| Avian leukosis is a major viral tumour disease in the aviculture, because of its varioussubtypes, widely distributed and vertical transmission, and therefore, there were somedifficulties in the prevention and decontamination of the disease. The ALV-infected becomemore serious in the local strains of chickens in China. In the past period, the relevantresearchers pay more attention about ALV-J, while ignoring the classic ALV-A/B, whichindirectly caused the widely spread of ALV-A/B in the local strains of chickens. Thepurification of ALV-A/B in the local strain chickens become a top priority. The developmentof monoclonal antibodies and establish a standardized test kit, to achieve rapid and accuratemonitoring of the disease is the primary task.This study was designed to prepare monoclonal antibodies against ALV-B. The samplescollected from Shouguang chickens in Shandong were tested for the p27antigen of ALV(ALV-P27-Ag), anti-ALV-A/B antibody (ALV-A/B-Ab) and anti-ALV-J antibody(ALV-J-Ab) via ELISA kits produced by IDEXX Company, the results showed antibodypositive rate reached70.37%(95/135). Necropsy and observe pathological changes of thechickens, the tissue of liver, spleen and other organs were collected to stain with haematoxylinand eosin and to observe these pathological changes. The disease material stored at ultra lowtemperature refrigerator, as the extraction of viruses and proviral DNA.Virus was extracted by using spleen of the infected chickens and inoculated to DF-1cellsfor amplification. Detected p27antigen in the cell culture supernatant via ELISA, todetermine the presence of the virus, and named it for ALV-B-SG12, stored them at ultra-lowtemperature refrigerator. Uesed the DNAiso Reagent bought from Takara Bio Company forextraction of proviral DNA, in order to obtain the target gene. The main distinction betweenthe subgroup of ALV were based on the different of the envelope protein-gp85, the differencesof sequences of each subgroups determine the specificity of the antigens. Gp85gene primerswere designed for the ALV-B, and took it for PCR, recovered and sequenced. The sequenceresults showed1038bp measured accurately. Compared it with12strains of ALV-A and6strains of ALV-B which were reported home and abroad, the homologous rate of ALV-A wasabout85%, and the rate was above90%for ALV-B, furthermore the rate reached up to96%for RAV-2, WB11080, RSV-SCHMIDT-RUPPIN_B and JS-B1203.The recycled products were connected into pET32a vectors, and the vectors weretransformed into expression host strain BL21to express the interest protein. The express condition has been optimized from three aspects, including IPTG concentration, temperatureand time, each test detected by SDS-PAGE. Ultimately determine the optimum conditions forthe induced expression: IPTG concentration of0.1mmol/L, temperature30℃, the inductiontime of8h. There were clear bands at53kDa showed in the SDS-PAGE of the purified proteinafter the supersonic lysate and purification. The purified proteins were refolded byTGE-dialysate, and finally got the production of the immunogenic specific active proteins.After the obtaining of purified protein, we established indirect ELISA method. Through aseries of comparative tests, the optimal reaction conditions were: the concentration ofantigens was3μg/ml, the dilution of serum was1:1000and the dilution of second antibodywas1:2000. Took50μg of purified protein per mouse each time for injection, at the1、15、30d,respectively Collected blood from caudal vein and separated the serum, the establishedmethod was used to detect it and the titer was reached up to1:26~29. After the immune, tookthe spleen cells of the mice and sp2/0cells for cell fusion, the proportion was3~5:1, after atotal of eight cell fusion work, the positive holes eventually detected by indirect ELISAmethod. Positive wells were amplified and frozen storage. The limiting dilution assays wasused to sub cloning of the positive cells, and screened the monoclonal antibodies. Use themonoclonal antibodies for IFA and the test of stability of antibody-secreting, the monoclonalantibodies was high specific to ALV-B and secrete stably. |
