Expression Of The Fusion Gene STⅠ-STⅡ-LTB Flanked By MARs In Alfalfa

In this report , first step, MARs were obtained from the genome of tobacco by PCR,β-glucuronidase (GUS) gene of plant expression vector pBI121 was flanked by the MAR to form a new plant expression vector. STI,STII gene were obtained from plasmid pGEM-5Zf (+)-STI and K88ab (LT+,ST+). The heat- stable toxin (STI,STII) were fused by TP-PCR , and then fused heat-labile toxin B subunit by restriction endonuclease. The 5′terminus of the gene encoding LTB was fused to the 3′terminus of the ST gene, a seven amino-acid linker were inserted between the ST and LTB gene. The fused gene STI-STII-LTB was cloned into plant expression vector pBI121-MARs to replace GUS gene, another plant expression vector was constructed without MAR sequence. Recombinant plasmids were amplified by PCR, restriction endo- nuclease analysis and DAN sequencing. The results indicate that the recombinant plasmids were correctly constructed.The recombinant plasmids pBI121-MARs–STI-STII-LTB, pBI121-STI-STII-LTB were transferred into Agrobacterium tumefaciens EHA105. By using co-cultivated method, the fused gene STI-STII-LTB and MARs were transferred into alfalfa cell. Transferred shoots were selected on solid medium containing Kanamycin. Transgenic alfalfa plants were found containing STI-STII-LTB gene by PCR and Southern blotting. These transgenic alfalfa plants, were confirmed expressing STI-STII-LTB recombinant protein through SDS-PAGE. Based on Western dotting, results showed that recombinant protein expressed by transgenic alfalfa plants could eliciting immunoresponse to LTB antibody.This project could offer science warrant for studying edible vaccine.