Development Of An Indirect ELISA Kit For Detection REV Antibody

Reticuloendotlheliosis is a group of pathological syndrome caused by the avian retrovirus genus reticuloendothelial hyperplasia virus. REV has been shown to cause immunosuppression, lymphomas and runting stunting syndrome, and is associated with sporadic outbreaks of chronic neoplastic disease in turkeys, and can cause significant losses in commercial turkey flocks. The virus can lead to different degrees of superinfection and multiple infections; decrease the immue response for various vaccines. There have not vaccines or medicine against it up to now. Many countries take elimination program to control this disease, such as eliminate the infected chicks, eradicate the chicks groups and so on.In order to further understand the prevalence of REV, and eliminate the infected chicks as soon as possible. The method of indirect—enzyme linked immnnosorbent assay (ELISA) is the best method to detect clinical samples. The patent of REV antibody test kit has not be seen in our country, and the specific detection of REV antibody test kit has been commercialized, but expensive, and difficult to a wide range of use. Therefore, the ELISA antibody test kit with our own intellectual property is particularly important.REV-env gene encoding the gp90C-terminal epitope on the surface of infected cellsontaining the sequence and conformational epitopes, the immunodominant protein of the virus, can stimulate the infected host to produce neutralizing antibodies; REV-ENV protein is considered aspecies used in the diagnosis good choice for REV. This research chose SNV strain as the standard strain, using specific primers obtain a1200bp relatively conservative sequence. The env gene of REV encodes SU protein and TM protein which contain more than95%antigen site. In order to obtain recombinant protein, expression plasmids pET-28a-env was constructed and transformed into E.coli BL21strain. SDS-PAGE indicated that the expressed fusion protein His-env existed in the patterm of inclusion body. The inclusion body was solved by ultrasound wave and8mol/L urea respectively, then the protein was dialyzed to restore protein natural conformation and activity.The purified and recombinant protein was used to coat the96pores ELISA board as antigen, which was used to coat a96well high affinity ELISA plate as antigen. A series trials were performed for the groped optimal reaction conditions of the indirect ELISA and the results were obtained as follow:the best coating quantity of antigen was400ng per well; Serum was diluted800times; The best coating conditions of recombinant protein was4℃overnight and the best coating liquid was carbonate buffer solution (liquid pH9.6); The best block solution was4%skimmed milk powder, the best block time was2h at37℃; The best reaction time of serum was1.5h; The best diluted time of enzyme-linked secondary antibodies was4000for reaction1h; the best reaction time of substrate was30min; the cut-off value of positive and negative result was0.340(Cn=X+3SD). Moreover, the established indirect ELISA method with the advantages of good repeatability and specificity offered a foundation to REV diagnostic kit.508chicken serums from some regions of Shandong were detected by this method. The results showed that the general positive rate was34.25%, including the lowest6.52%of commercial egg-thickener and the highest62.00%of local chicken. The results showed that REV was widespread in chickens of certain parts in Shandong province.The experiment built a foundation for REV antibody diagnosis kits. The kit will be more available for molecular epidemiology investigation of REV. Technical assistance was provided to support the development of poultry enterprise.