Study On The Immunogenicity Of VP2 And Establishment Of A Method For Detection Of Bovine Enterovirus

Bovine enteroviruses are members of the family Picornaviridae, genus Enterovirus.The virus was first reported by Moll and Davis in 1959.It has occurred and epidemic in the main cattle countries and regions around the world. In China bovine enteroviruses were also identified in Inner Mongolia, Shandong, Jilin, Heilongjiang and Beijing. The infection can cause mild diarrhea and mild respiratory symptoms, decreased appetite, bloody stools, milk yield sharply declined, causing serious economic losses to the cattle industry. At present, little information is available about the diagnosis and prevention of bovine intestinal diseases in China. Development of the diagnosis and subunit vaccines as soon as possible would contribute great significance to dairy industry.Bovine enterovirus genome is a nonsegmented single-stranded positive strand RNA, which can be directly used as mRNA translation of a multimeric protein. After a series of degradation, the multimeric protein produces 4 structural proteins(VP1、VP2、VP3 and VP4) and 7 non structural protein(2A、2B、2C、3A、3B、3C and 3D). It was reported that, epitopes locate to VP1, VP2, VP3 of the virus particle surface. And capacity to neutralizing of VP2 is best, thus the VP2 proteinit is the best choice of specific antigen of BEV. And the 3D region is conserved which can be used as the best candidate sequence of BEV molecular detection. Based on these, two aspects of research have been carried out in this paper.Firstly, study on the immunogenicity of VP2, which has laid a foundation for further study on the methods of BEV diagnosis and subunit vaccine. Firstly, the prokaryotic expression vector pET32a(+)-VP1 was constructed by using VP2 fragment from PCR product, and the recombinant protein expressed. Western-blot was used to examine the target protein. Then the purified recombinant protein was used to immunize New Zealand rabbits for preparing VP2 polyclonal antibody. At the same time, antibody titer of the antiserum was detected by iELISA and neutralization titer of the antiserum was detected by serum neutralization test. The molecular mass was estimated as 50 k Da by SDS-PAGE,which was consistent with the expected size. The result of i ELISA revealed that antibody titer of the prepared antiserum was 1:25600, and antibody neutralization titer of the antiserum was 1:107.4 by serum neutralization test. Preparation of immunogenic VP2 protein and high titer polyclonal antibody has laid a foundation for further study on the methods of BEV diagnosis and subunit vaccine.Secondly, establish molecular biology method for detection of bovine enterovirus, which laid a foundation for further study of BEV epidemiology and diagnosis.A specific, sensitive and rapid SYBR Green Ⅰbased real-time RT-PCR method was developed for detection and quantification of bovine enterovius(BEV). The assay was carried out using a pair of specific primers designed to amplify highly conserved sequence of the 3D gene of BEV in GenBank and a recombinant plasmid containing the target gene 3D was constructed as a standard control. Standard deviation and coefficient of variation was low for both intra-assay and inter-assay variability. The detection limit of the reaction was 7.13×101 plasmid copies/μl of initial templates, which was 10 times more sensitive than the conventional RT-PCR. Moreover, the assay was highly specific since all negative controls and other viruses of clinical samples failed to develop any positive results. The assay performance on field samples was evaluated on 44(41 diarrhea and 3 aerosol) samples and compared with the conventional RT-PCR assay. The results showed that 16 diarrhea samples were positive with the positive rate as 39.02%(16/41), 3 aerosol samples are all positive with the positive rate as 100%. Preliminary clinical detection results showed that the SYBR Green Ⅰreal-time PCR assay was highly sensitivity, specificity and reproducibility. The robustness and a high-throughput performance of the developed assay make it a powerful tool in epidemic diagnostic applications and in BEV research.