Functional Analysis Of The Intelectin (cITLN) Of Common Carp (Cyprinus Carpio L.)

Innate immunity is one of the most original defense and protection mechanisms to resist the invasion of foreign pathogens in vertebrates and invertebrates.It works by pattern recognition receptors(PRRs)to recognize specific carbohydrates on the surface of microorganisms.In the immune system,pattern recognition receptors play an important role in the immune response of invertebrates.Various forms of PRRS can recognize specific components on the surface of pathogen cells,which are called pathogen associated molecular patterns(PAMPs).PRR interacts with PAMP and triggers a series of innate immune responses.Lectins are a major subgroup of PRRs that can identify specific complex carbohydrates.In recent years,research has found that intestinal lectin(Intelectin,ITLN)is a new type of X-lectin,which was first discovered in Xenopus cortex granules(XL35).Later studies found that Paneth cells are abundantly expressed and named "intelectin".Since then,the presence of intelectin has been successively found in mammals,fish,amphibians,cephalochordatas,urochordatas,deuterostomias and invertebrates.In this experiment,the most common freshwater fish carp in China served as the research material.Using bioinformatics-related technologies,the c DNA encoding the intestinal lectin was retrieved from the carp genome homologous sequence and named cITLN.Analysis of the carp cITLN gene revealed that the cITLN sequence is comprised of 296 amino acids,about 63% similar to humans.The main sequence of cITLN is composed of the N-terminal fibrinogen domain(FRe D)and the C-terminal intelectin domain.Related calcium ion binding sites have a calcium ion-dependent agglutination effect on both Gram-positive and Gram-negative bacteria.The Real-time PCR method was used to extract RNA from normal and Aeromonas hydrophila stimulated tissues of carp to explore the expression and tissue distribution of cITLN at m RNA level.The results showed that cITLN was widely expressed in various tissues of carp,and highly expressed in the intestine and spleen.After the stimulation of Aeromonas hydrophila,the intestinal immune response to bacteria was the most rapid,up-regulated at 3h,while the immune response of spleen,liver and head kidney tissue to bacteria was slow,up-regulated at 6h.The expression of cITLN was up-regulated after stimulation.We speculated that cITLN might be involved in the immune defense caused by bacterial infection in carp and play a role in the innate immunity of carp.In order to further verify the function of carp cITLN,we induced the expression of cITLN in vitro.First,we constructed the expression vector p ET-29b-cITLN and successfully expressed the cITLN protein.The in vitro function of cITLN was verified in bacterial agglutination experiments,bacterial binding experiments,and polysaccharide binding experiments.Bacterial agglutination experiments showed that the recombinant cITLN was calcium-dependent on the agglutination of bacteria,and its ability to agglutinate E.coli was stronger than that of S.aureus.Bacterial binding experiments showed that: cITLN has a stronger ability to bind Gram-positive bacteria than Gram-negative bacteria.There is a significant difference between adding calcium ions and not adding calcium ions.Calcium ions can increase the degree of binding of recombinant proteins and bacteria.In addition,the binding of cITLN to polysaccharides was analyzed by ELISA technology.The results showed that the affinity of recombinant cITLN to peptidoglycan(PGN)was higher than that of lipopolysaccharide(LPS),and calcium ion also promoted binding.The above results shows that carp cITLN can recognize bacterial cell wall-specific components in the animal's innate immunity,thereby causing bacterial agglutination and participating in immune defense.Through the bioinformatics analysis of carp cITLN,carp bacteria stimulus model was initially constructed,the expression pattern of cITLN in carp,and the mechanism of natural immune defense processes such as bacterial agglutination and binding were analyzed,which will help deepen our participation in the intelectin gene family.The understanding of the immune defense mechanism caused by bacterial infection provides a reference and basis for our further comprehensive understanding of the role of carp cITLN in natural immunity.