Cloning And Immune Activity Study Of C-type Lectin Genes In Sinonovacula Constricta

Razor clam（Sinonovacula constricta）is an important maricultural species in China.Recently,bacterial infection was appeared more frequently in the razor clam farming.C-type lectins,a kind of pattern recognition receptor（PRR）,are believed have wide participation in shellfish immunity.In this study,two C-type lectins of S.constricta had been cloned and identified by their mRNA expression pattern,binding specificity,agglutinating activity and phagocytic promoting activity.1.Two fragments sharing sequence similarity with C-type lectins was identified from the transcriptome of S.constracta established in the early research.In this study,full length cDNA sequence of these two fragments were obtained by RACE-PCR.Results showed that full length ScCTL-1 consists of a 91 bp 5’-UTR,a 380 bp 3’-UTR,and an 1899 bp ORF encoding a 632-amino acid polypeptide with a signal peptide,a C-terminal transmembrane region and four CTLD.Full length ScCTL-2 consists of a 63bp 5’-UTR,a 238 bp 3’-UTR,and an 1893 bp ORF encoding a 630-amino acid polypeptide with a N-terminal transmembrane region and four CTLD.Two C-type lectins shared 30%similarity with each other.Six cysteines are completely conserved in eight CTLD of two C-type lectins,indicating that all are‘long form’types.The third CTLD of ScCTL-1 and the third,fourth CTLD of ScCTL-2 are predicted possess mannose-binding specific by their amino acid constitution forming chemical bond in carbohydrate-binding.2.The two S.constracta C-type lectins mRNA expression patterns were detected in tissues of healthy razor clams and hemocytes of post-injection clams with real-time PCR.The results showed that ScCTL-1 and ScCTL-2 have significantly higher expression in hepatopancreas than other tissues,and ScCTL-2 has a significantly higher expression in gills than other five tissues except hepatopancreas,indicating that two S.constracta C-type lectins are both immune-related.In the bacterial injection experiment,ScCTL-1 was up-regulated at 4h and raised for second time at 24h after Staphylococcus aureus injection.It was also up-regulated at 4h and raised for second time at 12h after Vibro anguillarum injection.ScCTL-2 was up-regulated from 4h to 24h after S.aureus injection,and at 8h and 12h after V.anguillarum injection.3.In this study,immune activity of two S.constracta C-type lectins was identified by recombinant proteins.The recombinant vectors of two S.constracta C-type lectins were constructed and expressed in E.coli BL21（DE3）（recombinant protein designed rScCTL-1 and rScCTL-2）.Results showed that rScCTL-2 possessing agglutinate activity for all the six microorganisms in this study.The agglutinate activity is strict Ca²⁺-dependent.ScCTL-1 possess binding and agglutinate activity for both S.aureus and V.anguillarum,the binding and agglutinate activity are Ca²⁺-dependent and consistent for one type of bacteria.pathogen associated molecular pattern（PAMP）binding assay showed that the PAMP-binding activity and bacteria-binding activity of rScCTL-1 are closely related.Phagocytic promoting experiment showed that ScCTL-1could enhance the bacterial phagocytic activity of hemocytes.The promoting was Ca²⁺-and bacteria-related,indicating that rScCTL-1 could promoting phagocytosis by bacterial binding activity of CTLD.The immune characteristic of rScCTL-1 is similar to invertebrate macrophage mannose receptors,which likely means ScCTL-1 could take part in immunity of razor clam by function similar to macrophage mannose receptors.