Study On Screened In Vivo-induced Antigen And Biological Function Of Brucella

Objective:In order to further understand the pathogenic mechanism of Brucella,in vivo induction antigen technology(IVIAT)was used to screen specific in vivo induction antigen of Brucella,verify its immunogenicity,and preliminarily analyze its role in the survival of Brucella.It provides a new idea for the study of Brucella infection mechanism.Methods:(1)A genomic expression library of Brucella was established.Using the treated serum as a probe and the characteristics of IVIAT technology,the differentially induced antigen genes of 16M strain and S2 vaccine strain of Brucella were specifically screened in vivo.(2)DK63₄26 and DK63₁0 genes were amplified from 16M genome by PCR and linked to pMD19-T vector.The recombinant expression plasmid pET-28a-DK63₄26(DK63₁0)was constructed after digestion and transformed into E.coli BL21(DE3)and induced by IPTG.The reactivity of DK63₄26 and DK63₁0 was detected by Western Blot after SDS-PAGE identification and Western Blot test wascarried out with 18 sera of 16M sheep,S2 sheep and negative blood collected in the laboratory,and the positive rate of each protein was counted.(3)Using the genome of 16M strain of Brucella melitensis as template,the upstream and downstream homologous arms of DK63₄26 and DK63₁0 genes were fused by fusion PCR technology and linked to the cloning vector.The 16MDK63₄26 deleted strain of 16MDK63₄26 gene of Brucella melitensis constructed by gene recombination technology,and the parents of 16MDK63₁0 deleted strain of DK63₁0 gene are observed.The growth trends of 16M and 16MDK63₄26 and 16MDK63₁0 were observed.RAW264.7 model of macrophages was established to detect the viability of parent and deleted 16MDK63₄26 in cells,the release of IL-6 and TNF-a cytokines,and the LPS of deleted 16MDK63₁0 was extracted and detected by Western-blot.Results:(1)The 16M genome of Brucella was 3 417 183 bp in size.The coverage rate was 95%and the clone insertion rate was 97%.The fragment size was concentrated in 500-5000 bp and the library capacity was 2.5*104.Five specific 16M induced antigen genes of Brucella were screened in vivo.(2)PCR amplification of DK63₄26 and DK63₁0 genes showed bands at 1598 bp and 1257 bp.Induced expression and purified SDS-PAGE showed that DK63₄26 and DK63₁0 fusion proteins were located at 63KD and 53KD.DK63₄26 and DK63₁0 only reacted with serum of Brucella 16M sheep and Western Blot showed bands.Bioinformatics Analysis of DK63₄26 and DK63₁0 Genes Related to LPS Synthesis.(3)Using the genome of 16M strain of Brucella melitensis as template,the upstream and downstream homologous arms of DK63₄26 and DK63₁0 genes were fused by fusion PCR technology and linked to the cloning vector.The 16MDK63₄26 deleted strain of 16MDK63₄26 gene of Brucella melitensis constructed by gene recombination technology,and the parents of 16MDK63₁0 deleted strain of DK63₁0 gene are observed.The growth trends of 16M and 16MDK63₄26 and 16MDK63₁0 were observed.RAW264.7 model of macrophages was established to detect the viability of parent and deleted 16MDK63₄26 in cells,the release of IL-6 and TNF-a cytokines,and the LPS of deleted 16MDK63₁0 was extracted and detected by Western-blot.Conclusion:(1)Five specific 16M inducible antigen genes of Brucella were successfully screened by IVIAT.(2)DK63₄26 and DK63₁0 proteins were successfully cloned and expressed,which proved that they had good reactivity and specific expression,which was consistent with the results of IVIAT immune screening technology and could provide targets for the diagnosis of Brucella.(3)The deletion of DK63₄26(DK63₁0)gene can affect the intracellular viability of Brucella and the expression of related inflammatory factors,laying a theoretical foundation for the study of Brucella infection mechanism.