[Construction of a eukaryotic expression plasmid containing gp120 gene of HIV-1 subtype B and its expression in HepG2 cells].

ABSTRACT

OBJECTIVE: To construct an eukaryotic expression plasmid containing gp120 gene of HIV-1 subtype B and obtain gp120 gene expression in HepG2 cells. METHODS: According to the published gp120 gene sequence in Genbank, a pair of primers was designed and synthesized. The PCR amplification product of gp120 gene was cloned into pMD-18T vector using TA cloning followed by BamHI and XhoI digestion and sequence analysis. The target gene was then subcloned into a highly efficient eukaryotic expression vector pcDNA3.1 (+). The recombinant plasmid was sequenced and identified by restrictive endonuclease digestion, and transfected into HepG2 cells via liposome. The expression of gp120 gene was analyzed by RT-PCR and Western blotting, respectively. RESULTS: Restriction endonuclease digestion and sequence analysis verified successful construction of the recombinant vector pcDNA3.1(+)/gp120. The target fragment gp120 was identical with U26942 in Genbank, and the expression of gp120 gene was detected in the lysate of the transfected HepG2 cells by RT-PCR and Western blotting. CONCLUSION: The eukaryotic expression plasmid for gp120 has been constructed successfully, which is capable of stable expression in HepG2 cells.