| Brucellosis is a human and livestock chronic infectious disease caused by Brucella,the Brucellosis is widespread in most parts of the world,causing serious economic losses to the world of animal husbandry.Therefore,the study of the pathogenic mechanism of Brucella infection,to screening the target and the efficient and safe vaccine development of Brucella and its cells are the key problems to be solved.Brucella virulence factors including cell adhesion virulence factors,intracellular transport virulence factors,evade and resist the host immune response virulence factors.Brucella outer membrane protein OMP10 is a key outer membrane protein that interacts with macrophages,but the specific interacts site is unknown.The aim of this study was to screen the interaction proteins on the macrophages that bind to the outer membrane protein OMP10,the mechanism of the Brucella OMP10 in the intracellular survival process of Brucella is revealed,and provides scientific basis for the development of the treatment of Brucellosis.Objects: In our study,to screen and identify the interacting proteins of Brucella OMP10 protein in mouse RAW264.7,the B.abortus 2308 OMP10 protein was selected as the research object.(1)Prokaryotic expression and purification of Brucella OMP10 protein and bioinformatic analysis the Brucella OMP10.(2)Screening of polypeptides specific binding to Brucella OMP10 protein from the M13 phage display library.(3)The polypeptide transfection into mouse RAW264.7 to verify the intracellular survival of Brucella.(4)Surface Plasmon Resonance technique in screening the interaction protein of Brucella OMP10 protein in mouse RAW264.7.(5)To analyze the expression of HspA8 and Rac1 gene and intracellular viability of Brucella,and determine the RNAi of HspA8 and Rac1 on intracellular survival.(6)To screen the interaction domain of Brucella OMP10 protein and Hsp A8 and Rac1 proteins,and to investigate the mechanism of OMP10 in intracellular survival of Brucella.Methods:(1)We used the genetic engineering methods to construct the prokaryotic expression vector pET30a-omp10 of Brucella OMP10 protein,expressed and purified by Escherichia coli DE3,SDS-PAGE analysis and Western blot identification;using IDEB online,bioinformatics analysis of Brucella OMP10 protein.(2)Using M13 phage,Brucella OMP10 protein as antigen,the peptide was screened from the Ph.D.?-C7 C Phage Display Peptide Library Kit through three rounds of biological panning,and the specific binding peptide of OMP 10 protein was screened.The interaction of Brucella OMP10 protein was screened by Blast homologous comparison method.(3)Synthesis of peptides by solid phase synthesis and the establishment of Brucella infected macrophage model to verify the effect of polypeptide on intracellular survival of Brucella.(4)Using Brucella OMP10 protein as bait and mouse RAW264.7 lysate as library,the Surface Plasmon Resonance technique was used to screen the proteins interacting with Brucella OMP10 protein.(5)According to the literature analysis,selection of Rac1 protein and Hsp A8 protein as an interacting protein of Brucella OMP10 protein;Brucella infected mouse RAW264.7 and detected the Rac1 and HspA8 gene transcription by qRT-PCR;RNA interference mouse RAW264.7 HspA8 and Rac1 gene,detection the intracellular survival of Brucella.(6)According to the HspA8 and Rac1 protein domain and divided into different truncated and connected to the pGADT7-vector,Brucella omp10 gene was connected to the pGBKT7-vector and then transformed into Y187 or Y2HGold;through the detection of their self activation and toxicity testing,pGBKT7-omp10 self activation activity and toxicity of OMP10 to the Y2 HGold yeast;the positive and negative control were constructed according to Clontech,as the positive and negative control group of yeast two hybrid;recombinant yeast Y187 and Y2 HGold of different truncated fusion,screening HspA8 and Rac1 and different truncated omp10-Y2 HGold recombinant yeast interactions,determine the interaction domain.Results:(1)The prokaryotic expression vector pET-30a-omp10 was successfully constructed and the 11.8 kDa OMP10 protein was obtained;OMP10 protein has certain immunogenicity;IDEB results online shows that the OMP10 protein has four advantages respectively is 20-72,92-97,103-103 and 107-118 amino acids.The advantage of section β-turns in the 20-60 amino acids,the advantage in section antigen index 100 th amino acid,hydrophilic segment advantage in 63-69,scores of 6.586,the advantage of flexibility in 21-30.(2)Sixty one polypeptide were screened from M13 phage display peptide library;through Clustalx software analysis of four representative peptides(NK-7,KL-7,NS-7,ST-7);Blast NK-7 and KL-7 of the interaction with the OMP10 protein for Brucella with the source of heat shock protein 71(HspA8).(3)Four peptide synthesis after transfection of mouse macrophage,the NK-7 and KL-7 polypeptidesof plays a role in blocking early stages of the survival of Brucella infection,the NS-7 and ST-7 had no effect on Brucella intracellular survival.(4)Surface Plasmon Resonance screening,Brucella OMP10 protein successfully captured 4261 polypeptides,identified 503 potential interacting proteins;through the analysis of bioinformatics screening to protein with 5 related functions,Eno1,HspA8,Ndrg1,S100-A6,Rac1.(5)According to HspA8 and Rac1 gene fragments in the design and interference screening to determine Hsp A8-mus-480 and Rac1-mus-414 for the best interference fragment;Brucella infecting mouse RAW264.7,expression of Rac1 and HspA8 gene increased gradually,reached the highest point at 4h and 8h respectively,then decreased in expression after infection;At 2h,4h,8h,12 h and 24 h the Brucella CFU count was significantly lower than that of the control group in mouse RAW264.7(P<0.05).(6)Constructed the yeast bait bacteria and yeast segments was detected bacteria bait no self activation activity of yeast and non-toxic;the yeast two hybrid screening results showed that pGADT7-HspA8(385-542)interaction with pGBKT7-omp10,pGADT7-Rac1(1-197)interaction with p GBKT7-omp10.Conclusion: T he results showed that the interaction between Hsp A8 and Rac1 protein with Brucella OMP10 during the Brucella infection mouse RAW264.7 process.OMP10 protein interaction with HspA8 385-542 subdomain,and interaction with the Rac1(1-197)protein,HspA8 and Rac1 is one of the most important sites and binding proteins of Brucella OMP10 in mouse RAW264.7. |
