Expression and purification of HIV-1 subtype C Gp120, and its antibodies preparation
10.3760/cma.j.issn.1003-9279.2009.02.006
- VernacularTitle:HIV-1 C亚型Gp120蛋白表达、纯化及其抗体制备的研究
- Author:
Hai-Ru YANG
1
;
Xia FENG
;
Shuang-Qing YU
;
Xiao-Guang ZHANG
;
Xiao-Mei ZHANG
;
Guo-Min CHEN
;
Ze-Lin LI
;
Yi ZENG
Author Information
1. 中国疾病预防控制中心病毒病预防控制所
- Keywords:
HIV;
Viral envelope protein;
Gene expression;
Antibodies,viral
- From:
Chinese Journal of Experimental and Clinical Virology
2009;23(2):94-96
- CountryChina
- Language:Chinese
-
Abstract:
Objective To prepare H1V-1 subtype C Gp120 protein and to produce its polyelonal antibodies.Methods 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-3Oa-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 CGp120-specific polyelonal antibodies, New-Zealand rabbit was immunized with the purified Gp120 protein. Serum samples were tested by enzyme-linked immunosorbont 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.Results HIV-1 subtype C Gp120 protein was successfully acquired and the titer of its polyclonal antibodies was 1 : 204 800. The polyclonal antibodies efficiently recognized Subtype C Gp160 protein expressed in COS-1 cells.Conclusion HIV-1 subtype C Gp120 fusion protein with high purity was obtained and its corresponding polyclonal antibodies with high titer were produced.
