刚地弓形虫多表位抗原基因在转基因番茄中的表达研究  

Expression of Multiepitope Antigenic Gene of Toxoplasma gondii in Transgenic Tomatoes

作　　者：周晓红[1] 黄小琴[1] 钟任佳[1] 朱巧珍[1] 赵莹[1] 郭瑜琪[1] 陈晓光[1] 

机构地区：[1]南方医科大学公共卫生与热带医学学院病原生物学系,广州510515

出　　处：《中国寄生虫学与寄生虫病杂志》2008年第6期432-437,共6页Chinese Journal of Parasitology and Parasitic Diseases

基　　金：国家自然科学基金(No.30080024;30400367;30872204);广东省自然基金(No.001052;5004747);广东省科技计划项目(No.2003c104041;73007)~~

摘　　要：目的获得稳定表达弓形虫多表位抗原基因(Tg-MAG)的转基因番茄植株。方法用植物组成型启动子E35S、番茄果实特异性E82.2启动子以及E35S-E81.1复合式启动子驱动弓形虫Tg-MAG的植物表达载体pC35MG、pCE2MG与pC35E1MG,以农杆菌介导的T-DNA转化法转化番茄子叶和下胚轴;经芽诱导、伸长以及生根的连续性抗性筛选和培育,获得Tg-MAG转基因番茄植株,练苗后移栽花盆中培育至开花、结果。用PCR、RT-PCR、蛋白质印迹(Western blotting)分别对Tg-MAG转基因植株进行DNA、RNA、蛋白水平的鉴定。结果获得的Tg-MAG转基因番茄植株经PCR和RT-PCR鉴定,得到预期360bp的Tg-MAG片段,经Western blotting筛选获得能正确表达预期相对分子质量(Mr)为11900的Tg-MAG重组蛋白,且8株具有免疫反应性,其中有2株于番茄果实中所表达的重组蛋白能与抗弓形虫速殖子主要表面抗原1(SAG1)单克隆抗体K7H3发生强免疫反应。结论获得Tg-MAG转基因番茄株系,在其果实中成功表达具有良好免疫反应性的Tg-MAG重组蛋白。Objective To obtain the transgenic tomato plant expressed muhiepitope antigenic gene of Toxoplasma gondii（Tg-MAG）. Methods Tg-MAG under the control of E35S promoter, E35S-E81.1 chimeric promoter and E8 2.2 promoter in the plant expression vectors was transferred into the tomato cotyledons and hypocotyls via Agrobocteriummediated T-DNA transformation. The candidate buds were obtained and elongated by kanamycin resistant screening. After further rooting selection, the selected tomato lines were trained and transplanted to soil and grown in a greenhouse till blossoming and bearing fruit.The transgenic tomato plants were identified using PCR, RT-PCR and Western blotting. Results Tg-MA G was introduced into the transgenie plant genomic DNA and transcribed correctly at expected size 360 bp identified by PCR and RT-PCR. Western blotting results indicated that the recombinant Tg-MAG from 8 transgenic lines, expressed in tomato fruits at expected size 11 900, 2 of them showed strong reaction band with HRP labeled McAb K7H3 against the major surface antigen 1 （ SAG1 ） of T. gondii tachyzoites. Conclusion Tg-MA G transgenie tomato lines were obtained.Tg-MAG recombinant protein with good immune activity was expressed successfully in transgenic tomato fruits.

关 键 词：刚地弓形虫 多表位抗原基因 转基因番茄 表达 

分 类 号：R382.5[医药卫生—医学寄生虫学]