Cell Receptor Identicifation And Nucleic Acid Vaccine Preparation Of Brucella BMEI0340

Brucellosis(brucellosis)is a serious chronic zoonotic disease caused by bacteria of the genus Brucella,which seriously threatens the development of breeding industry and human health.The type IV secretion system(T4SS)is an important virulence factor of Brucella that transports effector proteins into host cells,promoting bacterial infection and causing host pathological damage.Through previous studies,BMEI0340 was found to be a T4 SS effector protein,and after recombinant expression of BMEI0340,it was found to up-regulate TNF-α expression in macrophages.TNF-α is an important inflammatory factor that plays an important role in Brucella intracellular survival and pathogenic effects.Therefore,exploring the molecular mechanism by which the effector protein BMEI0340 upregulates TNF-a expression has an important promoting effect on understanding the infection and immune mechanism of Brucella.In this study,the cell receptor types of BMEI0340 were analyzed and identified and the nucleic acid vaccine of BMEI0340 was prepared,which laid a foundation for the research of brucellosis prevention and control techniques.The research contents are as follows:1.To analyze the type of PRRs that recognize the BMEI0340 protein.The recombinant vector pET32a-BMEI0340 was constructed,induced to express and purify,and the recombinant protein was obtained,emulsified and immunized to BALB/C mice to prepare murine polyclonal antibodies;murine macrophages RAW264.7 were incubated with endotoxin-depleted recombinant protein to determine the changes in the expression levels of cell receptors.The results showed that when the concentration of BMEI0340 protein increased,the relative expression values of receptors Nod1,TLR1,TLR5,TLR8,TLR9 and TLR10 increased,and the type of PRRs recognizing BMEI0340 protein was determined.The results lay an important foundation for the study of the pathogenic mechanism of Brucella.2.Preparation of Brucella BMEI0340 nucleic acid vaccine and evaluation of immune effect.The eukaryotic recombinant expression plasmid pcDNA3.1-BMEI0340 was constructed and transfected into 293-T cells,and protein expression was verified by indirect immunofluorescence and Western blot to immunize BALB/C mice.Lymphocyte transformation test and serum antibody titer were performed on the mice to detect the cellular and humoral immune levels.The results showed that the lymphocyte proliferation ability and serum antibody level of mice continued to increase after immunization,indicating that the eukaryotic recombinant expression plasmid can induce a certain degree of humoral and cellular immunity in the body and has the potential to be developed as a nucleic acid vaccine.In summary,this study validated the PRRs types recognizing macrophages under BMEI0340 protein-stimulated conditions and constructed the recombinant plasmid pc DNA3.1-BMEI0340 to prepare nucleic acid vaccines,indicating its potential for development as nucleic acid vaccines.