| Brucellosis is an important zoonotic infectious disease caused by Brucella,which causes obvious economic losses to cattle and sheep farming in China and poses a serious threat to the health of related practitioners.Accurate diagnosis is an important part of brucellosis prevention and control,but the infection is difficult to be detected in the early stages because there are no obvious signs of brucellosis after infection.Serological testing is the most common means of brucellosis diagnosis at home and abroad,but different testing methods have their own advantages and disadvantages in terms of ease of operation,sensitivity and specificity,and it is not yet possible to rely on one serological method alone to confirm the diagnosis of brucellosis.IFN-γ release test is a supplementary method recommended by WOAH for confirming the diagnosis of brucellosis,which has the characteristics of high sensitivity and good specificity,and there is an urgent need to establish the corresponding detection method in China.In this study,the sheep IFN-γ protein gene was ligated to the prokaryotic expression vector p Cold II,transformed into E.coli BL21(DE3),and then induced to be expressed after low temperature and IPTG.The fusion expression target protein gene CIFN-γ with m Cherry tag was ligated to the eukaryotic expression vector p Fast Bac HT B to construct a cellular expression system for Sf9 cells.The results of immunizing mice with the target proteins expressed by the 2 systems respectively and preparing anti-sheep IFN-γ monoclonal antibodies showed that the 2 expression systems constructed were able to express the target proteins effectively,the E.coli expression system was able to express sheep IFN-γprotein solubilized,and the purity of the target protein after purification by Ni-NTA column was above60%,and the sheep IFN-γ protein expressed by Sf9 cells was purified by Ni-NTA column and the purity reached above 70%.The target protein expressed by Sf9 cells was immunized in mice and five hybridoma cell lines that stably secreted anti-sheep IFN-γ monoclonal antibody were successfully screened,and the antibody isotype of four strains was identified as Ig G2 b and that of one strain was Ig M,among which the antibody potency of mouse ascites prepared by 4Z10 hybridoma fine was the highest at 1:32,000.Meanwhile,rabbit anti-sheep IFN-γ polyclonal antibody was prepared by immunizing rabbits with prokaryotic expression of sheep IFN-γ protein with a purified potency of1:64000.Using the prepared monoclonal antibody and rabbit anti-goat IFN-γ polyclonal antibody,the optimal action concentrations of the capture and detection antibodies were determined by the checkerboard method to optimize the ELISA coating and reaction conditions.The optimal Brucella abortus IFN-γ release stimulating agent was then screened and the method of in vitro IFN-γ release assay for brucellosis in sheep was refined.Finally,the established double antibody sandwich ELISA method was initially applied to the detection of sheep immunized with Brucella abortus S2 vaccine,and the results were compared with the Tiger Red plate agglutination test and the competition ELISA method.The main experimental results were as follows:(1)A sheep IFN-γ double sandwich ELISA assay was established using a 1:4000 dilution of multiple antibody for capture antibody and a 1:2000 dilution of HRP-labeled monoclonal antibody for detection antibody,with a minimum sheep IFN-γ concentration of 1.25 ng/m L.(2)Brucella abortus strain S2 hydrolysin is the best stimulant for sheep brucellosis IFN-γ in vitro release assay.(3)The established IFN-γ double antibody sandwich ELISA method for sheep brucellosis was applied to sheep immunized with S2 vaccine and showed the highest positive detection rate at the early stage of infection compared with Tiger Red plate agglutination test and competitive ELISA. |
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