Development And Identification Of Monoclonal Antibodies Against BoIL-2 And Ag85A Protein Of Mycobacterium Bovis

Bovine tuberculosis (bTB) is mainly caused by Mycobacterium bovis (M. bovis) which poses an infectious risk to cattles and human. After infected with M. bovis, the cattle can mainly produce Thl-type immune responses, and interleukin-2 is one of the main cytokines produced by Thl-type immune response. Bovine interleukin-2 (BoIL-2) is a kind of cytokine with multiple biological properties, which was mainly produced by stimulation of the specific antigen or mitogen stimulation it plays an important regulatory role in the immune system. Ag85 protein complex is a major antigen secreted by M. Tuberculosis, which is mainly composed of three kinds secreted proteins, Ag85A, Ag85B and Ag85C. M. tuberculosis Ag85A is a kind of protein with high secretion, some studies have shown that Ag85A can stimulate the body to produce Thl-type cellular immune responses, induce the secretion of IFN-y, TNF-a, IL-2 and other cytokines. Therefore, in this study, we prepared monoclonal antibodies (mAbs) against BoIL-2 and Ag85A respectivly, which provide an important biological material for the prevention and diagnosis of bTB.1. Prokaryotic expression of BoIL-2 and development of mAbs against BoIL-2The BoIL-2 gene was amplified by RT-PCR with the template of total RNA isolated from ConA-stimulated peripheral blood lymphocytes of healthy cows. And then the gene was inserted into prokaryotic expression vector pET-30a(+) and pGEX-6p-l respectively. Then the recombinant plasmids were transformed into BL21(DE3) and BL21 respectively. After induced by IPTG, the results of SDS-PAGE showed that the size of bands were 21 kDa and 41 kDa, which was consistent with the expected size. The Western Blot revealed that the recombinant proteins had good immunoreactivity. BALB/c mice were immuned with the purified protein rHis-BoIL-2. After three times of immunizations, the cell fusion was proceeded with the lymphocyte hybridoma technology. Indirect ELISA was used to screen positive cell clones with the purified protein rGST-BoIL-2 as detecting antigen. After two times of subclones, we obtained 11 hybridoma cell lines secreting mAbs against BoIL-2, which were named 1A3,1D3, 2G7,3C10,3D8,5D10,5F11,5G2,5G6,6D7 and 6E5 respectively. The subtype identification results showed that six mAbs were IgG1, besides 1A3 and 6E5 were IgG2b,2G7 was IgG2a, 3C10 and 5G2 were IgG3. The indirect ELISA results showed that, the cell supernatant titers of mAbs were higher than 1.0×104, besides 3C10 was 1:2560,2G7 and 5G2 were 1:5120, the ascite titers were 1.0×106-3.0×107. The 11 mAbs reacted with the BoIL-2 commercialization expressrd in E.coli and Yeast systems with good biological activity by ELISA analysis. Western Blot results showed that 11 mAbs could only react with fusion protein rHis-BoIL-2 and rGST-BoIL-2, but without pET-30a/BL21(DE3) and pGEX-6P-1/BL21 vector. The results showed all mAbs have good specificity,11 mAbs could only react with the corresponding fusion proteins, and they have no cross reactivity with other cytokines. The FACS results showed that 3D8,5D10,6D7 and 6E5 could react with natural BoIL-2.2. Development and identification of mAbs against Ag85A proteinIn this study, the purified protein rGST-Ag85A was used as immunogen to immunized BALB/c mice, and the purified protein rHis-Ag85A was used as detecting antigen. The lymphocyte hybridoma technology was used to prepare the mAbs against antigen Ag85A of M. Bovis. The positive cell clones were screened by indirect ELISA, and after 2 times of cell subclones,9 hybridoma cell lines which can steadily secrete M. Bovis Ag85A mAbs were detected, which were named 1C6,2E6,2F2,3B8,3D9,4B3,4F3,5B9 and 6B5 respectively. The substyle and titer identification assay results showed that 9 mAbs were all IgGl type, the ascite titers were 1.0×4106-1.0×108, and the cell supernatant titers were higher than 1.0×104. The ELISA results showed that the mAbs can both react with Ag85A protein in rBCG::Ag85A and wtBCG. Western Blot and IFA analysis revealed that 9 mAbs could react with rHis-Ag85A and rGST-Ag85A protein, and recognize with GFP-Ag85A expressed in eukaryotic systems, and also response to Ag85A in wtBCG and rBCG::Ag85A, besides 3D9 could react with GFP-Ag85B and GFP-Ag85C expressed in eukaryotic systems,4B3 recognize with GFP-Ag85C expressed in eukaryotic systems.In this study, we prepared 11 hybridoma cell lines secreting mAbs against BoIL-2 and 9 hybridoma cell lines secreting mAbs against M. Bovis Ag85A. And the results showed that both BoIL-2 mAbs and M. Bovis Ag85A mAbs have good reactivity with native antigen. All the mAbs provides important biological materials for detection and diagnosis of bTB in further research.