噬菌体展示技术筛选人隐孢子虫P23抗原表位

Cell epitope screening on P23 antigen of Cryptosporidium hominis by phage display technique

目的本研究利用噬菌体展示技术筛选人隐孢子虫P23抗原表位,为研制其抗原表位疫苗奠定基础。方法首先用人隐孢子虫重组P23抗原免疫动物以制备抗体,将该抗体与噬菌体展示随机肽库结合,通过ELISA和Western-blot方法鉴定阳性克隆,并对阳性克隆进行增殖,然后对其序列测定和抗原表位预测。结果噬菌体肽库结合抗体后,经ELISA方法可鉴定10株为阳性克隆,Western-blot检测可发现有67kD目的条带出现;序列分析结果可发现它们中具有3个不同结构表位序列,命名为ep1,ep2,ep3,且预测出具有良好的亲水性。结论所得序列是具有B细胞抗原表位。

In this study, phage display peptide technology was applied to screen cell epitope with anti-P23-IgG in order to develop its epitope vaccine. The recombinant P23 antigen was applied to immune animal to obtain antibody （Ab）, then the Ab was hybridized with phage display peptide library, and the positive clones were identified by indirect ELISA and Western- blot. After proliferating, the positive clones were sequenced. Finally, antigen index, hydrophilicity and secondary structure were analyzed by bio-informaties, and epitope sequences were deducted. The results indicated that 10 positive phage clones were identified by ELISA, and 67 kD proteins could be found by Western-blot. The analysis of sequence showed that three dif- ferent sequences could be found, named epl, ep2 and ep3, respectively. And the three peptides all had hydrophilicity in differ- ent degrees. It is concluded that these peptides can be considered as B cell epitope.