Antigenic Analysis For Capsid Protein VP1 Of Feline Calicivirus And The Establishment Of Indirect Enzyme Linked Immunosorbnent Assay

FCV is an important pathogen for feline species. It is a member of Caliciviridae Vesivirus, thenucleic acid of which is a single strand RNA molecule. FCV is a nonenveloped virus, only one serotype. FCV mainly infected kittens less than one year old and leads to acute oral and respiratory infections. The main symptoms of cats infected FCV are oral ulcers, rhinitis,conjunctivitis, upper respiratory tract infections, lameness. High virulent strains can cause systemic infection in cats and lead to death. Currently the diagnosis methods of FCV mainly include RT-PCR, immunofluorescence, neutralization test, agar diffusion test, sandwich enzyme linked immunosorbent assay(ELISA), indirect ELISA, but at present there is not yet commercially available ELISA kits. Indirect ELISA detection method can provide accurate diagnosis of FCV infection, and has the advantages of simple, rapid, high sensitivity. The study of the ELISA method is of great significance to epidemiological investigation, monitoring and prevention and control of the disease.VP1 protein is the major structural protein of FCV, divided into A, B, C, D, E, F six regions,containing more than one epitopes. In order to clarify the antigenic of VP1 FCV protein and obtain the ideal recombinant antigen, In this study, molecular biology software Protean was used to predict the epitopes of A, B, C, D, E and F in six regions, and the conservatism was analyzed by Megalign software; The primers were designed, the target gene was amplification by RT-PCR, then the the target protein was expressed in the prokaryotic expression system, which were analysed by Western-lot and serological analysis. The results are as follows. Protean software analysis revealed that B, E, F regions have a high antigen. Megalign software analysis showed that the amino acid conservation of B, E, F regions were 90.2%-100%, 67.7%-100%,89%-100%, respectively, and were relatively conservative. Western-blot proved that VP1-B,VP1-E, VP1-F protein has a good response, and further serological antigenic analysis proved that VP1-F protein has good immunogenicity.with a good antigen, the VP1-F was choosed as the coated antigen to establish indirect ELISA diagnostic method. The reaction conditions were optimized, the Cut off value was established,the specificity, repeatability and sensitivity were established. The established ELISA wascompared with the serum neutralization test and indirect immunofluorescence test, the clinical serum was detected with the established ELISA. The results were as follows. The working concentration of the VP1-F protein was 0.2μg / m L, the best antigen coating time is 2h in 37℃;the optimal dilution of serum to be tested was 1: 200, the optimal incubation time was 1h in 37℃;the best blocking buffer was 5% skim milk, the optimum blocking time is 1h in 37℃; the best secondary antibody dilution is 1: 5000, the optimum reaction time was 45 min in 37℃; The best reaction time of substrate was 4 minutes. The cut off value was 0.352. The established indirect ELISA had a good specificity because it had no cross reaction with other five positive serums,included feline herpes virus positive serum, feline panleukopenia virus positive serum, feline infectious peritonitis virus type I positive sera, feline infectious peritonitis virus type II positive serum. The quality control serums were tested with the same batch and different batches, the coefficient of variation was less than 10%, the indirect ELISA has good repeatability. The quality control positive serum of FCV was diluted to 1:6400, and the sensitivity was good. The coincidence rate of the ELISA with the serum neutralization test was 95%, and with the Indirect immunofluorescence test was 100%. The ELISA diagnostic method was used to detect the serum samples from 60 cats, which were collected from pet market in Harbin, and the positive rate was88.3%.The six regions FCV VP1 gene were anagetic prediction with the Protean software, and the conservation of which were analysed with the Megalign software. The recombinant protein VP1-A, VP1-B, VP1-CD, VP1-E and VP1-F were successful expressed by prokaryotic expression system, and VP1-F protein was proved the advantage in anagetic with Western-blot and serological antigenicity analysis. VP1-F protein was choosed as a package to establish the indirect ELISA diagnose mehod with good specificity, reproducibility, sensitivity, which laid the foundation.for the diagnosis and prevention of FCV.