The Expression Of Heat-labil Enterotoxin LT In Tobacco

Heat-labile enterotoxin (LT) excreted by enterotoxigenic Escherichia coli was an AB5-typed hexyl-polyprotein,which was one of the strongest immunogenicity and mucosal immunoadjuvanticity. However the known research found that amino terminal or carboxyl terminal of LTB could form natural pentyl-polyprotein and had affinity to acceptor .But the efficiency of the pentyl-polyprotein which was combined with cell surface and chemistry stability were influenced greatly.So it reduced the capability as transmitted carrier of antigen.The immunology experiment of animal had proved that immunity of LT full toxin was stronger evidently than pentyl-polyprotin of the B subunit..People studied influence of A,B subunit to adjuvanticity function.The result found after separeted LTB and targent antigen were orally taken at one time, the adjuvanticity function was not evident.But when micro-LT and antigen were orally taken together,they manifested stronger adjuvanticity function. Currently, expression of reduced-toxin LT had been studied in chloroplast of tobacco.But expression of LT in the plant cytoplast had not reported.A subunit and B subunit from strongly nosogenetic bacilli K88ac+ was cloned respectively .We constructed two kinds of bivalent plant expression vectors.One of them was that LTA and LTB were constructed to two expression regions.We named it as pCAMBIA2300-LTA-LTB.The other was that Ubiquitin and signal peptide sequence of pathogenesis-related protein 1a (PR1a) were recombinant together by TP-PCR in the research.The fusion gene was constructed to front of A subunit and B subunit respectively. The experiment proved that the exterior protein was accumulated effectively in the plant. Then a recombinant was constructed by a set of plant expression vectors,which was named as pCUPAB.At last the recombinants were transformed into tobacco.Transformed shoots were selected on solid medium containing Kanamycin. These transformants were checked by PCR ,SDS-PAGE and Western dot which validated if pentyl-polyprotin and hexyl-polyprotein was assembled.The Western dot indicated that only few transformants were positive.The results proved that LT could be assembled correctly. The research laid a foundation for being constructed plant vaccine with holotoxin-like chimeras.