摘要
目的针对抗虫转sck基因水稻中标记基因表达蛋白HPT的食用安全性开展初步研究,主要包括该蛋白在大米中的定性、定量研究、可能的膳食摄入量估计及其潜在致敏性评估等.方法将HPT的编码序列插入带有组氨酸标签(His-tag)的原核表达载体PBV222中进行融合表达,通过脲变性和镍柱亲和层析的方法纯化融合蛋白.用纯化后的6His-HPT蛋白免疫家兔和小鼠,分别制备相应的抗HPT多克隆和单克隆抗体,进一步将这两种抗体组成双抗夹心酶联免疫检测体系,用于检测HPT在转sck基因水稻中的表达水平.在致敏性评估方面,首先将HPT序列与已知的致敏原数据库进行比较,再利用模拟的胃肠液环境进行体外消化稳定性实验.结果重组体经表达纯化后获得了高纯度(95%)的6His-HPT融合蛋白,免疫动物后获得了高效价的抗HPT多克隆及单克隆抗体,经免疫印迹检测证明所制备的抗体可与转基因水稻中及纯化出的HPT蛋白形成特异结合带.由这两种抗体组成的双抗夹心酶联免疫检测体系用于检测HPT抗原时的灵敏度为30ng/ml,可检测出转sck基因水稻叶片中HPT蛋白的表达量约为80～150ng/ml;而在相应的转基因大米中未能检出.HPT序列经与已知的致敏原数据库进行比较未见同源性,但在体外消化稳定性实验中,HPT蛋白在模拟的胃肠液环境中均迅速降解.结论就目前的检测水平来看,转sck基因大米中所含的HPT蛋白水平极低,且对消化环境不稳定,推测该标记基因表达蛋白不易引起可观察到的食用安全性问题.但利用表达的HPT蛋白直接进行动物急性毒性及致敏性实验的工作仍有待进行,有关的结果将进一步报道.
Objective This article studied on the food safety of HPT protein （ Hygromycin B Phosphotransferase）, a kind of plant selectable marker gene expression product, which mainly included the quantity and quality detection of HPT in the SCK transgenic rice plants, the possible dietary intakes and its stability in the simulative digestive system. Methods HPT cDNA fragment was inserted into the prokaryotic expressing vector pBV222 for the expression of 6His-HPT fusion protein. The inclusion bodies were denatured by 8mol/L urea and purifiee with metal chelate affinity chromatography on Ni- NTA agarose under denaturing condition. The purified 6His-HPT were used to immunize rabbit and BALB/C mice for the preparation of the polyclonal and monoclonal antibodies, and the double-Ab sandwich ELISA to detect HPT was established then. Conceming the sensitivity assessment of HPT protein, the amino acid sequences of HPT was compared with the other allergens and the simulative digestive experiment in vitro was carried out too. Results 6His-HPT protein was obtained with the purity up to 95% . The specificity of antisera and ascites was detected by Westem blot, which showed specific binding reaction between the antibodies to the purified 6His-HPT proteins and their expressed product. The double-Ab sandwich ELISA system was established successfully with the sensitivity of 30ng/ml. The HPT expression level in the leaves of transgenic rice was proved in the range of 80 - 150ng/ml, yet we cannot detect HPT protein in the seeds of the same rice. There showed no comparability in the amino acid sequences between HPT and other allergens, and HPT protein was digested quickly in the simulated stomach and intestines liquids. Conclusion All the results showed that the expression level of HPT in the SCK transgenic rice plants is very low and they are not stable in the simulative digestive system. So we inferred that under the current detection level, HPT protein might not be able to induce any safety problem. Yet further study on the acute toxicitv and sensitivitv exoeriments bv the animals need to be carried out. and the results will be reported in the near future.
出处
《卫生研究》
CAS
CSCD
北大核心
2005年第5期549-553,共5页
Journal of Hygiene Research
基金
国家973基金资助项目(No.2001CB109007
2001CB10901)
863基金资助项目(No.2004AA212221
2002AA212041)
关键词
HPT蛋白
表达
纯化
抗体制备
食用安全性检测
HPT protein, expression, purification, antibodies preparation, food safety evaluation
作者简介
杨丽琛，女，博士，助理研究员
