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  

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

作　　者：李艳[1,2,3] 陈兆国[2,3] 聂奎[1] 米荣升[2,3] 

机构地区：[1]西南大学动物科技学院,重庆400714 [2]中国农业科学院上海兽医研究所农业部动物寄生虫学重点开放实验室 [3]中国农业科学院动物源性食品安全研究中心,上海200241

出　　处：《中国动物传染病学报》2010年第2期41-46,共6页Chinese Journal of Animal Infectious Diseases

基　　金：国家“十一五”高技术研究发展计划(863)项目(2006AA10A207);国家“十一五”科技支撑计划项目(2007BAD40B05);上海市科技兴农重点攻关项目(沪农科攻字[2005]第3-4号)

摘　　要：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 Tlymphocyte,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基础。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.

关 键 词：微小隐孢子虫 表位预测 多表位基因 原核表达 抗原性 

分 类 号：S852.42[农业科学—基础兽医学]