Molecular cloning, characterization, functional diversity, and specifically monoclonal antibody production of immunoglobulin M heavy chain (ighu) genes in bighead catfish (Clarias macrocephalus), african catfish (Clarias gariepinus) and their hybrids

Name: Anurak Bunnoy

LCSH: Kasetsart University -- Dissertations. Ph.D. (Aquaculture) 2020

Classification :.LCCS: QL638.C6

LCSH: Kasetsart University. -- Department of Aquaculture -- Dissertations

LCSH: Clarias gariepinus

LCSH: Catfishes -- Genetics

LCSH: Molecular cloning

LCSH: Monoclonal antibodies

LCSH: Immunoglobulin M

Abstract: Immunoglobulin M (IgM) is generally recognized as a key molecule crucially involved in the specific immune system of all jawed vertebrates to neutralize toxins or invading pathogens. In this study, the prospective molecular structure, splicing patterns, functions, diversification, expressions, cytogenetic mapping and homogeneity or heterogeneity between species of immunoglobulin M heavy chain (ighμ) genes of the bighead catfish (Clarias macrocephalus), African catfish (C. gariepinus) and their hybrids (C. macrocephalus x C. gariepinus) were firstly investigated. Besides, the production of recombinant protein and a monoclonal antibody specific to the IgM heavy chain molecules of these Clarias catfish was properly developed. Cloning and characterization resulted from RACE PCRs demonstrated that the general characteristic ighμ transcripts in bighead catfish, African catfish, and their hybrids are typically produced by specifically splicing patterns of mRNA reported in all teleost. The membrane-bound form encoded with three constant domain exons and two transmembrane domain exons [VDJ-VDJ-Cμ1-Cμ2-Cμ3-TM1-TM2] and secreted form encoded with four constant domain exons [VDJ-Cμ1-Cμ2-Cμ3-Cμ4]. Significant evidence was novelly founded in the bighead catfish by expression of two unusual ighμ transcripts of membrane-bound forms. Firstly, the molecule contains four constant region and two transmembrane domain-encoding exons [VDJ-Cμ1-Cμ2-Cμ3-Cμ4-TM1-TM2] similar to those molecules observed in all tetrapods, birds, and sharks and has been not reported in any teleosts so far. Secondly, an expressed in membrane form by additional spliced four ighδ exons within ighμ molecules [VDJ-Cμ1-(Cδ2-Cδ3-Cδ4-Cδ5)-Cμ2-Cμ3-TM1-TM2], which has never reported in vertebrates, is discovered in this research. Additionally, gene organization analysis by gene sequencing and cytogenetic mapping evidently indicated that all ighμ transcripts were produced from a single gene copy in individual bighead catfish and African catfish. Interestingly, the different ighμ transcripts, membrane and secreted forms of diploid hybrid catfish were made up of two different copies of ighμ genes from their parents. Tissue expression analysis by qRT-PCR revealed that the ighμ was predominantly expressed in head kidney, peripheral blood lymphocytes (PBLs), and spleen, which are primary lymphoid organs in teleost. The overall expression levels of ighμ of hybrid catfish are mostly similar to the African catfish and significantly higher than expression levels found in all organs of the bighead catfish. Diversification analyses of 100 cDNAs encoding VH domains of IgM of each catfish showed distinct structural variability in Clarias catfish and their hybrid. The number of VH families was 15, 15, and 17 in bighead, African catfish, and their hybrid, respectively. While a predicted number of JH were 9, 11, and 13, containing the putative DH numbers of 11, 11, and 8, respectively. Based on this finding, it could be suggested that these Clarias catfish might employ P- and N-nucleotides and randomly junctional combinations of these components to widely generate diversification of the VJH repertoire to recognize a huge number of antigens in their lives. Finally, a monoclonal antibody (mAb) specific to Cμ1 of IgMH of catfish (IgMHCμ1 mAb) was firstly developed in a rabbit model. The IgMHCμ1 mAb strongly recognized the IgM heavy chain of the bighead catfish, African catfish, and their hybrids, in immunological Western blot analysis and competitive ELISAs. Additionally, the IgMHCμ1 mAb strongly recognized IgM+ cells by detecting IgM molecules in both secreted and membrane-bound forms in peripheral blood leukocytes (PBLs). The IgMHCμ1 mAb was further used to quantify the percentage of IgM+ cells among PBLs through flow cytophotometry. The IgM+ cell percentages of healthy bighead catfish, African catfish, and their hybrids were 38.0-39.9%, 45.6-53.2%, and 58.7-60.0%, respectively. Furthermore, the IgMHCμ1 mAb showed no cross-reactivity with the IgM of zebrafish. These findings suggest that this mAb can be used as an immunological tool for monitoring the health, immune status, and immune development of cultivated Clarias catfish. In summary, the knowledge obtained from this study can be useful to get a better understanding of specific immune responses and the development of B cells in fish immunity. Additionally, it can be beneficially improved aquaculture of Clarias catfish by selection for specific resistance to their infectious diseases, helping to prevent the dissemination of fish diseases and developing effective vaccines specific to Clarias catfish that are beneficial effects to sustainable catfish aquaculture industries.

Kasetsart University. Office of the University Library

Address: Bangkok

Email: tdckulib@ku.ac.th

Name: Prapansak Srisapoome

Role: Thesis Advisor

Name: Uthairat Na-Nakorn

Role: Thesis CO-Advisor

Created: 2020

Modified: 2025-06-30

Issued: 2025-06-30

วิทยานิพนธ์/Thesis

application/pdf

URL: https://www.lib.ku.ac.th/KUthesis/2563/anurak-b

CallNumber: QL638.C6 .A58

eng

DegreeName: Doctor of Philosophy

Level: Doctoral Degraee

Descipline: Aquaculture

Grantor: Kasetsart University

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