泰泽隐孢子虫CP15基因的原核表达及抗血清制备

发布时间：2018-12-17 02:38

【摘要】：将泰泽隐孢子虫(Cryptosporidium tyzzeri)CP15基因从pMD18-T-CP15重组质粒中切下,连接至pET-28a(+)原核表达载体,经酶切和测序鉴定正确后,转化到大肠埃希菌BL21(DE3)中进行诱导表达,表达产物进行SDS-PAGE和Western blot分析,并将纯化的重组蛋白免疫ICR小鼠制备多克隆抗体。结果显示,重组表达载体pET-28a-CP15在大肠杆菌中以可溶形式表达,表达蛋白分子量约为17 kDa。Western blot显示重组蛋白能被泰泽隐孢子虫感染小鼠血清所识别。ELISA结果显示重组蛋白免疫小鼠血清能和泰泽隐孢子虫卵囊可溶性抗原特异性反应,表明获得的重组蛋白具有较好的抗原性。
[Abstract]:The (Cryptosporidium tyzzeri) CP15 gene of Cryptosporidium taizei was digested from the recombinant pMD18-T-CP15 plasmid and ligated to the prokaryotic expression vector of pET-28a (). The recombinant plasmid was transformed into Escherichia coli BL21 (DE3) after restriction endonuclease digestion and sequencing. The expressed product was analyzed by SDS-PAGE and Western blot, and the purified recombinant protein was immunized with ICR mice to prepare polyclonal antibody. The results showed that the recombinant expression vector pET-28a-CP15 was expressed in soluble form in Escherichia coli. The molecular weight of the expressed protein was about 17 kDa.Western blot which showed that the recombinant protein could be recognized by the sera of mice infected with Cryptosporidium taizei. The ELISA results showed that the recombinant protein immunized mice could react specifically with the soluble antigen of Cryptosporidium taizei oocysts. The result showed that the recombinant protein had good antigenicity.
【作者单位】：吉林农业大学动物科学技术学院;中国农业科学院上海兽医研究所农业部动物产品质量安全生物(性危害因子风险评估实验室(上海))农业部动物寄生虫学重点开放实验室;江苏省动物重要疫病与人兽共患病防控协同创新中心;山东英才学院;
【基金】：上海市科技兴农重点攻关项目(沪农科攻字(2015)第1-10号,(2005)第3-4号) 国家农产品质量安全风险评估项目(GJFP201600703,GJFP201700703) 国家自然科学基金项目(31302083) 中央级公益性科研院所基本科研业务费(2016JB13) 闵行区高层次人才科研项目团队资助
【分类号】：S852.723

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