伊氏锥虫PFR2蛋白的原核表达及多克隆抗体的制备  

Prokaryotic expression and preparation of polyclonal antibody of PFR2 protein from Trypanosoma evansi

作　　者：罗文蔚 罗金[2] 任巧云[2] 刘文阁[2] 王奇林 张高峰 刁沛文 曹润来 陈则宇 马永华 刘光远[2] LUO Wen-wei;LUO Jin;REN Qiao-yun;LIU Wen-ge;WANG Qi-lin;ZHANG Gao-feng;DIAO Pei-wen;CAO Run-lai;CHEN Ze-yu;MA Yong-hua;LIU Guang-yuan(College of Veterinary Medicine,Gansu Agricultural University,Lanzhou 730070,China;State Key Laboratory of Veterinary Etiological Biology/Key Laboratory of Veterinary Parasitology of Gansu Province/Lanzhou Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Lanzhou 730046,China)

机构地区：[1]甘肃农业大学动物医学院,甘肃兰州730070 [2]中国农业科学院兰州兽医研究所家畜疫病病原生物学国家重点实验室甘肃省动物寄生虫病重点实验室,甘肃兰州730046

出　　处：《中国兽医科学》2022年第10期1302-1307,共6页Chinese Veterinary Science

基　　金：国家重点研发计划项目(2019YFC1200500,2017YFD0501206);国家寄生虫资源库项目(NPRC-2019-194-30);国家肉牛牦牛产业技术体系项目(CARS-37);国家自然科学基金项目(32160843);江苏高校动物重要疫病与人兽共患病防控协同创新中心项目;甘肃省科技厅重点研发项目(18YF1FA080);甘肃省科技厅国际合作项目(144WCGA169);甘肃省现代丝路寒旱农业科技支撑项目(GSLK-2021-18);甘肃省自然科学基金项目(18JR3RA167);甘肃农业大学学科建设专项基金项目(GUA-XKJS-2018-073);甘肃农业大学青年导师基金项目(GAU-QDFC-2020-11);甘肃农业大学人才专项经费项目(2017RCZX-11);农业科技创新工程项目(ASTIP)

摘　　要：为了探究伊氏锥虫副鞭毛杆蛋白2(paraflagellar rod protein 2,PFR2)的分子特征和免疫特征,利用PCR扩增伊氏锥虫PFR2基因,将扩增产物插入p QE-80L载体,经原核表达后,将纯化后的重组蛋白免疫小鼠,制备多克隆抗体,利用Western-blot鉴定反应原性。结果表明,PFR2基因大小为1803 bp,编码含601个氨基酸残基的蛋白质,SDS-PAGE电泳显示该蛋白大小约为66 ku,主要以包涵体的形式表达。用间接ELISA方法检测小鼠抗PFR2多克隆抗体的效价为1∶25600;Western-blot检测显示重组PFR2蛋白具有较好的反应原性。本研究为进一步研究伊氏锥虫PFR2蛋白的功能奠定了基础。In order to explore the molecular and immune characteristics of paraflagellar rod protein 2(PFR2)from Trypanosoma evansi,the PFR2 gene was amplified by ordinary PCR,and then was inserted into the p QE-80L vector.After prokaryotic expression,the purified recombinant protein was used to immune mice,and the polyclonal antibodies were prepared,and the reactivity of the recombinant protein was identified by Western-blot.The results showed that PFR2 gene was 1803 bp in length,encoding a protein with 601 amino acid residues.SDS-PAGE showed that the recombinant PFR2 was about 66 ku in molecular weight and expressed in precipitation in the form of inclusions.The mouse anti-PFR2 polyclonal antibody titer was 1∶25600detected by indirect ELISA method.Western-blot showed that the recombinant PFR2 protein had good reactivity.This study laid a foundation for further research of the function of PFR2 protein from T.evansi.

关 键 词：伊氏锥虫 PFR2基因 原核表达 多克隆抗体 

分 类 号：S852.72[农业科学—基础兽医学]