Preparation Of The Special Monoclonal Antibody Against Porcine Circovirus Type 2

Porcine circovirus (PCV) which is suspected a potential zoonoses pathogen is one of the newly discovered animal viruses. According to the pathogenic and genomic characteristics, it can be divided into non-pathogenic type 1 (PCV1) and pathogenic type 2 (PCV2). Porcine circovirus type 2 (PCV2) is the primary causative agent of postweaning multisystemic wasting syndrome (PMWS) in pigs.it is involved in many diseases generation, such as poricine dermatitis nephropathy syndrome (PDNS), hyperplasia necrotizing pneumonia (PNP), porcine respiratory disease complex (PRDC), reproductive, congenital trembled, enteritis and other diseases, which has taken place and spreaded in the world, and mortality rate ranging from 10% to 30%. When outbreak of postweaning multisystemic wasting syndrome in more serious areaes, the farm's the death rate is as high as about 40%, so the harms and economic losses caused by the disease are clearly. At present,it has been publicly consider that the disease is the newly found important infectious disease of pigs after the porcine reproductive and respiratory syndrome (PRRS) by veterinary and raiser around the world, but there is no effective prevention and treatment measures. Therefore, how to control the disease and early detection of the disease in pig production has become the new problems.At present, traditional detection methods (for example, electron microscopy, immunofluorescence technique,in situ hybridization and immunohistochemistry method, polymerase chain reaction) which are applicable to laboratory tests can not be used in practice or large areas of serological investigation. Therefore, the establishment of a rapid, sensitive, specific methods has important significance for detecting farms-group and establishing a healthy herd.Porcine circovirus is a member of Circoviridae,which is a nonenveloped virus containing covalently closed, circular, single stranded DNA.Further researches have shown that PCV1 and PCV2 both contain two major open reading frames, which are ORF1 and ORF2. ORF1 encodes a putative protein involved in viral replication (Rep protein). Rep protein is the major reason of PCV1 and PCV2 have cross-reaction of antigenicity. ORF2-coding product is the major structural protein have no cross-reaction of antigenicity between them. only PCV2 ORF2 protein can be more resistant identification PCV2. Therefore, this study focused on the background to the use of PCV2 Jilin strains (JL 01) as the immunogen and PCV2 ORF2 prokaryotic protein is used as an indirect ELISA coating antigen to screening PCV2 specific monoclonal antibody, so this will provided foundation with establishing an accurate and rapid identification for detecting PCV 2. First,the PCV Jilin strains (JL 01 ) were inoculated to the PK-15 cell line, harvested cells from repeated freeze-thaw after 3 times, identified by PCR and purified by sucrose gradient centrifugation, and used as immunogen. According to the published ORF2 gene sequence of PCV2, in GenBank, a pair of primers that were specific to the ORF2 gene of PCV2 were designed and synthesized in the research. A fragment of 579bp of ORF2 gene was amplfied by PCR and cloned into pET-32a expression vector. The recombinant pET-32a-ORF2 plasmid was transformed into BL21 comptent cell of E.coli and induced by IPTG. SDS-PAGE analysis showed that the recombinant protein had been expressed and the expression of specific band of about plates was 40KD.The protein could react with polyclonal antibody against PCV2 by Westem-Blot test. The result declared that the expressing protein shared a good antigencity and could be used as envelope antigen. At the same time, immunizing BALB/c mice with purified PCV2 and establishing enzyme-linked immunosorbent assay method. At last,Two hybridoma cell strains against PCV2 ORF2 fusion protein named C4D2 and E3D4 respectively, were developed by indirect enzyme-linked immunosorbent assay method after fusion between Sp2/0 myeloma cells and spleen cells BALB/c mice immunized with purified PCV 2. The ELISA titers of culture supernatant were 1:1 024 and 1:512, and ascites were 1:204 800 and 1:102 400. The results of ELISA indicated that the two monoclonal antibodies were only against PCV20RF2 fusion protein but not reacted with PCV1, PPV and PRRSV indicative of its high specificity. This research provided a powerful tool for the research of PCV20RF2 gene and established the method for discriminating and diagnosing the two serotype of PCV.