Expression and identification of recombinant human C-reactive protein in Pichia pastoris
10.3969/j.issn.1671-8348.2013.28.022
- VernacularTitle:重组人C反应蛋白在甲醇酵母中的分泌表达及鉴定
- Author:
Junming LI
;
Heng LIN
;
Lichao ZHANG
;
Gaoshun GE
;
Xuejun HU
- Publication Type:Journal Article
- Keywords:
C-reactive protein;
pichia pastoris;
induced expression;
secretory expression;
immunity reactivity
- From:
Chongqing Medicine
2013;(28):3390-3392
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct the secretory expression vector of recombinant human C-reactive protein(rhCRP) for its se-cretory expression in Pichia pastoris ,rhCRP was expressed as a secretory protein and purified ,and the immunity reactivity of the purified protein was identified .Methods The DNA fragment of rhCRP which was designed and synthesized was cloned into pPICZαA vector .Recombinant plasmid pPICZαA/rhCRP was linearized by SacⅠand transformed into Pichia pastoris X-33 by elec-trotransformation .The rhCRP was secreted into the medium under the methanol induction .RhCRP was purified by Histamine affin-ity chromatography .The purified rhCRP was identified by SDS-PAGE and Western blotting ,and its immunity reactivity and stabili-ty was identified by indirect ELISA .Results The pPICZαA/rhCRP expression vector was successfully constructed .The rhCRP of 23 × 103 was inducted and successfully expressed as a secretory protein by the recombinant Pichia pastoris strains .The rhCRP was purified by one step up to 90 .42% purity ,and it was showed good immunity and stability by indirect ELISA .Conclusion The rh-CRP with higher purity and immunoreactivity was successfully obtained by using the Pichia pastoris expression system ,which pro-vided an important experimental basis for producing anti-human CRP antibodies and developing testing CRP reagent .
