Prokaryotic Expression,Polyclonal Antibody Preparation and Subcellular Localization of Theileria annulata GAPDH |
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| Authors: | ZHAO Hong-xi LIU Jun-long YANG Cong-shan ZHAO Shuai-yang LIU Juan LIU Guang-yuan YIN Hong GUAN Gui-quan LUO Jian-xun |
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| Institution: | 1. Key Laboratory of Veterinary Parasitology of Gansu Province, State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, China;2. College of Agriculture, Ningxia University, Yinchuan 750021, China;3. Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Disease, Yangzhou 225009, China |
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| Abstract: | 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. |
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| Keywords: | Theileria annulata glyceraldehyde-3-phosphate dehydrogenase polyclonal antibody |
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