Antigen Epitope Analysis and Establishment of Rapid ELISA for Phosphinthricin Acetyltransferase
LIANG Yu-Xin1,3, LI Zhong-Peng1, ZHANG Chun-Yu1, HOU Ji-Chao1,3, LI Xiao-Yu1,*, WANG Yong-Zhi1,2,*
1 Institute of Plant Protection, Jilin Academy of Agricultural Sciences, Gongzhuling 136100, China;
2 Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China;
3 College of Plant Protection, Jilin Agricultural University, Changchun 130118, China
Abstract：With the continuous expansion of genetically modified soybean market, Chinese citizens have become more and more disputable about the food safety and environmental risks of genetically modified soybean (Glycine max). More and more countries require labeling of genetically modified food, and some countries also have requirements on the content of genetically modified ingredients. Therefore, in order to establish a rapid and effective detection method for Bar-transgenic soybean quantitative detection, 18 strains of phosphinothricin acetyltransferase (PAT) monoclonal antibody (MAb) with good specificity were prepared in mice (Mus musculus) immunized with purified PAT protein, and a panel of monoclonal antibodies (9F5 and 3G12) that can be used for sandwich enzyme linked immunosorbent assay (ELISA) were screened using the segmented expression peptides of PAT expressed in Escherichia coli, and a rapid quantitative double antibody sandwich ELSIA (DAS-ELISA) for PAT was established based on this panel of monoclonal antibodies. ELISA plate coated with the capture antibody 9F5, and co-incubated with detector antibody 3G12 (2 μg/mL) labeled with HRP for 30 minutes at 37 ℃, color rendering at room temperature and away from light for 15~20 minutes. The detection limit was 1.69 ng/mL and detection range of PAT protein was 0.5~8.0 μg/mL. The coefficient of variation in the plate and between plates was less than 10%. With high sensitivity and stability, the detection could be completed in 50 minutes. This method provides technical support for detection of Bar-transgenic soybean.
[1] 白卫滨, 孙建霞, 姜桂传, 等. 2007. ELISA方法定量检测转基因大豆及其产品的研究[J]. 食品与发酵工业, 33(11): 103-106.
(Bai W B, Sun J X, Jiang G C, et al.2007. Study on the quantitative detection for genetically modified soybean and processed products by ELISA method[J]. Food and Fermentation Industries, 33(11): 103-106. )
[2] 高旭东, 王永志, 时圣凤, 等. 2013. 膦丝菌素乙酰转移酶的表达及单克隆抗体制备[J]. 细胞与分子免疫学杂志, 29(05): 500-503.
(Gao X D, Wang Y Z, Shi S F, et al.2013. Preparation of monoclonal antibody against phosphinothricin acetyltransferas[J]. Chinese Journal of Cellular and Molecular Immunology, 29(05): 500-503. )
[3] 李明. 1999. Bar基因[J]. 中山大学研究生学刊: 自然科学与医学版, 20(4): 1-4.
(Li M.1999. Bar gene[J]. Journal of the Graduates Sun Yat-Sen University (Natural Sciences Medicine), 20(4): 1-4. )
[4] 李小宇, 张春雨, 郭东全, 等. 2016. 双抗夹心ELISA检测转Bar基因抗除草剂大豆[J]. 食品科学, 37(04): 222-225.
(Li X Y, Zhang C Y, Guo D Q, et al.2016. Double-antibody sandwich ELISA for the detection of transgenic Bar gene herbicide-tolerant soybeans[J]. Food Science, 37(04): 222-225. )
[5] 李忠鹏. 2013. 转基因靶分子CP4-EPSPS单克隆抗体的制备及其ELISA检测方法研究[D]. 硕士学位论文, 吉林农业大学, 导师: 胡耀辉, pp. 27-28.
(Li Z P.2013. Preparation of monoclonal antibodies against transgenic target molecule CP4-EPSPS and study on its detection method by sandwich ELISA[D]. Thesis for M. S. , Jilin Agriculture University, Supervisor: Hu Y H, pp. 27-28. )
[6] 刘志浩, 高丽丽, 王新桐, 等. 2013. 转基因玉米中膦丝菌素乙酰转移酶双抗体夹心ELISA检测方法的建立[J]. 西北农林科技大学学报(自然科学版), 41(11): 45-50.
(Liu Z H, Gao L L, Wang X T, et al.2013. Development of monoclonal antibody based sandwich ELISA to detect PAT protein in genetically modified maize[J]. Journal of Northwest A & F University (Natural Science Edition), 41(11): 45-50. )
[7] 王敏, 张亮, 张威, 等. 2013. 转基因作物pat蛋白的双抗体夹心酶联免疫检测方法研究[J]. 分析化学, 41(10): 1555-1560.
(Wang M, Zhang L, Zhang W, et al.2013. Development of a sandwich enzyme-linked immunosorbent assay for phosphinothricin acethl transferrse protein of genetically modified crops[J]. Chinese Journal of Analytical Chemistry, 41(10): 1555-1560. )
[8] 王立平, 王东, 龚熠欣, 等. 2018. 国内外转基因农产品食用安全性研究进展与生产现状[J]. 中国农业科技导报, 20(3): 94-103.
(Wang L P, Wang D, Gong Y X, et al.2018. Research progress and production status of domestic and overseas transgenic agriculture products and GM food safety[J]. Journal of Agricultural Science and Technology, 20(3): 94-103. )
[9] 王永志, 苏颖, 米丽娟, 等. 2011. 山羊痘病毒p32基因的克隆、表达及单克隆抗体制备[J]. 中国畜牧兽医, 38(09): 101-105.
(Wang Y Z, Su Y, Mi L J, et al.2011. Clone, expression and monoclonal antibody preparation of p32 gene of GPV[J]. China Animal Husbandry & Veterinary Medicine, 38(09): 101-105. )
[10] 文静, 郭勇, 邱丽娟. 2020. 耐草甘膦转EPSPS/GAT大豆多重PCR检测体系的建立及应用[J]. 中国农业科学, 53(20): 4127-4136.
(Wen J, Guo Y, Qiu L J.2020. Establishment and application of multiple PCR detection system for glyphosate-tolerant gene EPSPS/GAT in Soybean[J]. Scientia Agricultura Sinica, 53(20): 4127-4136. )
[11] 文雪霞, 陈化兰, 熊永忠, 等. 2012. 抗原表位鉴定方法的研究进展[J]. 中国畜牧兽医, 39(07): 66-70.
(Wen X X, Chen H L, Xiong Y Z, et al.2012. Research advance in epitope identification methods[J]. China Animal Husbandry & Veterinary Medicine, 39(07): 66-70. )
[12] 吴曰程, 王玉斌. 2019. 中国转基因大豆进口及其影响分析[J]. 大豆科学, 38(04): 635-643.
(Wu Y C, Wang Y B.2019. The effect of China's GM soybean imports[J]. Soybean Science, 38(04): 635-643. )
[13] 徐晶晶, 李迪, 曹永生, 等. 2013. 辣根过氧化物酶标记兔抗丝状噬菌体衣壳蛋白抗体的制备与初步应用[J]. 黑龙江畜牧兽医, 03(05): 114-116.
(Xu J J, Li D, Cao Y S, et al.2013. Preparation and preliminary application of the rabbit antibody against capsid protein of the filamentous phage labeled with horseradish peroxidase[J]. Heilongjiang Animal Science and Veterinary Medicine, 03(05): 114-116. )
[14] 许文涛, 黄昆仑, 邓爱科, 等. 2006. 转基因产品中PAT蛋白的酶联免疫检测(英文)[J]. 农业生物技术学报, 14(2): 250-254.
(Xu W T, Huang K L, Deng A K, et al.2006. Enzyme linked immunosorbent assay for PAT protein detection in genetically modified rape[J]. Journal of Agricultural Biotechnology, 14(2): 250-254. )
[15] 姚东旻, 张磊, 张鹏远. 2020. 一样的科学, 不一样的政策—转基因产品标识政策差异的博弈分析[J]. 财经研究, 46(04): 63-78.
(Tao D W, Zhang L, Zhang P Y.2020. The same science, different policies:game analysis of the differences in the labeling policy of GM products[J]. Journal of Finance and Economics, 46(04): 63-78. )
[16] 尤晴, 李小宇, 张春雨, 等. 2016. 马铃薯Y病毒属三种病毒通用型单克隆抗体的鉴定[J]. 植物保护, 42(06): 76-79.
(You Q, Li X Y, Zhang C Y, et al.2016. Identification of universal monoclonal antibodies against three viruses of Potyvirus[J]. Plant Protection, 42(06): 76-79.)
[17] 查霆, 钟宣伯, 周启政, 等. 2018. 我国大豆产业发展现状及振兴策略[J]. 大豆科学, 37(03): 458-463.
(Zha T, Zhong X B, Zhou Q Z, et al.2018. Development status of china's soybean industry and strategies of revitalizing[J]. Soybean Science, 37(03): 458-463. )
[18] 张金荣, 刘岩. 2012. 风险感知: 转基因食品的负面性—基于长春市城市居民食品安全意识的调查分析[J]. 社会科学战线, (02): 218-223.
(Zhang J R, Liu Y. 2012. Risk perception: The Negative side of GENETICALLY modified Food: Based on the investigation and analysis of food safety awareness of urban residents in Changchun city[J]. Social Science Front, (02): 218-223. )
[19] 张秀芳, 张宪省. 2012. 城市居民对转基因食品的认知与消费: 鲁省调查[J]. 改革, (07): 146-151.
(Zhang X F, Zhang X S. 2012. Urban citizens recognition and consumption of GM foods: survey from Shandong province[J]. Reform, (07): 146-151. )
[20] 张忠民. 2015. 转基因食品标识阈值问题研究[J]. 食品科学, 36(09): 254-259.
(Zhang Z M.2015. Research on the Threshold of Labeling of Genetically Modified Food[J]. Food Science, 36(09): 254-259. )
[21] 张忠民. 2016. 转基因食品标识豁免制度研究[J]. 食品科学, 37(11): 262-269.
(Zhang Z M.2016. Labeling Exemption Regulation for Genetically Modified Foods[J]. Food Science, 37(11): 262-269. )
[22] Ao J, Li Q Z, Gao X J, et al.2011. A multiplex nested PCR assay for the simultaneous detection of genetically modified soybean, maize and rice in highly processed products[J]. Food Control, 22(10): 1617-1623.
[23] Datukishvili N, Kutateladze T, Gabriadze I, et al.2015. New multiplex PCR methods for rapid screening of genetically modified organisms in foods[J]. Frontiers in Microbiology, 6(6): 757.
[24] Farías M E, Marani M M, Ramírez D, et al.2020. Polyclonal antibody production anti Pc_312-324 peptide. Its potential use in electrochemical immunosensors for transgenic soybean detection[J]. Bioelectrochemistry, 131.
[25] Hérouet C, Esdaile D J, Mallyon B A, et al.2005. Safety evaluation of the phosphinothricin acetyltransferase proteins encoded by the pat and bar sequences that confer tolerance to glufosinate-ammonium herbicide in transgenic plants[J]. Regulatory Toxicology and Pharmacology, 41(2): 134-149.
[26] Shi X B, Fan X M, Nie S Y, et al.2019. Identification of a linear B-cell epitope on glycoprotein (GP) 2a of porcine reproductive and respiratory syndrome virus (PRRSV)[J]. International Journal of Biological Macromolecules, 139: 1288-1294.
[27] Thompson C J, Movva N R, Tizard R, et al.1987. Characterization of the herbicide-resistance gene bar from Streptomyces hygroscopicus[J]. The EMBO Journal, 6(9): 2519-2523.
[28] Verginelli D, Annalisa Paternò, Marchis M L D, et al.2019. Development and comparative study of a pat/bar real time PCR assay for integrating the screening strategy of a GMO testing laboratory[J]. Journal of the ence of Food and Agriculture, 100(5).
[29] Xiao X, Wu H H, Zhou X H, et al.2012. The combination of quantitative PCR and western blot detecting CP4-EPSPS component in roundup ready soy plant tissues and commercial soy-related foodstuffs[J]. Journal of Food Science, 77(6): 603-608.
[30] Xu W T, Huang K L, Zhao H, et al.2005. Application of immunoaffinity column as cleanup tool for an enzyme linked immunosorbent assay of phosphinothricin-N-acetyltransferase detection in genetically modified maize and rape[J]. Journal of Agricultural & Food Chemistry, 53(11): 4315-4321.
[31] Xu W T, Huang K L, Deng A K, et al.2006. Enzyme linked immunosorbent assay for PAT protein detection in genetically modifed rape[J]. Journal of Agricultural Biotechnology, 14(03): 177-181.
[32] Zhang Y Y, Li M, Cui Y, et al.2018. Using of tyramine signal amplification to improve the sensitivity of ELISA for Aflatoxin B1 in edible oil samples[J]. Food Analytical Methods, 11(9): 2553-2560.
