title: [Expression and purification of HIV-1 subtype C Gp120, and its antibodies preparation].
abstract: 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.
authors.affiliation: State Key Laboratory for Infectious Disease Prevention and Control, National Institute for Viral Disease Control and Prevention, China CDC, Beijing 100052, China.
authors.forename: Hai-ru
authors.initials: HR
authors.lastname: Yang
authors.forename: Xia
authors.initials: X
authors.lastname: Feng
authors.forename: Shuang-qing
authors.initials: SQ
authors.lastname: Yu
authors.forename: Xiao-guang
authors.initials: XG
authors.lastname: Zhang
authors.forename: Xiao-mei
authors.initials: XM
authors.lastname: Zhang
authors.forename: Guo-min
authors.initials: GM
authors.lastname: Chen
authors.forename: Ze-lin
authors.initials: ZL
authors.lastname: Li
authors.forename: Yi
authors.initials: Y
authors.lastname: Zeng
mesh_terms: 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
chemical_list: D000914:Antibodies, Viral; D015699:HIV Envelope Protein gp120; D011994:Recombinant Proteins