[Expression and purification of HIV-1 subtype C Gp120, and its antibodies preparation].
OBJECTIVE
To prepare HIV-1 subtype C Gp120 protein and to produce its polyclonal 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-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.


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.


CONCLUSIONS
HIV-1 subtype C Gp120 fusion protein with high purity was obtained and its corresponding polyclonal antibodies with high titer were produced.
Yang
Hai-ru
HR
State Key Laboratory for Infectious Disease Prevention and Control, National Institute for Viral Disease Control and Prevention, China CDC, Beijing 100052, China.
Feng
Xia
X
Yu
Shuang-qing
SQ
Zhang
Xiao-guang
XG
Zhang
Xiao-mei
XM
Chen
Guo-min
GM
Li
Ze-lin
ZL
Zeng
Yi
Y
D000818:Animals; D000914:Antibodies, Viral / Q000032:analysis*; D019556:COS Cells; D002522:Chlorocebus aethiops; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D015699:HIV Envelope Protein gp120 / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification*; D011817:Rabbits; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification
D000914:Antibodies, Viral; D015699:HIV Envelope Protein gp120; D011994:Recombinant Proteins