Preparation and Preliminary Application of Highly Immunogenic Recombinant CD2v Antigen of African Swine Fever Virus |
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| Authors: | ZHOU Xiaohui XIAO Jingjing ZHANG Xinyu ZHANG Quan XIA Xiaoli SUN Huaichang |
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| Institution: | 1. Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, College of Veterinary Medicine, Yangzhou University, Yangzhou 225009, China;2. The Key Laboratory for High-Tech Research and Development of Veterinary Biopharmaceuticals, Jiangsu Agri-Animal Husbandry Vocational College, Taizhou 225300, China |
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| Abstract: | To obtain the highly immunogenic recombinant CD2v antigen of African swine fever virus (ASFV), the amino acid sequence of CD2v was analyzed for antigenic index using bioinformatics software and the intracytoplasmic region with high antigenic index was expressed in E. coli as an elastin-like polypeptide (ELP) fusion protein. After optimization of the conditions for inverse transition cycling (ITC), ELP-CD2v fusion protein was purified by ITC in the presence of different concentrations of Triton X-100 and the ELP tag was cleaved with active inclusion bodies of tobacco etch virus (TEV) protease. The tag-free recombinant CD2v antigen was recovered by an additional round of ITC and identified by Western blotting. By using the recombinant CD2v antigen, an indirect ELISA was established and used to detect ASFV antibody-positive and antibody-negative sera in parallel with the multi-antigen ELISA. The results showed that ELP-CD2v fusion protein was expressed correctly in E. coli with an optimal transition temperature of 28 ℃ at 1.5 mol·L-1 NaCl. After one cycle of ITC in the presence of 0.2% Triton X-100, ELP-CD2v fusion protein was purified to 76.3% purity. The ELP tag was cleaved efficiently with the TEV protease and removed after an additional round of ITC. The recovered recombinant CD2v protein had a purity of 91.7%, which was recognized by pig anti-ASFV serum. For 30 serum samples detected by ASFV multi-antigen ELISA, recombinant CD2v ELISA showed that all of 15 antibody-negative sera were CD2v antibody negative and all of 15 antibody-positive sera were CD2v antibody positive. These data suggest that the presence of immune dominant epitopes in the intracytoplasmic region of CD2v protein and the potential application of the recombinant CD2v antigen for ASFV antibody detection. |
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| Keywords: | African swine fever virus CD2v protein highly immunogenic antigen preliminary application |
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