Molecular Cloning,recombinant Expression And Activity Analysis Of A C-type Lectin From Haliotis Discus Hannai

H.discus hannai is one of the major economic mariculture species.In recent years,frequent occurrence of diseases is threatening the sustainable development of the industry.Therefore,indepth study of the immune mechanism of H.discus hannai and the effective disease management are urgent to be carried out.Since the lack of acquired immunity,H.discus hannai mainly rely on the innate immunity when resisting the invasion of exogenous microorganisms.As an important pattern recognition receptors,C-type lectins can identify and remove exogenous microorganisms,and play an important role in the immune defense of H.discus hannai.In this study,a C-type lectin gene(Hd CTL)was cloned and analyzed by various Biological Softwares.The distribution of Hd CTL in the tissues and its expression pattern was detected by real-time PCR.Transetta competent cells were used as expression vector to construct the prokaryotic expression system,r Hd CTL protein was obtained through recombinant,and its activity was analyzed.This study aimed at exploring the effect of Hd CTL in the immune defense mechanism of H.discus hannai,and provide theoretical basis for further understanding of the innate immune mechanisms.The full-length c DNA of Hd CTL acquired in this study was 1933 bp,containing a 5’ untranslated region(UTR)of 9bp,3’ UTR of 430 bp with a polyadenylation signal AATAAA,and an open reading frame(ORF)of 1494 bp encoding a polypeptide of 497 amino acids.According to Ex Pa Sy analysis,the isoelectric point and estimated molecular mass of Hd CTL were 7.15 and 56.14 k Da respectively.The coding sequence of amino acids 1(M)-25(F)is a signal peptide,31(R)-153(F)is a CLECT domain,and 196(W)-463(C)is a Zona-pellucida domain.The results of the three-dimensional structure of Hd CTL showed that its encoding amino acids cotained a C-type lectin domain.The expression pattern of Hd CTL in the tissues of H.discus hannai and the hemocyte after stimulation with exogenous microorganisms were detected by real-time PCR.The results indicated that expression of Hd CTL was found in muscle,mantle,gill,hemocyte,hepatopancreas and gonad,with the highest expression in muscle,followed by blood cells and lowest in gills.After 6 hours of stimulation with Listonella anguillarum and Micrococcus luteus,the expression level of Hd CTL in blood was significantly increased.Being stimulated by L.anguillarum,the expression level increased significantly and reached the peak at 24 h,and decreased to the lowest at 48 h.The stimulation of M.luteus also induced the expression.The expression level was highest after 6 hours of induction,showed a decrease at 12 h and reached the peak at 24 h,and the high expression was sustained to 48 h.Summing up the above,Hd CTL may be involved in the H.discus hannai’s immune defense process,and play a role in resisting the invasion of different pathogenic microorganism.The Hd CTL was cloned into Peasy-E1 expression vector and transformed into Transetta expression system,expression with IPTG.The r Hd CTL recombinant protein was purified by nickel chelate agarose gel chromatography and renatured by gradient dilution method.The anti-r Hd CTL antibody was collected from immunizing mice.The specificity analyse of the antibody was performed by Western-bolt.The results showed that anti-r Hd CTL antibody could specifically recognize r Hd CTL.ELISA was using to detect the binding capacity of r Hd CTL to mannan,zymosan,LPS,PGN and β-glucan PAMPs.In vitro binding can happen between r Hd CTL with mannan and zymosan rather than LPS,PGN and β-glucan.The binding activity to zymosan increases with the increasing recombinant protein.These results demonstrate that r Hd CTL may mediate the immunological recognition of H.discus hannai for mannan and zymosan.The agglutination activity of r Hd CTL for exogenous microorganisms was further studied.It turned out that r Hd CTL had agglutination activity against Candida Albicans and Pichia pastoris,thus the identification of mannan and zymosan may be the molecular basis of microbial organisms such as fungi,which indicating that Hd CTL can be used as a pattern recognition receptor involved in the immune recognition and immune response of exogenous microorganisms.The effects of r Hd CTL on the phagocytosis and encapsulation of H.discus hannai were detected.The results showed that r Hd CTL can promote the phagocytosis of to hemocyte exogenous microorganisms.By detecting the effect of r Hd CTL on the promotion of hemocyte encapsulation by nickel agarose granules,we found the nickel agarose granules coated with r Hd CTL could promote the encapsulation of agarose granules in vitro.These results indicate that r Hd CTL may be involved in the ablation of exogenous microbes.In summary,a C-type lectin(Hd CTL)from H.discus hannai was cloned,and its m RNA expression pattern was detected and characterization of its encoded protein was analyzed.The results proved that r Hd CTL could bind to mannan and zymosan in vitro and showed agglutination activity against Candida Albicans and Pichia pastoris.Meanwhile,r Hd CTL could also promote the phagocytosis and encapsulation of hemocyte to Listonella anguillarum and Micrococcus luteus.The results of this study would enrich and develop the immunology of marine invertebrates,also provide the basis for the further study of the immune mechanism of mollusks as well as laid a theoretical foundation for the selective breeding of H.discus hannai.