Cloning And Expression Of Macaque IFN-γ Gene And Establishment Of Macaque IFN-γ Detection Assay

Tuberculosis (TB) is a zoonotic, chronic and consumptive infectious disease with the highest death in infectious diseases of human. It was caused by Mycobacterium tuberculosis complex (MTC). Macaque (Macaca mulatta, Mm) is widely distributed in China and put in the list of Grade II protectivewild animal species in our country. Furthermoreand it is of a high value as an experimental animal species in medical research. Macaques are the most susceptible species to TB among the non-human primates. The tuberculosis will lead to loss of monkeys and interfere experiments. In addition, monkey TB may transmit to human. Since there is not an effective vaccine for Macaque TB prevention, the only method to control macaque TB is "detection-slaughtering". Therefore, a diagnostic method for TB which has a good sensitivity and specificity is a prerequisite for the implementation of this policy.The most commonly diagnostic method for TB is the tuberculin skin test (TST). This method is cumbersome and subjectivity, often gives false positivedue to the cross-reactivity to the non-tuberculosis mycobacteria. Interferon-y(IFN-y) is a multifunctional glycoproteins. MTC infection can stimulate IFN-y production. Therefore, IFN-y in vitro release assays based on the IFN-y production by pheriperal blood cells in vitro after stimulation of Mycobacterium tuberculosis specific proteins are widely used in cattle TB and human TB detection in developed countries. Monkey IFN-y in vitro release assay is available in international market, but expensive and unavailable in Chinese market.Thus this paper was aimed to establish a monkey IFN-y in vitro release assay and provide an effective tool for monkey TB detection. The main contents include cloning and expressing the Macaque IFN-y (MmIFN-y) gene, making the purified recombinant MmIFN-y, preparing monoclonal antibodies and polyclonal antibodies of MmIFN-y, and finally establishing a indirect sandwich ELISA for detection of MmIFN-y.1. Cloning and expression of MmIFN-y in Escherichia coli and Pichia pastoris YeastBasing on the sequence of MmIFN-y cDNA saved in our lab, one pair of primers was designed and the MmIFN-y cDNA was cloned into the prokaryotic expression vector pET-30a. Then the recombinant vector was transformed it into E. coli BL21and the expression was induced by IPTG. The soluble protein was expressed with molecular mass of about24kDa. It is a fusion protein of his-tag and MmIFN-y. Basing on the sequence of MmIFN-y cDNA saved in our lab, another pair of primers was designed and MmIFN-y cDNA was cloned into yeast expression plasmid pPICZaA. The resulting recombinant vector was transformed by electroporation into yeast competent cells GS115. The expression of MmIFN-y was then induced by methanol and the resultant proteins were recombinant MmIFN-y of about17kDa and glycosylated MmIFN-y of22kDa.2. Preparation and characterization of MmIFN-y monoclonal antibodies and polyclonal antibodies.In this research, we used the recombinant MmIFN-y expressed in E. coli to immunize Balb/C mice. The protein was used to screen hybridoma cells which were able to produce monoclonal antibodies to MmIFN-y with an indirect ELISA. Finally10hybridoma cells secreting monoclonal antibodies were obtained, and designated as2A1,2C3,2F6,2H8,3A1,3B2,3E5,3F6,3G7and3H9respectively. The titers of the monoclonal antibodies ascites were between3.2×103(3G7) and6.5×106(2A1). The relative affinity of the monoclonal antibodies ranged between104and107. By using the indirect sandwich, it was showed that these10monoclonal antibodies can react to MmIFN-y expressed in yeast.At the same time, we used the MmIFN-y expressed in E. coli to immunize rabbits, then the rabbit hyperimmune antisera were produced. After purification, the polyclonal antibody IgG against MmIFN-y with the titer of5.12×104was obtained.3. Establishment and application of indirect sandwich ELISABy using McAb2F6as the captive antibody and PcAb as the detection antibody, an indirect sandwich ELISA was established. The sensitivity of this assay to detect MmIFN-y was125pg/mL. Meanwhile, this ELISA is able to detect the natural IFN-y produced by pheriperal blood cells after stimulated with ConA. Seven blood samples from TB negative monkeys were detected to be negative with this ELISA after stimulated with bovine PPD, while positive after stimulated with ConA. Furthermore, the detectable limit125pg/mL was corresponding to0.18of OD630. This was equal to value of Mean+3.5SD for the negative samples. Since the cutoff value is generally defined as Mean+2-3SD, this sensitivity of the ELISA can meet the requirement of clinical detection. In addition, it was reported that the pheriperal blood cells from infected macaques usually release IFN-y more than500pg/mL. Therefore, this home-made macaque IFN-y in vitro release assay can be used in the detection of macaque TB And Thereby this research provides a promising tool for macaque TB detection and thereby TB control.