人血清白蛋白-C肽融合蛋白在毕赤酵母中的分泌表达  

Secretory Expression of the Fusion Protein HSA-CP in Pichia Pastoris

作　　者：周利苹[1] 张莲芬[1] 雷楗勇[1] 张文婷[1] 蔡燕飞[1] 金坚[1] 

机构地区：[1]江南大学医药学院分子药理研究室江南大学工业生物技术教育部重点实验室,无锡214122

出　　处：《生物技术通报》2008年第3期71-75,80,共6页Biotechnology Bulletin

基　　金：国家"863"计划项目(2006AA02Z153)

摘　　要：目的构建重组表达人血清白蛋白(HSA)-C肽(CP)融合蛋白的毕赤酵母表达菌株。方法根据表达系统的密码子偏好性优化CP基因,酶切连接pBlue-HSA质粒(HSA1 800bp)和CP(100bp)基因,将HSA-CP融合基因双酶切后插入分泌表达载体pPIC9K中,重组质粒pPIC9K-HSA-CP经SalⅠ线性化后,电击转化毕赤酵母GS115,表型筛选Mut+转化子。PCR鉴定后,用甲醇诱导摇瓶分泌表达。结果融合基因约为1 900bp,序列测定正确。SDS-PAGE分析表明表达融合蛋白的相对分子量约为70kD,摇瓶培养表达量为140mg/L,Western blot鉴定显示表达的融合蛋白为HSA和CP的杂合分子。结论实现了HSA-CP融合蛋白在毕赤酵母中的分泌表达,细胞活性研究显示HSA-CP融合蛋白对人胚肾293细胞的生长具有一定的促进作用。Purpose：To development the recombinant expression of fusion protein HSA-CP,a potential long-acting CP Analog in Pichia postoris. Methods：In order to optimize HSA-CP production,the codon usage of the gene encoding for CP was optimized to Pichia postoris codon bias and lower repetitive AT and GC content. The optimized artificial CP gene was fused to the HSA cDNA in the same reading flame. Then the fusion gene HSA-CP was cloned into Pichia postoris expression vector pPIC9K. Digested by restriction enzyme Soil,the recombinant vector pPIC9k-HSA-CP was transformed into Pichia postoris strain GSll5 by electroporation. The Mut transformants were selected out from histidine-deficient medium plates and screening for Mut phenotype. Confirmed integration by PCR,the recombinant strains were induced to express fusion protein HSA-CP by methanol. Results：The expressed fusion protein has apparent molecular weight of 70kD observed by SDS-PAGE. It was specifically recognized by an anti-human HSA polyclonal antibody and CP monoclonal antibody in Western blot assay. The yield of the recombinant strain is 140mg/L determined by C-peptide Chenfiluminescence Immunoassay kit and Microalbuminuria Immunoassay kit. Conclusion：The fusion protein HSA-CP was expressed successfully in Pichia postoris and could effect on 293 cell proliferation.

关 键 词：C肽 人血清白蛋白 融合蛋白 毕赤酵母 

分 类 号：Q78[生物学—分子生物学]