Screening And Identification Of Recombinant Proteins As Stimulants In Mycobacterium Bovis Gamma Interferon Release Assay For Cattle

Bovine tuberculosis (bTB) which caused by Mycobacterium bovis (M. bovis) is considered a significant economical issue and an important health problem, and is deemed to be the one of the animal epidemic needed to inform by the Office International des Epizooties (OIE). Because cattle are the principal maintenance hosts among domestic animals, the disease presents a major trade barrier for animal-related products and livestock production, causing significant losses in rural economies worldwide. As a result, simple, rapid and accurate diagnostic assay for the prevention and control of this disease has a great significance.The interferon-gamma (IFN-y) assay is based on cell-mediated immunity (CMI) response, which depends on the response against purified protein derivative (PPD) tuberculin. Bovine PPD is a poor defined mix of proteins, which can explain the low specificity of the test and the variations in specificity and sensitivity from batch-to-batch. To overcome these issues, antigens specific to pathogenic mycobacteria including the M. tb and M. bovis are being studied. Taking ESAT6, CFP10and TB7.7(also known as Rv2654c) for example, because of their clear backgroud, easy preparation and quality control, they can improve the specificity of the detection.In this study, the fusion protein CFP10-ESAT6-TB7.7(hereinafter called CE-TB7.7) was expressed successfully by E.coli expression system and28fusion proteins were screened to evaluate their potential in IFN-γ release assay for the detection of bovine tuberculosis used as stimulants. 1. Prokaryotic expression, purification and identification of Mycobacterium tuberculosis CE-TB7.7fusion proteinThe fragments of lhp-esat6-linker and linker-TB7.7were amplified by PCR, recombinant plasmid pETT2a(+)-lhp-esat6-linker-Rv2654c was obtained and transformed into BL21(DE3). Results of SDS-PAGE showed that the fusion protein was expressed successfully with a relative molecular weight about45kDa after induced by IPTG, and purified by affinity chromatography assay from ultrasonic lysis supernatant. Western blot analysis showed that the rHis-CE-TB7.7fusion protein could react specifically to anti-ESAT6monoclonal antibody (MAb), anti-His MAb, anti-CFP10MAb and anti-TB7.7polyclonal antibodies respectively, which confirmed rHis-CE-TB7.7protein’s immunoreactivity.C57BL/6female mice aged6weeks were immunized subcutaneously after sufficient emulsification of rHis-CE-TB7.7and DDA adjuvant. After7days of second immunization, splenocytes were prepared from mice for the detection of cytokines and T cell proliferation, meanwhile, murine sera were prepared for IgG detection. Results of cytokine production level detected by sandwich ELISA showed that both IFN-y and IL-4produced by rHis-CE-TB7.7groups were significantly higher than the control groups (p<0.01), and also IFN-y secreting level was significantly higher than IL-4, which implied that the rHis-CE-TB7.7fusion protein could induce Thl type immune response. The significant T cell proliferation of rHis-CE-TB7.7group was observed by stimulated with CFP10, ESAT6and TB7.7. Test of IgG titers showed that specificity antibodies against to CFP10, ESAT6and TB7.7could be induced by the fusion protein, respectively. All these results suggested that these three components of rHis-CE-TB7.7had good biological activity.2. Screening of the stimulants for IFN-y release assay in bTB detection28different fusion proteins were screened to evaluate their potential usage as stimulator in IFN-γ release assay for the detection of bovine tuberculosis. Results from1542negative and1037positive samples tested showed that protein of CE, CE-TB7.7and MPT63displayed an advantaged positive correlation with PPDB-based IFN-γ release assay (Pearson’s r>0.7) compared to other25stimulants, then following by MPT64, MPT51, MPT83protein which also showed descending order potential to be stimulators (Pearson’s r>0.5). According to the results of stimulating the same samples with CE, CE-TB7.7and MPT63(n=72), these three proteins still showed a good correlation with traditional PPDB IFN-y release assay because Pearson’s r were up to0.9492,0.9284and0.8943, respectively. As for the others to stimulate the same samples, MPT51and MPT64showed a medium correlation (Pearson’s r were0.4605and0.4519) while MPT83had the lower with PPDB IFN-γ release assay (n=19). Therefore, protein of CE and CE-TB7.7displayed most and followed by MPT63, MPT51and MPT64obvious advantages as stimulants used in IFN-γ release assay for bTB detection.Furthermore, two group stimulants with different forms which including CE and CE-TB7.7as fusion form together with mixed form containing CFP10plus ESAT6, CE plus TB7.7and CFP10plus ESAT6plus TB7.7were also evaluated and the results showed high correlation coefficients between both fusion proteins and mixed ones with PPDB (Pearson’s r>0.9), which illustrated the equivalent effect of these two forms. However, it is seemed that mixed proteins form showed greater sensitivity and specificity than fusion proteins’because of the both higher positive and negative coincidence rate compared to PPDB in IFN-y release assay.