Development And Application Of Indirect ELISA For Pocrine Circovirus Type2and Preparation Of Monoclonal Antibodies Against ORF2

Porcine circovirus2is the main pathogen of postweaning multisystemic wastingsyndrome （PMWS）, which was divided into the family circovirus, genus circovirus. PMWSwas initially recognized in weaning piglets of a Canadian herd in1991, since then PMWS hasbeen detected in virtually all regions of the world where pigs are produced. PCV2hasincreasingly been associated with various disease syndromes in pigs, such as congenitaltrembled, porcine dermatitis nephropathy syndrome(PDNS), porcine respiratory diseasecomplex. What is worse, PCV2can depress the immune system, then induced the clinicalsecondary infection. The wide spreading of the diseases poseed huge loss in many nations andregions in the world.PCV2is a nonenveloped virus, containing a circular, single-stranded DNA genome. Thelength of DNA genome is1767bp or1768bp, contains eleven open reading frames. ORF2ofPCV2is702bp in length encoding a major capsid protein that contains234amino acids(Cap).Cap can be used an optimal antigen to detect the antibody against PCV2because of its goodimmunogenicity. A truncated ORF2gene (516bp) without nuclear localization signal wascloned into pET-28a (+) vector. The recombinant protein was expressed and purified as theantigen, then established the method of indirect ELISA for PCV2, meanwhile prepare themonoclonal antibodies specific for ORF2of PCV2. Our research porvided a powerful tools ofthe research of PCV2. This study included three parts:Part Ⅰ: Cloning andexpressing ORF2gene fragments in E.coli RosettaThe truneated ORF2(516bp) gene was expressed correctly in E.coli Rosetta system. Themolecular weight of the expressed recombinant protein was about25KD, and it could reactwith polyclonal antibody against PCV2.Part Ⅱ: The recombinant protein was used as the envelope antigen，established the indirectELISA The results demonstrated that optimal concentration of recombinant ORF2protein forcoating was1μg/ml, diluted in0.06M carbonate buffer.10%FBS in0.05/%PBS’T was usedas blocking buffer. The optimal coating condition of recombinant ORF2protein for ELISAwas incubated at4℃overnight, the dilution of serum was1:80, the work concentration ofHRP-labeled secondary antibody was1:2000. the best incubation period and temperature forboth the blocking, serum samples and the HRP-labeled secondary antibody were37℃for1h,the substrate for ELISA was at37℃for15min. Meanwhile, the results revealed that theindirect ELISA has good specificity and sensitivity for the detection of PCV2antibody inserum.Part Ⅲ:Preparation and application of monoclonal antibodies specific for ORF2protein ofporcine circovirus type2Splenocytes from6-8weeks BALB/c mice immunized with purified target protein werefused with SP2/0cells to produce MAbs, which were selected with indirect ELISA. MAbs(3H3and3F2) belonged to IgG1subtype were produced. Supernatant and ascitic of themonoclonal antibodies reacted with ORF2protein in ELISA at a titerof1：1600and1：51200.The two MAbs reacted with ORF2protein specifically in western blot. This research suppliedimportant tools for detecting and studying PCV2.