微小隐孢子虫抗原CTL细胞表位预测及多表位基因的原核表达

应用多个服务器对微小隐孢子虫（Cryptosporidium parvum）候选疫苗抗原的氨基酸序列进行分析，预测可能存在的CD8＋细胞毒性T细胞（CD8＋cytotoxic T lymphocyte，CD8＋CTL）表位。从6种常见的免疫效果较好的候选疫苗抗原基因中选出10个分值较高的表位基因串联在一起，形成一条多表位基因，进行人工合成，表位之间用柔性氨基酸GGGGS或GPGPG链接，命名为CpCTL10。将该多表位基因重组入高效融合表达载体pET-28a（＋），获得重组质粒pET-28a（＋）-CpCTL10，转化大肠杆菌BL21（DE3）进行诱导表达，表达产物进行SDS—PAGE、Western blot分析。结果显示，CpCTL10基因在大肠杆菌中主要以包涵体形式高效表达，表达的重组蛋白占菌体蛋白总量的55．3％，纯化的重组蛋白纯度达75．1％。Western blot分析显示表达产物能与感染隐孢子虫的鼠阳性血清发生特异性反应，表明表达的重组蛋白具有较好的抗原性，为多抗原多表位疫苗的研制打下某础．

PREDICTION OF CTL EPITOPES OF CRYPTOSPORIDIUM PARVUM ANTIGENS AND PROKARYOTIC EXPRESSION OF A MULTI-EPITOPE GENE

The CD8 ＋ cytotoxic T lymphocyte （ CD8 ＋ CTL） epitopes of Cryptosporidium parvum vaccine candidate antigens were predicted by bioinformatics softwares. A muhi-epitope gene CpCTLIO contained ten epitopes with high scores that were selected from 6 vaccine candidate antigens and were designed by linking flexible amino acids （ GGGGS or GPGPG ） between epitopes. The nucleotide sequence of above mnlti-epitope gene CpCTLIO was synthesized and linked into prokaryotic expression vector pET-28a（ ＋ ）. The recombinant plasmid pET-28a（ ＋ ）-CpCTL10 was transformed into Escherichia coli BL21 （DE3） competent cells with induction of IPTG. The expressed recombinant protein was analyzed by SDS-PAGE and Western blot. The results showed that the recombinant protein with molecular weight of 18 kDa was expressed mainly in inclusion bodies. The recombinant protein accounted for approximately 55.3 % of the total protein and 75.1% of the purified bacterial proteins.Western blot analysis showed that the expressed protein could be specifically recognized by the antisera from C. parvum infected mice, indicating its strong antigenicity.