论文部分内容阅读
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。
hCGβ-CTP37 (referred to as CTP) peptide is a unique part of hCG molecules, there is a specific epitope of hCG. In order to understand the expression of CTP in Pichia pastoris and to explore its feasibility as a regulator of fertility and anti-tumor vaccine, in this study, we recombined 2,3,4 CTPs into a multimer gene in a tandem manner , And then cloned into vector pPIC9K to obtain expression plasmid pPIC9K-Cα- (CTP) _n (n = 2,3,4), which was electrotransfected into yeast cells for expression. In the medium supernatant, we detected the target gene expression products, Westernblot results showed that their molecular weight were about: 20KDa, 30KDa, 40KDa, and with anti-hCG polyclonal antibody binding. In addition, we also predicted the structure of the CTP tetramer using the ANTHEPROT4.3 protein sequence analysis software package. The results showed that the antigenicity of CTP tetramer was significantly stronger than that of hCGβ-CTP37 monomer, CTP tetramer Similar to the secondary structure of hCGβ, the antigen specificity of CTP tetramers is superior to that of hCGβ. The successful expression of CTP multimers in Pichia pastoris and the preliminary analysis of the antigenicity of the expressed products laid the foundation for the future development of reproductive and antineoplastic vaccines by CTP.