High-throughput Screening And Identification Of Brucella Diagnostic Proteins

Brucellosis(brucellosis)is a worldwide zoonotic disease caused by Brucella,which brings huge harm to the development of animal husbandry and people's health.The methods listed in the national standard for the diagnosis of brucellosis are designed based on LPS antigens or whole cell proteins,and cannot distinguish the antibody reaction caused by vaccination and wild strain infection,which is not conducive to the removal of brucellosis.With the development of proteomics,it is possible to use high-throughput technology to screen protein molecules with diagnostic functions.In this study,immunoprecipitation(IP)was used to isolate serum reactive proteins from Brucella M28 and M5-90 inoculated sheep for 180 days,and mass spectrometry analysis was performed using high-throughput Label-free technology.The results showed that a total of 1940 effective proteins were detected.According to the criterion that the fold difference was greater than 1.5 and the serum persisted for 6 months,71 differentially expressed proteins were screened.M28 was up-regulated by 14 and down-regulated by 23 compared with M5-90 Only 19 were expressed in the M28 group and only 15 were expressed in the M5-90.The protein B0715,A0248,A0251,A0249,B0713,A0253 and A0225 were up-regulated 46.0,19.6,10.7,8.8,8.8,8.6 and 7.5 times in the M28 group compared with the M5-90 group within 6 months after Brucella inoculation.The results show that: 1.There is a cross-reaction between the tag proteins in the pMAL and pET32 a systems,but no cross-reaction in pET28 a.2.The initial identification of protein A0253 has the diagnostic function,and also has the function of identifying the infection of the virulent strain(M28)and the vaccine strain(M5-90).In order to detect the antibody reactivity of seven Brucella proteins with significant fold differences and whether the tag protein affects the antibody reactivity of the differential proteins,this paper selected three prokaryotic systems to express the differential proteins and combined them with iELISA to screen their functions as diagnostic proteins.sRNA plays an important role in regulating bacterial metabolism,virulence determination and environmental adaptation.In the previous research in this laboratory,the Brucella virulent strain M28 was taken as the object.Several new sRNAs were sequenced,screened and verified by transcriptome,and the virulence-reducing effect of Bmsr1 was proved one.In this study,homologous recombination was used to construct a Brucella Bmsr10 deletion strain,and the effect of Bmsr10 deletion on the virulence of Brucella M28 was analyzed through the detection of Brucella proliferative ability in vivo and in vitro,The results showed that the lack of Bmsr10 did not affect the growth of Brucella M28 on liquid-based TSB.Cell infection experiments showed that the intracellular proliferation ability of M28?Bmsr10 significantly decreased after 48 hours of infection with macrophages(p<0.05).The results of mouse experiments showed that the number of spleens of M28?Bmsr10 was significantly lower than that of M28 at 1,3,5 and 7 weeks after infection(p<0.01);the spleen weight of mice after 1,3 and 5 weeks of infection Both decreased significantly(p<0.001).It proved that the deletion of Bmsr10 significantly reduced the virulence of Brucella M28.In summary,this study analyzed 71 differentially expressed proteins in Brucella M28 and M5-90 by analyzing the differential proteins of M28 and M5-90 bound to Brucella sera,using iELISA A preliminary study was conducted on the reactivity of differential protein antibodies.The virulence effect of sRNABmsr10 on Brucella M28 was also studied.The results showed that: 1.Protein A0253 has the diagnostic function,and also has the function of identifying the infection of virulent strain(M28)and vaccine strain(M5-90).2.The deletion of sRNABmsr10 has no effect on the growth of Brucella maltophilia in vitro,but significantly reduces its proliferation ability in macrophages and mice.This study laid the foundation for the construction of a natural infection and vaccine immunodiagnostic reagent and target vaccine to distinguish between brucellosis and the identification of Brucella.