人血清白蛋白-血管钠肽融合蛋白在毕赤酵母中的表达  

Expression of HSA-VNP fusion protein in Pichia pastoris

作　　者：崔燕生[1] 姜曰水[1] 高昊[1] 雷楗勇[1] 钱凯[1] 陈蕴[1] 段作营[2] 金坚[1,2] 

机构地区：[1]江南大学药学院 [2]江南大学工业生物技术教育部重点实验室,无锡214122

出　　处：《工业微生物》2014年第3期49-52,共4页Industrial Microbiology

基　　金：国家重大新药创制专项资金资助项目(2009ZX09103);国家自然科学基金资助项目(81273437)

摘　　要：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。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.

关 键 词：心力衰竭 HSA-VNP 毕赤酵母 蛋白表达 

分 类 号：Q78[生物学—分子生物学]