| Subgroup J avian leukemia virus(Avian Leukosis Virus Subgroup J, ALV-J) was isolated from chickens in 1988, widely circulated in the world. The virus spreads in two ways of vertical and horizontal, inducing the tumor, slow growth, weight loss and death in poultry. In breeding chicken it reduces egg production, hatching rate as the main feature, meanwhile sick chicken bone marrow, thymus, bursa retarded resulting in the immune suppression, and chicken can’t produce enough antibodies to resist against disease, and thus vulnerable by disease, bringing huge economic losses in poultry industry. In recent years, this disease in our country presented popular trend, and the high quality native chickens, sanhuang chickens and commercial chickens have reported. There were no clinically effective drugs and vaccines can be used to treat the disease, for the reason, through the elimination ALV-J positive chicken, purification of ALV-J pathogen chickens is the only means to solve the problem of the current. Therefore, the establishment of double sandwich Elisa to detect the virus in the blood has great significance in controlling the disease epidemic, caring out the population purification.Due to the features that most viruses and bacteria are mainly through the digestive tract and respiratory tract mucous membrane into animal organism, thus the use of recombinant live carrier vaccine, adopt the way of oral immune to express the apes to mucosal tissues, inducing systemic immune response against outside invasion of the bacteria and viruses. Lactic acid bacteria, as a kind of widespread distribution in nature, and widely distributed in the animal body fungi has a good health care function. In recent years, lactobacillus oral vaccine has became a hot research, and which reorganization of lactic acid bacteria through the natural form of oral immune mediated mucosal immune is a safe and convenient method, not only mediates the local mucosal immune response, but also induces systemic immune response. When the reorganization of the exogenous gene expression vector containing lactobacillus as live carrier strains, it including the biological characteristics of lactic acid bacteria and immunogenicity of exogenous antigen gene, moreover the combination is not susceptible to the end toxin, and there are no inclusions, and it is easy to express to the outside of the cell. Recombinant strains is ease to save and propagation also conforms to the characteristics of new genetic engineering oral vaccinesThe purpose of this study is to restructure GP85 capsule membrane glycoprotein of lactobacillus casein oral immune mice, including mice to produce specific antibodies, weekly detection antibody level after vaccination. After 6 weeks by AGAR double diffusion method and Western blot, directly ELISA detection qualified mice resist chicken multi-antibodies. After the extraction, the antibody was marked as enzyme mark antibody. And user at monoclonal antibody in our labs a capture antibody ELISA plate, rat multi-antibodies as twoantibodies, setting up double sandwich ELISA method to detect ALV-J antigen in blood of chicken, and optimize the working conditions of the ELISA method indirectly. We detect the DF1 poultry ALV- J in the blood after subculture. The results show that the sealing fluid concentration is 5% skimmed milk powder; single package antibody by quantity for 1 mu g/ml; enzyme mark two- antibodies dilution degrees is 1:1600 and the effect time is 1 h; the substrate chromomeric time is15 mines. The specificity test, repeatability test, sensitivity experiments and threshold determined were conducted, and the results show that the method has good repeatability, specificity, sensitivity, and MD virus, infectious bronchitis virus, NDV has no cross reaction, and through a lot clinical testings, and then we can get more many scientific positive rates, providing the basis for clinical detection of ALV-J. |
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