| Maedi-Visna disease(MVD)is a chronic viral disease of sheep,which is caused by Maedi-visna Virus(MVV).At present,there is no effective treatment for MVD,and the infected animals with MVV can only be isolated and culled,Therefore,early detection and isolation as soon as possible is the most feasible method.However,a diagnostic method with high sensitivity and specificity plays an important role in the prevention and control of MVV,Therefore,the following experiments were carried out in this study:In this study,the antigenic regions of MVV CA protein and MVV TM protein were analyzed by DNA Star biological software.p Easy-E1-CA,p Easy-E1-TM and p EasyE1-CA-TM expression plasmids were constructed by Molecular cloning technology.Recombinant CA,TM and CA-TM proteins were obtained by prokaryotic expression,and rabbit polyclonal antibodies against recombinant CA and TM proteins were prepared.Their titers were determined by indirect ELISA.The antibodies were tested by Western blot and the rabbit polyclonal antibodies prepared from CA protein were preliminarily applied by immunohistochemical method,and then the prepared rabbit polyclonal antibodies were evaluated.Establishment of an indirect ELISA method for detection of Maedi-visna virus using CA-TM fusion protein as coating antigen.The detection effect was evaluated by specificity,repeatability,sensitivity and clinical application.The results showed that the expression plasmids of p Easy-E1-CA,p Easy-E1-TM and p Easy-E1-CA-TM were successfully constructed.SDS-PAGE results showed that the recombinant CA protein was expressed in soluble form and its molecular weight was about 27 k Da.Restructuring TM protein expressed in the form of inclusion body,its molecular weight is about 14 k Da;Restructuring the CA-TM protein expressed in the form of inclusion body,its molecular weight is about 40 k Da.The rabbit polyclonal antibodies against recombinant CA protein and recombinant TM protein were prepared,and their titers were 1:8192 and 1:4096,respectively.The results of Western blot specificity test showed that the rabbit polyclonal antibody could specifically recognize MVV CA protein and MVV TM protein.The results of IHC showed that the obvious brown positive signals were mainly located in the cytoplasm of macrophages in the lungs naturally infected with MVV.The indirect ELISA method of Maedi-visna virus was successfully established in this experiment.The specific results showed that the detection method established in this experiment could not detect common pathogens of sheep such as Foot-and-mouth disease virus(FMDV)and Mycoplasma ovipneumoniae(MO)except MVV,and could recognize both anti-CA protein antibody and anti-TM protein antibody produced in diseased animals.Sensitivity results show that the ELISA detection method can detect at least 16000 times diluted positive serum.The batch repeatability test results of the ELISA detection method show that the coefficient of variation is between 2.3%-8.5%.The blood samples of 69 suspected cases collected from the diseased sheep followed by the research group showed that 31 positive samples and 38 negative samples were detected by the ELISA method,37 positive samples and 32 negative samples were detected by ID Screen MVV/CAEV Indirect kit.After comparison,the positive coincidence rate of the ELISA detection method established in this test and the ID Screen MVV/CAEV Indirect kit was 83.7%,the negative coincidence rate was 100%,and the overall coincidence rate was 91.3%.In summary,the indirect ELISA detection method of MVV was successfully established in this experiment.This method not only reduces the influence of inaccurate serological detection caused by seroconversion,but also increases the detection range,not only of great significance for the prevention,control,surveillance and public health of MVD,but also lays a foundation for the diagnosis and purification of MVD in Inner Mongolia. |
PDF Full Text Request