微小隐孢子虫P23基因在毕赤酵母中的表达及初步应用  

PRELIMINARY APPLICATION AND EXPRESSION OF CRYPTOSPORIDIUM PARVUM P23 PROTEIN IN PICHIA PASTORIS

作　　者：黄燕[1] 米荣升[1] 周鹏[1] 曹薇[1] 杨晓娇[1] 王向佩[1] 王晓娟[1] 石凯[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：《中国动物传染病学报》2012年第6期56-62,共7页Chinese Journal of Animal Infectious Diseases

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);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：为将微小隐孢子虫(Cryptosporidium parvum)P23基因在巴斯德毕赤酵母(Pichia pastoris)系统中进行表达,利用表达蛋白初步建立隐孢子虫病间接ELISA诊断技术,设计引物从微小隐孢子虫基因组DNA中扩增P23基因序列,构建pPIC9K-P23重组质粒,在毕赤酵母中进行表达,用阴离子交换层析柱进行纯化。以重组P23纯化蛋白为抗原建立间接ELISA检测方法,对现场采集的猪血清样品进行检测。SDS-PAGE显示所表达的蛋白大小约为23 kDa。Western blot检测表明该蛋白能与兔抗P23蛋白血清特异性结合。用建立的间接ELISA技术对186份猪血清样品进行检测,阳性率为83.3%。本研究获得了真核表达的P23重组蛋白,初步建立了微小隐孢子虫病间接ELISA诊断技术,为隐孢子虫病的诊断和流行病学调查打下了基础。An indirect ELISA method was developed for diagnostics of cryptosporidiosis using P23 protein of Cryptosporidium parvum expressed in Pichia pastoris.The P23 gene of Cryptosporidium parvum was amplified using primers designed from genomic DNA.The recombinant plasmid pPIC9K-P23 was then constructed and transformed into Pichia pastoris under pre-determined expression conditions.The expressed product was purified with anion exchange chromatography and characterized in SDS-PAGE and Western blotting.The recombinant P23 protein with molecular weight of approximately 23 kDa specifically reacted with rabbit antiserum.Subsequently,P23 protein was used to develop an indirect ELISA for detection of swine serum samples.Out of tested 186 swine serum samples,the positive rate was 83.3%.Therefore,the indirect ELISA method using recombinant P23 protein was promising for use in diagnosis and epidemiology of cryptosporidiosis.

关 键 词：微小隐孢子虫 P23基因 巴斯德毕赤酵母 真核表达 

分 类 号：S852.723[农业科学—基础兽医学]