| Chinese sucker(Myxocyprinus asiaticus)is the only representative of family Catostomidae in Asia and only distributed in the the Yangtze and Minjiang river.Meanwhile,Chinese sucker is a species of potential economic value in aquaculture because of its delicious meat.Due to the destruction of environment,over fishing and late in sexual maturity,the the number of Chinese sucker is decreasing.At present,the species has been listed in the second class of protected animals in China.Artificial breeding is an important way of recoverying resources,but the mortality rate is high in larval period.The immune system plays an important role in immune response,so the research of immune system can provide basis for disease prevention.Adaptive immune system is the most important mechanisim for organism fighting with pathogen.Research demonstrated that fishes are the most primitive species with adaptive immune system in the lower vertebrates,which have a special position in research of evolution of immune system.So,the research of fish immune system can provide theroretical foundation for evolution law of immune system.In immune system,humoral immunity mediated by B lymphocytes can destory pathogens through antibody.The CD79 a and CD79 b,which locate on the surface of B lymphocytes,play an important role in signal transduction.In addition,CD79 a protein is present on the surface of B cells throughout their cycle life,and is absent on all other healthy cells,making it a reliable marker for B cells.However,the study of B lymphocytes accessory molecular is scare till now,only a related study in teleost fishes such as Ictalurus punctatus and Epinephelus coioides.The sequences of CD79 a and CD79 b were obtained using homologous alignment and rapid amplication of cDNA end from Chinese sucker.The cDNA of MaCD79 a consisted of 1059 bp,including a 5' untranslated region(UTR)of 33 bp and 3' UTR of348 bp containing polyadenyla signal(AATAAA),an open reading frame(ORF)of 678 bp encoding for 225 amino acids.Comparatively,the ORF of MaCD79 b comprised 636 bp encoding for 211 amino acids.The conserved features and important functional residues were found by sequences analysis,the cysteine residues known to form the intradomain disulfide bond were conserved in all species.Phylogenetic tree showed thatMaCD79 a and MaCD79 b cluster at high bootstrap values with their respective counterparts,especially with CD79 molecules of Danio rerio and Cyprinus carpio.RT-PCR analysis revealed that the transcripts of MaCD79 s were at detectable level in eggs,and became constantly detected after 30 days post hatching(dph).Tissue distribution analysis showed that MaCD79 s were most highly expressed in immune tissues,such as spleen,head-kidney and kidney,the relatively low levels were detected in heart,gill and liver,while the transcripts have not been detected in intestine,brain,skin and muscle.After challenged with A.hydrophila,the results showed that the expression levels of MaCD79 s were significantly up-regulated at all time points in head-kidney,especially 48 h,72 h,96 h and 7 d.In the case of spleen,change of transcripts level of MaCD79 s was slighter,and significant up-regulation of MaCD79 s was seen only at 48 h,72 h and 96 h.In kidney,no significant change occurred at the most time points.In order to explore the location of MaCD79 s expressing cells,in situ hybridization with DIG-labeled anti-sense and sense probes were performed on peripheral blood lymphocytes(PBLs),spleen,head-kidney,kidney,intestine,gill and skin from Chinese sucker.Positive signals of CD79 s and IgM were detected in PBLs,as well as spleen,head kidney and kidney in scattered or gathered form throughout the tissue.In the intestine,most positive cells expressing MaCD79 s were detected in the lamina propria(LP)and few positive cells in the lamina epithelialis(LE)of hind-intestine,not in anterior and posterior intestine.However,staining for the IgM was found in LP throughout the intestine,especially the anterior and posterior intestine.Additionally,the IgM positive cells were also identified in the LE of hind-intestine and the serosa layer of posterior intestine.Few positive cells for CD79 s and IgM staining were detected in gill filament epithelium.No CD79 s and IgM positive cells were detected in the skin.The open reading frame(ORF)except Signal Peptide(SP)of CD79 was amplified by PCR using the gene-specific primer containing restriction sites,then inserted into prokaryotic expression vector PET-28 a and PET-16b-GFP,respectively.The recombinant plasmids were transformed into E.coil BL21(DE3).The recombinant protein were induced by isoprophl-?-D-thiogalactopyranoside(IPTG)and purified by Ni-NTA affinity chromatography according to manufacturer's instructions.The molecular weight of the CD79 a and CD79 b was 30 KDa and 55 KDa,respectively.The recombianant proteins were used to immunized Balb/c mouse and New Zealand white rabbit for the production of antiserum.Finally,anti-CD79 a monoclonal antibody were obtain,strains of hybridoma cell lines named 1-A5B1?1-D12H10 and 1-C5A4.Western blotting demonstrated that the antibody can specifically react with head-kidney protein extract of Chinese sucker.Taken together,our findings provided further information regarding CD79 s gene and their role in adaptive immunity,which will contribute to the preservation and aquaculture of Chinese sucker. |
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