人血清白蛋白-血管钠肽融合蛋白在毕赤酵母中的表达

人工合成VNP基因,通过酶连构建HSA和VNP基因的融合基因,插入表达载体pPIC9K,电转至毕赤酵母GS115,构建成工程茵,甲醇诱导表达。重组表达质粒经双酶切验证构建正确;表达产物经SDS-PAGE分析分子量为69 000 Da,与理论值相符;Western blot鉴定产物兼有HSA和VNP免疫原性,说明其为杂合分子;兔胸主动脉环离体灌流实验证明融合蛋白具有舒张血管活性。本研究说明毕赤酵母适于HSA-VNP融合蛋白的表达,为进一步开发稳定的VNP药物提供了生物制备方法。

Expression of HSA-VNP fusion protein in Pichia pastoris

VNP genes were synthesized and spliced with HSA genes by enzyme and the fusion protein genes were inserted into expression vector pPIC9K. The constructed recombinant plasmid pPIC9K-HSA-VNP was transformed to Pichia pastoris GS115 by electroporation for expression under the induction of methanol. Restriction analysis proved that recombinant plasmid pPIC9K-HSA-VNP was constructed correctly. The expressed product was identified by SDS-PAGE with a molecular weight of 69 000 Da, consistent with the theoretical value ; the fusion protein was identified with HSA and VNP immunogenicity through Western blot, indicating it being hybrid molecule. It was suitable for expression of HSA-VNP fusion protein in Pichia pastoris and provided biological preparation for further development of stable VNP drug.