摘要
为表达弓形虫表面抗原糖蛋白相关序列(surface antigen glycoprotein 1-related sequences,SRS47D)并确定其在弓形虫中的定位,本研究将SRS47D基因序列插入原核表达载体pColdⅠ,构建原核表达质粒pColdⅠ-SRS47D,转入大肠杆菌BL21(DE3)中,经IPTG诱导后,SDS-PAGE分析重组蛋白表达情况,Western blot分析重组蛋白的反应原性;利用His柱纯化重组蛋白,免疫新西兰兔,制备抗血清,通过间接ELISA检测血清效价;利用免疫荧光法检测蛋白在弓形虫速殖子中的定位。结果显示:成功构建了原核表达质粒pColdⅠ-SRS47D,在大肠杆菌BL21(DE3)中以包涵体形式表达,表达产物约为55 kDa;重组蛋白能与感染弓形虫的小鼠血清发生特异性反应,免疫兔血清效价达到1∶51 200;免疫荧光检测结果显示,SRS47D蛋白分布在弓形虫速殖子表面,尤其前部和后部表膜。上述结果为进一步深入研究弓形虫SRS47D的特性和功能奠定了基础。
In order to prepare Toxoplasma gondii surface antigen glycoprotein 1-related sequence SRS47D and determine its location in tachyzoites,the prokaryotic expression plasmid pColdⅠ-SRS47D was constructed by inserting the SRS47D gene into vector pColdⅠand transformed into Escherichia coli BL21(DE3) for the prokaryotic expression with induction of IPTG.The recombinant SRS47D protein (rSRS47D) was detected by SDS-PAGE and Western blot and purified by affinity chromatography.The antibodies against rSRS47D were prepared by immunizing New Zealand white rabbits with the purified recombinant protein and detected in indirect ELISA.The localization of SRS47D in T.gondii tachyzoites was examined in immunofluorescence method.The results showed that the recombinant plasmid pColdⅠ-SRS47D was constructed and rSRS47D was expressed in E.coli BL21(DE3).The resulting rSRS47D had a molecular mass of appx.55 kDa and reacted with antisera from infected mice in Western blot.The rabbit antisera had titers at 1:51 200 in ELISA method.In addition,inmmunofluorescence analysis showed that the SRS47D protein was mainly located at the surface of T.gondii tachyzoites,especially at the outer membrane of the anterior and posterior ends.These results laid a foundation for further study on the characteristics and functions of the Toxoplasma SRS47D.
作者
刘辉
米荣升
贾海燕
王旭
黄燕
张烨华
张晓丽
杨衡
韩先干
陈兆国
LIU Hui;MI Rong-sheng;JIA Hai-yan;WANG Xu;HUANG Yan;ZHANG Ye-hua;ZHANG Xiao-li;YANG Heng;HAN Xian-gan;CHEN Zhao-guo(Laboratory of Quality and Safety Risk Assessment for Animal Products on Biohazards(Shanghai)of Ministry of Agriculture and Rural Affairs,Key Laboratory of Animal Parasitology of Ministry of Agriculture and Rural Affairs,Shanghai Veterinary Research Institute,CAAS,Shanghai 200241,China)
出处
《中国动物传染病学报》
CAS
北大核心
2020年第4期17-23,共7页
Chinese Journal of Animal Infectious Diseases
基金
国家重点研发计划(2018YFD0502305)
国家农产品质量安全风险评估计划项目(GJFP201900022)
国家自然科学基金项目(31302083)
上海市科委技术标准专项项目(16DZ0501900)
中央级公益性科研院所基本科研业务费项目(2019JB13)。
关键词
刚地弓形虫
表面抗原糖蛋白相关序列
原核表达
定位
Toxoplasma gondii
surface antigen glycoprotein 1-related sequence
prokaryotic expression
location
