人成纤维细胞生长因子21基因的克隆及在毕赤酵母中的表达

目的：以毕赤酵母(Pichia pastoris) GS115和SMD1168为宿主菌表达人源成纤维细胞生长因子21（hFGF21），为开发治疗糖尿病药物提供一定的实验基础。方法：根据毕赤酵母基因偏爱密码子和hFGF21氨基酸序列，设计多条寡核苷酸引物。利用融合PCR方法获得人工合成的成纤维细胞生长因子21(FGF21)基因，定向克隆到质粒pPIC9K中，电转化Pichia pastoris GS115和SMD1168。MD、MM平板筛选表型Mut⁺，YPD/G418平板进一步筛选高拷贝转化子。甲醇诱导表达，硫酸铵沉淀、分子筛、离子交换方法纯化目的蛋白。结果：PCR　扩增出约569 bp的特异性片段，测序分析正确；诱导表达上清经SDS-PAGE和Western blotting分析表明：FGF21在宿主菌GS115中不表达，而在SMD1168表达上清中出现相对分子质量为20 000的特异性条带，且与抗FGF21抗体产生特异性反应，阴性对照在该处无特异性带，证明hFGF21蛋白在SMD1168菌株中成功分泌表达。结论：成功克隆FGF21基因，并在Picha pastoris　SMD1168中获得表达。

Cloning and expression of human fibroblast growth factor-21 in Pichia pastoris

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.