安徽三黄鸡β-防御素2(gal-2)在毕赤酵母细胞内的表达

Intracellular Expression of Chicken Beta-defensin-2 (gal-2) Gene in Pichia pastoris

目的:构建安徽三黄鸡β-防御素2(gal-2)基因的真核表达载体,表达gal-2蛋白。方法:提取鸡肺组织中总RNA,应用RT-PCR技术获得鸡β-防御素2(gal-2)全长cDNA基因片段,经PCR获得gal-2的成熟肽基因片段,并将其克隆入真核表达载体pPIC3.5K中,构建重组表达质粒pPIC3.5K-gal-2;经测序鉴定正确后,电转化巴斯德毕赤酵母菌GS115。经甲醇诱导表达gal-2蛋白,对表达产物进行SDS-PAGE电泳分析。结果:通过电转化方法成功将经酶切线性化的重组表达质粒整合入宿主菌基因组中,并在甲醇诱导的条件下成功表达出了目的蛋白。结论:成功构建了重组真核表达质粒pPIC3.5K-gal-2;并借助巴斯德毕赤酵母细胞表达出了gal-2的蛋白,其分子量大约为4.1kDa。该研究为下一步应用基因工程和蛋白质工程技术进行gal-2蛋白的规模化生产奠定了基础。

[Objective]To construct a eukaryotic expression vector to express chicken beta-defensin-2 （gal-2）.[Methods]With total RNA extracted from chicken lung tissues as a template,full-length gal-2 cDNA was obtained by RT-PCR.The gene of mature peptide obtained by PCR was cloned into the eukaryotic expression vector pPIC3.5K to yield recombinant plasmid pPIC3.5K-gal-2 which was identified by DNA sequencing.The pPIC3.5K-gal-2 was then transformed into the competent Pichia pastoris GS115 and induced by methanol.The recombinant gal-2 protein was analyzed by SDS-PAGE.[Results]The lined recombinant plasmid DNA was successfully transformed into the competent Pichia pastoris GS115 and integrated into GS115 chromosome through electroporation and it expressed target protein with methanol induction.[Conclusion]The eukaryotic expression vector pPIC3.5K-gal-2 was constructed,and the 4.1 kDa chicken gal-2 protein was expressed in Pichia pastoris.The research provided a basis for further large-scale production of gal-2 protein by genetic engineering and protein engineering techniques.