Development Of An Indirect ELISA Method For Detecting The N Protein Of Peste Des Petits Ruminants Virus

Peste des petits ruminants is a highly infectious disease dangers by Peste des petits ruminants virus(peste des petits ruminants virus,PPRV)caused mainly against small ruminants.Since 2007 in Tibet region was first discovered Ali PPR epidemic so far,the country has 23 provinces there have been regional report appears PPR outbreak of the disease to domestic livestock caused serious harm and incalculable economic losses.Published by PPRV NCBI reference strain Nigeria75 /1 N is a nucleotide sequence of the gene,a pair of primers designed to amplify PPRV N gene 1578 bp,the amplified fragment was inserted into p MD-19-T vector,cloning vector p MD-19-T-N.After the sequencing was correct,it was transformed into the competent cells DH5α,and the restriction sites Eco R I and Sac I were selected.The correct digested product was ligated into the expression vector p ET-32a(+)to construct the prokaryotic expression vector For p ET-32a-N.The expression of p ET-32a-N vector was induced by SDS-PAGE at 28℃ and6 h,and the protein was purified and purified.Western blot analysis was performed.The results showed that the PPRV N gene was expressed and the recombinant N protein was soluble protein and had good immunogenicity.The recombinant PPRV N protein was expressed as a coating antigen by prokaryotic expression.First,the use of checkerboard method of coating is optimized antigen concentration and serum dilutions,to determine the optimal concentration of coating antigen and serum dilutions;simultaneously conditions,5% skim milk blocking time by optimizing antigen coating,incubation time and an antiserum dilution degree,HRP secondary antibody incubation times and dilutions,of TMB chromogenic time,determining the optimum operating conditions established indirect ELISA.Using the optimized operating conditions,select the intra-assay reproducibility for serum samples testing;simultaneous detection of different antibody titers of serum samples by indirect ELISA established inter-assay reproducibility test;ORFV last positive serum tested,verified by indirect ELISA methods other serum specificity.A total of 94 serum samples from Tianchang,Chuzhou,Chizhou,Bozhou,Guzhen,Chaohu,Feixi and other sheep farms were detected by ELISA.The positive rate of PPR antibody was 86.4%.The results of the test with the French ID.vet company ID Screen ?PPR Competition ELISA kit with the same batch of antibody detection,the compliance rate of 96.0%.In conclusion,this study PPRV N protein expressed in prokaryotic,indirect ELISA method established good specificity,sensitivity and reproducibility.Which provides a reference method for the detection of antibodies and the evaluation of immune effect.