小鼠IL-35基因毕赤酵母表达载体的构建及表达

Construction and expression of mouse IL-35 gene in Pichia pastoris vector

目的构建真核酵母表达载体pPIC9K与小鼠IL-35基因的重组质粒pPIC9K-mIL-35-His,在毕赤酵母GS115菌株中诱导表达,并对其进行鉴定。方法以pET-30a-mIL-35为模板,用PCR扩增出IL-35去信号肽基因全序列,并在3'端引入His标签,构建重组质粒pPIC9K-mIL-35-His,经SalⅠ线性化重组质粒后,用电穿孔方法将该基因转染毕赤酵母细胞内。经G418梯度筛选高拷贝转化子,筛选Mut+表型后经PCR进一步鉴定IL-35基因与酵母染色体是否整合。小量诱导表达筛选出高表达菌株,大量甲醇诱导表达,以SDS-PAGE及免疫印迹(Western blot)鉴定蛋白表达。结果小鼠IL-35基因真核酵母表达载体pPIC9K-mIL-35-His构建成功,并且在GS115中通过甲醇诱导表达,经SDS-PAGE及Western blot分析,可见相对分子质量约为65 000的目的蛋白。结论实验所构建的重组质粒pPIC9K-mIL-35-His可在毕赤酵母GS115中正确的表达小鼠IL-35基因。

Objective To construct a recombinant plasmid pPIC9K-mIL-35-His in combining eukaryotic expression vector pPIC9K with mouse IL-35 gene,express and induction of protein IL-35 in Pichia pastoris vector, and expressed IL-35 pro-tein was carried out a identification. Methods IL-35 sequence with removed of signal peptide sequence was amplified by using of pET-30a-mIL-35 as a template and His-tag was introduced in the 3 ＇end by PCR, by which the recombinant plas-mid pPIC9K-mIL-35-His was constructed. The recombinant plasmid pPIC9K-mIL-35-His was linearized by SalⅠ, and the gene was transformed into electroporated Pichia pastoris cells. The clones with high-copy were screened through a direct se-lection by geneticin 418,Mut＋ and Muts phenotypes were identified by PCR for DNA integration. The high protein expres-sion strains were selected by an inducion with a small amount of expression, and then methanol induction in a large quantity of culture. The IL-35 protein expression was identified by SDS-PAGE and western blot. Results The yeast expression vector pPIC9K -mIL-35-His was constructed. The expression of mouse IL-35 was induced by methanol and analyzed by SDS-PAGE and western bolt, by which the target molecule with a relative molecular quality 65 000 was identified. Conclu-sion The recombinant plasmid of mIL-35 gene was successfully constructed and the protein was corectly expressed in Pichia pastoris GS115.