Cloning And Protocaryotic Expression Of ETEC K88ac-STâ…¡ Fused Gene

To testify the ability of K88ac fibriae to display heterologous epitopes, overlap extention PCR was used to construct K88ac-STII fused gene. The fused gene was expressed in Echerichia.coli.According to K88ac structural gene,we devised a pair of primers,which included NcoI and XhoI restriction endonuclease,abtained 846 bp fragment by PCR,digested this fragment by BsaJI restriction endonuclease, extracted both 536 bp fragment and 310 bp fragment,individually .Synthesizing 87 base oligo strain,in which part of fragment encodes STII structural gene,we abtained K88ac-STII fused gene by another two PCR.Ligating the fused gene to pBS-T vector,the ligation mixture was use to transform E.coli DH10B.The transformants were screened on ampicillin/Xgal-IPTG plates,the recombinant, T-K88ac-STII,was identified with restriction endonucleases (XhoI and NcoI).Result of sequencing reveals sequenced gene was in correspondence with the object gene.Digesting both vector pET-28a and recombinant plasmid T-K88ac-STII,extracting 5.2 kb and 896 bp fragment individually,ligating 5.2 kb and 896 bp fragment,transforming the ligation mixture to E.coli DH10B,we abtained recombinant plasmid pET-K88ac-STII ,identifying by PCR and NcoI/XhoI digestion,we transformed recombinant plasmid pET-K88ac-STII to E.coli.BL21,inducing expression of bacterial protein by IPTG.We observed a special and high expression in 25-35Kd by SDS-PAGE,extracting protein deposit,activating inclusion, agar immunodiffusion assay proved expression of K88ac protein.We further proved K88ac fimbriae can be taken as an exposure vector for heterologous antigentic determinants.All those reveal that this strain may be used as a new way to prevent diarrheal disease in piglets.