玉米特异启动子驱动下结核hsp65基因植物表达载体构建及鉴定

以本实验室构建的pEGHLE为模板经聚合酶链反应(PCR)扩增出Hsp65基因，连接到含有玉米特异性启动子globulin-1的pCR2.1载体上；将globulin1-Hsp65联合片断切下连到含有抗除草剂基因bar的pCAMBIA1300载体中；电击法将重组质粒转化到农杆菌LBA4404中。结果表明：成功构建了pC1300GHsp65质粒，酶切鉴定得到3 Kb和8.6 Kb两条带，测序分析表明，克隆的Hsp65与NCBI上公布序列一致；成功转化到农杆菌中，酶切从农杆菌中所提的质粒条带大小与预期结果相符合。结果显示，已成功构建和转化了pC1300GHsp65质粒,为成功研制利用转基因植物生产抗结核口服疫苗奠定了基础。

Construction of Vector for Expression of hsp65 Gene Driven by Maize Endosperm-specific Promoter

The DNA of Hsp65 amplied from pEGHLE by polymerase chain reaction (PCR) were cloned into the vector pCRG. The combine fragment of promoter globulin and the target gene Hsp65 which got from doubled enzymes digestion of the recombined plasmid pCRGHsp65 was inserted into the plant express vector pCAMBIA1300 which contain the bar gene for herbicide resistance. The recombinant plasmid was analyzed by restriction enzyme digestion and the inserted target genes in the pC1300GHsp65 were verified by nucleotide sequencing. Then was transformed pC1300GHsp65 vector into Agrobacterium Tumerfaciens LBA4404 by electroporation. The results indicated that the binary expression plasmid, which could express Hsp65, was correctly constructed after detected by restriction enzyme. The result showed that the recombinant vector containing Mycobacterium tuberculosis Hsp65 was constructed successfully and transformed into LBA4404, and laid a foundation for further study on its immunity effectiveness against MTB.