Preparation and Identification of Polyclonal Antibody Against the p38MAPK of Nile Tilapia (Oreochromis niloticus)
HU Ping-Ge1, 2, ZOU Zhi-Ying1, 2, ZHU Jing-Lin2, YU Jie2, XIAO Wei2, LI Da-Yu2, YANG Hong1, 2, *, MA Yin-Hua1, 2, CHEN Bing-Lin1, 2
1 College of Wuxi Fishery, Nanjing Agricultural University, Wuxi 214081, China; 2 Freshwater Fisheries Research Center of Chinese Academy of Fishery Sciences, Key Laboratory of Freshwater Fisheries and Germplasm Resources Utilization, Ministry of Agriculture and Rural Affairs, Wuxi 214081, China
Abstract P38 mitogen-activated protein kinase (p38MAPK) plays an important role in the process of biological autoimmunity regulation. To explore the role of p38MAPK in the immune response of Nile tilapia (Oreochromis niloticus), the p38MAPK partial coding sequence was prepared by gene synthesis. It was cloned into pET-28a to construct prokaryotic expression plasmid pET-28a-p38MAPK. And expression was successfully induced in Escherichia coli after purification by Ni-NTA chromatography column, the p38MAPK polyclonal antibody was prepared by immunizing New Zealand white rabbits (Oryctolagus cuniculus) with purified fusion protein. Using the purified polyclonal antibodies, the p38MAPK protein expression was detected in different tissues of Nile tilapia. The results showed that the fusion protein obtained by the expression induction of the prokaryotic vector had a molecular weight of about 23 kD, and mainly existed in the form of inclusion body. The titer of antibody was estimated as high as 1∶256 000 dilution ration detected by ELISA and Western blot, and it could specifically recognize the p38MAPK protein. The tissue expression analysis showed that p38MAPK was expressed in most tested tissues, with the highest levels in ovary, brain and muscle; moderate levels in head kidney and blood; low levels in skin and gill; the lowest levels in liver, spleen, intestine and heart. This study provides foundation for further exploring the disease-resistant immunity of Nile tilapia.
HU Ping-Ge,ZOU Zhi-Ying,ZHU Jing-Lin, et al. Preparation and Identification of Polyclonal Antibody Against the p38MAPK of Nile Tilapia (Oreochromis niloticus)[J]. 农业生物技术学报, 2020, 28(3): 553-561.
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