Expression Of Bovine Interferon-gamma Gene And Its Preliminary Application In The Therapy Of Bovine Mastitis

Interferon-gamma is a pleiotropic cytokine characteristically prouduced by natural killer cells and T helper 1 type cells in response to antigenic or mitogenic stimulus. Apart from its capacity to directly exert anti-viral activity and anti-tumor activity, it plays many crtitical roles in the modulation of immune reponses. Due to its important biological activity, IFN- Y now is a focus among the medical and biological fields, and recombinant IFN- Y produced by recombinant technology has been successfully implemented for the therapy and prevention of some human diseases. In comparison with human IFN- Y , animal IFN- Y have only recently been investigated. Since it first reported by Cerretti et al in 1986, bovine interferon-gamma (BoIFN- r) gene has been expressed in some prokaryotic and eukaryotic expression systems, and recombinant bovine interferon-gamma (rBoIFN- Y ) has been applied for the control of bovine mastitis at abroad, there is few report in china. Traditional means of mastitis control involve antibiotic therapy and vaccination, which often company with some defects such as antibiotic residues, drug resitance and immune failure, therefore, new and innovative approaches for mastitis control are needed. In this paper, We described the selection of highly active rBoIFN- Y expressed in three different expreesion systems and its preliminary application in the treatment of bovine mastitis.1. Expression of bovine interferon-gamma gene in soluble form in Esherichia Coli and preparation of specific antisera to recombinant bovine interferon-gammaBovine interferon-gamma gene without signal peptide encoding region was amplified by reverse transcription polymerase chain reaction (RT-PCR) from total RNA of bovine spleen lymphocytes in response to Phytohemagglutinin (PHA). The products of RT-PCR were cloned into pUCm-T vector. Postive recombinant clone named BoIFN-Y -Tl was identified by restriction enzyme digestion and sequencing. The fragment of BoIFN- Kin the pUCm-T vector was subcloned into pGEX-6P-1. Partly soluble 42 kDaGST-BoIFN- Y fusion protein was expressed after induced by EPTG, which was confirmed by SDS-PAGE and accounted for 25 % of total soluble bacterial proteins detected by Tic-scanning. Recombinant fusion protein showed some anti-viral activity in the cytopathic effect inhibition assay with VSV. The interferon titer in the supernatant of bacterial sonicates was up to 1.6384x104U per milligram.Fusion protein GST-BoIFN- Y separated by SDS-PAGE was extracted from the gel and injected into BALB/C mice once a week for five times. The antisera was collected from the immunized mice, and its specificity to GST-BoIFN- Y was identified by indirect ELISA. The above results suggested that the specific antisera can be used as a useful tool in preparing a specific monoclonal antibody to rBoIFN- Y and identifying the expression of rBoIFN- Y in the next eukaryotic expression system.2. Development and identification of the monoclonal antibody against recombinant bovine interferon-gammaGST-BoIFN- Y fusion protein in the gel was used to immunize the BALB/C mice. Two hydromas secreting specific monoclonal antibodies against rBoIFN- Y were developed by cell fusion between SP2/0 cells and spleen cells from the BALB/C mice immunized with GST-BoIFN- Y fusion protein extracted from the gel. In direct ELISA, these two monoclonal antibodies named as 4A3 and 4G5, respectively, only reacted with sonic supernatant of E. coli expressing GST-BoEFN- Y, and not reacted with sonic supernatant of E. coli expressing GST protein, the tilers of the cultured supernatant and ascites of 4A3 can achieve 1:25600 and 1:1.3x107 respectively. Subclasses of these two McAbs were identified by capture ELISA as IgG2b and IgM respectively. McAb 4A3 showed some potent neutralizing activity against E. coli derived rBoIFN- Y. The hereby reported two McAbs will provided useful tools for detection and purification of rBoIFN- Y expressed in eukaryotic expression systems.3. Expression of bovine interferon-gamma gene in COS-1 cellsBoIFN- rg...