Identification And Functional Study Of The Immune-related Gene Pellino,and The Functional Analysis And Application Prospects Of Anti-lipopolysaccharide Factor(ALF)from Kuruma Prawn(marsupenaeus Japonicus)

Kuruma prawn,Marsupenaeus japonicus,which is the crucial species of farmed shrimp in the world,is of high economic value.However,with the continuous and rapid development of the shrimp cultivation industry of M.japonicus,there is a consequent occurrence of diseases such as white spot syndrome(WSS),which in turn seriously impact shrimp cultivation.The TLR/NF-κB innate and immune signaling pathway plays an important role in shrimp innate resistance to viral and bacterial infection.This research was focused on the study of anti-bacterial/anti-virus function and immune mechanism of the key regulatory gene of TLR/NF-κB signaling pathways,Pellino,and a downstream effect factor anti-lipopolysaccharide factorMjALF-D by making use of sequence and structure identification,tissue distribution,immune stimulation induction analysis,protein functional verification and optimization of LBD region transformation of antimicrobial peptide.Below are the key research results:1.Identification and functional study of Pellino gene in M.japonicusFirstly,the P gene from M was identified.The MjP m RNA sequence showed that has a length of 2139-bp and contains a 114-bp(1-114)5’-untranslated region(UTR)and a 726-bp(1414-2139)3’-UTR,and the ORF from positions 115 to 1413(1299-bp)encodes a 432-amino-acid protein.Secondly,the q RT-PCR assay showed the presence of MjPellino in all eight tested tissues,including hemocytes,muscle,hepatopancreas,eyestalks,stomach,heart,intestine and gill.It’s worth noting that the expression of gill was the highest.Thirdly,the transcript level of MjPellino was analyzed,and was highly present in both the gill and hemocytes after challenge with WSSV,Vibrio parahaemolyticus,Staphylococcus aureus,LPS,and Poly(I:C).Moreover,the function of MjPellino was further studied at the protein level.Results of the three-dimensional(3D)modele analysis,the protein docking analysis and a GST pull-down assay revealed that the MjPellino protein might be able to bind to the WSSV envelope protein VP26.The study also found that knockdown of MjPellino in vivo could significantly decrease the expression of MjALF-D and two other AMPs.These results showed that MjPellino is likely to play an important role in the immune response of the kuruma prawn.2.Identification of MjALF-D of M.japonicus and immune-stimulus analysisThis study identified and cloned a new ALF of group D from M.japonicus,MjALF-D.The full-length c DNA of MjALF-D was 756 bp,encoding 124 amino acids which contained a 26-residues signal peptide and an LPS-binding domain(LBD).It was discovered that the structure of MjALF-D contains three α-helices,fourβ-sheets and random coils.q RT-PCR analysis revealed that MjALF-D expression was primarily observed in the stomach.MjALF-D was generally upregulated in both the gill and stomach after the challenge by LPS,V.parahaemolyticus and WSSV.The results indicated that MjALF-D possessed the potential involvement in the innate immune response of M.japonicus.3.The optimization of induction expression and purification conditions of MjALF-D recombinant protein(rMjALF-D)The prokaryotic expression system of MjALF-D recombinant protein was successfully set up and the induced expression and purification process of MjALF-D recombinant protein were optimized.A large amount of MjALF-D recombinant protein was obtained after 6 hours of induction at the final IPTG concentration of 0.3m M at an induced temperature of 28℃.The purified rMjALF-D was confirmed by western blot and mass spectrum identification.4.Analysis of antibacterial activity and antibacterial mechanism of rMjALF-DThe antibacterial spectrum of recombinant protein was detected using the liquid antibacterial method,thereby indicating that rMjALF-D reacted against V.parahaemolyticus and other bacteria.In addition,bactericidal reaction depended on time and concentration.The antimicrobial mechanism of rMjALF-D was studied using the SEM,TEM and bacterial binding assay,and it was discovered that rMjALF-D has a potential of destroying the bacterial membrane,leading to cytoplasmic leakage.Furthermore,rMjALF-D showed distinct binding after direct incubation with bacteria had an effect on its protein expression.5.Detection of Mmodification design synthesis and activity detectionreaction of lipopolysaccharide binding domain with LBD short peptide.The physical and chemical properties of MjALF-D LBD area(LBD-ALF)were analyzed as well as the basis of the design of the corresponding three short peptides i.e.LBD transformation LBD-K,LBD-Q,LBD-S.The antibacterial reaction of these three short peptides were analyzed and evaluated using bacteriostasis and the results indicated increase in their antibacterial reaction,compared to LBD-ALF,especially the LBD-Q which was chosen for subsequent in vitro antibacterial reaction as well as the detection and analysis of in vivo and in vitro antibacterial and antiviral reaction.The results indicated that its antibacterial reaction was dependent on time and concentration.Moreover,LBD-Q had been shown to bind to bacterial genomic DNA,and when assayed by SEM and TEM,formed a cluster of bacteria wrapped by a plastic coating which ruptured and killed them.In addition,co-injection with LBD-Q and V.alginolyticus,S.aureus or WSSV in vivo could improve the survival of M.japonicus and reduced the WSSV virus replication,indicating that LBD-Q plays an important role in resistance to bacterial and WSSV in vivo infection.