Expression and purification of HIV-1 subtype C Gp120, and its antibodies preparation / 中华实验和临床病毒学杂志

ABSTRACT

<p><b>OBJECTIVE</b>To prepare HIV-1 subtype C Gp120 protein and to produce its polyclonal antibodies.</p><p><b>METHODS</b>A C-terminal fragment of gp120 gene was amplified by PCR from a plasmid expressing full-length HIV-1 subtype C gp160 gene. The length of the subtype C gp120 fragment was 612 nt and it encodes 204 amino acid residues. The resulting DNA construct was cloned into a prokaryotic expression vector (pET-30a) and recombinant pET-30a-gp120 was expressed in Escherichia coli BL21 (DE3) as an insoluble protein. The vector also contained a six-histidine (His6) tag at the C-terminus for convenient purification. To produce subtype C Gp120-specific polyclonal antibodies, New-Zealand rabbit was immunized with the purified Gp120 protein. Serum samples were tested by enzyme-linked immunosorbent assays (ELISA) to determine the level of antibodies. And Western blotting was used to further verify whether the polyclonal antibodies could specifically recognize subtype C Gp160 protein expressed in mammalian cells.</p><p><b>RESULTS</b>HIV-1 subtype C Gp120 protein was successfully acquired and the titer of its polyclonal antibodies was 1204 800. The polyclonal antibodies efficiently recognized Subtype C Gp160 protein expressed in COS-1 cells.</p><p><b>CONCLUSION</b>HIV-1 subtype C Gp120 fusion protein with high purity was obtained and its corresponding polyclonal antibodies with high titer were produced.</p>