人脑钠肽与人血清白蛋白融合蛋白在毕赤酵母中的分泌表达及其活性

目的在毕赤酵母中表达重组人脑钠肽(BNP)与人血清白蛋白(HSA)融合蛋白,并检测其活性。方法重叠PCR法拼接BNP二联体与HSA基因,插入表达载体pPIC9K,电穿孔法转化毕赤酵母GS115,甲醇诱导表达,并对表达产物进行SDS-PAGE分析、Western blot鉴定及活性检测。结果融合基因(BNP)2-HSA经测序正确,重组表达质粒经双酶切鉴定构建正确。诱导72h,融合蛋白表达量最高,可达200mg/ml,且具有良好的反应原性,其活性为rhBNP标准品的1%。结论已在毕赤酵母中成功表达了具有一定生物活性的(BNP)2-HSA融合蛋白,为开发BNP长效药物奠定了基础。

Secretory Expression of (BNP)2-HAS Fusion Protein in Pichia pastoris and Activity of Expressed Product

Objective To express recombinant human natriuretic peptide(BNP)and human serum albumin(HSA)fusion pro-tein in Pichia pastoris and determine its activity. Methods The (BNP)2 and HSA genes were spliced by overlapping PCR and in-serted into expression vector pPIC9K. The constructed recombinant plasmid pPIC9K(-BNP)2-HSA was transformed to P. pastoris GS115 by electroporation for expression under induction of methanol. The expressed product was identified by SDS-PAGE and West-ern blot and determined for activity. Results Sequencing result showed that fusion gene (BNP)2-HSA was spliced successfully. Re-striction analysis proved that recombinant plasmid pPIC9K(-BNP)2-HSA was constructed correctly. After induction for 72 h, the fu-sion protein reached a maximum expression level of 200 mg / ml and showed good reactogenicity, and its activity was equivalent to 1% of that of rhBNP standard. Conclusion The(BNP)2-HSA fusion protein with a certain biological activity was successfully expressed in P. pastoris, which laid a foundation of developing long-acting BNP.