人碱性成纤维细胞生长因子在植物毛状根和毕赤酵母中的表达

目的在植物毛状根和毕赤酵母中表达人碱性成纤维细胞生长因子(Human basic fibroblast growth factor,hbFGF),并对发酵条件进行优化。方法将hbFGF基因分别克隆至表达载体pCAMBIA1301和pPICZα上,构建重组表达质粒pCAMBIA1301-hbFGF和pPICZα-hbFGF。通过发根农杆菌介导,将pCAMBIA1301-hbFGF质粒转入大豆毛状根;采用电转化法将质粒pPICZα-hbFGF转入毕赤酵母X-33菌株,PCR法筛选阳性转化子,诱导表达。筛选高效稳定表达株,发酵培养并优化发酵条件,SDS-PAGE和Western blot分析表达产物,并进行纯化。结果经酶切及测序证明两个重组表达质粒构建正确;经PCR鉴定证明hbFGF基因已整合入大豆毛状根及毕赤酵母基因组中;筛选出了在两种体系中发酵培养的最佳条件;hbFGF在大豆毛状根和毕赤酵母中的表达量分别占总蛋白的31%和42%,且均具有良好的反应原性;经纯化后,均可见相对分子质量约18000的单一条带。结论 hbFGF能够在大豆毛状根和毕赤酵母中高效表达,为其大规模工业化生产奠定了实验基础。

Expression of Human Basic Fibroblast Growth Factor in Plant Hairy Root and Pichia pastoris

Objective To express human basic fibroblast growth factor(hbFGF)in plant hairy root and Pichia pastoris and optimize the condition for fermentation.Methods The hbFGF gene was cloned into expression vectors pCAMBIA1301 and pPICZα to construct recombinant plasmids pCAMBIA1301-hbFGF and pPICZα-hbFGF respectively.Recombinant plasmid pCAMBIA1301hbFGF was transformed to soybean hairy root in the mediation of Agrobacterium rhizogenes,while pPICZα-hbFGF to P.pastoris X-33 strain by electrotransformation.Positive transformants were screened by PCR for expression under induction.The strains expressing hbFGF highly and stably were screened and cultured by fermentation,based on which the condition for fermentation was optimized.The expressed products were analyzed by SDS-PAGE and Western blot then purified.Results Both restriction analysis and sequencing proved that recombinant plasmids pCAMBIA1301-hbFGF and pPICZα-hbFGF were constructed correctly.PCR showed that hbFGF gene was integrated to both the genomes of soybean hairy root and P.pastoris.The hbFGF expressed in soybean hairy root and P.pastoris contained 31% and 42% of total somatic proteins respectively,and both showed good reactogenicities.Either of the expressed hbFGF showed a single band with relative molecular mass of about 18 000 on SDS-PAGE profile.The conditions for fermentation in the two systems were optimized.Conclusion Human bFGF may be highly expressed in both soybean hairy root and P.pastoris,which lays an experimental basis of large-scale production of bFGF.