Prokaryotic Expression And Functional Analysis Of Antheraea Pernyi C-type Lectin ImmunlectinA

As invertebrates,insects do not possess the complement activation pathway of vertebrates,and T cells and B cells do not exist in their immune systems.With the in-depth study of insect immune mechanism,it is found that the innate immune system is the main means for insects to resist pathogenic microorganisms.The innate immune system of insects mainly relies on the recognition and combination of pattern-recognition receptors(PRRs)and pathogen-associated molecμLar patterns(PAMPs)in their bodies to play immune functions.C-type lectins(C-type lectins,CTL),namely Ca2+-dependent lectins,are one of the important members of PRRs.Like most PRRs,it activates the Toll receptor signaling pathway(Toll)and the immune deficiency signaling pathway(IMD)by recognizing PAMPs,releasing immune active substances such as antimicrobial peptides and antiviral factors.At the same time,it activates the prophenoloxidase cascade reaction to produce melanin,reactive oxides,etc.,which promotes the formation of cell melanin and cell nodules,and finally completes the immune defense against pathogenic microorganisms.At present,the research on CTL in higher animals has made important progress,but the research on CTL in insects is still in its infancy.In this paper,we designed and synthesized primers based on the conserved sequence of insect lectin genes,and successfully cloned the Antheraea pernyi CTL gene immunlectinA.The gene is 927 bp in length and encodes a total of 309 amino acids.The target gene was cloned into p ET-28 a prokaryotic expression vector,and the recombinant plasmid was constructed for heterologous expression in E.coli BL21(DE3).After affinity purification,prokaryotic expression of immunlectinA recombinant protein was obtained.The results of functional analysis showed that the recombinant protein of immunlectinA could induce agglutination of various pathogenic microorganisms including E.coli,B.cereus and Monilia albican.The results of the glucose coagulation inhibition test showed that after incubation with different carbohydrates such as glucose,mannose and maltose,the agglutination effect of the immunlectinA recombinant protein was inhibited.Consistent with this,similar results were obtained in the enzyme-linked immunosorbent assay to verify the binding ability of immunlectinA to various PAMPs.This suggests that a variety of carbohydrates and their derivatives are also a type of PAMPs on the surface of pathogenic microorganisms,which can act as immune target molecules to interact with CTL to induce immune responses.Furthermore,this paper found that lipopolysaccharide(LPS)and peptidoglycan(PGN),as the PAMPs with the most abundant surface content of pathogenic microorganisms,had the strongest binding ability to immunlectinA recombinant protein.The antibacterial experiment found that the immunlectinA recombinant protein could inhibit the growth of Bacillus methylotrophicus and P.aeruginosa,but had no obvious inhibitory effect on the growth of other pathogenic microorganisms in the experiment.The results of antiviral experiments showed that the recombinant protein of immunlectinA could not effectively inhibit the Antheraea pernyi nuclear polyhedrosis baculovirus(Ap NPV).To sum up,this paper successfully cloned and purified the Antheraea pernyi CTL immunlectinA recombinant protein in the prokaryotic expression system.The functional analysis results showed that immunlectinA has a certain antibacterial activity.These results provide a theoretical and experimental basis for insect lectin-related research.