Full length articleProduction, characterization and application of monoclonal antibody against immunoglobulin D heavy chain of flounder (Paralichthys olivaceus)
Introduction
Immunoglobulins (Igs) are important molecules in the innate and adaptive immune systems of jawed vertebrates. Igs are useful in protecting against a wide range of pathogens [1], [2]. In mammals, five Ig isotypes were identified based on the constant domains of their H chains. In teleost fish, there are three major Ig isotypes: IgM, IgD and IgT, defined by the heavy chains μ, δ and τ, respectively. Among the three Ig types, IgM is the earliest identified Ig isotype, which presents as the main antibody in serum and is secreted into the mucus via the polymeric Ig receptor (pIgR) [3], [4]. IgZ/T was found to play an important role in mucosal immunity similar to IgA in mammals [5], [6]. In contrast, IgD has remained an enigmatic isotype since its discovery over 50 years ago. IgD is a primitive class of immunoglobulin present in most jawed vertebrates and conserved through evolution [7]. Initially, IgD was only detected as a membrane-bound form in teleosts, but a secreted form was subsequently discovered in catfish [8] and rainbow trout [9]. In recent decades, research on IgD in teleost fish has attracted much attention, and many IgD genes have been cloned and characterized, such as cod (Gadus morhua) [10], [11], Japanese flounder (Paralicthys olivaceus) [12], fugu (Fugu rubripes) [13], Atlantic salmon (Salmo salar) [14] and zebrafish (Danio rerio) [15]. Teleosts demonstrate remarkable plasticity in their δ genomic arrangement, with many species possessing repeated blocks of exons encoding Cδ2–4 as well as duplicated δ loci [16], [17], [18].
To date, information regarding the immunological properties and biological functions of IgD is very limited. In mammals, IgD was reported to function as an antigen receptor optimized for efficient recruitment of B cells in antigen-driven responses [19]. Moreover, IgD can largely increase in B cells, in which IgM function is suppressed, suggesting that IgD is largely able to substitute for IgM functions [20]. In addition, previous studies have demonstrated that the association of secretory IgD with basophils and mast cells results in the production of antimicrobial factors and the enhancement of respiratory immune resistance [8], [21]. Taken together, these facts demonstrate a crucial role for IgD in coordinating the immune surveillance of the host at the intersection of the innate and adaptive immune systems. Teleost IgD is thought to play similar roles to mammalian IgD in the immune system [22]. However, in teleost fish, the available information about IgD is mainly its gene structure and expression profile after stimulation [23], [24]. Flounder is an economically important fish popularly cultured in Asian countries, and accumulating studies on its immune system have been conducted in recent years. The cDNA encoding flounder IgD has been cloned and analysed previously [12], though no further research was performed at the protein level.
In the present study, the conserved δ1−δ4 region was recombinantly expressed and purified, which was then used as an immunogen to produce MAbs against the H chain of flounder IgD. Furthermore, the produced MAbs were used to investigate the distribution of IgD and IgM molecules by the double immunofluorescence assay test (DIFAT), and the percentage of IgD+ and IgM + lymphocyte subsets were determined by flow cytometric immunofluorescence analysis (FCIA).
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Expression and purification of recombinant IgD
According to the gene sequence encoding the IgD heavy chain of flounder (GenBank No. AB052658), specific primers were designed to amplify the δ1−δ4 region of flounder IgD (F: 5′-CGGGATCCAAAAGTCGGGTTGTCTCTCC-3’; R: 5′-CCAAGCTTTGAGCAGAGGCTGATATTC-3′). The PCR product was purified and digested with specific restriction enzymes and subsequently ligated into the pET-28a vector to construct recombinant plasmids. The recombinant plasmid pET-28a-IgD was transformed into E. coli BL21 (DE3) cells. The
Expression and purification of rIgD
The δ1−δ4 region of flounder IgD was expressed in E. coli BL21 (DE3) cells with the pET-28a system. SDS-PAGE revealed that the δ1−4 region of IgD was successfully expressed after IPTG induction with a distinct band at approximately 45 kDa (Fig. 1, lane 2), which is in accordance with the predicted molecular mass of the δ1−δ4 region of flounder IgD. After purification with a Ni2+ affinity chromatography, high purity rIgD was obtained (Fig. 1, lane 3).
Production and selection of MAbs
A total of 97 hybridomas were observed under
Discussion
IgD is considered to be an enigmatic molecule because of its low level of expression, the difficulty of detecting its antibody activity and its diverse gene structures among species. In the past several decades, the production of monoclonal antibodies against marine and freshwater fish Igs has greatly advanced studies of the immune system of teleost fish. MAbs against fish Igs provide a powerful tool for investigating the immunological functions of different Igs and the immune response of
Acknowledgements
This study was supported by the National Natural Science Foundation of China (31302216; 31672685; 31672684; 3142295), the Taishan Scholar Programme of Shandong Province, Science and Technology Development Project of Shandong Province (2014GNC111015) and the Scientific and Technological Innovation Project Financially Supported by Qingdao National Laboratory for Marine Science and Technology (No.2015ASKJ02).
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