ABSTRACT
This study was aimed to develop a sandwich ELISA kit for the diagnosis of bovine tuberculosis.And it was applied and evaluated in the quarantine of bovine tuberculosis.We established a bovine IFN-γ release method in vitro and developing three batches of kits.The sensitivity,repeatability and retention period of the kit were all evaluated.Totally 961 serum samples were tested using the developed sandwich ELISA kit tuberculin skin test and a commercial ELISA kit.Our results showed that the detection limit of this ELISA was 8.21 mg/mL.The repeatability tests showed good reproducibility in the intraassay and inter-assay.At the same time,the retention period of the kit was more than 12 months.Compared with the tuberculin skin test,the positive coincidence rate was 70.59％ and the negative coincidence rate was 99.20％,while the total coincidence rate was 98.44％.And compared with the BOVIGAMTM kit,the positive coincidence rate was 91.30％ and the negative coincidence rate was 99.78％,while the total coincidence rate reached 99.58％.At the same time,the sensitivity and specificity of the sandwich kit were 85.00％ and 100％,respectively.We established a bovine IFN-γ release method in vitro and developing corresponding kits successfully have a good application prospect.
ABSTRACT
Flagellin can be expressed in monomeric or polymeric form based on assembly. The difference of these two forms of flagellin is less studied. In this experiment, recombinant plasmid pET-fliC/M2e2 was transferred into Escherichia coli BL21(DE3) and Salmonella SL5928 to express chimeric flagellin, mfliC/M and pfliC/M, respectively, and then their assembly characteristics were analyzed. Sodium dodecyl sulfate polyacrylamide gel electrophoresis results indicated that the two recombinant bacteria could successfully express chimeric flagellin. The transmission electronic microscope observation showed that no flagella were found on the surface of recombinant E. coli, whereas it was found for recombinant Salmonella. After purification, distinct circular dichroism spectra between them were found and pfliC/M showed the similar structure as wild-type flagellin, but not for mfliC/M. The dynamic light scattering assay also indicated that the polymerization of mfliC/M was much lower than that for pfliC/M. Three hours after transfection into mouse peritoneal macrophages, both could induce interleukin 1β secretion, but mfliC/M is stronger than pfliC/M. These data will be helpful for the selection of expression form of flagellin.
ABSTRACT
We established an ELISPOT for bovine interferon-gamma (BoIFN-γ), and applied it in the diagnosis of bovine tuberculosis (bTB). Monoclonal antibodies that can bind with native BoIFN-γ were screened as the coating antibody and detecting antibody. After optimization of detecting conditions including coating antibody concentration, cell number, and detecting antibody concentration, the ELISPOT assay was established. Peripheral mononuclear cells (PBMCs) isolated from 30 cows were co-cultured with PPD, and detected with the ELISPOT assay. The optimal conditions of ELISPOT assay were 2.5 μg/mL coating antibody 2G5, 2.5 x 10(5) cells/well, and 1 μg/mL detecting antibody Bio-5E11. In these 30 cows tested both with the ELISPOT assay and the BOVIGAM kit, 11 cows were proved to be positive in ELISOPT assay with the sensitivity of 78.6%, and 12 cows were proved to be negative in ELISOPT assay with the specificity of 75%. The ELISPOT assay for BoIFN-γ could be used to detect bTB efficiently and it might be an alternative method for the diagnosis of bTB.
Subject(s)
Animals , Cattle , Female , Antibodies, Monoclonal , Enzyme-Linked Immunospot Assay , Interferon-gamma , Sensitivity and Specificity , Tuberculosis, Bovine , DiagnosisABSTRACT
Bovine interferon-gamma (BoIFN-gamma) gene was amplified by reverse transcription polymerase chain reaction (RT-PCR) from total RNA of bovine spleen lymphocytes stimulated with ConA. The products of RT-PCR were cloned into pVAX1 vector, positive recombinant clone was identified by restriction enzyme digestion and sequencing. The recombinant plasmid pVAX1-BolFN-gamma was transfected into COS-7 cells mediated by lipofectine, indirect immunofluorescent assay analysis confirmed that rBoIFN-gamma was expressed in COS-7 cells. BoIFN-gamma gene (without signal peptide) was cloned into pET-30a(+) and pGEX-6p-1 vector, and transformed into the Escherichia coli cells. After optimizing the induction condition, SDS-PAGE analysis showed that the expression products were all found in soluble form and had a molecular weight of 23 kDa and 43 kDa respectively. BoLFN-gamma precursor gene (with signal peptide) was cloned into transfer vector pFastBac 1, and transformated into DH10Bac E. coli cells. By site-specific transposition, BoIFN-gamma gene was integrated into shuttle vector Bacmid, and transfected into the Sf9 insect cells mediated by lipofectine to produce recombinant baculovirus. Indirect immunofluorescent assay analysis confirmed that rBac-BoLFN-gamma was expressed successfully in Baculovirus vector system. The antiviral activities of rHis-BoIFN-gamma, rGST-BoIFN-gamma and rBac-BoIFN-gamma were up to 8.389 x10(7) U/mg, 6.554 x10(5) U/mg and 4.096 x 10(4) U/mL respectively, which were analyzed in MDBK/VSV system. A sandwich ELISA was established using monoclonal antibodies 3E6 and 5G4, which can detect BoIFN-gamma in quantity and provide a useful method for the clinical practice and research of BolFN-gamma.