密码子优化的人成纤维细胞生长因子-21在毕赤酵母中的表达

Expression of hFGF-21 with Optimized Codon in P.pastoris

目的在毕赤酵母中表达密码子优化的人成纤维细胞生长因子-21(hFGF-21),为探讨hFGF-21在糖尿病治疗方面的作用奠定基础。方法经密码子优化合成hFGF-21基因,构建表达载体pPICZαA-hFGF-21,电转化毕赤酵母GS115,斑点免疫印迹法筛选工程菌株,甲醇诱导表达,SDS-PAGE和Western blot分析表达产物,并对诱导条件进行优化。结果重组表达质粒经PCR、双酶切及测序鉴定,证明构建正确。筛选出5株阳性菌株,诱导上清经SDS-PAGE分析,可见相对分子质量约为25000的目的蛋白条带,目的蛋白的表达量约为6μg/L。Westernblot显示该蛋白具有良好的反应原性。诱导96h和培养液pH值为4.0时,目的蛋白的表达量最高,甲醇浓度对表达量无影响。结论已成功构建了密码子优化的hFGF-21真核表达载体,并在毕赤酵母GS115中分泌表达了hFGF-21。

Objective To express human fibroblast growth factor-21（hFGF-21）protein with optimized codon in P.pastoris and lay a foundation of study on role of hFGF-21 in therapy of diabetes mellitus.Methods Human FGF-21 gene was synthesized by optimization of codon and inserted into vector pPICZαA,and the constructed recombinant plasmid pPICZαA-hFGF-21 was transformed to P.pastoris GS115 by electrotransformation.Recombinant P.pastoris strain was screened by dot immunoblot for expression under induction of methanol.The expressed product was identified by SDS-PAGE and Western blot,and the condition for induction was optimized.Results PCR,restriction analysis and sequencing proved that recombinant expression vector pPICZαA-hFGF-21 was constructed correctly.Five positive clones of recombinant P.pastoris strains were screened.SDS-PAGE showed that the target protein with a relative molecular mass of 25 000 was expressed in supernatant of recombinant P.pastoris after induction,and the expression level reached 6 μg /L.Western blot showed good reactogenicity of expressed protein.The expression level of target protein reached a peak value after induction of recombinant P.pastoris at pH 4.0 for 96 h.However,methanol concentration showed no significant effect on expression level.Conclusion A eukaryotic expression vector for hFGF-21 with optimized codon was constructed successfully,and hFGF-21 was expressed in a secretory form in P.pastoris GS115.