| Porcine parvovirus (PPV) causes reproductive failure in sow especially first farrowing sow, manifested as sterility, abortion, stillbirth, abnormal embryo, embryonic resorption, fetal mummification and weak born pigs. The virus is omnipresent in swine all over the world. It has caused great economic losses to pig industry. It is essensial to develop a specific and sensitive method to help the epidemiology survey of PPV in our country. The PPV structural protein VP2 is the major immunogenicity protein. It can be used as a perfect diagnostic antigen. Nonstructural protein 1(NS1) is the major nonstructural (NS) protein produced during PPV infection. Developing an assay to differentiate vaccinated pigs from infeceted ones based on the NS1 will be very useful.In this reaserch, the genes encoding the major epitope domain of VP2 and NS1 were expressed in Escherichia coli expression system respectively, then the purfied recombinant VP2 and NS1 proteins were obtained. The VP2-ELISA was developed by using the purified recombinant VP2 protein as antigen. The main study contents were summarized as following:1. Prokaryotic Expression of Major Epitope Domain of Porcine Parvovirus Structural Protein VP2 and Nonstructural Protein NS1.According to the published sequence of the PPV China strain, the genes encoding the major epitop domain of VP2 and NS1 were amplified by PCR using synthetic oligonucleotide primers. The expected 509bp and 698bp fragments were amplified and cloned into pGEM-T-Vector separately, then subcloned into the downstream of T7 prometer of an expression plasmid, pET-32(a). After induced by IPTG, the fusion proteins were highly expressed in Escherichia coli BL21 (DE3). The expression products were about 39.1KDa and 45KDa proteins. The recombinant fusion protein VP2 and NS1 occupied 65.2% and 53.1% to the total bacterial protein respectively. SDS-PAGE and Western-blot identified that the purified fusion proteins could both react with the pig serum containing antibody against PPV. The expression products were purified from bacterial inclusion bodies by His·Bind affinity chromatography. The concertration of purified expression product was 1.53mg/ml and 0.35mg/ml respectively.2. The establishment of indirect VP2-ELISAThe indirect VP2-ELISA was established by using the recombinant fusion protein VP2 as antigen. The reaction condition of the VP2-ELISA were explored and optimized. The optimal coating concentration was 306ng per well, the optimal coating condition was at 4℃over night, the serum sample was diluted to 1:50, serum sample and HRP-labeled rabbit anti-porcine IgG should be incubated at 37℃for 30 minutes and 15 minutes respectively. The substrate was added and incubated at 37℃for 10 minutes before terminated with stopping solution. The cutoff was determinated to be 0.2×ODP+0.8×ODN. The results were negative when the antiserum against PCV, PRV, HCV, JEV and PRRSV were detected by the optimal procedures. The results of reproducibility intraassay shows the coefficient of variation (CV) were less than 10%. All these results indicated that this indirect VP2-ELISA has high specificity and good repeatability.Swine serum samples were detected by VP2-ELISA and the Ceditest? PPV ELISA Kit respectively. The sensitivity and pecificity of indirect VP2-ELISA estabilished in this study were 93.8% (61/65) and 81.1% (30/37) repectively. The coincidence rate between these two ELISA was 89.2% (91/102). All results indicated that the indirect VP2-ELISA estabilished in this study has high sensitivity and specificity, and can be used as a diagnostic assay to detect antibody against PPV.
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