Cloning Of Foot-and-Mouth Disease Virus Integrin Receptor β₁ Subunit And Antibody Production To Its Ligand-Binding Domain

Objective:Laid foundation for exploring foot-and-mouth disease virus how to utilize integrinαvβ1 and knowing what function thatβ1 play,theβ1 Gene of foot-and-mouth disease virus integrin receptor was cloned from bovine lung,then constructed its euokaryotic expression recombinant plasmid.Expressed ligand-binding domain ofβ1 Gene in E.coli BL21(DE3)and purified,then immunized rabbits preparing of its polyclonal antibody,and the immunoreactivity and immunogenicity of the recombinant protein was analyzed.Method:1. Designed and synthesized two pairs of specific primers referring to the beta1 gene in Genebank,the required fragment was produced by reverse transcription polymerase chain reaction (RT—PCR) from bovine lung.The amplified fragment cloned into pGEM-T easy vector, confirmed by sequencing. Then designed a pair of euokaryotic expression primers on the basis of sequence–resoults,amplified expression fragment from clonal vector pGEMbeta1, digested and purified beta1 gene subcloned into euokaryotic expression vector pCDNA3.1zero(+) ,constructed euokaryotic expression recombinant plasmid pCDNAβ1.2. The immuno-dominant epitope ofβ1 gene was chosen by computer analysis and then syncretized ligand-binding domain of ecytoplasm with six histidine, expressed LBD protein massly in E.coli BL21(DE3), and identified by SDS-PAGE . The fusion protein was purified with Ni-NTA affinity chromatography and immunized New Zealand rabbits preparing of its polyclonal antibody .Antibodies titers to LBD in serum were detected by indirectted ELISA and determined antigentiy of the fusion protein. Salt out to purified antibody. Specificity of the antibody was examined by western blot.Results:The result of 1% gelose gel electrophoresis about Products of RT-PCR was disperse ,followed by a nested PCR with another pair primers amplifying 2400bp fragment; The complete coding sequence for the bovineβ1 subunit comprised 2,394 nucleotides coding for a 798-amino-acid-residue protein, consisting of a 20-residue signal peptide, a 708-residue ectodomain, a 29-residue transmembrane domain, and a 41-residue cytoplasmic domain, amino acid similarities between mammal integrinβ1 subunits exceed 94 percent; results of PCR,restricted enzymes and sequencing showed that successfully constructed euokaryotic expression recombinant plasmid pCDNAβ1;Analysed antigentiy ofβ1 gene by Expasy software and referred foreign interrelated reseraches,then ascertain 346-843bp to be ligand-binding domain and syncretized with six histidine ;The SDS–PAGE resoult confirmed that a body-bagged fusion protein with molecular weight about 26KDa was attained after expression and purification. Specific response were elicited after introducing LBD in New Zealand rabbits and the specific antibody titer was above 1:12800 detected by indirect ELISA:The result of Western blot showed that this antibody could be recognized by LBD antigen.Conclusion:1. cloned the completeβ1 Gene of foot-and-mouth disease virus integrin receptor and successfully constructed its euokaryotic expression recombinant plasmid pCDNAβ1.2. Gained molecular weight near 26KD recombinant LBD protein and its polyclony antibody.3. The LBD has excellent immunongenicity and can efficiently induce the humoral responses in New Zealand rabbits.