| GIFT strain of Nile tilapia（Oreochromis niloticus）, which one of the most importantfish species in South China and all over the country, is of considerable food value andeconomic value. In recent years, with the rapid development of intensive marine fishfarming and deterioration of the aquaculture environment, outbreaks a variety ofpathogenic bacteria have affected the GIFT strain of Nile tilapia aquaculture causing hugeeconomic losses. Immunoglobulin is a very important humoral factor plays the key role infish immune system response. In order to study Ig gene in the host immune responsemechanisms involved in the process of outside pathogen, the nonspecific immune genesIgM and mIgD was cloned from Nile tilapia and expression and characteristics wereinvestigated for Ig as well.1. GIFT strain of Nile tilapia（Oreochromis niloticus）, induced by inactivatedStreptococcus agalactiae, was used to construct the head kidney SMART cDNA library.The full-length sIgM and mIgD cDNA of Nile Tilapia was cloned using homologicalcloning and rapid amplification of cDNA ends (RACE) methods. Results showed thefull-length of sIgM cDNA was1921bp, containing a5’untranslated region (5’-UTR) of41bp, a3’-UTR of140bp and an open reading frame of1740bp encoding579amino acidswith an estimated molecular weight of64.26ku and an estimated isoelectric point of5.36.In the N-terminal of sIgM exists a signal peptide structure. The phylogenetic treesconstructed showed that sIgM of nile tilapia shared the closest relationship with thecorresponding proteins of Paralichthys olivaceus and Rachycentron canadum. Thefull-length of mIgD cDNA was3347bp, containing an open reading frame of3015bpencoding1004amino acids with an estimated molecular weight of110.9ku and anestimated isoelectric point of6.24. The mIgD consists of variable and constant region. Thevariable region has a typical VDJ structure and the constant region consists serially of a μ1domain,seven CH domains(CH1to CH7),and a TM region.BLAST result based on themIgD heavy chain amino sequence of CH1-CH7domain showed that tilapia mIgD sharedhigh similarites with Hippoglossus hippoglossus (64%), with CH1to CH7having53%、56%、45%、57%、63%、69%、69%，and the TM domain having54%identity,respectively. 2. The expression profiles of Nile tilapia of sIgM and mIgD transcripts in differenttissues of healthy fish and the fish immunized by inactivated Streptococcus agalactiaewere analyzed by fluorescent quantitative real-time PCR technology. The results showedthat in healthy Nile tilapia, the sIgM transcripts were mainly detected in intestine, spleen,head kiney and gill, and were not expressed in muscle, brain and heart. The mIgDtranscripts were mainly detected in thymus, head kidney and spleen, but no expressed inthe muscle, gonad, brain and heart. After vaccinated with inactivated Streptococcusagalactiae36h later, the sIgM and mIgD mRNA expression levels were significantlyup-regulated in most studied tissues of Nile tilapia, and the expression level in someimmune organs or tissues such as head kidney, spleen, thymus is significantly higher thanhealty tissues. In the experimental period, the expression patterns of sIgM and mIgD werevery similar. They all expressed in intestine, skin, gill first and reached the highest levelwithin24h. In other immune tissues, however， after vaccinated with inactivatedStreptococcus agalactiae36h later, the mRNA expression levels were reached the peak.3. The constant segment of sIgM and mIgD gene were amplified and inserted into thepET-28a(+) and pET-32a(+) vector to construct the prokaryotic expression plasmidET28-μ-M and pET32--D．The recombinant sIgM and mIgD fusion proteins wereoverexpressed in E. coli BL21(DE3) cells in the presence of IPTG．The recombinant sIgMand mIgD fusion proteins was purified by His TrapTMHP column, and then identified bywestern blot using His-Tag Mouse mAb. Polyclonal antibody against sIgM fusion proteinwas raised in a New Zealand pedigree white rabbit immunized with the purified SIGMfusion protein, and the antibodytiter reached1:256000.4. For further study on the distribution of sIgM in immune organs in Nile tilapia,immunohistochemistry analysis was carried out using the prepared SIGM polyclonalantibody. The results show that, in the intestine and gill tissues, the positive signal exist inthe epithelial cell surface, but no exist in the goblet cells；While in spleen and head kidneytissues, the SIGM protein mainly distributed in lymphocytes in this organs. In order todetermine the location of SIGM in subcellular level, this experiment using theimmunoelectron microscopy technique to analysing the intestine epithelial cells、gillepithelial cells and spleen lymphocytes in Nile tilapia. The results showed that SIGMmainly exist in the vicinity of intestinal epithelial cell membrane and the surfacemembranes of the epithelial cells in intestinal microvilli. In the gill epithelial tissue, SIGMmainly exist in the part of the red blood cells and the epithelial cells which is surroundingthe gill filaments and gill-amella. The presence of colloidal gold particles in a large numberof secretory vesicles spleen lymphocytes at internal Golgi apparatus. All the results suggested sIgM played a critical role in the muscosal immunity of teleost. These provideinsights into the roles of fish sIgM in the mucosal immunity. |
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