Expression Of Env Gene Of REV In E. Coli And Preparation And Application Of Its Monoclonal Antibodies

A cDNA encoding env gene was amplified from total cDNAs of REV by PCR. DNA sequencing indicated that the open reading frame encoding 400 amino acid residues. In order to obtain recombinant env, an expression plasmid pGEX-4T-3-env was constructed and transformed into E.coli BL21strain.SDS-PAGE indicated that the expressed fusion protein GST-env existed in the shape of cytorrhyctes. The cytorrhyctes was broken into pieces and solved by ultrasound wave and 8mol/L urea respectively.The expressed fusion protein GST-env was purified by SDS-PAGE and digested with thrombin, and the digestion mixture was subjected to SDS-PAGE again to recover env.Based on the antigenic analysis of Reticuloendotheliosis virus (REV) envelop glycoprotein (env) protein, an alternative indirect enzyme- linked immunosorbent assay (ELISA) for detection of REV antibody was developed using a truncated envelop glycoprotein(env) protein of REV produced in Escherichia coli. This assay was validated by comparison with an indirect immunofluorescence assay (IFA) and a REV-based ELISA. The diagnostic sensitivity (DSN), specificity (DSP) and accuracy of the ienv-ELISA were 87.9%, 95.2% and 92.7%, compared with IFA on 96 field serum samples, and 90.6%, 92.19% and 91.7%, compared with the REV-based ELISA on 96 field sera, respectively. Cross-reactivity assay showed that this assay was REV-specific. Repeatability tests revealed that the coefficients of variation of positive sera within and between runs were less than 15%. This ELISA is simpler to produce and perform, time-saving and suitable for large scale surveys of REV antibody at low cost. The 8-week-old female Babl/c mice were immunized with env protein. Spleen cells collected from immunized mice were fused with SP2/0 myeloma cells after the fourth immunization. The indirect ELISA test was used to screen hybridoma cells. Sixteen strains (0.5-2E12,3-2A11,3-2B3,3-2F3,1-2E3,3D2,1-2D8,1-2E9,1-1A4,1-2B5,1-2B4,1-2C1,1-2C2,1-2D1,1-2D11,1-2H8) of hybridoma secreted moloclonal antibodies (McAbs) were obtained after several subcloning.The titera of obtained McAbs from the mouse ascetic fluid were over 105 in indirect ELISA.The Western blot analysis showed the 16 strains McAbs were all specific to REV env protein. IFA determined that six strains (0.5-2E12,3-2B3,1-2E3,3D2,1-2D8,1-2H8) could recognization the natural env protein of REV.The sandwich ELISA was developed using REV env protein specificity monoclonal antibody sa detecting antibody for REV detection. The optimal conditions were determined as follows, anti-sera was diluted to 1:800,REV specificity McAb was diluted to 1:4000, and the enzyme-labeled antibody was diluted to 1:5000. The best coating buffer was CB.The cutoff value of the method was 0.274. The sandwich ELISA also had excellent specificity and repeatability.