旋毛虫脱氧核糖核酸酶Ⅱ蛋白的制备及其定位研究  

Preparation and Localization of Deoxyribonuclease Ⅱ Protein from Trichinella spiralis

作　　者：赵冰 阿曼古丽·斯拉依 米荣升[2] 程龙[2,4] 刘旗 龚海燕 张烨华[2] 贾海燕 韩先干[2] 黄燕[2] 陈兆国[2] 赵权[1] ZHAO Bing;SILAYI Amangui;MI Rongsheng;CHENG Long;LIU Qi;GONG Haiyan;ZHANG Yehua;JIA Haiyan;HAN Xiangan;HUANG Yan;CHEN Zhaoguo;ZHAO Quan(College of Animal Science and Technology,Jilin Agricultural University,Changchun 130118,China;Key Laboratory of Animal Parasitology of Ministry of Agriculture and Rural Affairs,Laboratory of Quality and Safety Risk Assessment for Animal Products on Biohazards(Shanghai)of Ministry of Agriculture and Rural Affairs,Shanghai Veterinary Research Institute,CAAS,Shanghai 200241,China;Ili Kazak Autonomous Prefecture Center for Animal Disease Control and Diagnosis,Yining 835000;Shanghai Key Laboratory of Regulatory Biology,Institute of Biomedical Sciences and School of Life Sciences,East China Normal University,Shanghai 200241,China)

机构地区：[1]吉林农业大学动物科学技术学院,长春130118 [2]中国农业科学院上海兽医研究所,农业农村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)农业农村部动物寄生虫学重点实验室,上海200241 [3]伊犁哈萨克自治州动物疾病控制与诊断中心,伊宁835000 [4]华东师范大学生命科学学院,上海市调控生物学重点实验室,上海200241

出　　处：《中国动物传染病学报》2023年第6期11-19,共9页Chinese Journal of Animal Infectious Diseases

基　　金：上海市科技兴农项目(No.2019-02-08-00-08-F01151);新疆少数民族科技人才特殊培养计划科研项目(2020D03030);国家农产品质量安全风险评估计划项目(GJFP2019022);上海市科技创新行动计划科普专项项目(20DZ2304800)。

摘　　要：为获得旋毛虫(T1)脱氧核糖核酸酶Ⅱ(DNase Ⅱ)蛋白,观察其在肌幼虫虫体中的分布,以旋毛虫(T1)肌幼虫cDNA为模板,利用RT-PCR技术扩增旋毛虫脱氧核糖核酸酶Ⅱ(TsDNase Ⅱ)蛋白的编码基因tsdnase Ⅱ,将其克隆到原核表达载体pCold-TF中,转化至大肠杆菌BL21(DE3)感受态细胞中,以IPTG诱导表达,表达产物进行SDS-PAGE电泳分析;利用酶联免疫吸附试验(ELISA)及Western blot分析重组Ts DNaseⅡ(rTsDNase Ⅱ)蛋白的反应原性;将rTsDNase Ⅱ蛋白免疫6-8周BALB/c小鼠,制备其多克隆抗体;制作旋毛虫肌幼虫冰冻切片,利用免疫荧光试验观察TsDNase Ⅱ蛋白在虫体中的分布。结果,成功地扩增出了长度为942 bp的旋毛虫tsdnase Ⅱ基因片段,构建了其原核表达质粒pCold-TF-tsdnase Ⅱ;转化了重组表达质粒的大肠杆菌BL21(DE3)成功可溶表达了rTsDNase Ⅱ,大小为86 kDa;ELISA与Western blot分析结果显示,rTsDNase Ⅱ蛋白能与感染旋毛虫的小鼠血清发生特异性反应;冰冻切片免疫荧光分析显示,TsDNase Ⅱ分布于旋毛虫肌幼虫整个虫体。研究结果为进一步分析TsDNase Ⅱ的特性与功能、利用该蛋白研发旋毛虫病新型防控策略打下了基础。In order to obtain the deoxyribonuclease Ⅱ(DNase Ⅱ)protein of Trichinellaspiralis(T1)and observe its distribution in T.spiralis muscle larvae,tsdnaseIIgene was amplified by RT-PCR using T.spiralis muscle larva cDNA as template and cloned into prokaryotic expression vector pCold-TF.The recombinant plasmid was then transformed intoEscherichiacoliBL21(DE3)competent cells and induced by IPTG.The expressed product was analyzed by SDS-PAGE electrophoresis.The reactogenicity of recombinant TsDNase Ⅱ(rTsDNaseⅡ)protein was analyzed by ELISA and Western blot.BALB/c mice aged 6-8 weeks were immunized with rTsDNase Ⅱ to prepare the polyclonal antibodies.The frozen sections of T.spiralis muscle larvae were analyzed by immunofluorescence assay to observe the subcellular distribution of TsDNase II.The results showed that the tsdnaseIIgene fragment was successfully amplified and its length was 942 bp.The prokaryotic expression plasmid pCold-TF-tsdnase Ⅱ was successfully constructed and theE.coliBL21(DE3)transforming with the recombinant expression plasmid successfully expressed the rTsDNase Ⅱ,which was about 86 kDa.ELISA and Western blot analysis showed that the rTsDNase Ⅱ protein could specifically react with the sera of mice infected with T.spiralis.The frozen sections ofT.spiralismuscle larvae were analyzed by immunofluorescence and the results showed that TsDNase Ⅱ was distributed on the throughout body of T.spiralismuscle larvae.These results laid a foundation for further analysis of the characteristics and functions of TsDNase Ⅱ,as well as the development of new prevention and control strategies for trichinellosis.

关 键 词：旋毛虫(T1) 脱氧核糖核酸酶Ⅱ 原核表达 ELISA 定位 

分 类 号：S852.7[农业科学—基础兽医学]