Expression Of S1and M Gene Of IBVZZ2004Strain Isolated From Duck In Sf9Insect Cells And The Antigenicity Study Of Expressed Protein

The virus named IBVZZ2004strain was isolated from the diseasedduck body,with the main symptoms of growth and immune suppression. The virushas the pathogenicity to the chicken and ducks. This experiment has carried on theexpression of S1and M genes of IBVZZ2004strain isolated from duck in sf9insectcells,and tested the antigenicity of expressed protein, which provide the basicinformation for the the research on the biological function of this two proteins andthe genetic engineering vaccine.According to the complete genome sequence of IBVZZ2004strain,andrespectively,analyze S1gene and M gene sequence,the specific primers of S1andM genes were designed.A1.6kb fragments of S1gene and700bp fragments of Mgene were amplified by RT-PCR, and identified through the agarose gelelectrophoresis,which is consistent with the expected resμL ts.Using restriction enzymes to cut S1gene, M gene and the transfer vectorpFastbacHTA,respectively,then connected in the16℃water bath pot for night;the connected product were transformed into E.coli DH5competentcells,constructing restructuring transfer vector.After identified by enzyme cutting,PCR and sequencing, The sequencing of the right recombinant transfer vectorspFastBacHTA-S1and pFastBacHTA-M were,respectively, transformed into E.coliDH10Bac competent cells, the positive restructuring of the Bacmid-S1andBacmid-M were screened by blue-white method,recombination shuttle vectorBacmid-S1and Bacmid-M were constructed successfμL ly through the proof of PCRidentification; sf9insect cells in logarithmic growth phase were transfected withrecombination Bacmid-S1and Bacmid-M, respectively,after three times purificationtraining,then harvesting the recombinant bacμL ovirus and lesions cells to detect theexpression of S1and M genes; The SDS-PAGE and Western-blot tests get thespecificity of the protein strip,with the size of64kDa and31kDa, cells infected byBacmid-S1and Bacmid-M were specific fluorescence though the detection ofimmune fluorescent. The resμL ts of the study show that S1and M genes ofIBVZZ2004strain isolated from Duck were successfμL ly expressed in sf9insect cells, the S1and M protein both have antigen activity and coμL d be used as immunedetection antigen.