| The Qihe crucian carp(Carassius auratus),as a triploid freshwater fish,is one of unique and high quality aquatic products in Henan Province.However,in recent years,with the increase of the breeding density and number of diseases,the immunity of Qihe crucian carp has decreased significantly,resulting in extensive economic losses.The specific immunity is not perfect in Qihe crucian carp,which is a lower vertebrate,and non-specific immunity is particularly important.The study on the mechanism of non-specific immunity in Qihe crucian carp can control effectively the occurrence of fish diseases.Lectin plays a crucial role in the non-specific immunity of fish.Protein-carbohydrate interactions which are mediated by lectin have been recognized as the important components of innate immunity in fish.To understand the role of lectin in the non-specific immunity and antibacterial effect of fish,it will provide a great significance to control effectively the occurrence of fish diseases.In the present study,the Qihe crucian carp was used as the experimental fish.Three kinds of lectin,including CaNTC,CaCLR and Ca Gal-9 were cloned in the C.auratus,as well as the molecular characteristics,tissue distribution,expression response and immune function of lectins were systematically studied.The main results are as follows:(1)The study of gene clone,tissue expression and immune function of C-type lectin(CaNTC)in Qihe crucian carpThe full-length cDNA of CaNTC was composed of 776 bp,with a 152 bp 5?-untranslated region,a 492 bp open reading frame encoding a 163-aa protein,and a 132 bp 3?-untranslated region.The deduced amino acid sequence of CaNTC contained a signal peptide,and a single carbohydrate recognition domain(CRD) which had four conserved disulfide-bonded cysteine residues(Cys57-Cys150,Cys126-Cys142),and an EPN-WND motif required for carbohydrate-binding specificity.The tissue distribution of CaNTC transcripts were analyzed using the method of q RT-PCR.The m RNA transcripts of CaNTC could be detected in all tested tissues,among which it was the most abundant in liver,moderately abundant in head kidney,spleen,gill,and blood,and relatively lower level in kidney,skin,brain,intestine,heart,and muscle.The temporal expressions of CaNTC were obviously up-regulated in gill,liver,spleen,kidney and head-kidney after challenged by Aeromonas hydrophila and Poly I:C,respectively.The recombinant CaNTC(rCaNTC)was purified from Escherichia coli BL21(DE3),and mice were immunized with rCaNTC to obtain polyclonal antibodies.On the basis of obtaining rCaNTC and polyclonal antibody,it was found that rCaNTC exhibited strong binding ability with LPS and PGN,as well as all tested bacteria(A.hydrophila,E.coli,Vibrio anguillarum,Bacillus subtilis,Micrococcus lysodeikticus and Staphylococcus aureus)using the method of ELISA and Western blot,respectively.With regard to the agglutinating activity of rCaNTC,rCaNTC was able to agglutinate rabbit erythrocytes and three kinds of bacteria(E.coli,A.hydrophila and S.aureus)in a Ca2+-dependent manner.In vivo,it was investigated that rCaNTC promoted the elimination of A.hydrophila.These findings collectively demonstrated that CaNTC,as a pattern-recognition receptor(PRR),could bind with,agglutinate and clear pathogenic bacteria through recognizing the LPS and PGN on the surface of pathogenic bacteria and played an important role in immune defense of C.auratus.(2)The study of gene clone,tissue expression and immune function of C-type lectin receptor(CaCLR)in Qihe crucian carpThe full-length cDNA of CaCLR was composed of 1130 bp,with a 792 bp open reading frame encoding a 263-aa protein.The deduced amino acid sequence of CaCLR contained a transmembrane region and a single CRD.The tissue distribution of CaCLR was analyzed by q RT-PCR.The m RNA transcripts of CaCLR could be detected in the 11 tissues tested,among which it was the most abundant in head-kidney.The temporal expressions of CaCLR were obviously up-regulated in gill,liver,spleen,kidney and head-kidney after challenged by A.hydrophila and Poly I:C,respectively.The tissue distribution of CaCLR in the protein level was detected using the method of Western blot.The CaCLR protein could be detected in all tested tissues,among which it was the most abundant in head-kidney and liver,moderately abundant in spleen,kidney and blood,and relatively lower level in intestine,heart and brain.The recombinant CaCLR was purified from E.coli,and mice were immunized with rCaCLR to obtain polyclonal antibodies.With regard to the agglutinating activity of r CaCLR,r CaCLR was unable to agglutinate rabbit and Qihe crucian carp erythrocytes,as well as seven kinds of microorganism in the assay.However,it was found that r CaCLR exhibited strong binding ability with LPS,PGN,?-glucan and mannan by ELISA.And the result of Western blot indicated that,r CaCLR exhibited binding ability with all tested bacteria except B.subtilis and S.aureus.In the experiment of antibody blockage,it was found that CaCLR could promote the phagocytosis of leukocytes.These findings collectively indicated that CaCLR,a C-type lectin receptor,which functioned as a PRR in the process of pathogen infection.It was able to recognize the carbohydrate structures on the surfaces of pathogenic bacteria,and performed opsonization function by promoting the phagocytosis and scavenging effect of pathogenic bacteria.(3)The study of gene clone,tissue expression and immune function of galectin(Ca Gal-9)in Qihe crucian carpThe full-length cDNA of Ca Gal-9 was composed of 1318 bp,with a 58 bp 5?-untranslated region,a963 bp open reading frame encoding a 320-aa protein,and a 297 bp 3?-untranslated region.The deduced amino acid sequence of Ca Gal-9 contained two tandem CRDs(CRD-1 and CRD-2)without the signal peptide or transmembrane region.The tissue distribution of Ca Gal-9 was analyzed using the method of q RT-PCR.The transcripts of Ca Gal-9 could be detected in all tested tissues,among which it was the most abundant in spleen,relatively lower level in gill,skin,blood,heart and brain,and the lowest level in muscle.The temporal expressions of Ca Gal-9 were obviously up-regulated in five kinds of immune-related tissues after challenged by A.hydrophila and Poly I:C,respectively.The tissue distribution of Ca Gal-9 in the protein level was detected by Western blot.The Ca Gal-9 protein could be detected in all tested tissues except blood,among which it was the most abundant in intestine,moderately abundant in spleen and kidney,and relatively lower level in gill,liver,head-kidney,heart and brain.The recombinant Ca Gal-9,CRD-1 and CRD-2 was purified from E.coli and mice were immunized with r Ca Gal-9 to obtain polyclonal antibodies.The r Ca Gal-9,r CRD-1 and r CRD-2 was able to agglutinate rabbit erythrocytes and all tested bacteria(Saccharomyces cerevisiae,A.hydrophila,E.coli,V.anguillarum,B.subtilis,M.lysodeikticus and S.aureus)in a Ca2+-independent manner.The inhibition experiments of carbohydrates on the agglutinating activity of rabbit erythrocytes showed that,r Ca Gal-9,r CRD-1 and r CRD-2 also had the binding ability with D-Galactose,N-acetyl-D-glactosamine,?-Lactose,LPS,PGN and?-Glucan.And through the method of Western blot,it was found that r Ca Gal-9 and r CRD-1 exhibited strong binding ability with all tested bacteria,and r CRD-2 bound with all tested bacteria except B.subtilis and M.lysodeikticus.The Ca Gal-9,CRD-1 and CRD-2 possessed hemaggulation,carbohydrate recognition and binding,microbial binding and agglutination activity.However,compared with single CRD,Ca Gal-9was endowed with a broader specificity and higher affinity for carbohydrate and bacteria.The r Ca Gal-9 had obvious antibacterial activity to A.hydrophila and E.coli.In vivo,it was investigated that r Ca Gal-9 enhanced the elimination of A.hydrophila and the survival rate of Qihe crucian carp infected by A.hydrophila.In the present study it was shown that Ca Gal-9,as a PRR,recognized the sugar structures on the surface of A.hydrophila,combined with and agglutinated A.hydrophila,inhibited their growth,and enhanced the elimination of A.hydrophila.In this study,three kinds of lectin,including CaNTC,CaCLR and Ca Gal-9,were selected from the transcriptome of Qihe crucian carp infected by A.hydrophila,because that the m RNA expression levels of three lectins were obviously up-regulated after challenged by A.hydrophila.The full length of cDNA was obtained by RT-PCR and RACE,and three lectin genes were systematically studied in the m RNA and protein level.The results showed that they possessed different characteristics in the molecular structure and immune function in the process of pathogenic microorganism infection.The research of three kinds of lectin on the molecular characteristics,tissue expression and molecular mechanism of pathogenic microorganisms recognition,agglutination and clearance,which provided a great help to understand the molecular basis of non-specific immunity in fish,and possessed important theoretical significance and application value for improving fish immunity level and preventing fish from disease. |
PDF Full Text Request