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

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，Western blot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ANTHEPROT 4．3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。

Expression of HCGbeta-CTP polymeride by the methylotropic yeast, pichia pastoris and structure prediction of the expressed products]

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 pastoris expression vector pPIC9K to construct pPIC9K-(hCGbeta-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 suggested 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 hCGbeta, 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 anticancer vaccines basing on hCGbeta--CTP37.