变异链球菌表面蛋白PAcP与霍乱毒素B亚单位融合基因在转基因番茄中的表达  

Expression of Streptococcus mutans surface protein PAcP and cholera toxin B subunit fusion gene in transgenic tomato

作　　者：顾瑜[1,2] 刘建国[1,2] 关薇薇[1] 陈筑[1,3] 白国辉[1,2] 唐琳[1,3] 管晓燕[1,2] 田源[1,2] 韩琪[1] 

机构地区：[1]遵义医学院口腔学院,贵州遵义563000 [2]贵州省高等学校口腔疾病研究特色重点实验室,贵州遵义563000 [3]贵阳市口腔医院,贵州贵阳550002

出　　处：《上海口腔医学》2014年第3期261-265,共5页Shanghai Journal of Stomatology

基　　金：国家自然科学基金(30160086);贵州省科技支撑计划项目[黔科合SY字(2012)3086号];贵州省第六批科技创新人才团队资助项目[黔科合人才团队(2013)4026号];贵州省科学技术基金项目[黔科合J字LKZ(2011)41号]~~

摘　　要：目的:在获得含编码变异链球菌表面蛋白PAcP和霍乱毒素B亚单位融合基因的转基因番茄原代种子的基础上,应用分子生物学技术检测外源基因在转基因植株中的表达,测定目的蛋白的含量。方法:PCR筛选含编码变异链球菌表面蛋白PAcP与霍乱毒素B亚单位融合基因的转基因番茄植株;提取番茄果实总蛋白,用BCA试剂盒测定番茄果实总蛋白含量;通过Western印迹检测外源蛋白的表达情况,并用ELISA法对外源目的蛋白含量进行测定。结果:PCR扩增分析可见1.6 kb特异性扩增条带,出现特异性条带的植株占总检测植株的55.6%;转基因番茄总蛋白含量为3.93 mg/mL,Western印迹结果显示,在PVDF膜上,约60 kD处出现特异性条带;ELISA测得表达的目的蛋白占番茄可溶性总蛋白的0.18%。结论:含编码变异链球菌表面蛋白PAcP和霍乱毒素B亚单位融合基因的转基因番茄子代植株能有效表达外源蛋白。PURPOSE ：To detect the protein expression level of foreign fused gene in the first generation of genetically modified tomatoes transformed with fused gene of antigen epitope PacP in surface protein of Streptococcus mutans and cholera toxin B subunit. METHODS： The total DNA in tomatoes was extracted and PCR was applied to screen the first generation of transgenic tomatoes carrying Streptococcus mutans surface protein coding PAcP with cholera toxin B subunit fusion gene. Total proteins were extracted and quantitatively tested with BAC kit. Foreign fused protein expression level was analysed by Western blotting and ELISA. RESULTS： Ten DNA samples with the full length of 1.6 kb foreign fused DNA fragment was detected by PCR among all 18 samples. The crude protein in the transgenic tomato fruit tissue was 3.93 mg/mL. Compared with the normal tomatoes, Western blotting showed that the transgenic tomato protein contained a distinct protein band with a molecular weight about 60 kD. The results of ELISA suggested that fused foreign protein level was up to 0.18% of the total soluble protein in genetically modified tomato fruit. CONCLUSIONS： The target protein encoded by fused foreign gene of antigen epitope PacP in surface protein and cholera toxin B subunit was expressed ingenetically modified tomatoes fruit.

关 键 词：变异链球菌 霍乱毒素B亚单位 转基因番茄 可食用防龋疫苗 

分 类 号：R780.2[医药卫生—口腔医学]