Expression, purification of nattokinase in Pichia pastoris and preparation of its polyclonal antibody
- VernacularTitle:纳豆激酶基因在毕赤酵母中的表达纯化及抗体制备
- Author:
Litao CAI
;
Xiang XU
;
Tingting WANG
;
Meixing YU
;
Yanyan YANG
- Publication Type:Journal Article
- Keywords:
Nattokinase;
Pichia pastoris;
fibrinlytic activity;
polyclonal antibody
- From:
Chinese Journal of Biochemical Pharmaceutics
2010;31(1):10-13
- CountryChina
- Language:Chinese
-
Abstract:
Purpose To indicate the expression of nattokinase (NK) in Pichia pastoris , an emulsion was prepared with the purified NK to prepare polyclonal antibody. In order to establish sandwich enzyme-linked immunosorbent assay (ELISA) to assay NK in organism, furthermore to lay the foundation for researching in vivo metabolism and function of NK. Methods The NK gene was cloned into a Pichia pastoris expression vector pHBM905A to construct the recombinant plasmid pPRONK.The recipient cell of Pichia pastoris GS115 was transformed with pPRONK which had been cut by restriction enzyme Sal I , under the induction of methanol. The expressed production is purified by salting out and ultrafiltration membrane. An emulsion was prepared with the purified NK and injected into rabbits to prepare polyclonal antibody. Results NK was expressed and identified by SDS-PAGE.The molecular mass of expressed production is about 27 kD.The fibrin plate assay indicated that the NK protein can cleavage fibrin effectively. ELISA analysis indicated that the polyclonal antibody titer is about 1:8 000. Western blot demonstrated that there was a special strap nearby 27 kD. Conclusion NK was successfully expressed in Pichia pastoris , the production can cleavage fibrin effectively and it had great immunogenicity.
