Development Of IELISA For Detection And Application Of Brucella Abortous Virb8

Brucellosis is a zoonotic disease which is caused by Brucella. It obstacls livestockdevelopoment and is harmful to the human health. The disease is characterized by abortionand fever. Brucellosis is a worldwide public health problem, it is hard to cure completelywhen infected. The monitoring results showed that the situation of disease had been bad inrecent years, so prevention and detection must be strengthened.In order to explore the possibility of Virb8iELISA for practice,Brucella Virb8wasselected to construct a prokaryotic expression cloning vectors. When the recombinant proteinwas purified, an iELISA which used the purified recombinant protein as coating antigen wasestablished for detecting antibodies to Brucella abortus. Serum samples were also tested bythe RBPT as reference.According to the sequence of Brucella Virb8gene(Gen Bank:AF226278.1), one pair ofspecific primer was designed. BamHI restriction site in the upstream was introduced inprimers and also HindⅢ restriction site in the downstream. Using the method of boiling,Brucella abortus vaccine strain S19genomic DNA was extracted. The Virb8gene fragmentwas amplified using the total genomic DNA as template, and the size is720bp. The amplifiedgene fragment was cloned into pEASY-T3, then transformed into DH5α to construct thecloned carrier pEASY-T3-Virb8. The recombinant plasmid and the pET-32a were doubledigested with BamHI and HindⅢ. The target gene fragment was ligated into the expressionvector by using T4ligase, then transformed into DH5a, and constructed the recombinantexpression plasmid pET-32a-Virb8. After sequencing, the restructuring expression plasmidwas transformed into E.coli BL21(DE3), then expressed by1mM/L IPTG, identified bySDS-PAGE and Western-blot. The recombinant protein was purified by the Ni-NTA Spin Kit.When the recombinant protein was purified, an iELISA which used the purified recombinantprotein as coating antigen was established for detecting235serum samples. During thedevelopoment, sunch as the optimal coating condition of recombinant protein and the optimal dilution of peroxidise were determined. Serum samples were also tested by the RBPT asreference.In this sdudy, Brucella Virb8was successfully cloned and high purified protein wasobtained. The iELSIA established was stable and sensitive.The coincidence of iELISA withRBPT was97.02%. The iELISA could be used as a method for serological diagnosis ofbrucellosis and could be convenient to practice.