Cloning Of TLR13 Gene From Nibea Albiflora And Exploration Of Related Immune Mechanism

With the rapid development of aquaculture for Nibea albiflora and the expansion of its aquacultural scale,the disease problem that has gradually emerged,especially the disease infected by vibrios,causes the large-scale death of Nibea albiflora,becomes the key factor affecting the healthy and sustainable development of this species’ aquaculture,and has caused serious economic losses at the same time.Therefore,it is particularly important to elucidate the immunobiological characteristics and disease resistance mechanism of Nibea albiflora.The innate immunity mediated by the Toll-like receptor family is the first line of defense against disease in fish,and acts as a bridge between innate immunity and acquired immune response.As a poorly known member of the TLR family,TLR13 can participate in the immune and inflammatory responses of the body.Therefore,based on the current situation of Nibea albiflora being threatened by pathogens during aquaculture,the c DNA sequence of TLR13 gene in N.albiflora(named as NaTLR13,Gen Bank accession number:MT701899)was obtained.The expression of NaTLR13 and myeloid differentiation factor(named NaMyD88,Gen Bank accession number: MN384261.1,obtained in the previous experiment)in Nibea albiflora infected with V.parahaemolyticus,V.alginolyticus,and Poly(I:C)were detected.Eukaryotic expression vectors were constructed to explore the cell localization and co-localization of the two molecules;the prokaryotic expression systems of NaTLR13-LRR and NaTLR13-TIR were constructed,and the induced expression conditions of the recombinant proteins were optimized.After the recombinant proteins were purified,and the binding activities of NaTLR13-LRR and NaTLR13-TIR proteins with bacteria were detected and analyzed.The molecular mechanism of NaTLR13 mediated antipathogen infection was preliminarily explored from the above experiments.The main research results were as follows:1.The identification and analysis of NaTLR13.Based on the transcriptome data of Nibea albiflora,the full-length c DNA sequence of NaTLR13 was obtained by homologous cloning and RACE technology,which was 4210 bp,and the open reading frame(ORF)was2886 bp encoding 962 amino acid residues.The molecular weight of the protein was 110.37 k Da and the theoretical isoelectric point(p I)was 9.08.It contains 15 leucine-rich repeat sequences(LRRs),one Toll-IL-1 receptor domain(TIR),one LRR-CT terminal domain,two LRR-TYP domains and two transmembrane domains.Multi-sequence alignment and phylogenetic analysis indicated that Nibea albiflora TLR13 shared high similarity with Larimichthys crocea and Collichthys lucidus(88.79% and 87.02%,respectively)and branched into the same cluster,which suggested that had similar immune responses.2.The spatiotemporal expression of NaTLR13 and NaMyD88.The results of real-time quantitative PCR(RT-q PCR)showed that NaTLR13 and NaMyD88 m RNA were expressed in all the tissues examined,the highest m RNA expression of NaTLR13 was in the spleen,followed by the liver,kidney,gills,heart,intestine and muscle,and the stomach was the lowest(P < 0.05).The m RNA expression of NaMyD88 was also the highest in spleen,followed by liver,kidney,gill,intestine,stomach,muscle and heart(P < 0.05).After being infected by V.alginolyticus,V.parahaemolyticus and Poly(I:C),the expression of NaTLR13 in spleen increased firstly,reached the highest at 24 h,12 h and 12 h respectively(P < 0.05),then decreased over time and finally stabilized.As the control group the expression of NaTLR13 for PBS injection was maintained at a relatively stable level,and the highest expression in V.alginolyticus infection group was significantly higher than that in V parahaemolyticus and Poly(I:C)groups.The expression of NaMyD88 in spleen significantly up-regulated after being stimulated by V.alginolyticus,V.parahaemolyticus and Poly(I:C),and all peaked at 12 h(P < 0.05),then decreased with time.The above results suggested that NaTLR13 and NaMyD88 might play an important role in the immune response of the spleen in N.albiflora for infection.3.The expression,purification and antibacterial binding of recombinant proteins of NaTLR13-LRR and NaTLR13-TIR.The prokaryotic expression system of the recombinant protein was constructed,and the molecular weights of the obtained NaTLR13-LRR and NaTLR13-TIR proteins were 81.69 k Da and 47.58 k Da,respectively.The optimal expression conditions were at 37 ℃ with the concentration of IPTG of 0.5 mmol/L after the bacteria being cultured for 4 h.SDS-PAGE showed that the target protein existed in the supernatant after ultrasonic fragmentation.After being Purified by Ni-NAT Superflow resin kit,the purified renatured proteins were incubated with V.alginolyticus,V.parahaemolyticus and V.harveyi respectively for in vitro binding experiments.Western blot results implied that NaTLR13-TIR and NaTLR13-LRR could bind to vibrio sp.bacteria respectively,which indicated that NaTLR13 had the ability to identify bacteria..4.Subcellular localization analysis and the co-expression of NaTLR13 and NaMyD88.The p EGFP-NaTLR13 and p Ds RED-NaMyD88 fusion vectors were constructed,respectively.The detection showed that NaTLR13-EGFP exhibited non-uniform dotted signal distribution at the cytoplasm,and NaMyD88-Ds RED was detected evenly distributed in the cytoplasm.The co-expression positions of the two were consistent,showing an uneven dot-like signal distribution in the cytoplasm.Based on the above results,it can be speculated that there may be an interaction relationship between NaMyD88 and NaTLR13.In this study,the reaction mechanism and antibacterial activity of NaTLR13 in the body’s innate immunity were preliminarily explored in Nibea albiflora,which proved that there might be a TLR-NF-κB innate immune signaling pathway depending on MyD88 in N.albiflora.Therefore,the results of this experiment would allow a reference for studying the functional mechanism of natural immune molecules,and at the same time,it would provide basic data for the study on the immune function of the Nibea albiflora and a theoretical reference for the scientific prevention and control of economic fish diseases.