Construction and expression efficiency of the recombinant plasmid pBI-Eg95 of Echinococcus granulosus in the agrobacterium tumefaciens LBA4404 strain / 重庆医科大学学报

ABSTRACT

Objective:To construct the recombinant plasmid pBI-Eg95 of E.granulosus,and to study its expression efficiency in the Agrobacterium tumefaciens(At) LBA4404 strain.Methods:Total RNA was extracted from hydatid cyst protoscoleces shattered by supersound.The Eg95 coding gene was amplified by RT-PCR,and then was cloned into the plant expression vector pBI121 to construct the recombinant plasmid pBI-Eg95.The recombinant plasmid was electroporated into At(LBA4404) strain.The recombinant At(rAt) was induced by IPTG for 0,1,3,5,7,9,11or13 hour,respectively.The expression product was identified by SDS-PAGE and Western blot assay.Results:The 471bp Eg95 coding gene was successfully amplified,and the recombinant pBI-Eg95 plasmid was successfully constructed,which was confirmed by restriction endonuclease digestion and PCR.The recombinant plasmid pBI-Eg95 was expressed successfully in the rAt,and the expression product with an approximately Mr of 16.5 kD could be detected by sera from mice infected by E.granulosus,and the expression efficiency was about 20 percent of the total bacterial protein.Conclusion:The recombinant plasmid could provide the basis for further research of the transgenic plant vaccine of E.granulosus.