弓形虫SAG1基因截短片段植物表达载体的构建  

Construction of the plant expression vector for the truncated fragments of SAG1 gene of Toxoplasma gondii

作　　者：周晓红[1] 陈晓光[1] 卢丽琴[2] 李林[2] 吴优[2] 咸鹏[2] 言慧[1] 吴琨[1] 

机构地区：[1]第一军医大学寄生虫学教研室,广州510515 [2]第一军医大学临床医学本科参加课外科研组

出　　处：《中国人兽共患病杂志》2004年第8期662-665,共4页Chinese Journal of Zoonoses

基　　金：国家自然科学基金 (No .3 0 0 80 0 2 4) ;广东省"十五"重大科技专项(No .2 0 0 1A10 90 2 0 5 ) ;广东省科技攻关项目 (No .2 0 0 3c10 40 41) ;广州市重大科技攻关项目 (No .2 0 0 2Z3 -T2 40 11)

摘　　要：目的　将弓形虫SAG1基因截短片段 (t SAG1) ,分别用植物组成型E35S启动子和番茄果实特异性E8启动子驱动 ,构建t SAG1植物表达载体。方法　自本室构建的pET32a t SAG1载体中PCR扩增得到t SAG1基因 ,克隆进T载体为pMD T t SAG1,酶切鉴定后进行测序确认。用BamHⅠ单酶切连接方式将t SAG1分别亚克隆进 pB35MG、pB35E1MG、pBE2MG载体 ,分别构建中间载体 pB35 t SAG1、pB35E1 t SAG1、pBE2 t SAG1载体。其中 ,pB35 t SAG1以E35S驱动t SAG1,3′端携带NOS3′终止子序列 ,组成E35S/t SAG1/NOS3′操纵子结构单元。pB35E1 t SAG1含E35S和番茄果实特异性E8启动子核心序列E81.1双启动子驱动t SAG1,组成E35SE81.1/t SAG1/NOS3′操纵子结构单元。pBE2t SAG1以番茄果实特异性E8启动子全长E82 .2驱动t SAG1,组成E82 .2 /t SAG1/NOS3′操纵子结构单元。pB35 t SAG1、pB35E1 t SAG1、pBE2 t SAG1经酶切证实后 ,分别将其中E35S/t SAG1/NOS3′、E35S -E81.1/t SAG1/NOS3′、E82 .2 /t SAG1/NOS3′操纵子结构单元以HindⅢ单酶切连接方式亚克隆进pCAMBIA2 30 0质粒中 ,构建植物表达载体 pC35 t SAG1、pC35E1 t SAG1、pCE2 t SAG1,酶切鉴定。含三种启动子驱动t SAG1的植物表达载体转化根癌农杆菌LBA4 4 0 4感受态细胞后 ,The plant expression vector for the truncated fragments of SAG1 gene (t-SAG1) of Toxoplasma gondii was constructed under control of the constitutively expressed cauliflower mosaic virus(CaMV) 35S promoter and the tomato fruit-specific E8 promoter.The t-SAG1 was amplified from pET32a-t-SAG1 vector by PCR and subcloned into pMD-T vector.After identification by restriction enzyme digestion and sequencing,the t-SAG1 was cleaved from pMD-T-t-SAG1 and ligated into pB35MG,pB35E1MG and pBE2MG vectors to construct the transfer plasmids pB35 t-SAG1,pB35El t-SAG1 and pBE2 t-SAG1 carrying the t-SAG1 that were driven by E35S promoter,double promoter E35S-E81.1 and E82.2 promoter respectively.The operon fragments E35S/t-SAG1/Nos3',E35S-E81.1/t-SAG1/Nos3' and E82.2/t-SAG1/Nos3' were digested out with HindⅢ from pB35 t-SAG1,pB35E1 t-SAG1,pBE2 t-SAG1,then subcloned into the pCAMBIA2300 plasmid to construct the plant expression vectors pC35 t-SAG1,pC35El t-SAG1 and pCE2 t-SAG1 respectively.After identification by enzyme digestion,pC35 t-SAG1,pC35El t-SAG1,pCE2 t-SAG1 were transformed into Agrobacterium tumefaciens strain LBA4404 competent cells and verified by PCR.The experimental results showed that all the recombinant vectors had the inserts with expected length of the target fragments,and the amplified products of PCR had a predicted legth of 700bp.The open reading-frame of t-SAG1 was completely corrected as shown by the results of sequencing.It is concluded that the transfer vector pB35 t-SAG1,pB35E1 t-SAG1,pBE2 t-SAG1 and their corresponding plant expression vectors pC35 t-SAG1,pC35El t-SAG1,pCE2 t-SAG1 are successfully constructed in the present study,and these results can provide basis for the further studies on the development of the transgenic tomato oral vaccines.

关 键 词：刚地弓形虫 SAGl基因截短片段 番茄果实特异性启动子 植物表达载体 

分 类 号：R382.5[医药卫生—医学寄生虫学]