Establishment And Preliminary Application Of Indirect ELISA Method For Detection Of Porcine Parvovirus Antibodies Based On NS1 Protein

Porcine parvovirus（PPV）infection can cause Porcine parvovirus infection（PPI）,sow infection can cause infertility,abortion,malformed fetus,mummified fetus and even stillbirth,resulting in the death of newborn piglets,piglet infection will lead to piglet dermatitis,diarrhea and so on.Porcine parvovirus disease has a wide epidemic in China,and the infection rate is on the rise,which seriously endangers the healthy development of the breeding industry.At present,there is no reliable serological detection method for this disease,and timely detection of antibodies to this pathogen plays an important role in the prevention and control of the disease.Therefore,an indirect ELISA method for the detection of porcine parvovirus antibodies was established,and 496 clinical porcine serum samples were initially tested.The following results were achieved:1.Successful cloning of NS1 gene of porcine parvovirus Shandong isolate.The nucleotide sequence of NS1 gene was homologous similar to other porcine parvovirus NS1gene,which was 99.4%～100%.Genetic evolution analysis showed that the NS1 gene of porcine parvovirus Shandong isolate was closely related to the Korean strain N108（MH566237.1）,and the homology was the highest with the Chinese strain 16WS（KM268633.1）and the Korean strain 18KWB13（MW711829.1）,which was 100%.The amino acid sequence derived from the nucleotide sequence of NS1 gene does not contain signal peptides,has no transmembrane region domain,and has strong hydrophilicity,and the secondary structure includes 94α-helixes,37 extended chains,19β-rotation angles,162random curls,and a large number of antigenic determinants.2.Successful expression of porcine parvovirus NS1 protein.The recombinant plasmid p ET-28a-NS1 was constructed,the induction conditions were optimized,and finally a large number of NS1 proteins with a size of 45 ku were obtained by purification at 37°C at a final concentration of 0.8 mmol/L IPTG for 6 h.Identified by Western blot,it was shown that the recombinant NS1 protein had good reactogenicity.3.Successful establishment of an indirect ELISA detection method for porcine parvovirus antibodies based on NS1 protein.The antigen coating amount was 300 ng per well;serum to be tested 1:100 dilution,enzyme-labeled secondary antibody 1:5 000 dilution;The optimal blocking time,serum incubation time,and enzyme-labeled secondary antibody incubation time were 90 minutes.TMB color rendering 30 min;The critical OD_450nm value of seronegative positive was 0.317,the intra-batch coefficient of variation was between 5.1%～9.1%,and the inter-batch coefficient of variation was between 2.9%～3.6%.The specificity,sensitivity and reproducibility of this method were good.The indirect ELISA detection method of porcine parvovirus antibody was used to detect 496 porcine serum samples randomly collected clinically,of which 185 were positive,with an average positive rate of 37.30%.In summary,the NS1 gene of Shandong parvovirus isolate were first cloned and sequenced in this experiment.A recombinant plasmid expressing NS1 protein prokaryote was constructed,and an indirect ELISA detection method for porcine parvovirus antibody using NS1 protein as coated antigen was established to provide technical support for the laboratory diagnosis of porcine parvovirus.