| Avian Leukosis and reticuloendotheliosis are both diseases can cause avian tumors and immunosuppressive. Avian leukosis virus Subgroup J is retrovirus identified in 1990s'by Payne, which causes serious economic losses of meat-type chickens. There are some reports about the morbidity of ALV-J in some parts of China. The Reticuloendohetliosis virus (REV) is a group of oncogenic and immunodepressive type C avian retroviruses which cause reticuloendotheliosis. REV is known to cause economically important immunodepression in infected chicken, and is associated with sporadic outbreaks of chronic neoplastic disease in turkeys, and can cause significant losses in commercial turkey flocks. Both of these two viruses can lead to different degrees of superinfection and multiple infections; decrease the immue response for various vaccines. There have not vaccines or medicine against them up to now. Many countries take preventive measures to control this disease,such as eliminate the infected chicks, eradicate the chicks groups and so on.The surface protein (SU) encoded by gp85 gene is the important component of envelope protein which contains many antigen site of ALV-J virus. It is the main bases of subgroups distinguish with other subgroup of ALV, so it is the optimum antigen which used to develop subgroup specific antibody. Env gene of REV encodes SU protein and TM protein whiche contain more than 95% antigen site. A cDNA encodes env gene was amplified from total cDNAs of REV by PCR. DNA sequencing indicated that the open reading frame encodes 400 amino acid residues. In order to obtain recombinant protein, expression plasmids pProEXHTB-gp85 and pET-28a-env was constructed and transformed into E.coli BL21strain and BL21 (DE3), respectively. SDS-PAGE indicated that the expressed fusion protein His-gp85 and His-env existed in the shape of inclusion body. The inclusion body was solved by ultrasound wave and 8mol/L urea respectively, then the protein was dialyzed to restore protein natural conformation and activityNew Zealand white rabbits were immuned by purified His-gp85 and His-env for each 300μg to prepare polyclonal antibody. The titers of the polyclonal antibody against the His- gp85 and His-env protein were about 1: 1×106 and 1:2×106 by ELISA, Titers were about 3200 and 12800 analysed by IFA.The purified His-gp85 and His-env was inoculated on Balb/C mice, immune 40μg each mouse every time, totally four times. Indirect ELISA method and IFA method were developed to determine the blood serum antibody titer. The spleen cells were taken and fused with SP2/0 bone myeloma cells by PEG1500. Cultured the hybridoma with HAT and HT, then detected the antibody activity of supernatant when hybridoma covered with a quarter or half of the well. Positive clones were subcloned to get monoclonal cell lines by limit dilution, repeat 3times. We got two positive monoclones against ALV-J named 1D4 and 3B1; and one positive monoclone against REV named 4D5. They were frozen and prepared ascites at the same time. Titers of ascites and supernatants were detected by ELISA and IFA. The supernatants were also detected by SDS-PAGE and Western blot, the results showed that the monoclonal antibodids have good specificity and sensitivity.The experiment built a foundation for ALV-J and REV antigen/antibody diagnosis kits. These kits will be more available for molecμlar epidemiology investigation of ALV-J and REV. Technical assistance was provided to support the development of poultry enterprise. |
