Expression and purification of the outer shell proteinVP2 of the 4th serotype Bluetongue virus,and preparation of monoclonal antibodies against this protein |
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| Institution: | 1. College of Veterinary Medicine, Northeast Agricultural University, Harbin, PR China;2. Northeastern Science Inspection Station, China Ministry of Agriculture Key Laboratory of Animal Pathogen Biology, PR China;1. College of Marine Life Science, Ocean University of China, Yushan Road, No. 5, Qingdao, China;2. Department of Biology, Shantou University, Shantou 515063, China;3. Laboratory for Marine Biology and Biotechnology, Qingdao National Laboratory for Marine Science and Technology, 266003, China;1. School of Pharmacy, State Key Laboratory of Microbial Metabolism, Engineering Research Center of Cell & Therapeutic Antibody, Ministry of Education, Shanghai Jiao Tong University, Shanghai 200240, PR China;2. School of Life Science and Biotechnology, State Key Laboratory of Microbial Metabolism, Shanghai Jiao Tong University, Shanghai 200240, PR China;1. Yildiz Technical University, Faculty of Science and Letters, Department of Chemistry, 34210, Istanbul, Turkey;2. Gebze Technical University, Department of Chemistry, 41400 Gebze, Kocaeli, Turkey;3. Gebze Technical University, Department of Molecular Biology and Genetics, 41400 Gebze, Kocaeli, Turkey;4. Laboratory of Enzyme Technology, Department of Biotechnology, School of Food, Biotechnology and Development, Agricultural University of Athens,75 Iera Odos Street, GR-11855 Athens, Greece;5. Gebze Technical University, Department of Bioengineering, 41400 Gebze, Kocaeli, Turkey |
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| Abstract: | Bluetongue (BT) is an arbovirus transmitted disease by bites of the genus Culicoides and infects wild and domestic ruminants particularly in sheep. As an important outer shell protein which defines BTV serotypes, VP2 has been shown to be an ideal target antigen for identification of different BTV serotypes. In order to prepare a monoclonal antibody (mAb) against the VP2 protein of BTV-4, the corresponding encoding gene L2 was divided into three segments and then cloned into pET-28a (+) and pMAL-c5X vectors to generate recombinant plasmids, which were expressed in Escherichia coli BL21 (DE3) as histidine (His)-tagged (His-4A/4B/4C) and maltose-binding protein (MBP)-tagged (MBP-4A/4B/4C) fusion proteins. After affinity purification of His-4A/4B/4C with Ni-NTA agarose and MBP-4A/4B/4C with amylose resin, His-4A/4B/4C were used to immunize BALB/mice and MBP-4A/4B/4C were used to screen for mAb-secreting hybridomas. Five hybridoma cell lines stably secreting mAbs against different VP2 segments were obtained, in which 4A-1G7 and 4B-1B6 could recognize BTV-4 and also cross-react with other BTV serotypes. With the joint action of the two mAbs, BTV-4 and BTV-20 infection would be distinguished from other BTV serotypes. The successful preparation of recombinant VP2 segments and mAbs provides valuable materials that can be used in serological diagnosis of BTV-4. |
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| Keywords: | Bluetongue virus Group-specific antigen Serotype-specific antigen Prokaryotic expression Monoclonal antibody |
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