环形泰勒虫3-磷酸甘油醛脱氢酶的原核表达、多克隆抗体制备及亚细胞定位  

Prokaryotic Expression,Polyclonal Antibody Preparation and Subcellular Localization of Theileria annulata GAPDH

作　　者：赵洪喜[1,2] 刘军龙[1] 杨聪山 赵帅阳[1] 刘娟[1] 刘光远[1] 殷宏[1,3] 关贵全[1] 罗建勋[1] 

机构地区：[1]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,兰州730046 [2]宁夏大学农学院,银川750021 [3]江苏省动物重要疫病与人兽共患病防控协同创新中心,扬州225009

出　　处：《中国畜牧兽医》2016年第2期304-310,共7页China Animal Husbandry & Veterinary Medicine

基　　金：国家重点基础研究发展计划(973计划)(2015CB150300);国家自然科学基金项目(31372432;31402189);农业科技创新工程(ASTIP);PiroVAC(KBBE-3-245145);948项目(2010-S06);国家肉牛牦牛产业技术体系(NBCIS CARS-38)

摘　　要：为了研究环形泰勒虫3-磷酸甘油醛脱氢酶(glyceraldehyde-3-phosphate dehydrogenase,GAPDH)的功能,本试验利用PCR技术从环形泰勒虫裂殖体cDNA中扩增环形泰勒虫GAPDH(TaGAPDH)基因,将扩增产物克隆到原核表达载体pET-30a(+)中,并转入大肠杆菌BL21(DE3)中进行融合蛋白诱导表达,融合蛋白纯化后免疫家兔,制备多克隆抗体,分别用间接ELISA和Western blotting检测抗体的效价和特异性;利用激光共聚焦荧光显微镜观察TaGAPDH蛋白在环形泰勒虫裂殖体中的亚细胞定位。结果显示,克隆得到了TaGAPDH全长基因,大小为1 020bp;SDS-PAGE分析结果显示,融合蛋白大小约44ku,且以包涵体形式存在。制备的多克隆抗体效价高达1∶12 800,具有很高的特异性;亚细胞定位显示TaGAPDH蛋白主要分布于环形泰勒虫裂殖体细胞质内。以上结果表明本试验成功克隆表达了TaGAPDH基因,并制备了针对TaGAPDH蛋白的兔多克隆抗体,为筛选环形泰勒虫病疫苗、药物靶点及研究环形泰勒虫能量代谢奠定了基础。In order to study the function of the Theileria annulata（T.annulata）glyceraldehyde-3-phosphate dehydrogenase（GAPDH）,the T.annulata GAPDH（TaGAPDH）gene was amplified by PCR from the cDNA of T.annulata,and cloned into the pET-30a（＋）vector and expressed in Escherichia coli BL21（DE3）.After purification,the fusion protein was injected into rabbits to produce polyclonal antibodies.Specificity and titers of the polyclonal antibodies were determined by ELISA and Western blotting,and subcellular localization of TaGAPDH protein was observed by confocal fluorescence microscope.The results showed that TaGAPDH gene wasapproximately 1 020bp;SDS-PAGE analysis showed that the fusion protein was expressed in BL21（DE3）with 44 ku in molecular mass,and highest expressed level was detected in the inclusion.The titer of the polyclonal antibodies was more than 1∶12 800.The results of western blotting indicated that the polyclonal antibodies possessed good specificity,and TaGAPDH protein was predominantly present on the cytoplasm of T.annulataschizont by subcellular localization.This study laid the foundation for screening and identifying candidate antigens of vaccination and drug targets as well as studying the energy metabolism of T.annulata.

关 键 词：环形泰勒虫 3-磷酸甘油醛脱氢酶 多克隆抗体 

分 类 号：Q55[生物学—生物化学]