hCGβ-CTP37多聚体在巴斯德毕赤酵母中的表达及其表达产物的结构预测  

EXPRESSION OF HCGβ-CTP POLYMERIDE BY THE METHYLOTROPIC YEAST, PICHIA PASTORIS AND STRUCTURE PREDICTION OF THE EXPRESSED PRODUCTS

作　　者：喻光荣[1] 孙学铮[1] 沈卫英[1] 侯理理[1] 冯文华[1] 蒋俶[1] 申庆祥[1] 

机构地区：[1]上海市计划生育科学研究所

出　　处：《实验生物学报》2005年第2期157-163,共7页Acta Biologiae Experimentalis Sinica

基　　金：国家自然科学基金(30472087);上海市科委科技攻关重大项目(03DZ19229)

摘　　要：hCGβ-CTP37(简称CTP)肽段是hCG分子的特有部分,存在hCG的特异表位。为了解CTP利用毕赤酵母进行表达的情况及探索其作为研制调节生育和抗肿瘤疫苗的可行性,本研究中我们将2、3、4个CTP以头尾串联的方式重组成多聚体基因,然后克隆入载体pPIC9K得到表达质粒pPIC9K-Cα-(CTP)_n(n=2,3,4),电转染入酵母细胞进行表达。在培养基上清中我们检测到了目的基因表达产物,Westernblot结果显示它们的分子量分别约为:20KDa、30KDa、40KDa,且能与抗hCG多克隆抗体结合。另外,我们还利用ANTHEPROT4.3蛋白序列分析软件包对表达产物CTP四聚体的结构进行了预测分析,结果表明CTP四聚体的抗原性明显强于hCGβ-CTP37单聚体,CTP四聚体与hCGβ二级结构较为相似,但CTP四聚体的抗原专一性要优于hCGβ。CTP多聚体在毕赤酵母系统中的成功表达及对表达产物抗原性的初步分析为今后利用CTP研制调节生育和抗肿瘤疫苗奠定了基础。To investigate the expression of CTP encoding gene in the methylotropic yeast, pichia pastoris and the possibility of CTP acting as an antifertility vaccine or anti-cancer vaccine, we strung two, three or four CTP cDNA to construct CTP polymeric cDNA in order to enhance the immunogenicity of the CTP. Then, the recombinant genes were subcloned into a pichia pas- toris expression vector pPIC9K to construct pPIC9K-(hCGβ-CTP37)_n(n=2,3,4). After identified by restriction endonuclease digestion and DNA sequencing, the recombinant vectors were linearized and transferred into GS115 by electroporation. The induced culture supernatant was precipitated by PEG6000 and the precipitate was washed by 75% alcohol. SDS-PAGE and RIA analysis sug- gested GS115 expressed the recombinant genes successfully and the recombinant protein had anti- hCG antibody binding activity. In addition, ANTHEPROT 4.3 software was used to analyze the protein structure of CTP quadrigeminum. We found that CTP quadrigeminum had similar secondary structure with hCGβ, but the speciality of antigen better than that of the latter. Therefore, we conclude that this study prepared basic necessary data for developing antifertility vaccines or anti- cancer vaccines basing on hCGβ-CTP37.

关 键 词：hCG 巴斯德毕赤酵母 多聚体 结构预测 Western 基因表达产物 肿瘤疫苗 CTP 多克隆抗体 分析软件包 四聚体 表达质粒 酵母细胞 blot 预测分析 二级结构 初步分析 酵母系统 抗原性 培养基 分子量 专一性 生育 研制 载体 转染 

分 类 号：R346[医药卫生—基础医学]