共表达蛋白二硫键异构酶对IFNβ-HSA融合蛋白在毕赤酵母中表达的影响

Effect of Coexpressing Protein Disulfide Isomerase on Expression of IFN β-HSA Fusion Protein in Pichia pastoris

目的探讨共表达蛋白二硫键异构酶(PDI)对干扰素β与人血清白蛋白(IFNβ-HSA)融合蛋白在毕赤酵母中表达的影响。方法根据GenBank公布的毕赤酵母PDI序列设计引物,利用PCR方法从毕赤酵母基因组中扩增目的基因片段,插入表达载体pPICZαA,并整合入融合蛋白(IFNβ-HSA)基因工程菌中,筛选共表达PDI的重组酵母菌,甲醇诱导表达,SDS-PAGE鉴定表达产物,ELISA法检测IFNβ-HSA的表达量。结果经PCR鉴定,重组表达质粒pPICZαA-PDI已转入毕赤酵母KM71/pPIC9K-IFNβ-HSA中。经SDS-PAGE分析,PDI在菌体内大量表达,原始菌株和共表达菌株均明显表达融合蛋白IFNβ-HSA,共表达PDI菌株表达量提高了60%,ELISA法测定达(22.49±3.52)mg/L。结论共表达PDI能促进外源蛋白的分泌表达,为进一步研究在毕赤酵母中过量表达分子伴侣对其分泌外源蛋白的影响奠定了基础。

Objective To investigate the effect of coexpressing protein disulfide isomerase（PDI）on the expression of IFN β-HSA fusion protein in Pichia pastoris. Methods Primers were designed according to the PDI gene sequence of P. pastoris reported in GenBank, with which the target gene fragment was amplified from the genome of P. pastoris by PCR and inserted into expression vector pPICZαA. The constructed recombinant plasmid pPICZαA-PDI was transformed to recombinant P. pastoris KM71 / pPIC9K-IFNβ-HSA, and the positive transformants for coexpressing PDI were screened and induced by methanol. The expressed product was identified by SDS-PAGE, and the expression level of IFNβ-HSA was determined by ELISA. Results PCR proved that recombinant plasmid pPICZαA-PDI was transformed to recombinant P. pastoris KM71 / pPIC9K-IFNβ-HSA. SDS-PAGE showed that PDI was expressed in a large quantity, and IFNβ-HSA fusion protein was expressed significantly in both P. pastoris KM71 / pPIC9K-IFNβ-HSA and P. pastoris KM71 / pPIC9K-IFNβ-HSA / pPICZαA-PDI. However, the expression level of IFNβ-HSA fusion protein in P. pastoris KM71 / pPIC9K-IFNβ-HSA / pPICZαA-PDI was 60% higher than that in P. pastoris KM71 / pPIC9K-IFNβ-HSA, which reached （22. 49 ± 3. 52）mg / L. Conclusion Coexpressing PDI promoted the secretory expression of foreign protein, which laid a foundation of further study on effect of over-expression of chaperones in P. pastoris on secretion of foreign protein.