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Construction and identification of a single stranded cDNA clone containing full-length genome of hepatitis G virus
1Department of Microbiology, Second Military Medical University, Shanghai 200433, China. qizt@smmu.edu.cn
Aim:
To construct a single cDNA clone with full-length genome of hepatitis G virus (HGV) could be transcribed and expressed in vitro.
Methods:
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.
Results:
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.
Conclusion:
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.