Study On The MLVA Distribution,the Fusion Expression And Immunogenicity Of Recombinant Flagellin In Brucella

Objective:(1)To study the molecular epidemiological characteristics of Brucella isolated in multiple countries and regions in the world through multiple-locus variable-number tandem-repeat analysis(MLVA);(2)To explore the correlation between immune related factors interleukin 2(IL-2),interleukin 8(IL-8),tumor necrosis factor a(TNF-a)and brucellosis By detecting serum levels of IL-2,IL-8 and TNF-a in patients with brucellosis;(3)construct prokaryotic expression vectors containing the target genes FlgE,BLS-FlgE,FlgL and BLS-FlgL of the Brucella genome,express and purify the recombinant protein,And evaluate the diagnostic value of FlgE and BLS-FlgE protein in brucellosis.Methods:(1)Eleven variable-number tandem-repeat(VNTR)sites were selected,using BioNumerics software,using Unweighted Pair Group Method with Arithmetic Mean(UPGMA),for the period from 1953 to 2013 Brucella VNTR data isolated from 48 countries and regions in the world for cluster analysis,mapping phylogenetic tree and minimum spanning tree(MST),describing the epidemic distribution characteristics of strains;(2)ELISA Measure the levels of IL-2,IL-8 and TNF-a in serum of patients with brucellosis and normal people,and analyze the relationship between immune-related factors and brucellosis;(3)Obtain Brucella flagellin gene FlgE,FlgL and 2,4 Dioxotetrahydrodopteridine synthase gene(BLS)sequence,design primers,use PCR to amplify the target gene,connect with vector pET30 a,transform Ecoli BL21 competent cells,IPTG induce protein expression,identificate by SDS-PAGE and Western blot,protein purification using His-tagged protein purification kit.The purified FlgE flagellin and BLS-FlgE fusion protein were used to establish indirect Enzyme Linked Immunosorbent Assay(iELISA)to discriminate sera of positive patients from brucellosis and sera from healthy persons,and calculate the sensitivity and specificity of the iELISA method.Results:(1)The evolutionary relationship of the Brucella phylogenetic tree is basically consistent with the classic biotyping method,but the type 5 strain of pig species is far from the type 1,2,3 and 4 strains of other species;whale Brucella species is also divided into two parts,and has a far-reaching relationship.Cluster analysis of Brucella increased the lineages of East Africa,West Africa,South Africa and Asia.China is a brune-prone area,the main prevalent strain is Brucella ovis,followed by Brucella bovis,Brucella suis mainly appears in southern provinces of China,and Brucella canis is only found in animals.not in humans.(2)Serum IL-2 levels of patients with brucellosis were lower than normal people,while IL-8 and TNF-a levels were higher than normal people,the difference was statistically significant(P <0.05).Spearman correlation analysis showed a positive correlation between IL-8 and TNF-a(P <0.05)(3)FlgE flagellin,FlgL flagellin,BLS-FlgE fusion protein and BLS-FlgL fusion protein was cloned Successfully by optimizing expression Conditions.The target protein has a large amount of expression and immunoblotting results showed that it can be recognized by the serum of positive patients.The sensitivity and specificity of the iELISA method with FlgE protein were 70.83% and 41.67%,respectively,and the sensitivity and specificity of the iELISA method with BLS-FlgE protein were 68.75% and 52.08%.Conclusions:(1)Brucella has the characteristics of species phylogenetic tree,and has the specificity of separation time,region and host;(2)Brucellosis is related to the levels of immune cytokines IL-2,IL-8 and TNF-a.It is speculated that IL-8 and TNF-a have a synergistic effect.(3)Successful expression of Brucella FlgE flagellin,FlgL flagellin,BLS-FlgE fusion protein and BLS-FlgL fusion protein.FlgE flagellin and BLS-FlgE fusion protein have high sensitivity and low specificity as diagnostic antigens.It has early diagnosis value.