Studing On The Curative Effect Of Canine Parvovirus VP2 Protein Immunized Serum

Canine parvovirus disease is a highly contagious disease,it was caused by canine parvovirus(CPV),it is characterized by acute hemorrhagic enteritis and acute myocarditis,the morbidity and mortality of the disease were high.the clinical treatment is mainly by injecting CPV high immune serum into the sick dog,the main prevalent CPV strain of Guiyang is CPV-2a subtype,the antiserum against of CPV used in clinic is mainly prepared by CPV-2 subtype strain,although,the cure rate of canine parvovirus disease has improved,the phenomenon of treatment failure is also common.In order to explore the therapeutic effect of serum in this study on the treatment of canine parvovirus disease in Guiyang,we carried out this experiment,to provide basic data for the treatment of canine parvovirus disease.1.Prokaryotic expression and purification of canine parvovirus 2a subtype VP2 proteinThe recombinant plasmid of CPV-2a subtype VP2 gene was used as template,was inserted into the prokaryotic expression vector p ET-32a(+)by double enzyme cutting,the p ET-32a-VP2 recombinant prokaryotic expression vector was successfully constructed.p ET-32a-VP2 is transferred into E.coli BL21(DE3)competent cells for expression,the optimal expression conditions of IPTG induction concentration,induction time and induction temperature were explored.SDS-PAGE electrophoresis results show that the induced recombinant strain could express 64 ku His-VP2 recombinant protein;The best expression condition was 1 mm IPTG?37 ??5 h;The recombinant protein mainly exists in the form of inclusion body.The expressed His-VP2 recombinant protein was hanged on a nickel column by affinity chromatography,elution at different imidazole concentrations of 50 mmol/L,400mmol/L.The results of SDS-PAGE electrophoresis showed that,the purified recombinant protein appeared a band at the size of 64 ku;A single destination band was detected at 64 ku by Western Blottting.2.Preparation of immune serum against canine parvovirus 2a subtype VP2 proteinThe His-VP2 recombinant protein was was dialytic and refolding,Subunit vaccine was prepared by equal volume emulsification with Freund's adjuvant,use the BCA method determines concentration(the protein concentration is about 500 ?g/m L),5 healthy dogs were immunized by subcutaneous multipoint injection,immunized 3 times,the dose of each immunization is 1 m L(protein content is about 250 ug),the whole blood of dog was collected in 13 days after each immunization to detect ELISA antibody concentration;antibody specificity.The results of ELISA antibody test showed that: the antibody levels of the immunized dog 1,2,3,5 were the highest on the 13 th day after the second immunization,the antibody level of immunized dog 4 was the highest on the 13 th day after the third immunization.When the antibody level of 5 dogs was the highest,a large amount of immune serum was prepared;The antibody was detected by Western Blottting appeared a single target band at about 64 ku.3.study on curative effect of immune serumCanine parvovirus inoculated into cat kidney cells simultaneously and blind pass three generations,collect venom after 90% cytopathic effect(CPE),it was identified by HA and PCR method;The virus TCID50 was determined by Reed-Muench method.The results showed that the hemagglutination titer of CPV was 29;TCID50 is 107.42/100 u L;CPV NS1 gene was successfully amplified by PCR,2 007 bp in size.According to the dose of 1 m L/kg,the dogs were artificially infected with CPV by oral way.after the dog became sick,use the prepared immune sera to treat,at the same time,commercialized serum was set up as a control,the dosage was 1 m L / kg,count the cure rate of sick dogs.The results showed that vomiting,dilation and other clinical symptoms occurred in dogs after drug attack;3 dogs were treated with self-made serum,2 was cured and 1 died;3 dogs were treated with commercial serum,1 was cured and 2 died,Compared with commercial serum,the cure rate of self-made serum was higher.