| With the rapid development of shellfish culture industry in China,a series of health problems caused by pathogen invasion have emerged,which seriously restrict the sustainable development of marine ecology.Mining the key factors of shellfish response to pathogen stress can provide reliable data support for the study of immune defense mechanism of shellfish,at the same time,it is of great theoretical significance for the disease control and health management in the process of breeding.In this study,two types of immune-related molecules,C-lectin(VpClec-1,VpClec-2),were cloned from Venerupis philippinarum,and their sequences were analyzed.Real-time fluorescence quantitative PCR(qRT-PCR)was used to analyze the tissue distribution after Vibrio anguillarum stimulation,the correspondingly recombinant proteins were constructed to study their immune activities.The results of this study were as follows:(1)The gene sequence analysis showed that the full-length cDNA sequence of VpClec-1 gene of Venerupis philippinarum was 588 bp,the open reading frame(ORF)was 489 bp,encoding 162 amino acids,the isoelectric point was p I = 4.26.the predicted molecular weight of the protein was 15.98 kDa;The full-length cDNA sequence of the VpClec-2 gene was 688 bp,the ORF was 555 bp,encoded 184 amino acids,the isoelectric point was p I = 8.40,and the predicted molecular weight of the protein was 18.74 kDa.The homology analysis and multiple sequence comparison of the amino acid sequences of VpClec-1 and VpClec-2 genes revealed that both genes were similar to the C-type lectin sequence and structural characteristics and were the new members of the C-type lectin family.(2)Real-time fluorescent quantitative PCR(qRT-PCR)results showed that the mRNAs of VpClec-1 and VpClec-2 genes were both expressed in tissues of hemocytes,hepatopancreas,gill,mantle and muscle of healthy Venerupis philippinarum.After Vibrio anguillarum stimulation,the expression levels of VpClec-1 and VpClec-2 genes were both up-regulated.The VpClec-1 gene expression was significantly up-regulated after Vibrio anguillarum stimulation and reached a peak value at 24 h.However,VpClec-2 showed acute upregulation in 6 h(8.25 times,p < 0.01),12 hours(16.7 times,p < 0.01),and 24 hours(24.2 times,p < 0.01),and both closed to normal levels at 48 h.It is speculated that the up-regulation of mRNA expression of VpClec-1 and VpClec-2 genes could be related to the involvement of immune response against the pathogenic microorganisms in Venerupis philippinarum.(3)Both rVpClec-1 and rVpClec-2 possessed binding activity towards LPS,glucan and zymosan,and the binding abilities of rVpClec1 and rVpClec-2 were dose-dependent.Both rVpClec-1 and rVpClec-2 showed strong agglutination activities towards Vibrio.harveyi,Vibrio.splendidus,Vibrio.anguillarum,Enterobacter.cloacae and Aeromonas.hydrophila,while no obvious agglutination activities against Staphylococcus.aureus was observed in both recombinant proteins.Compared with rVpClec-2,rVpClec-1 showed higher antibacterial/agglutination activities against V.anguillarum,E.cloacae and A.hydrophila,but not S.aureus or V.splendidus.In addition,phagocytosis of the hemocytes could be enhanced by both rVp Clec-1 and rVpClec-2 incubation,especially rVpClec-2 had obvious ability to induce phagocytic activity of lymphocytes,and rVpClec-1 could promoted the chemotactic ability of lymphocytes,rVpClec-2 had no obvious effect on its chemotactic ability;meanwhile,rVpClec-1 and rVpClec-2 clould also promote the encapsulation ability of blood lymphocytes,which showed that both of them had some immunomodulatory effects in the innate immune response of Venerupis philippinarum.In a word,lectin VpClec-1 and VpClec-2 in Venerupis philippinarum,as pattern recognition receptors,played an indispensable role in the immune defense process of shellfish.The research results could provide certain theoretical support for the deeper understanding of the innate immune defense mechanism of shellfish. |
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