Expression Of Fusion Gene Of Enterotoxigenic E.coli K88-STII-LTA2/LTB In Transgenic Tobacco

Enterotoxigenic Escherichia coli (ETEC) included heat-labile enterotoxin and heat-stable enterotoxin . Heat-labile enterotoxin (LT) was an AB5-typed mixed six-polyprotein,which was one of the strongest immunogenicity and mucosal immunoadjuvanticity. A subunit has the ADP ribosylation enzyme activity. A subunit through its A2 fragment inserted the hole of B subunit five-polyprotein, which composed a complete LT.LT-B had the ability of combining with the small intestine mucous epithelium ganglioside GM1 acceptor, which caused the virulent A subunit intermality and absorption, thus induced the whole body and the partial immune antibody response.LT holotoxin-like fused protective antigen have two functions of carrier of delivery antigen and immunoadjuvant.In this reach, with gene engineering technology the fusion gene which was antigenic determinant (CEHYRQIAKESCKKGF)of heat-stable toxin STII and K88ac protein was cloned. The fusion gene EcoR I,Sac I sites were site-specific mutated by overlap extension of PCR technique.The high performance two-gene plant expression vector Pr1as-K88ac-STII-LTA2/UP-LTB was constructed,which included ubiquitin and signal peptide sequence of pathogenesis-related protein 1a (PR1a). Recombinant plasmids were amplified by PCR, restriction endonuclease analysis and DAN sequencing. The results indicated that the recombinant plasmids were correctly constructed. The recombinant plasmid was transferred into Agrobacterium tumefaciens GV3101 respectively using the freezethaw method. Transgenic tobacco plants recovered from leaf discs applying Agrobacterium mediated gene were selected on solid medium containing Kanamycin. They were detected by PCR analysis, the results showed that the foreign gene have been integrated into tobacco genome.RT-PCR indicated the fusion gene of transgenic plantlet has expressed at the transcriptional level. Based on Western dotting, results showed that recombinant protein expressed by transgenic tobacco plants could eliciting immunoresponse to LTB and LT antibody.The results proved that the fused genes K88-STII-LTA2/LTB could expressed correctly in transgenic tobacco and had immunogenity. The study will lay a foundation for further study of new plant edible vaccines to diarrhea in alfalfa.