| At the end of 2010,outbreaks of epidemic diarrhea occurred in many provinces and cities in China,causing the death of millions of piglets and huge economic losses to the pig industry in China and the world.At the same time,clinical epidemiological studies have found that there are still high levels of morbidity and mortality in vaccine-immune pig farms.In order to understand the genetic variation of the prevalent PEDV strains,specific primers were designed based on the genome sequence of the PEDV classical CV777 strain in Gene Bank.RT-PCR was used to amplify the S gene and ORF3 gene in clinically suspected PEDV samples.Cloning and sequencing,and splicing to obtain 8 S gene and ORF3 gene sequences.Further genetic evolution and homology analysis results showed compared to the classic CV777 strain,S and ORF3 genes of the strains in this study ranged from 93.6 to 94.1% and 95.7% to 99.6% nucleotide homology,respectively.The deduced amino acid sequences of the S protein showed that there were 91 amino acid mutations in the S1 region,accounting for 61.9%(91/147)of the total,suggesting that the S1 region of PEDV is more prone to mutation than S2 region.There were amino acid insertions and deletions in the S1 and S2 regions.There are 17 amino acid mutations in ORF3 protein,and no deletion or insertion is observed.These results showed that the current PEDV epidemic strains have been mutated and it has revealed to a certain extent the reason why the immune pig herd is still onset in China.Studies have shown that the S1D gene in the 636~769 amino acid region of the S protein gene can induce the production of neutralizing antibodies,and the prepared immune sera can neutralize the S natural protein of PEDV.The author successfully expressed a target protein with a molecular weight of approximately 21 k Da through the prokaryotic expression system p ET-28 a.The fusion protein was successfully obtained after optimization of the inducing conditions of the recombinant protein and purification by chromatography.Using porcine-derived PEDV whole virus positive serum as primary antibody,Western-blot results showed that the recombinant protein had a target band on the NC membrane,and the empty plasmid expressing bacteria control was established,indicating that the protein has good reactogenicity.After the purified recombinant S1D protein was emulsified with Freund’s adjuvant and immunized with New Zealand white rabbits three times,rabbit polyclonal serum against recombinant PEDV S1D protein was obtained.Western Blot showed specific responses.After PEDV whole virus was used as the coating antigen,the titer of the polyclonal sera was determined by the indirect ELISA method established by the square array method to be higher than 1:12800,indicating that the rabbit polyclonal serum was prepared and has good specificity and S1D protein has good immunogenicity.Recombinant S1D protein was used as coating antigen,serum to be tested was used as the primary antibody,and rabbit Anti-pig Ig G/HRP was used as the secondary antibody.An indirect ELISA method for detecting PEDV antibody was established by optimizing the reaction conditions.The amount of recombinant S1D protein as coating antigen was 1 μg/well,and it was stored at 37°C for 2 hours and then coated overnight at 4°C.The optimal blocking condition was 1% BSA 37°C for 2 hours,and the serum to be detected(200-fold dilution)was used at 37°C for 1 h,the secondary antibody was diluted(6000-fold dilution)at 37℃ for 1 h.TMB colouring solution was incubated at 37°C for 15 min.The OD450 of 30 PEDV-negative sera detected by indirect ELISA was established.The positive and negative serum critical values were the mean values of negative sera plus three times the standard deviation(X+3SD=0.310)and the mean of negative sera.Plus 2 times the standard deviation(X+2SD=0.279),during which it is doubtful to redetermination.No cross-reactions were found between positive sera and porcine transmissible gastroenteritis virus,hogcholera virus,pseudorabies virus,porcine reproductive and respiratory syndrome virus,and porcine circovirus,showing that the method is good specificity.The coefficient of variation in the repeated test in batch was less than 5% and the coefficient of variation between batches was less than 10%,indicating that the established indirect ELISA method had good repeatability and stability.The indirect ELISA method was applied to the detection of PED antibody from 69 clinical swine sera from pig farms in Henan provinces.Fifty-five samples for PED antibody were detected to be positive.The positive rate was 79.71%(55/69).In summary,the indirect ELISA method established in this study was specific,and can be used for the detection of PED antibody in swine serum,providing a practical and feasible technical means for clinical sera-epidemiological investigation of PED in pigs and vaccine efficacy assessment. |
