Establishment Of Indirect ELISA For Detecting NSP7 Of Porcine Reproductive And Respiratory Syndrome Virus And Development Monoclonal Antibodies Product

Porcine reproductive and respiratory syndrome (PRRS) is caused by porcine reproductive and respiratory syndrome virus (PRRSV). It is an important infectious disease of swine which is characterized by severe reproductive failure in sows and respiratory distress in piglets and growing pigs. PRRS disease was first recognized in the USA in 1987 and in Europe in 1990 and since then the disease has spread widely throughout many pig-producing countries. PRRSV is classified in the genus Arterivirus, family Arteriviridae, order Nidovirales. The PRRSV genome is a single-stranded, positive-sense RNA, which is approximately 15.4 kb in length and contains nine open reading frames (ORFs). ORF 1a and ORF 1b comprise 75% of the genome size and encode a polyprotein which is co- and post-translationally processed by autoproteolytic cleavage into 14 nonstructural polypeptides (Nsps). The other six ORFs (ORF2-7) are translated into structural proteins (gp2, gp3, gp4, gp5, M and N).To further study the structure, function and immunogenicity of porcine reproductive and respiratory syndrome virus (PRRSV) Nsp7, a pair of primer for Nsp7 gene of PRRSV was designed according to the published genome sequences of PRRSV HuN4 strains available in GenBank. The Nsp7 gene of PRRSV was amplified by RT-PCR from PRRSV HuN4 strains and cloned into the prokaryotic expression vector pET30a (+). The recombinant plasmid pET-Nsp7 DNA was transformed into E. coli competent cells BL21 for expression with induction of IPTG. Expression of fusion protein in E. coli was observed by western blot.Using the recombinant nsp7 as an antigen and optimizing various conditions. The Nsp7 ELISA can be used as a differential test with high levels of sensitivity and specificity for PRRSV serology. Though detecting 70 positive serums and 50 negative serums which backgrounds are known confined that this method threshold limit value, cut off value is 0.09, sensitivity and specificity are 99.2% and 100%, respectively by using ORC software analyzed detecting result. The clinical inspection of the 260 serum samples were detected with both the established Nsp7-ELISA and the import of PRRS antibody detection kit. Result displayed that Nsp7-ELISA and IDEXX PRRS X3 antibody detection kit relevance higher than LSIVET SUIS PRRS A/S and IDEXX PRRS X3Nsp7 immunization with purified BALB/c mice were immunized mice spleen cells with myeloma SP2/0 cell fusion, through an indirect ELISA screening, limiting dilution cloning, IFA authentication, access to three hybrid antibody secreting Nsp7 cell Supernatant. The indirect ELISA results showed that three antibody (25D2, 31A8 and 43B2) titers of cell supernatant were 1︰512, 1︰1 024, and 1︰512. The titers of ascites were 1︰2.56×10~4, 1︰1.024×10~5 and 1︰5.12×10~4.