Development Of An Indirect Elisa For Detection Of Porcine Reproductive And Respiratory Syndrome Virus And Preparation Of Monoclonal Antibodies Against Nsp2 Protein

Porcine reproductive and respiratory syndrome (PRRS) is a serious infectious disease of swine and characterized by severe reproductive failure in sows, and respiratory distress in piglets and growing pigs. Since it was first reported in the southern of United States in 1987, PRRS has existed all over the world and caused economic losses in the swine industry.The disease of swine, characterized by high and continuous fever, and high-mortality, occurred in the pigs in main pig-producing provinces in China in 2006, caused economic losses in the swine industry of China. It was confirmed that the highly pathogenic PRRS V mutants were the major pathogens of the disease by constantly exploration and lots of research in China.This virus belongs to the Arteriviridae family. It has a 15-kb positive-sense, which belongs to single-stranded RNA genome and contains nine known open reading frames. ORF1 encodes Non-structural polypeptides. The length of Nsp2 gene is about 2.9-kb. Because the strains have different sequences, so they are not the same at the length. Previous studies reveal that Nsp2 gene encoding Non-structural protein is the most variable protein-coding gene of PRRSV and including multiple B cell epitopes in the virus. The area of onncentration epitopes have stronger hydrophilic. During infections, Nsp2 can also stimulate the body to produce specific antibodies differenced. In this study, we performed the immunogenicity of Nsp2 as the research object. The main research works were as following:1. Development of an indirect elisa for detection of porcine reproductive and respiratory syndrome virusWe performed the the Nsp2 of PRRSV as the research object and truncated the C terminal hydrophobic transmembrane domain sequence of Nsp2, which may not affect of the immunogenicity. The major antigen region of Nsp2 was successfully amplified by RT-PCR from a highly pathogenic PRRSV (WUH3 strain), and inserted into pET28a(+) vector. The insoluble protein pET28a-Nsp2 with molecular weight of 83 kDa was expressed in E.coli and showed a strong reaction with the PRRSV positive sera in Western blot assay.The present experiment was performed with the objective of establishing an indirect ELISA for detection of PRRSV with the mixed Nsp2 fusion protein and N fusion protein as coating antigen.2. Preparation of monoclonal antibodies against Nsp2 proteinSix to eight-week-old BALB/c mice were hypodermically immunized three times at 2-week interval with 100μg of the Nsp2 protein emulsified in complete freund’s adjuvant. Two hybridoma cell strains against Nsp2 protein were screened by indirect enzyme-linked immunosorbent assay method after fusion between Sp2/0 myeloma cells and spleen cells BALB/c mice immunized with purified recombinant protein. One Hybridoma cell strain was designated as A2F1 after subcloning by three times. The results of Western-blot and IFA indicated that the monoclonal antibody can reacted with Nsp2 protein and PRRSV WUH3 strain specifically, but not with the PRRSV CH-la strain. The McAb belongs to IgGl isotype, and the light chain belongsκtype.the Elisa titer in cell cultural supernatant was 1:8000, meanwhile the Elisa titer of A2F1 in the ascites was 1:128000. The McAb will study the functions of Nsp2 gene in further and may be a useful tool that contribute to and a specific, sensitive and rapid detection methods of PRRS in future studies.