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目的 构建弓形虫多表位基因(TGMG)植物表达载体。方法①将TGMG亚克隆人pBAC55构建中间载体pB35MG,再将其中E35S/TGMG/NOS3′结构单元亚克隆人pCAMBIA2300构建植物表达载体pC35MG。②将番茄果实特异性启动子E81.1插入pB35MG构建中间载体pB35E1MG,再将其中E35SE81.1/TGMG/NOS3′结构单元亚克隆人pCAMBIA2300构建植物表达载体pC35E1MG。③将番茄果实特异性启动子E82.2插入pB35MG构建中间载体pBE2MG,再将其中E82.2/TGMG/NOS3′结构单元亚克隆人pCAMBIA2300构建植物表达载体pCE2MG。测序鉴定pB35MG、pC35E1MG、pCE2MG中的TGMG序列。④pC35MG、pC35E1MG、pCE2MG转化根癌农杆菌LBA404。结果 重组质粒用酶切鉴定均得到预期片段,pB35MG、pC35E1MG、pCE2MG测序结果正确。结论 成功构建TGMG中间载体pB35MG、pB35E1MG、pBE2MG,以及植物表达载体pC35MG、pC35E1MG、pCE2MG。并将3种植物表达载体导入根癌农杆菌。
Objective To construct Toxoplasma gondii multi-epitope gene (TGMG) plant expression vector. Methods ① The TGMG was subcloned into pBAC55 to construct the intermediate vector pB35MG, and then the plant expression vector pC35MG was constructed by subcloning human pCAMBIA2300 with the E35S / TGMG / NOS3 ’structural unit. ② The tomato fruit-specific promoter E81.1 was inserted into pB35MG to construct the intermediate vector pB35E1MG, and then the plant expression vector pC35E1MG was constructed by subcloning human pCAMBIA2300 with the E35SE81.1 / TGMG / NOS3 ’structural unit. (3) The tomato fruit-specific promoter E82.2 was inserted into pB35MG to construct the intermediate vector pBE2MG, and then the plant expression vector pCE2MG was constructed by subcloning human pCAMBIA2300 with the E82.2 / TGMG / NOS3 ’structural unit. Sequencing identified TGMG sequences in pB35MG, pC35E1MG, pCE2MG. ④ pC35MG, pC35E1MG, pCE2MG transformed Agrobacterium tumefaciens LBA404. Results The recombinant plasmids were identified by restriction enzyme digestion. The sequencing results of pB35MG, pC35E1MG and pCE2MG were correct. Conclusion TGMG intermediate vectors pB35MG, pB35E1MG, pBE2MG and plant expression vectors pC35MG, pC35E1MG and pCE2MG were successfully constructed. Three plant expression vectors were introduced into Agrobacterium tumefaciens.