Expression Of IBDV VP2 Gene In Insect Cells And Establishiment Of Antibodies Detection Method

Infectious bursal disease?IBD?is caused by infectious bursal disease virus?IBDV?.IBDV can cause damage of immune organs and immune cells——bursa of fabricius and B lymphocytes.VP2 is the main structural protein and a detection marker of IBDV,which carries the major neutralizing epitope of IBDV.In this study,the VP2 gene of IBDV DQ strain was cloned and expressed by the baculovirus expression system in order to establish an antibody ELISA detection method for IBDV.The plasmid of pFastbac1-GP67-VP2,including GP67 signal gene and IBDV VP2 gene,was transformed into the strain of E.coli JM109 and identified by polymerase chain reaction?PCR?,double enzyme assay and gene sequencing.This plasmid was transformed into the host strain E.coli DH10Bac.The recombinant bacmid rBacmid-VP2,including GP67 signal gene and IBDV VP2 gene,was obtained by site-specific recombination,triple resistance screening,blue-white screening,and identified by PCR.rBacmid-VP2 was transfected into the Spodoptera frugiperda cell?Sf9?by liposome transformation.SDS-PAGE and Western-blot were used to identified recombinant protein.The titer of recombinant baculovirus was expandedand and the expression conditions was optimized to obtain the recombinant VP2protein.The recombinant protein rVP2 was purified by Ni-NTA affinity chromatography and analyzed by colloidal gold immunochromatography test strip.The results showed that the GP67 signal gene and the IBDV VP2 gene were successfully cloned.Due to glycosylation of the recombinant protein,SDS-PAGE and Western-blot showed that bands with molecular weight 49 ku and 51 ku can specifically react with IBDV positive serum.IBDV colloidal gold immunochromatography test strips confirmed that rVP2 protein can react with IBDV monoclonal antibody.These results indicated that the recombinant protein were successfully expressed and has a good biological activity.The purified rVP2 protein and the serum were used as a coating antigen and standard serum to establish an IBDV antibody ELISA assay.First,IBDV positive and negative serum were developed.14-day-old chickens?IBDV antibody was negative?were immunized by IBDV S706 vaccine and challenged with IBDV NB strain.On 14-day challenge,the titer of serum was tested and the serum was collected from the chickens after boost immunization twice.The chicken negative serum was developed at the same time and tested by infectious bursal disease virus antibody test kit.Second,the purified rVP2 recombinant protein was used as the coating antigen,and the IBDV positive and negative serum were used as standard serum to establish an IBDV antibody ELISA assay.The experiment conditions was optimized and 30negative serums was determined by the ELISA method established in this study to determine the detection critical value.Finally,10 IBDV positive serum were detected by the ELISA method to establish the critical value of the effective antibody and to draw the maternal antibody growth curve.The serum identification results showed that the AGP titer were1:32 and the results of negative serum antibody test was negative.It indicates that the prepared serum has good reactivity and can be used as IBDV standard positive serum and standard negative serum.The optimal coating concentration of antigen is 0.2?g·mL^-11 and the blocking time is 1 h.The dilution of the serum to be tested is 1:400 and the incubation time is 30 min.The optimal dilution of the secondary antibody was 1:3000 and the optimal incubation time was 1 h.The cut-off value tests showed that the negative cut-off value is OD₄₅₀?0.4 and the positive cut-off value is OD₄₅₀?0.49.The positive threshold of immunoassay is S/P?0.22 and the sample dilution is higher than 1:400.If the sample was above the condition,it can be judged to be positive.So the sample is considered to have an effective antibody against IBDV.In a word,the IBDV VP2 protein was successfully expressed by the baculovirus expression system.An ELISA assay for IBDV antibody was established,which provides the material foundation for antibody level monitoring and vaccine evaluation of IBDV.