The Study Of Immune Function And Molecular Recognition Mechanism Of Manila Clam Ruditapes Philippinarum C-type Lectins

As one of the main members of the lectin family,C-type lectin plays an important role in recognizing carbohydrate groups on the surface of foreign microorganisms and activating immune factors in body fluids.In this study,molecular biology,bioinformatics and immunology were used to study the structure and function of C-type lectin domain in Manila clam Ruditapes philippinarum,and the effects of environmental factor stress such as pathogen infection,low temperature and air exposure on C-type lectin in aquaculture environment and during transportation,discussed its role in the immune system of the Ruditapes philippinarum.Six lectin C type lectin genes（CTL-1,CTL-2,CTL-3,CTL-4,CTL-5 and CTL-6）were identified,and the expression distribution in different tissues and the spatiotemporal expression pattern of CTLs in hepatopanpancreas of R.philippinarum under low temperature stress were analyzed.The results showed that the open reading frame length of the six C-type lectin genes was 588 bp,408 bp,462 bp,1017 bp,840 bp and 471 bp,respectively,and the predicted molecular weight range was 15.47-39.28kDa,with the PI between 4.33-6.17.It was speculated that these sequences were composed of 4-6 exons and 3-6 introns.The secondary structure of the protein was predicted to consist of 3-9 coils,3-9 chains,and 3-8 coils.To evaluate structural domain and discovered that besides CTL-2 and CTL-3,CTL in CTL1-4,CTL-5 and CTL-6identified the signal peptide,and they have a single open reading frame of CRD,four of them completely conservative participation within the CRD disulfide formation of the two cysteine residues and Ca²⁺reliance on carbohydrates in combination with the base sequence EPN and WND also found in the ORF.The qRT-PCR technique was used to detect the health R.philippinarum CTLs expression level in different tissues,the results showed that CTLs express level are significant expressed in the gills and hepatopancreas,and the expression level of CTLs in R.philippinarum after low-temperature stress were significantly induced in one or two time points,suggests that low-temperature stress was induced the R.philippinarum the C-type lectin gene expression.The full length of RpCTL gene was cloned from R.philippinarum by RACE technique.The cDNA sequence of the RpCTL gene GenBank（accession number:MH368785）.The full-length cDNA of RpCTL was 802 bp,and consisted of a 5′untranslated region（UTR）of 58 bp,a 3′UTR of 153 bp,and an ORF of 591 bp,including the stop codon（TGA）.The ORF encoded a polypeptide of 196 amino acids with an isoelectric point of 5.19 and a predicted molecular weight of 22.36 kDa.The deduced amino acid sequence of RpCTL has a signal peptide of 16amino acid residues and a single RpCTL CRD.Several important signatures of the CTL family were detected in RpCTL:the four completely conserved cysteine residues（Cys 30–Cys 104,Cys 124–Cys 132）involved in the formation of the internal CRD disulfide bonds and the EPD（Glu 94–Pro 95–Asn 96）motif,which determines the specificity of ligand binding.To evaluate the molecular evolutionary relationships between RpCTL and other CTLs,RpCTL clustered most closely with the CTLs of Crassostrea virginica and C.gigas,and then with those of other mollusks,including Azumapecten farreri,Haliotis discus discus,and Meretrix meretrix.The vertebrate CTLs formed another cluster,including the fish CTLs,mammal CTLs,amphibian CTL,and bird CTL.the phylogenetic relationships of RpCTL amino acid sequence are consistent with the traditional classification of the clam.RT-PCR was used to detect expression distribution in the mantel,gill,siphon,adductor muscle,and foot of RpCTL in two populations（Northern cultured population and wild population）and three strains（Zebra,White Stork and White Zebra）.In this study,the RpCTL transcripts were widely expressed in the various tissues of the different strains,including in the mantle,gill,siphon,adductor muscle,hepatopancreas,and foot.In the cultured clams,the expression of RpCTL was significantly higher in the siphon（4.38-fold）,gill（7.44-fold）,and hepatopancreas（11.52-fold）than in the foot（P<0.05）.In the wild clams,the RpCTL transcripts were 3.89-,4.16-,and 49.00-fold higher in the gill,siphon,and hepatopancreas,respectively,than in the foot（P<0.05）.In the white clams,RpCTL was highly expressed in the mantle,gill,siphon,and hepatopancreas（5.70-,12.24-,3.21-,and40.72-fold higher than in the foot,respectively;P<0.05）.In the white–zebra clam,RpCTL was highly expressed in the gill,siphon,and hepatopancreas,at 3.79-,6.83-,and 13.67-fold higher than in the foot,respectively（P<0.05）.In conclusion,RpCTL is mainly distributed in the gills and hepatopancreas of R.philippinarum.Inducing RpCTL protein by recombinant prokaryotic expression and purification method,and RpCTL had an apparent molecular mass of 23kDa.The fusion protein was purified with Ni–NTA affinity chromatography using different concentrations of imidazole（20,50,80,100,150,200,or 300mM）from the precipitates of Transetta cells.SDS-PAGE showed that the RpCTL concentration was higher after elution with 80,100,or 150mM imidazole.A considerable amount of RpCTL was obtained with affinity chromatography on an Ni–NTA column using eluent containing 100mM imidazole.The transparent inhibitory zones on the E.coli,V.anguillarum,and S.aureus plates treated with RpCTL were larger than those on the B.subtilis plates,implying that the inhibitory effects of RpCTL on E.coli,V.anguillarum,and S.aureus were stronger than that on B.subtilis.The inhibition of E.coli and B.subtilis cell growth by RpCTL at concentrations of 20 and 60μg/ml was weaker than that at 180μg/ml RpCTL.After clams were injected with RpCTL,they were injected with V.anguillarum,and their survival rates were observed.The survival rate of the injected RpCTL+V.anguillarum group was higher than that of the V.anguillarum+PBS group,but lower than that of the PBS+PBS combined with the PBS+RpCTL group.