微小隐孢子虫CP15-P23-CP15/60基因的融合表达及抗血清的制备  

Prokaryotic Expression of Cryptosporidium parvum CP15-P23-CP15/60 Fusion Gene and Preparation of Its Antiserum

作　　者：秦培兰[1] 米荣升[1] 黄燕[1] 周鹏[1] 张国恩[1] 苏庆美[1] 呼高伟[1] 曹薇[1] 陈兆国[1] 

机构地区：[1]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中国农业科学院动物源性食品安全研究中心,上海200241

出　　处：《动物医学进展》2012年第7期54-59,共6页Progress In Veterinary Medicine

基　　金：国家科技重大专项项目(2012ZX10004220-008);中央级公益性科研院所基本科研业务费专项资金项目(2012JB16);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：以微小隐孢子虫基因组DNA为模板,PCR扩增获得子孢子表面抗原CP15、P23和CP15/60基因。利用重叠延伸PCR(SOE PCR)将该3段基因片段串联在一起,各基因片段之间引入柔性氨基酸接头(GGGGS)编码基因。将串联基因克隆到原核表达载体pET-28a(+)上,构建重组表达载体,转化到大肠埃希菌BL21(DE3)中进行诱导表达,将纯化的重组蛋白免疫Balb/c小鼠制备多克隆抗体。结果表明,获得了CP15-P23-CP15/60融合基因,并在大肠埃希菌中高效表达,Western blot显示重组蛋白能被牛抗微小隐孢子虫阳性血清识别,制备的多克隆抗体能被重组蛋白特异性识别,表明获得的重组蛋白具有较好的抗原性。The three sporozoite surface antigen CP15, P23 and CP15/60 gene fragments were amplified from the genomic DNA of Cryptosporidium parvum by PCR, and connected by overlap extension PCR（SOE PCR） through a flexible linker （GGGGS） encoding gene inserted between each other. The fusion gene was subcloned into the prokaryotic expression vector pET-28a（＋） and the recombinant plasmid pET-CP15-23- 60 was transformed into E. coli BL21（DE3） for induction expression. The purified recombinant fusion pro- tein by prokaryotic expression was collected and used to immunize Balb/c mice to raise the corresponding polyclonal antibody. The results showed that the CP15-P23-CP15/60 fusion gene was obtained successfully and expressed efficiently in E. coli. Western blot analysis indicated that the recombinant fusion protein could be recognized by antiserum from cattle infected with C. parvum, and the raised polyclonal antibody from recombinant protein immunized mice. Our work revealed that the fusion protein had high reactogenic- ity and immunogenicity.

关 键 词：微小隐孢子虫 CP15-P23-CP15/60融合基因 原核表达 重叠延伸PCR 

分 类 号：S852.42[农业科学—基础兽医学] Q786[农业科学—兽医学]