Identification,Expression And Function Analysis Of MASP1?MASP2 And CL-11 Gene Of Complement Lectin Pathway From Half-smooth Tongue Sole (Cynoglossus Semilaevis)

In this study,the mannose-binding lectin-related serine protease gene 1(CsMASP1),mannose-binding lectin-related protein 34(CsMASP2)and collectin-11(CsCL-11)of the half-smooth tongue sole were cloned,expression level detection,prokaryotic expression and verification of the antibacterial activity of the recombinant protein.The following results we obtained:The full-length cDNA of CsMASP1 gene is 2507 bp,with a 82 bp 5?-UTR,a 142 bp3?-UTR,as well as a 2283 bp CDS region which encoded 760 amino acids.The predicted relative molecular weight and the theoretical isoelectric point of the CsMASP1 is 84.95 kD and 5.33,respectivly.SMART domain prediction analysis showed that CsMASP1 protein contains six domains: two like Clr/Cls protein domains(CUB)(47-168 aa,215-327aa),one calcium-binding epidermal growth factor-like domain(EGF-CA)(169-212aa),two complement control protein domains(CCP)(331-392 aa,397-461aa)and a serine protein kinase Tryp-SPc domain(SP)(478-752aa).CsMASP1 contains 13 exons and is located on chromosome 4.Homology comparison showed that CsMASP1 had the highest similarity(76%)with Lates calcarifer,while it had the lowest similarity(42%)with Cricetulus griseus.CsMASP1 showed expression pattern in 11 healthy tissues(blood,brain,gill,ovary,heart,head kidney,intestine,liver,skin,spleen and trunk kidney)at different degrees.The relative expression level in liver tissue is the highest(185.4).After infection with Vibrio anguillarum,CsMASP1 was up-regulated in all six immune-related tissues(blood,gill,head kidney,intestine,liver and spleen).Among these tissues,the up-regulated expression level in spleen and liver are the most obvious.The relative expression in spleen and liver was 15.4 and 12.4respectively.The full-length cDNA of CsMASP2 is 1457 bp,the 5? UTR,3? UTR and CDS region is136 bp,406 bp,and 915 bp respectively.The CDS region encoded 304 amino acids.The predicted relative molecular weight and the theoretical isoelectric point of the CsMASP2 is34.4 kD and 5.83,respectivly.SMART domain prediction analysis showed that the first 18 amino acid residues of CsMASP2 were signal peptides,the 18-136 and 183-294 amino acid residues were two CUB domains,and the 137-180 amino acid residues were EGF-CA domain.CsMASP2 contains 6 exons and is located on chromosome 10.Homology analysis showed that CsMASP2 had a high similarity with MASP2 of Lates calcarifer(79.28%).CsMASP2 expressed in 11 kinds of healthy tissues(the same as above)at different level,and the highest expression was found in liver(230.4).After infected by pathogenic bacteria V.anguillarum the expression of CsMASP2 was up-regulated in all six immune-related tissues(the same as above),Among these tissues,the up-regulated expression level in spleen and head kidney are the most obvious,and the relative expression in head and kidney was 11.7,and in spleen was 17.0.Prokaryotic expression vector pET-32a-CsMASP2 was constructed to induce protein expression,The antimicrobial activity of recombinant protein CsMASP2(rCsMASP2)was analyzed.The antibacterial experiments showed that rCsMASP2 had obvious antibacterial activity against V.anguillarum,V.Parahemolyticus,Edwardsiella ictaluri,Shewanella putrefaciens,Escherichia coli and Staphylococcus aureus,and the antibacterial activity of V.Parahemolyticus was the most obvious.The concentration of rCsMASP2 was 0.01 mg/mL and the bacteriostasis rate was 35.6%.The concentration of rCsMASP2 was 0.05 mg/mL and the bacteriostasis rate was 87.9%.These results revealed that CsMASP2 plays important role in the immune response of half-smooth tongue sole.The full-length cDNA of CsCL-11 gene is 1314 bp,with a 199 bp 5?-UTR,a 275 bp3?-UTR,as well as a 840 bp CDS region which encoded 279 amino acids.The predicted relative molecular weight and the theoretical isoelectric point of the CsCL-11 is 30.0 kD and5.23,respectivly.SMART domain prediction analysis showed that CsCL-11 contained a Collagen domain(55-114 aa)and a CLECT domain(159-274 aa).CsCL-11 contains 7 exons and is located on chromosome 1.Homology comparison showed that CsCL-11 had the highest similarity(86.4%)with Lates calcarifer.CsCL-11 expressed in 11 kinds of healthy tissues(the same as above)at different level,and the highest expression was found in ovary(12000).After infected by pathogenic bacteria V.anguillarum,the expression of CsCL-11 was up-regulated in all six immune-related tissues(the same as above).Among these tissues,the up-regulated expression level in liver and intestine are the most obvious.The relative expression in liver and intestine was 25.5 and 25.3 respectively.Prokaryotic expression vector pET-22b-CsCL-11 was constructed to induce protein expression.The antimicrobial activity of recombinant protein CsCL-11(rCsCL-11)was analyzed.The antimicrobial experiments showed that rCsCL-11 had broad-spectrum antimicrobial activity.It had good antimicrobial activity against V.anguillarum,V.Parahemolyticus,Pseudomonas aeruginosa,Escherichia coli and Staphylococcus aureus,among which the bacteriostasis effect on V.parahaemolyticus was the most significant.The concentration of rCsCL-11 was 0.01 mg/mL and the bacteriostasis rate was 51%.The concentration of rCsCL-11 was 0.05 mg/mL and the bacteriostasis rate was 96%.The results indicated that CsCL-11 was involved in the immune response of the half-smooth tongue sole.In conclusion,the above results indicate that CsMASP1 gene,CsMASP2 gene and CsCL-11 gene play an important role in the immune response of half-smooth tongue sole,which lays a theoretical foundation for the study of complement lectin pathway related genesof half-smooth tongue sole.