中国兽医科学2017，Vol.38Issue(8)：969-974,6.DOI:10.16656/j.issn.1673-4696.2017.08.006
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
Prokaryotic expression of Toxoplasma gondii protein phosphatase 1 gene and its location in tachyzoites
摘要
Abstract
In order to express Toxoplasma gondii protein phosphatase 1(TgPP1) gene,determine the location of TgPP1 in T.gondii tachyzoites,TgPPl gene was redesigned based on the bacteria favor,artificially synthesized and inserted into expression vector pET-30a(+).The positive recombinant plasmid pET30a-TgPP1 was transformed into Escherichia coli BL21(DE3) and induced with IPTG.The recombinant TgPP1(rTgPP1) protein was detected by SDS-PAGE and Western-blot,and purified by affinity chromatography.By immunizing Kunming mice with the purified recombinant protein,the antiserum for rTgPP1 was prepared and its titer was detected by indirect ELISA.The localization of TgPP1 in T.gondii tachyzoites was detected with the antiserum by immunofluorescence.The results showed that the recombinant vector pET30a-TgPP1 was constructed and expressed in Kcoli BL21(DE3) successfully.The recombinant protein was about 36 ku in molecular weight.Western-blot analysis showed that the target protein could react with sera from mice infected with T.gondii.Immunofluorescence analysis showed that the TgPP1 protein was mainly located at the top of T.gondii tachyzoites.This study laid the foundation for the further studies on functions of TgPP1 in the invasion and survival of T.gondii in host cells and their mechanisms.关键词
刚地弓形虫/Ⅰ型蛋白磷酸酶/原核表达/抗血清/定位Key words
Toxoplasma gondii/TgPP1/prokaryotic expression/antiserum/location分类
农业科技引用本文复制引用
程龙,詹婷婷,周鹏,黄燕,米荣升,宋悦,游艳敏,韩先干,陈兆国..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[J].中国兽医科学,2017,38(8):969-974,6.基金项目
国家自然科学基金项目(31302083) （31302083）
国家重点研发计划项目(2017YFD0500400) （2017YFD0500400）
国家农产品质量安全风险评估重大专项(GJFP201700703) （GJFP201700703）
上海市科委技术标准专项(16DZ0501900) （16DZ0501900）
中央级公益性科研院所基本科研业务费专项(2016JB13,2017JB03) （2016JB13,2017JB03）
