| Bovine tuberculosis is a relevant zoonosis, which caused by Mycobacterium bovis. It can spread to humans through inhalation of infectious droplet nuclei and by ingestion of raw milk. Prevention and complete eradication of TB require sensitive and effective diagnosis method. The skin test and IFN-γrelease assay with the stimuli of PPD which are wildly used are lack of specificity. Recently, scientists are focus on searching stimuli which can substitute for PPD. The RD (the regions of difference) genes are difference by compare the genome of pathogenic and nonpathogenic mycobacteria, Therefore, the proteins of RD zone are expected to replace PPD to distinguish pathogenic and nonpathogenic mycobacteria mycobacterial infection.TB10.4 is the RD Region protein of Mycobacterium bovis, We designed two pairs of primers. PCR-F1/R1 contained the initial codon and stop codon, while PCR-F2/R2 did not contain the initial codon and stop codon. The gene TB10.4 was amplified from M. bovis strain ValleeⅢby PCR technique. PCR product was cloned into pET-32a(+) plasmid and pET-28a(+) plasmid, respectively. Then, we got three recombinant-plasmids as follows: pET-32-TB10.4-1, pET-32-TB10.4-2 and pET-28-TB10.4-3. After transformation, expression and purification of the recombinants, we successfully obtained the recombinant-proteins rTB10.4-1, TB10.4-2 and rTB10.4-3., which were 30ku,30ku and 12ku as their theoretical value respectively.In this study, the T-cell activity of these three recombinants were detected by IFN-γrelease assay, Real-Time PCR and guinea pig skin test. 1) We screened three M. bovis-infected cattle and three heath cattle according to TST and IFN-γrelease assay (BOVIGAM). Then, collected whole blood through jugular vein into a blood collection tube containing heparin, dispense 750μL heparinised blood from each animal into wells of a 48-well tissue cultue tray. Add 50μL of either rTB10.4-1, TB10.4-2, rTB10.4-3, 32a, rCE or PPD-B to appropriate wells containing the blood. After incubated for 20 h at 37℃, collected the plasma and detected the quatity of IFN-γrelease using Mycobacterium bovis Gamma Interferon Test Kit For Cattle. 2) Separated bovine peripheral blood lymphocytes (PBMC) from heparinized blood of each cattle, Added 50μL of either rTB10.4-1, TB10.4-2, rTB10.4-3, 32a, rCE or PPD-B and incubated for 6 h at 37℃. Extracted total RNA of stimulated PBMC using TRIZOL,β-actin gene as reference, PBS- stimulated PBMC as a control sample to detect the quatity of IFN-γmRNA using Real-Time PCR method.3) guinea pigs were infected with inactivated M.bovis. Used recobinant rTB10.4-1, rTB10.4-2, rTB10.4-3, rCE, protein Tag 32a and PPD-B, respectively, as stimulus for skin test; or equimolarly mixed the three recombinants with rCE, respectivily as stimulus for skin test. The diameter of lesions were measured at stimulated of 24 h, 48 h and 72 h. The results showed that the T cells activity of these recombinant-proteins were very well, so these proteins could be a candidate antigen for rCE in IFN-γrelease test; when the recombinant proteins were used s as stimuls in TST, the results showed that the single TB10.4 had no significant reaction, while it mixed with rCE, the lesions can be seen at 24 h, and the diameter of lesions were similar to PPD-B stimulated, indicating that the proteins mixture can be used as stimuli in TST. The B cell activity of the recombinans were detected by Western blot and indirect ELISA method, and the results showed that all of the proteins had B cell activity, but rTB10.4-1 and rTB10.4-2 showed stronger activity than rTB10.4-3.In summary, the TB10.4 recombinant protein expression in this study has a good T-cell activity and B cell activity, The successful expression and purification of TB10.4 recombinant protein has laid a foundation for the subsequent establishment of specific, high sensitive and reproducible diagnosis kit for bovine tuberculosis.
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