| Avian leukosis (Avian Leukosis) is an easily infectious neoplastic disease caused by the avian leukosis virus (ALV). ALV causes flocks suffering from neoplastic diseases, which can not only directly do harm to the flocks, but also lead to the chicken immune failure, secondary infection, production capacity decline and so on. It has brought huge losses to China’s poultry industry. Up to now, against the disease, we have not yet developed an available vaccine and effective therapy. The only way to control the disease is to eradicate the chicks groups. The capsid P27protein is a group of specific antigen. Using ELISA to detect the P27antigen is now a common method to monitor ALV. In this study, we used prokaryotic expression technical to prepare recombinant P27protein and established indirect ELISA method to detect the P27antibody. Simultaneously, we used the P27protein as the immunogen to immune the Balb/c mouse and Japanese white rabbit to prepare monoclonal and polyclonal antibodies. Based on this, we established a double-antibody sandwich ELISA method to detect P27antigen, in order to provide a rapid, specific and easily performed technique for ALV detection.The content of this thesis includes:1. Prokaryotic expression of P27geneALV P27gene was cloned into the prokaryotic expression vector pET28a to build the pET28a-P27recombinant plasmid. Then the plasmid was transformed into Escherichia coli BL21(DE3), being induced by IPTG at16℃overnight to obtain highly expressed soluble P27protein. nickel affinity chromatography was used to purificate the P27protein. The purification effect and immunogenicity to ALV chicken positive serum was verified well by SDS-PAGE and western blot.2. The establishment of the ALV P27protein indirect ELISAP27recombinant protein as the coating antigen, horseradish peroxidase (HRP) labeled goat anti-chicken IgY as the secondary antibody, we established indirect ELISA for detecting avian leucosis virus antibody. By checkerboard titration experiment and exploring the optimal conditions, the P27protein coating concentration was ultimately defined as0.183μg/mL, the dilution of the serum and the HRP-labeled goat anti-chicken IgY were1:500and1:60000. Through series of tests, the method showed specificity, high sensitivity and good repeatability. Besides, it could be used to detect all ALV subsets antibodies and displayed the result of coincidence rate of85.7%comparing with IDEXX AB subsets and IDEXX J subset antibody test kit. 3. Preparation of monoclonal antibodies against P27proteinSix to eight week old Balb/C mouse was immunized with recombinant P27protein. Then its spleen cells were fused with SP2/0cells. Six hybridoma cell lines that secreted specific antibody against prokaryotic expression of the P27protein were obtained through cells culture and limited dilution. Biological characteristics analysis showed that the hybridoma chromosome numbers are all about90and indirect immunofluorescence confirmed that2F3,5C2and5C7could specifically bind to ALV.4. Preparation of polyclonal antibodies against P27proteinJapanese white rabbit was immunized with recombinant P27protein and its immune titer was1:64000ultimately. Then the rabbit blood serum was collected to prepare polyclonal antibodies. We demonstrated that the polyclonal antibodies could specifically bind to ALV P27protein.5. The establishment of the ALV P27protein sandwich ELISA2F3McAb as the capture antibody, P27PcAb as the detection antibody, HRP labeled goat anti-rabbit IgG as the secondary antibody, we established double-antibody sandwich ELISA for detecting avian leucosis antigen. By checkerboard titration experiment and exploring the optimal conditions, the2F3McAb coating concentration was ultimately defined as0.5μg/mL, the concentration of the PcAb was1.75μg/mL and the dilution of the HRP-labeled goat anti-rabbit IgG was1:7000. Through series of tests, the method showed specificity, high sensitivity and good repeatability and displayed the result with coincidence rate of99.3%comparing with IDEXX antigen test kit. |
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