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


CONCLUSIONS
The eukaryotic expression plasmid for gp120 has been constructed successfully, which is capable of stable expression in HepG2 cells.
Li
De-liang
DL
Institute of Genetic Engineering, Southern Medical University, Guangzhou 510515, China. lidlcn@yahoo.com
Ma
Wen-li
WL
Shi
Yong-xia
YX
Li
Ling
L
Zhang
Bao
B
Zheng
Wen-ling
WL
D016915:AIDS Vaccines / Q000096:biosynthesis / Q000235:genetics; D001483:Base Sequence; D006528:Carcinoma, Hepatocellular / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D005057:Eukaryotic Cells / Q000378:metabolism*; D015870:Gene Expression; D015699:HIV Envelope Protein gp120 / Q000096:biosynthesis / Q000235:genetics*; D015497:HIV-1 / Q000235:genetics*; D006801:Humans; D008113:Liver Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics*; D014162:Transfection; D019444:Vaccines, DNA / Q000096:biosynthesis / Q000235:genetics
D016915:AIDS Vaccines; D015699:HIV Envelope Protein gp120; D019444:Vaccines, DNA