Preparation Of Single-Chain Antibody Against Canine Parvovirus

Canine Parvovirus(Canine parvovirus,CPV),a single-stranded DNA virus,can cause a potent infectious diseases,so that the dogs show hemorrhagic colitis and non-suppurative myocarditis and other symptoms.For the treatment of diseases,industry preclude the use of specific treatment,and symptomatic treatment,supportive care to be assisted.Symptomatic treatment and supportive treatment can reduce major vomiting,diarrhea,dehydration and other symptoms,but can not eliminate the virus.Preclude the use of more specific therapy hyperimmune serum,canine parvovirus monoclonal antibody treatment,but hyperimmune serum titer is unstable,and the preparation of monoclonal antibodies has a complex process and here is a heterologous protein response.All these make the treatment of the disease limited.Therefore,the test aimed at preparation of single chain antibody on the basis of monoclonal antibodies.Preparation of monoclonal antibodies: Concentrated the virus by PEG,then purified by sucrose density gradient centrifugation,and injected it into the mice,after 3 times,determined the titer.Three days later,spleen cells from immunized mice were fused with SP2/0 myeloma cells by PEG1 500.Screen the hybridoma cells by ELISA,the positive cell lines were subcloned by limiting dilution method.We obtained 5 secreting monoclonal antibody-producing hybridomas,named 1G4,1E12,2C3,2H9,3F9.Detected characteristics of each cell line,including titer determination,subclasses identified,immunofluorescence,and activity.Subclass identification results showed that 1E12,1G4,3F9 were belonged to IgG1,2C3,and 2H9 were belonged to IgG2 a.The results of activity identification showed that 3F9 could neutralize canine parvovirus.Therefore,choose 3F9 to prepared the single-chain antibody.Extraction of total RNA from 3F9,reverse transcription to obtain cDNA strand,and amplified the heavy chain variable region(VH)and light chain variable region gene(VL)gene by RT-PCR.Using SOE PCR method to connect VH gene and VL gene with(Gly4Ser)3 linker peptide sequence,the single-chain antibody(ScFv)genes have 765 bp bases.ScFv genes connected with the prokaryotic expression vector pET-32a(+).Then transformed to BL21(DE3)competent cells,and induced by IPTG at 37 ℃ condition to obtain the fusion protein.SDS-PAGE analysis the fusion protein was about 46 kD,consistent with the expected results.Western-blotting result show that protein can recognized by monoclonal antibodies with anti-His-tagged specifically.Indirect ELISA and neutralization test results show that,ScFv can bind to canine parvovirus and has the activity to neutralize canine parvovirus.Five cell lines stably producing monoclonal antibody specifically bound to CPV were obtained,and the relative ScFv gene was constructed and successfully expressed in E.coli.These results may play an important role to explore the basis for the prevention and control of CPV in further.