| Diarrhoea is one of the important reasons of causing the death, it gives the industry caused great economic loss. In recent years, there have been new pathogenesis have been detected in pigs,pig crest virus(Porcine kobuvirus, PKV) is a new virus detected in developed diarrhea diseaseof pigs in recent years, and its positive detection rate is higher, so it caused the wide attention of scholars both at home and abroad. Crest swine virus(porcine kobuvirus, PKV) belong to the small RNA virus family picornaviridae crest viruses, it is a single strand is chain without capsule membrane RNA viruses. In domestic crest to the pig virus related research is less, and detection method is less, so we need to further perfect. In order to establish the pig crest the indirect ELISA detection of the virus, this study analyzed the sequence to login in GenBank 16 strains of pig crest virus VP1 gene and VP3, pick out the suitable to establish indirect ELISA detection method of gene, and then establish a pig crest the indirect ELISA detection method of the virus. The research content includes:1. Crest virus VP1 gene cloning and sequence analysis of pigThis study through RT-PCR method successfully cloned pigs CH441 crest virus strains VP1 gene. Consistency of VP1 gene sequence analysis, nucleotides, according to the results of the analysis of the comparative pig CH441 crest virus strains VP1 gene and other 15 strains of swine crest virus VP1 gene nucleotide, its similarity in 81.5% ~ 90.2%, suggesting that there was obvious variation VP1 gene. Amino acid homology analysis results showed that the amino acid sequence similarity was 86.6% ~ 96.9%, the results significantly higher than the nucleotide similarity, suggesting that part of the VP1 gene mutation to nonsense mutations.2. Cloning, analysis and prokaryotic expression of pig ridge virus VP3 geneThis study cloned pigs CH441 crest virus strains VP3 gene by RT-PCR method. Consistency of VP3 gene sequence analysis, nucleotides, according to the results of the analysis of the porcine CH441 crest virus strains VP3 gene and other 15 strains of pig crest virus VP3 gene nucleotide, similarity in 84.0% ~ 90.7%. Amino acid homology analysis results show that, the pig crest virus VP3 gene amino acid sequence similarity was 91.5% ~ 99.1%. The results show that both nucleotide similarity and similarity, amino acid pig crest virus VP3 gene similarity are bigger than VP1 gene, suggest VP3 gene variation is smaller than VP1 gene, more suitable for the candidate as a diagnostic methods of protein. Then using prokaryotic expression system to express VP3 protein, and with high purification VP3 protein to preparation of porcine serum.3. Indirect ELISA detection method of establishing swine crest of the virusPig crest virus diagnostic testing method is not perfect enough, is usually detected using RT-PCR technique, the detection method is mainly for virus nucleic acid detection. Although found the virus in pig serum, but has not yet been established mature serological method to test the virus antibody.This experiment will be purified pig crest virus VP3 protein as envelope antigen, successfully established indirect ELISA method of testing pig crest virus antibody, has high specificity and stability, for the pig crest virus diagnosis provides a new means. Through VP3 antibody detection method in this study, the clinical collection of 44 diarrhea pig serum samples for testing, VP3 antibody positive rate was 63.64%, the test results show that in gansu area of diarrhoea sick pigs group of pigs in the crest virus has high positive detection rate. |
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