Generation And Identification Of Monoclonal Antibodies Of Porcine Reproductive And Respiratory Syndrome Virus N Protein Recombinant Antigen

PRRSV is the main antigen that causes porcine reproductive and respiratory syndrome, two main PRRSV genotypes: European (EU) type and American (NA) type. The European type has been torturing mainland China since the end of 1995;PRRS bring great loss to the domestic pig raising industry. Tian Kegong reported in 2011 that the European type PRRSV was found in mainland China for the first time. Since then, precautions and controlling measures to finally eliminate such disease became a major challenge in China. Therefore, accurate and timely examination of the pathogeny is the key to preventing such disease from spreading. Accurate and specific monoclonal antibodie (McAb) is widely adopted in the examination of this disease for its cheap and easy operation. The Purpose of this study was to develop and identify a specific McAb which can react with both genotypes of PRRSV, laying a firm base for the up-coming development of rapid diagnostic kit.This experiment has successfully cloned NA type ORF7 gene and linked it to the express vector pET-30a(+). Then expressed it in the system of BL21(DE3) E.coli. Soluble analysis showed that the recombinant N protein was existed in inclusion body, which was proved to have good antigenicity through western boltting after renaturation. The recombinant N protein was subsequently purified by Ni2+-NTA chelating affinity chromatography. After three times immune (subcutaneously) and once fortified immune (intraperitoneally) in Balb/c mouse, two McAbs against PRRSV N protein have been obtained, designated as NA N002 and NA N008. Both of McAbs were finally identified by the indirect ELISA and western blotting using recombinant N protein and PRRSV as antigens. The subtypes of the Both of McAbs were lgG2b. The Chromosome numbers of both McAbs were 100 and 105, respectively. The ascites obtained after purification were 1:40960 and 1:20480.Specific detection was discovered that the two McAbs could simultaneously identify the two genotypes of PRRSV,with high specificity and no cross reactions to FMDV, CSFV and PCV. ELISA double antibody binding system found that both of McAbs identified the same antigenic site. We divided the NA ORF7 in two paragraphs, linked to the express vector pGEX-6p-1. The indirect ELISA detection found that these two McAbs only react to upper half of ORF7. After review of the relevant references and Sequence Alignment, we positioned the antigenic site at 25-30aa or 51-58aa, preliminarily.