Construction of Vector for Expression of Hsp65 and Esat-6 Genes Driven by a Maize Endosperm-specific Promoter

To construct the plant expression plasmid containing Mycobacterium tuberculosis Hsp65 and Esat-6 genes,and transform the recombined vector into Agrobacterium tumerfaciens LBA4404.The fusion DNA fragment of Hsp65 and Esat-6 were amplied from pEGHLE by polymerase chain reaction(PCR)were cloned into the vector pCRG.The combine fragment of promoter globulin-1 and the target gene HLE which get from doubled enzymes digestion of the recombined plasmid pCRGHLE was inserted into the plant expression vector pCAMBIA1300 which contain the gene bar for herbicide resistance.The recombinant plasmid was analyzed by restriction enzyme digestion and the inserted target genes in the pC1300GHLE were verified by nucleotide sequencing.Then transformed pC1300GHLE vector into Agrobacterium tumerfaciens LBA4404 by electroporation.The binary expression plasmid,which could express HSP65 and ESAT-6,was correctly constructed.The recombinant vector containing Mycobacterium tuberculosis Hsp65 and Esat-6 genes is constructed successfully and transformed into LBA4404,and lays a foundation for further study on its immunity effectiveness against MTB.