关键词: 人成纤维生长因子21；毕赤酵母；pPIC9K；基因克隆

Abstract:

Abstract:Objective To express the human fibroblast growth factor-21 (hFGF21) in Picha pastoris (P.pastoris) GS115 and SMD1168,and futher provide a basis for treatment of diabetes.Methods According to P.pastoris biased codon and the sequence of hFGF21,the gene full-length coding sequence,which gained by seven rounds of PCR from fourteen 55-59nt oligoes,was cloned into pPIC9K to obtain a recombinant vector pPIC9K-FGF21.And the plasmid pPIC9K-FGF21 was transformed into P.pastoris GS115 and SMD168 by electroporation.The recombinant P.pastoris strains ( His⁺ Mut⁺⁾were obtained by means of MD and MM plates,and high-copy transformants were further screened by increasing G418 concentration and identified by PCR.The positive transfomants were induced by methanol and the highest expressive strain was screend under the optimal conditions of pH6.5 and 0.8% methanol.The expression product was purified by the protocols including ammonium sulfate precipitation,Sephadex G-50 gel filtration,DEAE-Sepharose ion-exchange chromatography. Results The specific fragment of 569 bp was amplified by PCR,sequence analysis was   correct.The results of  SDS-PAGE and Western blotting demonstrated that the culture supernatant of GS115 strains and the negative control strains had no specific band,on the contrary,a specific molecular weight band at about 20 000  appeared in the culture supernatant of SMD1168 strains.Western blotting analysis also showed that the expressed protein could specifically react with anti-hFGF21 antibody,and the hFGF21 was successfully secreted in SMD1168. Conclusion The gene FGF21 has been cloned successfully,it is  expressed and purified in P.pastoris SMD1168.

Key words:  fibroblast growth factor -21;Pichia pastoris;pPIC9K;gene clone