Brucella Protein BMEI0340 Regulates The Expression Of Inflammatory Factors And Immunogenicity

Brucellosis caused by brucella is a natural epidemic infectious disease,which can cause a variety of animal and human infections,and the harm is severe.The pathogenicity of brucella mainly manifests as inflammatory damage caused by inflammatory factors.Among them,the type ? secretion system(T4SS)plays a vital role,but secreted proteins mediate the function of T4 SS.Earlier studies proved that BMEI0340 protein is a secreted protein of T4 SS.Through comparat?e analysis,it was found that the protein is homologous to the lipoprotein Pal,which regulates the expression of inflammatory factors in various bacteria,so it is inferred that the protein may be related to the regulation of inflammatory factor expression.Also,a large number of studies have shown that: lipoprotein Pal has good immunogenicity.In this study,it was experimentally determined whether the BMEI0340 secreted protein is involved in regulating the expression of inflammatory factors.Then its vital functional regions were identified,and the immunogenicity of the protein was studied.This research was carried out from the following two aspects,and the results are as follows:1.Determine whether the secreted protein BMEI0340 is involved in regulating the expression of inflammatory factors and identify its key functional areas.The BMEI0340 protein coding gene sequence was used to design primers,PCR amplify the BMEI0340 gene,construct a recombinant expression plasmid to transform competent cells,and induce expression of the protein.After the expression was verified by WB,the BMEI0340 protein was purified to obtain high-purity protein to stimulate mouse-der ? ed macrophages,and the expression level of inflammatory factors was determined.The results showed that BMEI0340 protein can up-regulate the expression of cytokine TNF-?,which is closely related to the occurrence and development of brucellosis.n order to determine the key functional regions that upregulate the expression of TNF-?,the BMEI0340 protein was truncated and expressed,and the cells were incubated again to determine theexpression level of cytokines.The results showed that the key functional region for up-regulating TNF-? expression was within the 70-129 amino acid range encoding BMEI0340 protein.It provides an important theory and research basis for the screening of therapeutic drugs for brucellosis and future prevention and treatment.2.Immunogenicity study of secreted protein BMEI0340The purified BMEI0340 protein was used to immunize mice.After immunization,the immune level of mice was detected.The antibody level was detected by ELISA.The results showed that: BMEI0340 protein is antigen-react?e and can cause a strong humoral immune response.MTT method was used to determine the level of cellular immunity in mice after protein immunization.The results showed that the protein can cause a certain degree of cellular immunity.It can be seen that the BMEI0340 protein has the potential to be developed as a subunit vaccine for brucellosis.