构建以受体为靶点的基因重组酵母系统检测β2-受体激动剂残留研究

β2-agonists are well-known for their ability to improve growth rate when misused to farm animals. The residues of the misused drugs that accumulated in animal tissues can cause symptoms of acute poisoning in humans. During the monitoring process, various analytical methods have been reported. Because of the relatively complicated operation, the methods for the detection of β2-agonists can’t satisfy entirely residue monitoring. So it is necessary to establish a new analytical method for residue detection of β2-agonists in the animal food. This study is to conducted to construct the recombinant pig β2-adrenoceptor(pβ2AR) gene fluorescenct yeast cell of NNY19/pβ2AR-FUS1-yEGFP. The recombinant cell is a kind of yeast cell sensor, whose yEGFP reporter gene regulated by pβ2AR. When β2-agonists binded to pβ2AR, the expression level of yEGFP gene will increase markedly. So we can monitor β2-agonists in sample by measuring fluorescenct intensity of yEGFP. Validation parameters for quantification of 30 β2-agonists are obtained under the optimal conditions. The accuracy and precision of the method are examined by the interday and intraday reproducibilities using spiked blank samples. This method will be applied to real samples including animal edible tissue, feed and urine samples. This method is especially suitable for large samples screening during routine monitoring. Overall, the proposed method is convenient, fast, reliable and sensitive, and can be applied in large scale supervision of illegal usage of β2-agonists.

以“瘦肉精”为代表的β2-受体激动剂在食品动物违规应用，造成的残留严重威胁消费者健康，且当前违规使用β2-受体激动剂的手段和品种不断翻新，现有检测方法不能满足监测需要。以受体为靶点的基因重组酵母技术在环境污染物监测中已发挥重要作用，但尚未在兽药残留检测领域应用。本实验室已完成酿酒酵母的遗传改造，使其能准确模拟外源受体功能。我们计划将猪β2-受体基因和绿色荧光蛋白报告基因导入该酵母，筛选出具有可靠反应特性的细胞株，并通过对β2-受体基因进行突变，优选出反应特异性低(可与多种激动剂反应)、灵敏高(检出限低)的表型，用于构建β2-受体激动剂的基因重组酵母检测系统，运用该系统检测动物可食性组织、饲料及尿液三类样品中多种β2-受体激动剂残留。与其他检测方法相比，本方法检测范围广、灵敏度高、成本低，其样品对象多样、采集方便，可实现活体检测，适用于经常性、大规模的动物性食品安全监控。

首先，构建猪的β2肾上腺素能受体 （β2AR）基因及其突变体真核表达载体，并鉴定其在HEK293T细胞中的表达。结果显示，猪β2AR基因已正确重组入pcDNA3.1(+)载体中；经测序鉴定，猪β2AR的第130位天冬氨酸和第285位半胱氨酸分别成功突变为天冬酰胺和丝氨酸。并证明所构建的表达载体均可在HEK293T细胞中表达。其次，将猪β2AR基因野生型和突变体表达载体转染HEK293T细胞，再用不同激动剂和拮抗剂分别以不同浓度进行孵育，提取蛋白进行Western Bolt检测相关信号通路蛋白表达水平。结果表明，随着激动剂（拮抗剂）浓度增加，各相关信号通路蛋白表达水平均出现相应变化，成功构建了猪β2AR野生型的真核表达载体及两个单点突变和一个双点突变的突变体。并证明突变体D130N处于易被活化状态，更适合用于β2受体激动剂受体分析残留检测。第三，将野生型猪β2AR和报告基因 (Lac Z)构建的质粒pRR- pβ2AR /Lac Z转入酵母细胞。利用4种不同的β2受体激动剂刺激重组酵母细胞，验证其反应活性并优化刺激条件。其次，再应用随机点突变技术，筛选相比于野生型猪β2肾上腺素能受体对激动剂反应活性升高的突变体，并利用冷冻干燥技术探究保存重组酵母细胞的条件和时限，成功构建了重组β2受体基因酵母细胞检测系统。最后，在江苏部分地区养殖场采集饲料、尿样等样品，前处理后用重组酵母细胞进行初筛，测定克伦特罗当量，应用HPLC-MS方法对检出的阳性样品进行β2受体激动剂鉴别和定量分析，以探究重组酵母细胞在环境β2受体激动剂残留检测中应用的准确性。本研究成功构建优化了重组β2受体基因酵母细胞检测系统，并证明可用于环境、饲料、尿样中β2受体激动剂的残留检测，该检测系统除具有快速、高效、灵敏的特点外，重复性和稳定性亦达到检测要求，为建立便捷、准确的环境中激素类药物残留检测方法奠定了基础。