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

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1 020 bp;SDS-PAGE分析结果显示,融合蛋白大小约44 ku,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.

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

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 was approximately 1 020 bp;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.annulata schizont 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.