弓形虫Ⅰ型蛋白磷酸酶基因的原核表达及蛋白的定位研究

发布时间：2018-06-10 12:30

本文选题：刚地弓形虫 + Ⅰ型蛋白磷酸酶　；参考：《中国兽医科学》2017年08期

【摘要】：为表达刚地弓形虫Ⅰ型蛋白磷酸酶(Toxoplasma gondii protein phosphatase 1,Tg PP1)基因、确定Tg PP1在弓形虫速殖子上的定位,将全基因合成的Tg PP1基因克隆到表达载体p ET-30a(+)中,构建重组载体p ET30a-Tg PP1,转化入大肠杆菌BL21(DE3)感受态细胞中,经IPTG诱导,利用SDS-PAGE和Westernblot检测重组蛋白表达情况及其免疫学活性。通过亲和层析获得Tg PP1重组蛋白,制备抗血清,采用间接ELISA检测血清效价、免疫荧光法检测Tg PP1蛋白在弓形虫中的定位。结果显示,成功构建了重组表达载体p ET30a-Tg PP1并实现了在大肠杆菌中的表达,表达产物的分子质量约为36 ku。Western-blot检测结果显示,重组蛋白能与弓形虫感染小鼠阳性血清发生特异性反应,具有良好的反应原性。免疫荧光检测结果显示,Tg PP1主要定位于弓形虫速殖子顶端。上述结果为进一步研究Tg PP1在弓形虫入侵和繁殖过程中的作用及其机制打下了基础。
[Abstract]:In order to express the Toxoplasma gondii type I protein phosphatase (Toxoplasma gondii protein phosphatase 1G PP1) gene and to determine the location of TgPP1 in Toxoplasma gondii Tachyzoites, the TgPP1 gene synthesized by the whole gene was cloned into the expression vector pET-30a (). The recombinant vector pET30a-TgPP1 was constructed and transformed into E. coli BL21DE3) competent cells. The expression and immunological activity of the recombinant protein were detected by SDS-PAGE and Western blot induced by IPTG. The recombinant protein TgPP1 was obtained by affinity chromatography and the antiserum was prepared. The titer of TgPP1 protein was detected by indirect Elisa and the localization of TgPP1 protein in Toxoplasma gondii was detected by immunofluorescence. The results showed that the recombinant expression vector pET30a-TgPP1 was successfully constructed and expressed in Escherichia coli. The molecular weight of the expressed product was about 36 ku.Western-blot. The recombinant protein can react specifically with the positive serum of Toxoplasma gondii infected mice. Immunofluorescence analysis showed that TgPP1 was mainly located at the top of Toxoplasma gondii tachyzoites. These results provide a basis for further study on the role and mechanism of TG PP1 in the invasion and reproduction of Toxoplasma gondii.
【作者单位】：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;
【基金】：国家自然科学基金项目(31302083) 国家重点研发计划项目(2017YFD0500400) 国家农产品质量安全风险评估重大专项(GJFP201700703) 上海市科委技术标准专项(16DZ0501900) 中央级公益性科研院所基本科研业务费专项(2016JB13,2017JB03)
【分类号】：S852.7

【相似文献】