| Porcine reproductive and respiratory syndrome virus(PRRSV)is the causative agent of porcine reproductive and respiratory syndrome(PRRS)which is characterized by severe reproductive disorders in sows and respiratory disease in pigs of all ages.PRRSV is a spherical,enveloped,single-stranded,positive-sense RNA virus that the diameter of the virus particles with various shapes is about 50 to 65 nm.PRRSV belongs to Arterviridae family with the genus Arterivirus,order Nidovirales.As an enveloped virus,PRRSV survival outside the host is affected by temperature,p H,light and medium.The infection of PRRSV decreased with the increase of temperature because of poor stability.PRRSV maintained stability in p H6.5 ~ 7.5 and the virus’ s ability to infect declined in other conditions.Therefore,PRRSV purification work is difficult and factors such as temperature,p H must be strictly controlled.At present,sedimentation ultracentrifugation and density gradient ultracentrifugation remains the most commonly used method for PRRSV purification.However,this technique has notable drawbacks including long processing time,limited processing volume in each run and the higher requirements of instruments and equipment.In addition,ultra centrifugation sedimentation and sucrose of higher viscosity and higher permeability damaged virus particles which led to virus infection ability decline and envelope protein loss for enveloped viruses.To overcome these limitations,a scalable process was developed.A method,with the differential centrifugation,tangential flow filtration and 4 Fast Flow Sepharose gel chromatography,was established to purify the porcine reproductive and respiratory syndrome virus(PRRSV).Then the purification effect was analyzed by detecting virus particles and protein contents,and further analyzed by SDS-PAGE and western-blot.The results showed that the TCID50 of virus purified by 4FF was increased by 50 times and the removal efficiency of impurity protein was 99.68 %.In addition,SDS-PAGE showed that virus purified by 4FF were no obvious impurity protein bands.The BALB/C female mice were immunized with the purified virus and then immunogenicity was detected by immune peroxidase monolayer assay(IPMA).The results of IPMA indicated that virus purified by4 FF had better immunogenicity,high specificity,which eliminated the nonspecific reaction between the serum of the immunized mice and the protein of Marc-145 cell.According to the conventional hybridoma technology,Prepare hybridoma cell lines by the spleen cells and mouse myeloma cells(SP2/0)fusion after strengthen the immune once.The positive wells were screened by IPMA on CRL-2843-CD163 cell lines and Marc145 cell lines and 15 hybridized cell clones against PRRSV were obtained.Five hybridoma cell strains secreting monoclonal antibodies for PRRSV were got and named 1D1,9D4,6E7,7G8 and 7F12,respectively..Meanwhile the IPMA valence of abdominal dropsy were over1:106.ELISA and Western blotting test results showed that 1D1,9D4 and 7g8 three strains were against nucleocapsid N protein of PRRSV epitopes but E6E7 and 7F12 were against the other structural proteins of PRRSV epitopes.The significance of this study : Firstly,a virus purification method for PRRSV was established,which lay the foundation for the preparation of monoclonal antibodies and large-scale purification of PRRSV vaccine.Secondly,The positive wells were screened by IPMA on CRL-2843-CD163 cell lines and Marc145 cell lines established which provided experimental materials for the study of the antibody dependent enhancement of PRRSV(ADE)and the mechanism of invasion and immunologic mechanism.Thirdly,E.Coli system of expressing PRRSV recombinant N protein was successfully established and anti N protein monoclonal antibody were screened by ELISA.It lay the foundation for developing for detecting PRRSV antigen and antibody diagnostic kit and colloidal gold immunochromatographic rapid test strip and colloidal carbon rapid test strip to provide the technical means for the clinical epidemiological investigation. |
