重组人血清白蛋白-hGCSF突变体在毕赤酵母中的表达  

Expression of mutant HSA-hGCSF in Pichia pastoris

作　　者：朱书峰 张莲芬 陈蕴 储敏 李英 金坚 

机构地区：[1]江南大学医药学院细胞与分子药理研究室,江苏无锡214122

出　　处：《中国生化药物杂志》2008年第5期289-293,共5页Chinese Journal of Biochemical Pharmaceutics

基　　金：国家863计划项目(2006AA02Z153)

摘　　要：目的通过定点突变,提高融合蛋白HSA-hGCSF的生物活性。方法结构模拟显示,对hG-CSF中5个氨基酸残基加以突变可以提高其生物活性。通过限制性酶切方法将该突变基因与HSA基因的3′连接,融合基因插入到毕赤酵母分泌型表达载体pPIC9K中,置于启动子AOX1之后,重组质粒pPIC9K-HSA-hG-CSFm经SalⅠ线性化,电击转化毕赤酵母GS115,摇瓶中进行分泌表达。用MTT比色法测定发酵液上清中融合蛋白的hG-CSF活性。结果PCR鉴定得到约为2300bp的融合基因,测序结果正确。SDS-PAGE和Western blot分析表达的融合蛋白HSA-hGCSFm,表明该蛋白相对分子质量约为85000,且具有HSA的抗原性,生物活性为1.5×106IU/mL。结论分子设计实现了突变体融合蛋白生物活性的提高。Purpose To enhance the biological activity of fusion protein human serum albumin （HSA）-hu- man granulocyte colony stiumlating factor （hG-CSF）by mutation. Methods Homology modeling indicated that the bioactivity of HSA-hGCSF would be higher after 5 amino acids of which were mutated. Restriction enzymes were used to splice the genes of mutated hGCSF and HSA in vitro. The fusion gene was located after the AOX1 promoter of Pichia pastoris secretory vector pPIC9K. The recombinant plasmid pPIC9K/HSA-hGCSFm was linearized by restriction enzyme Sal I and transformed into Pichia pastoris GS115 by electroporation. The bioaetivity of the product was determined by MTT. Results The gene of HSA-hGCSFm was about 2 300 bp with eletrophoresis and the nuclear acid sequence of gene was correct. SDS-PAGE and Western blot analysis of the fusion protein HSA-hGCSFm showed that the expressed fusion protein with an apparent 85 000 molecular weight had the HSA antigenicity. The specific human granulocyte colony stimulating activity of culture supernatant was about 1.5 ×10^6 IU/mL. Conclusion Bioactivity of fusion protein was successfully promoted by molecular design of HSA-hGCSF.

关 键 词：hG—CSF 定点突变 融合蛋白 毕赤酵母 

分 类 号：Q786[生物学—分子生物学]