The cDNA sequence of the Japanese flounder (Paralychthys olivaceus) IgD has been previously reported (GenBank accession no. AB052658) and this was followed by the detection of IgD mRNA expression in some flounder organ tissues. However, it has not been determined whether the flounder IgD gene is vir...
The cDNA sequence of the Japanese flounder (Paralychthys olivaceus) IgD has been previously reported (GenBank accession no. AB052658) and this was followed by the detection of IgD mRNA expression in some flounder organ tissues. However, it has not been determined whether the flounder IgD gene is virtually expressed into IgD protein. To characterize the flounder immunoglobulins utilized in elucidating the mechanism, evolution and diversity of the flounder immune system, antibodies specific to IgD and IgM were necessary. In the present study, partial flounder recombinant IgD (rIgD), IgM (rIgM) and the conserved regions of IgD and IgM (rCIg) were produced by cloning the cDNA sequence using isotype specific primers which were designed to produce unique fragments of IgD and IgM specific amino acid sequences. The production of recombinant Igs was ascertained by SDS-gel electrophoresis and immunoblot analysis using anti-T7$\cdot}$Taq antibody. The produced recombinant Igs were purified using affinity columns, and used as immunogens. Antibodies specific to the isotype of flounder Igs were generated by immunizing rabbits with rfIgs and the antibodies produced were identified by enzyme-linked immunosorbent assay (ELISA) and immunoblotting. Specificities of the generated antibodies were evaluated by testing cross-reactivity between recombinant IgM and IgD. By ELISA, rabbit antibodies against the rfIgD fragment (anti-rfIgD) failed to recognize any kind of flounder serum Igs, whereas respective antibodies against rfCIg (anti-rfCIg) and rfIgM fragments (anti-rfIgM) reacted with serum Igs. Likewise, in immunoblot assays, though anti-rfIgD did not, both anti-rfCIg and anti-rfIgM bound with the ~85 kd flounder IgM heavy chain. By flow cytometry analysis, anti-rfCIg, anti-rfIgD and anti-rfIgM reacted with 6%, 3% and 6.5% of cells, respectively, suggesting that flounder IgD is not secreted in serum but expressed on flounder B-like cell surfaces as in mammals. Antibodies produced against recombinant flounder Igs could be used to develop sandwich assay systems for detecting flounder Igs and for further investigating the flounder immune system.
The cDNA sequence of the Japanese flounder (Paralychthys olivaceus) IgD has been previously reported (GenBank accession no. AB052658) and this was followed by the detection of IgD mRNA expression in some flounder organ tissues. However, it has not been determined whether the flounder IgD gene is virtually expressed into IgD protein. To characterize the flounder immunoglobulins utilized in elucidating the mechanism, evolution and diversity of the flounder immune system, antibodies specific to IgD and IgM were necessary. In the present study, partial flounder recombinant IgD (rIgD), IgM (rIgM) and the conserved regions of IgD and IgM (rCIg) were produced by cloning the cDNA sequence using isotype specific primers which were designed to produce unique fragments of IgD and IgM specific amino acid sequences. The production of recombinant Igs was ascertained by SDS-gel electrophoresis and immunoblot analysis using anti-T7$\cdot}$Taq antibody. The produced recombinant Igs were purified using affinity columns, and used as immunogens. Antibodies specific to the isotype of flounder Igs were generated by immunizing rabbits with rfIgs and the antibodies produced were identified by enzyme-linked immunosorbent assay (ELISA) and immunoblotting. Specificities of the generated antibodies were evaluated by testing cross-reactivity between recombinant IgM and IgD. By ELISA, rabbit antibodies against the rfIgD fragment (anti-rfIgD) failed to recognize any kind of flounder serum Igs, whereas respective antibodies against rfCIg (anti-rfCIg) and rfIgM fragments (anti-rfIgM) reacted with serum Igs. Likewise, in immunoblot assays, though anti-rfIgD did not, both anti-rfCIg and anti-rfIgM bound with the ~85 kd flounder IgM heavy chain. By flow cytometry analysis, anti-rfCIg, anti-rfIgD and anti-rfIgM reacted with 6%, 3% and 6.5% of cells, respectively, suggesting that flounder IgD is not secreted in serum but expressed on flounder B-like cell surfaces as in mammals. Antibodies produced against recombinant flounder Igs could be used to develop sandwich assay systems for detecting flounder Igs and for further investigating the flounder immune system.
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문제 정의
The aim of the present study was to overexpress flounder IgD fragment protein and to raise antibodies against that could be as a tool for studies on the development of the fish immune system. Detailed knowledge of immune system function is an essential part of devising disease prevention strategies, such as, the development of vaccines and for monitoring therapeutic processes in microbially infected fishes.
제안 방법
To produce polyclonal antibodies against the purified rfIg fragments, CIg, IgD, and IgM, New Zealand rabbits were subcutaneously immunized with 100 µg of the rfIg fragments emulsified 1 : 1 in Freund’s complete adjuvant (FCA), and made up to a total volume of 1 ml per rabbit. Boosters injections were administered on days 7, 14 and 21 at the same rfIg fragment dose in Freund’s incomplete adjuvant (FIA). Final booster injects were administered on day 28 at the same rfIg fragment doses in 1 ml of sterile Hank’s balanced
AB052658 and AB052744, respectively). The three kinds of flounder Ig PCR products produced were adjusted to about 0.7 kb by each specific primer set, and cloned into an expression vector which carried an integrated copy of the T7 · Taq RNA polymerase gene under the control of inducible lac UV5 promoter (Fig. 1). The sequences of the insert and frame were confirmed by DNA sequencing analysis (Korea Basic Science Research Institute, Daejon, Korea).
The three DNA inserts eluted from agarose gel were ligated to pET28a vector restricted with BamHI and EcoRI. To confirm that the pET28a constructs were correctly made, the constructs were treated with restriction endonucleases and this was followed by agarose gel electrophoresis. As shown in Fig.
To investigate whether rabbit antibodies against rfIg fragments recognize natural flounder Igs in normal flounder serum, ELISA and immunoblot assays were conducted. By ELISA, rabbit antibodies against rfIgD fragment (anti-rfIgD) failed to recognize any kind of flounder serum Igs, whereas the respective antibodies against rfCIg (antirfCIg) and rfIgM fragments (anti-rfIgM) did react with serum Igs (Fig.
대상 데이터
To amplify the coding region of flounder IgD, IgM and the conserved sequence of both Igs (CIg) by PCR, specific primers were designed to introduce BamHI and EcoRI restriction sites at both ends of the PCR product since the expression vector, pET28a contains the same restriction enzyme sites. Specific primers were prepared (as detailed in Table 1) based on flounder IgD and IgM cDNA sequences (GenBank Accession nos. AB052658 and AB052744, respectively). The three kinds of flounder Ig PCR products produced were adjusted to about 0.
이론/모형
The resulting lysates containing rfIg fragments (CIg, IgD or IgM) were purified by T7 · Taq polymerase antibody-labeled affinity chromatography (Novagene, Germany) according to the manufacturer’s instructions. Purified rfIg fragments were dialyzed against PBS (pH 7.4) and protein concentrations were determined by the Bradford method (Bradford, 1976).
성능/효과
IgD cDNA of 3244 bp and IgM cDNA of 1877 bp coded 999 and 579 amino acid residues, respectively. The nucleotide sequences of flounder IgD and IgM used in this study showed 95% and 97% homologies, respectively, with previously registered sequences (Accession nos. AB052658 and AB052744) (data not shown). The homology of the conserved sequences of Japanese flounder IgD and IgM (CIg) was 97% (data not shown).
참고문헌 (22)
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