An Indirect ELISA Established For Detection Of Anti-BEV-3D Antibodies Used For The Application Of Screening Positive Individuals Infected Bovine Enterovirus

The bovine enterovirus is a member of the family Picornaviridae which belong to the genus Enterovirus with the appearance of spherical, icosahedral structure, without capsule, 25-30 nm in diameter in electron micrographs. BEV virions contain a naked capsid that surrounds a core of positive-sense, single-stranded RNA that about 7.5kb long. The first BEV was isolated from stools of healthy cattles by Kunin and Mc Ferran in 1957, respectively. Then, many virus isolated have been reported in the world, but the epidemiology of the virus rarely reported in our country. The natural hosts of bovine enterovirus are more extensive, including sheep, goats, people and so on. But the etiology of the virus and the pathogenic mechanism remains unclear. Clinical symptoms were diarrhea, reproductive disorders, and sometimes the clinical syndrome caused by invading other organs.We designed a specific primers for 3D sequence which named BEV-3531 saved by our lab(Wang Junwei’lab), amplified the gene fragment of 3D(1382bp)sequence induding the whole 3D of bovine enterovirus using PCR. Then the amplified product was cloned into p Bs K, verified by sequencing. The correct sequence fragment verified then cloned into the expression vector p ET-30 a. The results of SDS PAGE showed that the recombinant protein was highly expressed in E.Coli after the recombinant plasmid was transformed into host bacteria Rosetta and induced by IPTG. In Ni-NTA affinity chromatography purified recombinant protein 3D, we received high titer polyclonal antibody after initial expression product was used for immunizing rabbits. The analysis of western blotting and indirect immunofluorescence was that 3D protein has good antigenicity.The purified 3D protein as coating antigen, an indirect ELISA for the detection of BEV antibody was developed and optimized. The test by reproducibility and specificity proved that the indirect ELISA method established in this study had good reproducibility and specificity. In addition, 425 serum samples of censorship from Harbin were detected by the indirect ELISA method, and the results showed 3D protein could react with serums from infected herds and the positive rate was 41.41%.In order to reveal correlation between virus and dynamic of 3D antibody after animals infected bovine enterovirus, we detected dynamics of antibodies in mice infected with virus strains named BEV-3531 by BEV-3D-ELISA method which we built in the test, as well as the virus nucleic acid of intestinal tissue in mice infected by the same virus with the RT-PCR. In addition, 42 serum samples from a dairy herd of Harbin City were detected by BEV-3D-ELISA. At the same time the 42 corresponding stool samples dealed with PBS were inoculated in MDBK cells and observed the lesions. Then we extracted the supernatant RNA of occuring cytopathic and conducted RT-PCR, ligation, transformation and sequencing, at last identified morphological characteristics of the virus through scanning electron microscopy(SEM). To prevent accidental, 22 serum and stool samples from the same herd of Harbin were tested using the same method. The results indicated that the dynamic variation of the virus was positive correlation with antibody trends between growth and decline in 3D, and all increased at first and then decreased. The clinical tests showed that 10 of 42 seums from the first samples and 4 of 22 seums from the second were positive by BEV-3D-ELISA method. The RT-PCR results showed that 9 of 42 stools from the first samples and 4 of 22 stools from the second were positive. Both rates of positive compliance were 90% and 100%, respectively. And the sequencing and morphological analysis results showed that the virus was bovine enterovirus. These results confirmed the compliance between BEV 3D antibodies could be detected and the viral nucleic acid could be detected was good.By the results of all these tests, BEV-3D-ELISA method can be screened positive individuals infected bovine enterovirus, used for further isolation of pathogen, also had good prospects in the etiology of bovine enterovirus survey applications.