携带结核杆菌Ag85B基因的植物表达载体的构建及鉴定

Construction and Identification of the Expression Plasmid Containing Ag85B Gene of Mycobacterium tuberculosis

构建包含结核杆菌Ag85B基因的植物表达载体,并转化农杆菌LBA4404。以pcDNA3-Ag85B为模板,通过常规PCR克隆出Ag85B基因,定向克隆至含有玉米特异性启动子globulin-1的pCR2.1载体上,然后将globulin-1-Ag85B的融合片段酶切下来,并连接到经过加工修饰的含有抗除草剂基因bar的双元表达载体pCAMBIA1300上,将重组质粒转化至农杆菌LBA4404中。重组质粒双酶切可见0.8bp和10kb的两条特异性条带,与预期大小相一致。重组质粒测序表明克隆的Ag85B基因序列与Genbank上相一致,酶切从农杆菌中所抽提的质粒,片段大小与预期相一致。成功构建与转化了携带结核Ag85B基因的植物双元表达载体,为利用植物反应器生产口服结核疫苗奠定基础。

To construct the plant expression plasmid containing Mycobacterium tuberculosis Ag85B genes, and transform the recombinant plasmid into Agrobacterium tumerfaciens LBA4404. We used pcDNA3-Ag85B as template to amplify AgSgB gene by polymerase chain reaction （PCR）, and cloned Ag85B gene into the vector pCR2.1. The fusion fragment of promoter globulin-1 and the target gene Ag85B which get from double enzymes digestion of the recombi- nant plasmid pCRGAg85B was inserted into the plasmid pCAMBIA1300 which contains the bar gene for herbicide re- sistance,transformed recombinant plasmid pCAMG-Ag85B into Agrobacterium tumerfaciens LBA4404. Restriction en- zyme digestion demonstrated that the inserted gene fragments were 0.8 bp, DNA sequence analyzing revealed that the Ag85B gene sequences were totally consistent with the GenBank reported. We have successfully constructed expression plasmid containing Ag85B gene and transformed it into LBA4404,and lays a foundation for further study a better vac- cine against MTB.