| Since an outbreak of PED in China on October 2010, it has resulted in a large number of death of piglets and serious economic losses. Due to the high mortality rate of PED, the farmers of southern area commonly used vaccine immunity to prevent PED since the outbreak, but the result has shown a persistent infection after the outbreak in southern of China. In order to understand its causes, this experiment conducted detection of PEDV、TGEV and Rota V antigen for epidemic materials that a total of 214 which are have the performance of the diarrhea disease around the city in south China sent to tested during 2013 and 2014, select 30 PED-positive samples and were amplified by RT-PCR、cloned and sequenced for its M、ORF3 and partial S gene, and analysis the changes of sequence、changes in position of amino acid sites、 phylogenetic relationship; A primer system which is used to express the partial S gene was designed according to the CV777 sequence in Genbank, and purifing protein as the coating antigen to establish a indirect ELISA method of an detection of PEDV-Ab, at last provided the method to detect serum antibodies in pigs.The detection results of antigen show an average positive rate of PEDV is 74.8%, the average positive rate of TGEV is 7.0%, the average positive rate of Rota V is 12.1%; among the positive rate of PEDV in summer is significantly higher than in winter and spring seasons. The analysis results of sequence alignment、changes in position of amino acid sites、phylogenetic relationship show that the homology of M gene is 97.5% to 100%, the homology of amino acid is 97.3% to 100% between the sample strain, only a few amino acid site’s position has a mutation, this prove that M genes are highly conserved, the analysis results show that M gene has a close relative relationship with strains previously submitted in China, the results prove that the current strains in M gene originated from China local strain; The homology of nucleotide is 95.1% to 100%, the homology of amino acid is 96.4% to 100% between the 30 samples ORF3 gene, in this experiment 30 ORF3 gene encodes 225 amino acids, amino acid deletion does not appear, during two Interval that are10 to 30 and 70 to 90 happen more amino acid mutations, there are seven strains occur an amino acid mutation(L ' I) at 85, all of the amino acid sequence do not have contiguous amino acid mutations, showing that ORF3 gene is very conservative, phylogenetic analysis shows the experimental strains selected mostly are in two subtype, the same to southern subtypes previously submitted in south china, proving that ORF3 gene is continuous variation in southern; The results of S gene sequence alignment show that the homology of nucleotide is 96.5% ~100 % between the 30 samples,homology with the reference vaccine strains is between 88.3% and 90.5%, indicating that S gene has a larger mutation, phylogenetic analysis shows that S gene of epidemic strains in southern China has a closer genetic relationship with Thailand, Vietnam Reference strains. The results show that PEDV is prevalent in southern China, the gene is also constantly changing.Designing a primer system which is used to amplify the partial S gene in PEDV according to the CV777 sequence in Genebank, amplifying purpose fragment from diarrheal disease positive feed fragment with the RT-PCR method, and epitope-containing fragment which was cloned into the prokaryotic p ET32 a, constructing an expression vector called p ET32a-S which comprising a portion of the S gene prokaryotic expression vector, the correct p ET32a-S recombinant plasmid was transformed into an expression bacteria which is called BL21. Expression bacteria BL21 containing the recombinant plasmid after the medium was added IPTG and induced expression under the conditions of 37℃,the SDS-PAGE electrophoresis results show that the expression target protein is 46 k Da, and in the inclusion body protein form. Western-blot analysis results show that the expressed protein has a good reactogenicity. Establishing an indirect ELISA detection method for the detection of PEDV-Ab by the plates of purified S protein. The phalanx titration method to determine the optimal concentration of coating antigen was 2.5μg/m L, the best dilution of serum is 1:50, the best dilution of second antibody is 1:6000, criteria for the positive is test sample OD450 values≥0.24. According to ELISA method established in accordance with this method, comparing the test results between random sample of 94 serum samples and the commercial kit, the positive rate detected by this method is 79.7%, the rate in line with is 87%. The conclusion is that the indirect ELISA method established in this experiment can be used as an effective method for detecting PEDV-Ab Clinical Immunology monitored. |
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