的pHGVqz进行物理图谱分析，发现与我们预期的一致，其序列已提交至GenBank，登录号为AF081782。对能够在体外转录和表达的全长序列分析表明，这个单一克隆包含9373个核苷酸（编码2873个氨基酸），与其他已比较的HGV分离株具有高度同源性。
本研究首次成功构建了全长基因组HGV cDNA克隆，它能够进行表达和转录。这个单一的cDNA克隆有望在HGV复制和致病性研究中发挥重要作用。
