Preparation Of Monoclonal Antibody Against GE Protein Of Pseudorabies Virus And Establishment Of A Competitive ELISA Method

Pseudorabies is a kind of acute infectious disease caused by pseudorabies virus(PRV)in pigs,which caused neurological disorder and death in the newborn pigs,reproductive disorders in the pregnant sows,silent infection in the finishing pigs.PRV has only one serotype and PRV vaccines exhibit high protection efficacy in vivo.At present,gene deleted live vaccines are widely used in pigs,especially the gE gene deleted vaccine.In view of this,it is particularly important to establish a diagnostic method for differentiation of infected and vaccinated animals.In this study,based on the PRV strain(PRV-TX)newly isolated by our laboratory,we expressed and purified of gE protein,and prepared the monoclonal antibody(McAb)against gE protein of PRV strain,then a competitive ELISA detecting antibodies against PRV gE was established using selected McAb,which have significant value for prevention and control of the pseudorabies.1.Prokaryotic expression of gE gene of PRVAccording to the nucleotide sequence of the PRV gE genes,one pair of primer were designed to amplify the gE gene fragment.The fragment of gE was 500 bp,which encoding the main antigenic domains of PRV gE.The amplified gE gene was cloned into expression plasmid pET-32a to form recombinant prokaryotic plasmid pET-32a-gE.The recombinant prokaryotic plasmid was transferred to the competent cell BL21.Soluble recombinant gE protein of 45kDa was obtained under the condition of 16?,220 rpm,and 1 mM IPTG induction.After purified,the concentration of recombinant protein was 2.6 mg/ml.2.Preparation of monoclonal antibody against gE proteinBALB/c mice were immunized with purified recombinant His-gE fusion protein.Spleen cell of immunized mice were fused with myeloma cell(SP2/0)after the forth immunization.Indirect enzyme linked immunosorbent assay(ELISA)were used to detect the positive hybridoma cell,and limiting dilution method was performed to subclone positive hybridoma cells.The results showed that six hybridoma cell lines secreting anti-PRV gE protein were obtained,named as 2B4?3E5?4E6?5C5?5D6 and 5F9.The titers of six McAbs in ascites detected by indirect ELISA were 1:64000?1:32000?1:64000?1:16000?1:16000 and 1:32000 respectively.Western blot analysis showed that the six McAbs could specifically react with PRV.Indirect immunofluorescence assay(IFA)detection showed that the McAbs 2B4?5C5?5D6?5F9 and 4E6 could specifically react with the PK-15 cells infected with PRV.3.Establishment of a competitive ELISA methodPRV was proliferated in PK-15 cell.The harvested cell cultures were purified by sucrose density gradient ultracentrifugation.The McAb 2B4 was labelled with HRP as detection antibody and the purified virus as coating antigen,the competitive ELISA was established for detection antibody against gE.The reaction conditions were optimized for competitive ELISA,the optimal concentration of coating antigen was 0.313 mg/ml,the optimal dilution of HPR-2B4 was 1:800,the optimal dilution of the serum sample was 1:16,the optimal serum reaction time was 60 min,the optimal blocking solution was 2%BSA,and the optimal reaction time of the chromogenic liquid was 10 min.The cutoff value for positive sample was over 40%of inhibition rate.This method was used to detect the positive serum samples for PCV-1,PRRSV,PPV,CSFV,PEDV and PCV-2 respectively,all samples were negative,indicating that the method has a good specificity for the antibody of PRV.90 PRV gE antibody positive sera,60 PRV gB antibody positive sera and 70 PRV antibody negative sera were detected by the established competitive ELISA method.Compared with the results detected by the IDEXX ELISA kit,the coincidence rate of PRV gE antibody positive serum was 96.67%,and the coincidence rate of PRV gE antibody negative positive serum was 93.85%.These data indicated that the established competitive ELISA method is very specific and can be used for the detection of PRV gE antibodies,it is can also distinguish the antibody from vaccination or field virus infection,which provided an effective method for the clean-up of the pseudorabies.