| Feline Panleukopenia is an acute and highly contagious viral disease caused by Feline Panleukopenia virus (FPV). The main characteristics of this disease are high fever, vomiting, clehydration, serious decrease of the white blood cells and haemorrhagic enteritis. Under the natural conditions, multiple kinds of carnivores, such as tigers, leopards, lions minks and raccoons, can be infected by FPV with a high mortality rate; and which has posed a serious threat to the feeding and protection of big cats including both domestic and wild animals of the Felinedae. Therefore, a common diagnostic method is badly needed to set up to test FPV of various animals simultaneously on a large scale.VP2 protein is the main component of the capsid and major structure protein of FPV which is exposed to the surface the capsid protein. Furthermore, it is also the main immune protein antigen which is able to induce the body to produce antibodies. Thus, VP2 protein is the best for the research of the preparation of FPV diagnostic antigens. At the moment, there haven't researches of whole VP2 gene expression by prokaryotic expression system been reported.FPV strain was isolated from drops of diarrhea tigers from a wildlife zoo in China and identified by culturing on the CRFK cell line. VP2 gene of FPV was cloned and analyzed. The amplified VP2 gene was subcloned into the prokaryotic expressing vector pGEX-6p-1, and then transferred into E.coli BL21(DE3) for expression under IPTG treatment. The purified recombinant VP2 protein was analyzed by Western Blot. An indirect enzyme-linked immunosorbent assay (ELISA) for target antibodies detecting was developed by the purified recombinant VP2 protein to study its characteristics of Common Diagnostic Antigen of VP2 protein. Since the purified recombinant VP2 protein is a superior diagnostic antigen, an ELISA using SPA as conjugate (SPA-ELISA) was developed to detect FPV antibodies in clinical samples of cats and tigers.30 clinical serumal samples of domestic cats and 86 serumal samples of tigers were tested by SPA-ELISA, HI and indirect ELISA to compare total coincidence ratio. And then, monoclonal antibody (McAb) against recombinant VP2 protein of tiger feline panleukopenia virus were developed by fusing myeloma cell line SP2/0 with splenocytes of BALB/c mice which was immunized with prokaryotic expressed VP2 protein. Positive hybridoma cells were sifted by ELISA with tiger FPV. The results are as follows:The FPV-HLJ2 was isolated from drops of diarrhea tiger by culturing on the CRFK cell line successfullyï¼›sequencing analysis of VP2 gene showed that the full length of VP2 gene was 1755 bp and encoded 584 amino acids, and FPV-HLJ2 isolate shares 100% identity with FPV-HLJ isolate (Feline Panleukopenia Virus tiger strain), while the host tropism amino acid changes occurred in the same point. VP2 gene was highly effectively expressed in E. coli. SDS-PAGE and Western blotting assays revealed that the fusion protein of about 84 ku was obtained from the prokaryotic expression system, including a 58 ku VP2 protein and a 26 ku GST-tag protein, and had good immunoreactivity. The indirect ELISA assay indicated a good antigenic specificity of the expressed VP2 protein and the feasibility to be used as diagnostic antigen for the diagnosis of FPV infection in cats. Some variation of sites AA on the VP2 Protein didn't affect the antigenic nature of FPV. The potential of recombinant VP2 protein to be used as Common Diagnostic Antigen.for the diagnosis of FPV infection in felids was revealed.Based on the purified recombinant VP2 protein of tiger FPV, an ELISA using SPA as conjugate (SPA-ELISA) was developed to detect FPV antibodies in clinical samples of domestic cats and tigers. The assay was optimized. Variation coefficient of intraassay and interassay about SPA-ELISA were all less than 10%. The comparing detection among the 30 serumal samples of domestic cats and 86 serumal samples of tigers showed that the detection rate by SPA-ELISA was higher than HI, and the result was close to indirect ELISA. The total coincidence ratio of the three detecting methods were 96.7% by detecting 30 serum samples of cats. The coincidence ratio of the SPA-ELISA and HI was 94.2% by detecting 86 serumal samples of tigers. All these results indicated that SPA-ELISA could be a good method for serological detection of FPV antibodies in many species of felines.Monoclonal antibody 3C4 against recombinant VP2 protein of tiger FPV were developed by fusing myeloma cell line SP2/0 with splenocytes of BALB/c mice which was immunized with prokaryotic expressed VP2 protein. Positive hybridoma cells were sifted by ELISA with tiger feline panleukopenia virus. The antibody titers of ascites for the hybridoma lines were 1:12800. The subtype of monoclonal antibodies was determined as IgG2a. Indirect immunofluorescent test indicated that the monoclonal antibody could specifically react with FPV, and it may be used as a valuable tools for diagnosis of FPV.The difference between FPVs from two different kinds of mammals was initially known through the study, and the preliminary use of FPV VP2 Protein come true. The way of SPA-ELISA based on FPV VP2 protein, solving the problem that the present detections are too simple or can't detect a large amount of samples at one time, is likely to be used to test FPV antibodies in animals' serum such as leopards, lions, pandas, ocelots, raccoons, minks, lynx, etc. There will be great significance in realizing quick diagnosis, epidemic observation, epizootic research and detection of antibody potency. The neutralizing monoclonal antibody of FPV VP2 Protein based on FPV VP2 provides a more sensitive and different reagent for FPV, CPV and MEV diagnosis. |
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