| Classical swine fever virus(CSFV)is an enveloped,positive strand RNA virus,and member of the genus Pestivirus within the family Flaviviridae.Porcine parvovirus(PPV)is a non-enveloped single-stranded linear DNA virus,which belongs to the genus Parvovirus within the family Parvoviridae.Both CSFV and PPV can cause sow reproductive obstacles,threaten China's pig industry,and once culled,it will bring serious economic losses to the pig industry.At present,no effective antiviral drugs have been found for these two diseases.Therefore,the effective control of the two diseases is still based on immunity and prevention.As a new type of laboratory detection technology,the protein chip has the advantages of high throughput,high sensitivity,easy operation,and rapid diagnosis.It can be used to detect the antigen and antibody levels in pigs' blood and effectively monitor the health status of the pigs.In this study,two important non-structural proteins of CSFV NS3 and NS5B and nucleocapsid VP2 protein of PPV were expressed and purified in E.coli.,CSFV anti-NS5B polyclonal antibodies,CSFV anti-NS3 polyclonal antibodies and PPV anti-VP2 polyclonal antibodies were prepared,protein chip technology for the diagnosis of PPV antibodies was established.The main work of this study is as follow:1.Prokaryotic expression and purification of major proteins of classical swine fever virus and porcine parvovirusIn this study,the amino acid genes 239-479 of CSFV NS5B,optimized-NS3 codons,and optimized-amino acide at 156-438 in VP2 of PPV were cloned into prokaryotic expression vector pColdI and transformed into E.coli Rossetta 2(R2).The recombinant bacteria were induced with 0.1 mmol/L IPTG at 18? for 16 hours.The successfully expressed recombinant protein was purified using high-affinity nickel magnetic beads.The results of SDS-PAGE showed that the recombinant protein CSFV NS3,CSFV NS5B,and PPV VP2 were successfully expressed and purified.2.Preparation and identification of polyclonal antibodies against classical swine fever virus and porcine parvovirusIn this study,the recombinant proteins NS3,NS5B and VP2 were immunized respectively with 50-100?g/dose with Freund's complete adjuvant(1:1).On the 14th day after the first immunization,the recombinant protein was emulsified with Freund's incomplete adjuvant(1:1)for the second immunization and the third immunization.7 days after third immunization,blood was collected from the eyelid venous plexus of the mice,serum can be separated to obtain polyclonal antibodies.Western blot results showed that the three antiserum can not only recognize the prokaryotic and eukaryotic expression of NS3,NS5B and prokaryotic expression of VP2 protein,but also can identify CSFV,PPV infected cells and produce specific bands.The results of indirect immunofluorescence showed that both of the anti-blood of CSFV could react with CSFV in cytoplasm,and also recognized the eukaryotic expression of NS3 and NS5B proteins in host cells;the anti-blood of PPV could react with PPV in cytoplasm.3.Establish protein chip technology to diagnose porcine parvovirus antibodiesThe prokaryotic expressed and purified recombinant protein VP2 as a diagnostic antigen protein printed to the epoxy-coated glass slides,hybridization,blocking,washing,adding the test serum to be incubated,washing,adding a labeled secondary antibody incubation and other steps,optimization the concentration of antigen protein,incubation time of primary and secondary antibodies,optimal serum dilution and other conditions,the visible protein chip technology and Cy3-labeled protein chip technology were established successfully.The sensitivity of the visible protein chip technology can be diluted up to 6000 folds,and the sensitivity of the Cy3-labeled protein chip technology can be diluted to 12800 folds.The two protein chip methods were compared with commercial porcine parvovirus ELISA antibody detection kits,the positive detection rate,sensitivity and specifity of the two kind of protein chip chips were higher than ELISA. |
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