重组人神经生长因子真核表达载体构建及其在毕赤酵母中的表达与纯化  

Construction of Recombinant Human Nerve Growth Factor Eukaryotic Vector and Its Expression and Purification in Yeast Pichia Pastoris

作　　者：黄志立[1] 蒋伟[1] 周烨[2] 沈丽[1] 王妍[1] HUANG Zhili;JIANG Wei;ZHOu Ye;SHEN Li;WANG Yan(School of Applied Chemistry and Biological Technology,Shenzhen Polytechnic,Shenzhen,Cruangdong 518055,China;Key Laboratory for Molecular Enzymology ＆ Engineering,Jilin University,Changchun,Jilin 130012,China)

机构地区：[1]深圳职业技术学院应用化学与生物技术学院,广东深圳518055 [2]吉林大学分子酶学工程教育部重点实验室,吉林长春130012

出　　处：《深圳职业技术学院学报》2018年第5期41-46,67,共7页Journal of Shenzhen Polytechnic

基　　金：广东省教育厅产学研结合资助项目(2012B091100408);广东省深圳市创新委新兴产业公共技术服务平台资助项目(GGJS20130331152344401)

摘　　要：研究构建了重组人神经生长因子(Recombinant human nerve growth factor,rhNGF)的一种真核表达载体,并转化毕赤酵母GS115.在h NGF基因序列基础上对其进行毕赤酵母偏好密码子分析与改造,然后构建重组质粒p PIC9K-h NGF,经PCR、酶切及测序验证后,转化酵母,利用遗传霉素筛选出高效表达rhNGF的重组酵母.表达产物经SDS-PAGE分析,结果见一条与h NGF分子质量15.7kDa相当的条带,蛋白表达量约为52.86%.将表达产物经初步纯化和进一步精提后,纯度超过98%.分别以鼠NGF产品和人NGF标准品为对照,利用TF-1细胞/MTS比色法检测NGF活性,结果表明rh NGF具有良好的生物学活性,其活性和比活性分别为5.0U/m L和253U/mg,高于鼠NGF产品(2.6 U/mL,130U/mg),与hNGF标准品相当(5.0 U/mL,250 U/mg).In this study, a recombinant plasmid vector was constructed, which can efficiently express human nerve growth factor （rhNGF） in Pichiapastoris GS115. First, gene sequences ofhNGF were modificated based on codon usage bias of Pichia pastoris. The recombinant plasmid of pPIC9K-hNGF was constructed and its gene sequences were confirmed by PCR, restriction endonuclease digestion and sequencing. SDS-PAGE analysis revealed that the expression product owned a molecular weight of about 15.7 kDa with a protein express amount of 52.86%. After being purified by multiple separate processes, the hNGF protein was yielded with a purity of more than 98%. Biological activities of rhNGF were detected by TF-1 cells/MTS method in contrast with mouse NGF product and hNGF standard sample. Results showed that both biological activities and specific activities of rhNGF （5.0ng/mL, 253U/mg） were superior to mouse NGF product （2.6 ng/mL, 130U/mg） and similar to hNGF standard sample （5.0ng/mL, 250U/mg） .

关 键 词：重组 人神经生长因子 真核载体 毕赤酵母 生物活性 

分 类 号：R346[医药卫生—基础医学] Q78[生物学—分子生物学]