Preparation And Application Of Monoclonal Antibodies Against N And Nsp7 Proteins Of Porcine Reproductive And Respiratory Syndrome Virus

Porcine Reproductive and Respiratory Syndrome(PRRS)is an acute contagious disease caused by porcine reproductive and respiratory syndrome virus(PRRSV),which is characterized by obvious dyspnea and sow reproductive disturbance.It is also known as"blue ear disease" due to the peculiar ear infection of pigs.Clinical manifestations such as birth,mummification,weak offspring and stillbirth have caused serious economic damage to pig industry worldwide.The N gene of PRRSV is about 372bp long,and the N protein has strong immunogenicity.It is the earliest and largest antibody produced by PRRSV infected organism,but the antibody produced by N protein does not have neutralization activity.The length of PRRSV Nsp7 gene is about 777bp.Antibody against Nsp7 is produced earlier than that against N protein.It can be used for early detection of PRRSV infection.In this study,N and Nsp7 proteins were expressed and purified,and used as antigen to immunize mice to prepare monoclonal antibodies.An indirect ELISA method was established to detect PRRSV.The specific contents are as follows:1.Concentration and Purification of Prrsv and Prokaryotic Expression and Purification of N and Nsp7 ProteinIn this study,the recombinant expression plasmids pET-28a-N and pET-28a-Nsp7 containing PRRSV N and Nsp7 genes were transformed into E.coli BL21(DE3)respectively.The recombinant proteins of N and Nsp7 were induced by IPTG and purified by affinity chromatography.SDS-PAGE,Western blot identification and Quantity One software analysis showed that the target protein had good antigenicity,and the purity of the proteins were all above 90%.PRRSV was amplified and centrifuged by sucrose gradient.Observation showed that there were obvious layers of virus in sucrose gradient centrifuge tube.Western blot was used to identify each layer of virus.The results showed that the virus mainly existed between 30%and 40%sucrose concentration.Purified virus was prepared and purified,which provided necessary materials for the subsequent preparation of monoclonal antibodies and the establishment of ELISA antibody detection method.2.Development of Monoclonal Antibodies Against N and Nsp7 Proteins of Porcine Reproductive and Respiratory Syndrome VirusBALB/c mice were immunized with PRRSV N protein and Nsp7 recombinant protein.Four monoclonal antibodies against N protein and seven monoclonal antibodies against Nsp7 were prepared by lymphoma cell fusion technique and indirect ELISA method.The specificity of the monoclonal antibodies was identified by Western blot and IFA.Four monoclonal antibodies against N protein belonged to IgG1 subclass and all light chains were kappa chains.One of the seven monoclonal antibodies against Nsp7 belonged to IgG2a subclass,the light chain was kappa chain,the other six belonged to IgG1 subclass,and the light chain was kappa chain.The results of ELISA showed that the superposition coefficients of the four monoclonal antibodies against N protein were all above 50%.Three major antigenic epitopes of N protein were selected for ELISA detection,and the antigenic epitopes identified by the four monoclonal antibodies were all 30-52aa.The epitopes of 7 strains of Nsp7 monoclonal antibody were preliminarily identified,which provided important materials for the diagnosis of the disease and the study of viral protein structure.3.Establishment of an Indirect ELISA Method for Detection of Nsp7 Protein of Porcine Reproductive and Respiratory Syndrome VirusAn indirect ELISA method for Nsp7 was established by using purified recombinant protein as coating antigen.The optimum reaction conditions were as follows:the optimal coating concentration of Nsp7 was 1 ug/mL,the coating time was 37℃ for 2 hours and 4℃ overnight,the sealing liquid was 5%defatted milk,the sealing time was 37℃ for 3 hours,the optimum dilution of serum to be tested was 1:100,the optimum action time was 37℃ for 1 hour,and the optimum dilution multiple of enzyme-labelled antibody to be tested was 1:10000,the optimal action time was 37℃ for 45min,the optimum reaction time of TMB was 37℃ for 10 min respectively.The P/N value was calculated according to the OD450 nm value of the microplate readers.It was judged to be positive when the P/N value was more than 0.43,and negative when the P/N value was less than 0.36.The ELISA method showed no cross reaction with the antibodies against classical swine fever virus,pseudorabies virus,porcine circovirus type 2,swine foot and mouth disease virus,swine brain myocarditis virus,swine senivirus and haemophilus.Compared with the commercial PRRSV antibody detection kit of IDEXX,the relative sensitivity,specificity and coincidence rate of the method were 90.36%,88.23%and 92%,respectively.It proves that this method has high specificity and sensitivity and can be used for diagnosis and epidemiological investigation of PRRSV.