Research Article: Characterization of a high-affinity polyclonal antibody against the Cyprinus carpio (Linnaeus, 1758) intestinal tight junction protein occluding
J.C. Feng , X.R. Guo , X.L. Chang , Y.R. Zhang , X.L. Meng , R.H. Lu , M.Y. Huang , G.X. Nie , J.X. Zhang *
College of Fisheries , 450938518@qq.com
Abstract: (2184 Views)
Tight junction protein, occludin, plays an important role in intestinal health of fish. To study the function of occludin in the intestinal barrier at the protein level, a rabbit occludin polyclonal antibody was prepared against heterologously expressed Cyprinus carpio A fragment of the occludin gene containing antigenic determinants was first ligated into the pET-21a, which is an expression vector and transformed into E. coli BL21 (DE3) strain. Expression of the target fusion protein was induced by isopropyl-beta-D-thiogalactopyranoside (IPTG ). The purified fusion protein was used as an antigen to immunize New Zealand long-eared rabbits (Oryctolagus cuniculus ) through ear margin vein and subcutaneous injection to obtain rabbit anti-carp polyclonal antibodies against occludin. Enzyme-linked immunosorbent assay (ELISA ) was used to evaluate the antibody titre, and the antibody was used to determine the distribution and expression of occludin in the intestine of carp after infection with Aeromonas hydrophila . The target fusion protein had a molecular weight of approximately 31.7 ku, the antibody titre was 2.4 × 106 , and the integrity of occludin protein was lower in various intestinal segments after A. hydrophila infection . The results indicated that the prepared antibody had a high titre, affinity, and specificity and can be applied to study the expression and distribution of occludin in C. carpio . The availability of this polyclonal antibody laid the foundation for the systematic study of the intestinal barrier of Cyprinus carpio . Additionally, this polyclonal antibody could also be used for explorative studies of the biological function of occludin in other fishes.
Keywords: Cyprinus carpio , Occludin , Prokaryotic expression , Antibody titre , Immunohistochemistry
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Type of Study: Orginal research papers |
Subject:
aquaculture Received: 2018/07/3 | Accepted: 2019/10/16 | Published: 2021/03/20
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