抗克伦特罗单克隆抗体的研制与鼠β_2肾上腺素能受体基因的克隆及表达
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摘要
克伦特罗临床上作为呼吸促进药物，主要用于治疗人兽哮喘。在畜牧生产中将克伦特罗作为生长促进剂使用具有明显提高肉品瘦肉率、降低脂肪含量的作用。克伦特罗在被机体吸收、转化、代谢的过程中，虽然大部分被代谢或排泄，但仍有相当量残留在肝脏、肌肉、视网膜等组织中。此残留对人畜的健康造成巨大威胁和危害，并由此在国内外引发多起食物中毒事件。研究克伦特罗药物残留检测相关技术及开展有克伦特罗样作用的安全药物的研究显得十分有意义。
本论文从研究药物残留检测相关技术与寻找安全药物两方面为导向，其研究内容包括克伦特罗偶联物的制备与鉴定、抗克伦特罗单克隆抗体的研制和鼠β_2肾上腺素能受体基因的克隆及表达。
因为克伦特罗等药物为小分子，它们只具有反应原性而无免疫原性，所以此类药物与大分子的偶联成功是产生抗体的关键，同时有关半抗原偶联物的鉴定工作少见报道，此研究以期积累鉴定药物偶联物的经验，更好作好药物免疫分析工作。在本论文中，在制备好克伦特罗的偶联物后，利用红外光谱、紫外光谱、FPLC、SDS-PAGE及免疫鉴定，确定偶联成功。本文对偶联物鉴定的几种方法作一比较与评价。通过实验，得出FPLC是以上几种鉴定偶联物的最佳方法。
抗克伦特罗单克隆抗体的研制采用常规单克隆抗体制备技术，其中包括动物免疫与血清效价的测定、骨髓瘤细胞的培养、饲养细胞的制备、免疫脾细胞的获得、细胞融合与阳性克隆的筛选、阳性克隆的单克隆化等步骤。在经过以上步骤以后，获得了分泌抗克伦特罗单克隆抗体的细胞并对初步获得的抗体进行了效价测定。单克隆抗体的研制成功将有助于克伦特罗检测试剂盒的研制。同时，有助于克伦特罗样品的前处理。
安全药物有两种思路：建立药物筛选模型筛选生物体内易分解的活性分子，从受体的抗体着手制备抗受体的抗体以及抗克伦特罗单抗的抗独特型抗体。而所有研究的前提是获得β_2肾上腺素能受体基因和抗克伦特罗单克隆抗体，所以此研究的基础工作之一是完成β_2肾上腺素能受体基因的克隆与表达工作。
鼠β_2肾上腺素能受体基因的克隆及表达实验内容包括：PCR扩增与产物回收、回收产物与载体连接、连接产物的转化、蓝白斑筛选、阳性克隆的鉴定与基因测序、序列的分析比较。通过鉴定与序列比较，确定该基因鼠β_2肾上腺素能
受体基因被克隆。在完成鼠p／肾上腺素能受体基因的克隆后，将其进行原核表
达。将表达的蛋白进行SDS－rxGE电泳以检测基因的表达情况。这些为寻找安
全药物及受体药物分析奠定了基础。
本实验的研究意义在于：为药物残留检测及食品安全提供技术支持；为寻找
安全药物奠定基础。
Clenbuerol is a potent respiratory stimulant used in human and animal medicine. This drug is , however , used illegally as a growth promoter in production animals ,because its use leads to carcass which are fat-free and which have an extraordinary development of muscular mass. While much of the Clenbuterol will be excreted or metabolized, there is still a considerable amount retained by the animals especially in the liver, muscles, and retina. As a results , the residues rendered a great danger to consumer and animal ,furthermore ,several food poisoning accidents related to the consumption of illicit β 2 agonist in liver was happened in China and other country. There is a great demand about techniques related to the detection of residue and safety drug which have Clenbuerol positive effect and no lexicological characteristics.
According to this, the content in this paper is included as follow : Synthesis and Identification of Clenbuterol -carrier protein conjugate, the Preparation of Monoclonal Antibody against Clenbuterol and Cloning & expression of (32 Adrenergic Receptor Gene of Mouse.
For the reason that small moleculars liking Clenbuterol have their immunoreactivity but no immunogenicity, thus they couldn't induce animal to produce antibody until they have been conjugated to macromolecular ,such as bovine serum albumin, human serum albumin. Unfortunately ,little study has been done to make sure that small molecular ,hapten ,has been conjugated with carrier protein. As a result, no effective method has been put forward . In this paper, some work have been done and tried to solve this problem . A conjugate prepared by the direct coupling of diazotized Clenbuterol to bovine serum albumin was identified by IR, UV , SDS-PAGE v FPLC and immunological experiment . Compared with each other, a brief assessment of each analytical method is given. A conclusion was draw that among all the analytical methods, FPLC is the best one.
Monoclonal antibody was prepared by classical hybridomas technique . The whole process included the preparation of antigen(enough for
immunization and screen),planning a screening assay, developing the screening assay, immunization the mouse, preparation the sp2/0 cell, plasma cells in spleen ,feeding cell, the fusion of the sp2/0 cell and plasma cells in spleen cell , screening to get positive clone and cloning ,colony growth. After the whole operation , positive clone which could secrete antibody against Clenbuterol was obtained, furthermore the antibody concentration was measured. Monoclonal antibody could use as standard reagents in the residue immunoassay and reagents in the treatment of sample.
There are two way to obtain safety drug , on the one hand, when a pharmaceutical screening model has been established , easy-to-catabolism bioactive molecular could be approached . on the other hand, antibody against β sadrenoceptor, which is binding with the receptor just as β 2 adrenergic stimulant, may have the same bioactivity, anti-idiotypic antibodies against anti-Clenbuterol Monoclonal Antibody could Mimics the bioactive ofβ2 adrenergic stimulant. Above all, the basis of these study is that the β 2 adrenergic receptor gene of mouse must have been cloned and the monoclonal antibody against Clenbuterol must been prepared successfully.
β2 Adrenergic receptor Gene of Chinese Kunming mouse was amplified by PCR and it was ligated with pUCm-T vector by T4 ligase . The ligation product was then transfered into competent E.coli DH5α . After screening with α-complementarity, positive clone was identified by PCR ,restrict enzyme characterization and DNA sequencing . Furthermore ,the gene was blast in NCBI . A conclusion was draw that β2 adrenergic receptor gene of mouse had been cloned. Then,β2 Adrenergic receptor gene was expressed in E.Coli and fusion protein was detection by SDS-PAGE .
All in all , The meaning all work in this paper is that providing technique associated with residue detection , lay a basis for the screening safety drug .
本论文从研究药物残留检测相关技术与寻找安全药物两方面为导向，其研究内容包括克伦特罗偶联物的制备与鉴定、抗克伦特罗单克隆抗体的研制和鼠β_2肾上腺素能受体基因的克隆及表达。
因为克伦特罗等药物为小分子，它们只具有反应原性而无免疫原性，所以此类药物与大分子的偶联成功是产生抗体的关键，同时有关半抗原偶联物的鉴定工作少见报道，此研究以期积累鉴定药物偶联物的经验，更好作好药物免疫分析工作。在本论文中，在制备好克伦特罗的偶联物后，利用红外光谱、紫外光谱、FPLC、SDS-PAGE及免疫鉴定，确定偶联成功。本文对偶联物鉴定的几种方法作一比较与评价。通过实验，得出FPLC是以上几种鉴定偶联物的最佳方法。
抗克伦特罗单克隆抗体的研制采用常规单克隆抗体制备技术，其中包括动物免疫与血清效价的测定、骨髓瘤细胞的培养、饲养细胞的制备、免疫脾细胞的获得、细胞融合与阳性克隆的筛选、阳性克隆的单克隆化等步骤。在经过以上步骤以后，获得了分泌抗克伦特罗单克隆抗体的细胞并对初步获得的抗体进行了效价测定。单克隆抗体的研制成功将有助于克伦特罗检测试剂盒的研制。同时，有助于克伦特罗样品的前处理。
安全药物有两种思路：建立药物筛选模型筛选生物体内易分解的活性分子，从受体的抗体着手制备抗受体的抗体以及抗克伦特罗单抗的抗独特型抗体。而所有研究的前提是获得β_2肾上腺素能受体基因和抗克伦特罗单克隆抗体，所以此研究的基础工作之一是完成β_2肾上腺素能受体基因的克隆与表达工作。
鼠β_2肾上腺素能受体基因的克隆及表达实验内容包括：PCR扩增与产物回收、回收产物与载体连接、连接产物的转化、蓝白斑筛选、阳性克隆的鉴定与基因测序、序列的分析比较。通过鉴定与序列比较，确定该基因鼠β_2肾上腺素能
受体基因被克隆。在完成鼠p／肾上腺素能受体基因的克隆后，将其进行原核表
达。将表达的蛋白进行SDS－rxGE电泳以检测基因的表达情况。这些为寻找安
全药物及受体药物分析奠定了基础。
本实验的研究意义在于：为药物残留检测及食品安全提供技术支持；为寻找
安全药物奠定基础。
Clenbuerol is a potent respiratory stimulant used in human and animal medicine. This drug is , however , used illegally as a growth promoter in production animals ,because its use leads to carcass which are fat-free and which have an extraordinary development of muscular mass. While much of the Clenbuterol will be excreted or metabolized, there is still a considerable amount retained by the animals especially in the liver, muscles, and retina. As a results , the residues rendered a great danger to consumer and animal ,furthermore ,several food poisoning accidents related to the consumption of illicit β 2 agonist in liver was happened in China and other country. There is a great demand about techniques related to the detection of residue and safety drug which have Clenbuerol positive effect and no lexicological characteristics.
According to this, the content in this paper is included as follow : Synthesis and Identification of Clenbuterol -carrier protein conjugate, the Preparation of Monoclonal Antibody against Clenbuterol and Cloning & expression of (32 Adrenergic Receptor Gene of Mouse.
For the reason that small moleculars liking Clenbuterol have their immunoreactivity but no immunogenicity, thus they couldn't induce animal to produce antibody until they have been conjugated to macromolecular ,such as bovine serum albumin, human serum albumin. Unfortunately ,little study has been done to make sure that small molecular ,hapten ,has been conjugated with carrier protein. As a result, no effective method has been put forward . In this paper, some work have been done and tried to solve this problem . A conjugate prepared by the direct coupling of diazotized Clenbuterol to bovine serum albumin was identified by IR, UV , SDS-PAGE v FPLC and immunological experiment . Compared with each other, a brief assessment of each analytical method is given. A conclusion was draw that among all the analytical methods, FPLC is the best one.
Monoclonal antibody was prepared by classical hybridomas technique . The whole process included the preparation of antigen(enough for
immunization and screen),planning a screening assay, developing the screening assay, immunization the mouse, preparation the sp2/0 cell, plasma cells in spleen ,feeding cell, the fusion of the sp2/0 cell and plasma cells in spleen cell , screening to get positive clone and cloning ,colony growth. After the whole operation , positive clone which could secrete antibody against Clenbuterol was obtained, furthermore the antibody concentration was measured. Monoclonal antibody could use as standard reagents in the residue immunoassay and reagents in the treatment of sample.
There are two way to obtain safety drug , on the one hand, when a pharmaceutical screening model has been established , easy-to-catabolism bioactive molecular could be approached . on the other hand, antibody against β sadrenoceptor, which is binding with the receptor just as β 2 adrenergic stimulant, may have the same bioactivity, anti-idiotypic antibodies against anti-Clenbuterol Monoclonal Antibody could Mimics the bioactive ofβ2 adrenergic stimulant. Above all, the basis of these study is that the β 2 adrenergic receptor gene of mouse must have been cloned and the monoclonal antibody against Clenbuterol must been prepared successfully.
β2 Adrenergic receptor Gene of Chinese Kunming mouse was amplified by PCR and it was ligated with pUCm-T vector by T4 ligase . The ligation product was then transfered into competent E.coli DH5α . After screening with α-complementarity, positive clone was identified by PCR ,restrict enzyme characterization and DNA sequencing . Furthermore ,the gene was blast in NCBI . A conclusion was draw that β2 adrenergic receptor gene of mouse had been cloned. Then,β2 Adrenergic receptor gene was expressed in E.Coli and fusion protein was detection by SDS-PAGE .
All in all , The meaning all work in this paper is that providing technique associated with residue detection , lay a basis for the screening safety drug .
引文
1) 陈继明，龚晓明，陆承平等．兔异源蛋白与自身蛋白作为载体制备克伦特罗抗血清[J]．南京农业大学学报．1998，2181～88
2) 陈继明，黄日新，陆承平等．酶联免疫吸附测定样品中克伦特罗含量[J]．中国兽药杂志．1999，33(2)：19～22
3) 陈新建，陈梅英，赵会杰．免疫学在植物科学中的应用[M]．中国农业大学出版社
4) 陈勇，周光宏，刘清．猪β2肾上腺素能受体基因的克隆与鉴定[J]．畜牧兽医学报．2002，33(1)：36～39
5) 陈勇，周光宏．猪β2-AR基因重组质粒的快速筛选．生物技术[J]．2002，12(5)：5～6．
6) 陈杖榴，杨桂香，孙永学等．兽药残留毒性与生态毒理学研究进展[J]．华南农业大学学报．2001，22(1)：88～91
7) 程大敦，陶爱群，姚杏苏．喘敌素和克喘素的分析[J]．药物分析杂志．1986，6(3)：153～155
8) 方炳虎，陈杖榴，黄显会．克伦特罗在猪体内的生物利用度及药物动力学研究[J]．畜牧兽医学报．1997，28(3)：233～237
9) 冯振卿，李玉华，仇镇宁等．日本血吸虫单克隆抗独特型抗体NP30诱导抗卵免疫的研究[J]．中国人兽共患病杂志．1999，15(1)：17～20
10) 管晓红，马磊．日本血吸虫单克隆抗独特型抗体NP30的建株与初步鉴定[J]．中国寄生虫学与寄生虫病杂志．1991，9(4)：261～263
11) 黄传书，金伯泉，汪美先等．一种用于制备MCAB的体内和体外联合免疫诱导方法[J]．单克隆抗体通讯，1993，9(1)：54～56
12) 黄永科，刘清．猪抗克伦特罗抗血清的制备及其IGg纯化与鉴定[J]．畜牧与兽医．2001，33(3)：6～9
13) 姜金霞，钟建平，张胜权．HPLC法测定皮肤渗透液中克喘素含量[J]．药物分析杂志．1992，12(2)：118～119
14) 孔科，袁宗辉，范盛先．Development of a screening assay Of Clenbuterol residue in pig liver by ELISA Kit[J]．华中农业大学学报．2000，19(3)：252～255
15) 李明远，肖玉，肖丽英等。抗HEL抗独特型抗体准备与酶功能模拟初探[J]．中华微生物和免疫学杂志．1996，16(4)：292
16) 李述文．实用有机化学手册[M]．上海：上海科技出版社，1981，429-430
17) 罗道栩，邓国东，肖田安．CHARMⅡ检测在兽药残留分析中的应用[J]．中国兽药杂志．2003，37(2)：1～4
18) 吕宝璋，田英．受体学概论[M]．北京：科学出版社，1991．
19) 吕贵霞，刁有祥，许胜勇．建立完善的兽药残留留监测体系势在必行[J]．中国兽药杂志．2002，36(9)：7～9
20) 马颖哲．一次脾内直接注射免疫动物制备单克隆抗体[J]．白求恩医科大学学报．1993，19(4)：409～410
21) 马有福，马春艳．孕马血清促性腺激素单克隆抗体的研制[J]．畜牧兽医学报．1996，27(2)：125～130．
22) 缪晓辉，ELISA技术的某些改进[J]，上海免疫学杂志，1994，14(4)229
23) 农业部公告，105号．允许使用的饲料添加剂管理条例[Z]
24) 农业部公告，168号．关于分布食品动物饲料药物添加剂使用规范[Z]
25) 农业部、卫生部、国家药品监督管理局公告，176号．禁止在饲料和动物饮用水中使用的药物品种目录[Z]
26) 农业部文件，农牧发(2002)1号．关于分布食品动物禁用的兽药及其它化合物清单的通知[Z]
27) 农业部文件，农牧发(1997)3号[Z]
28) 秦路平．生物活性物质的高通量筛选[M]．第二军医大学出版社．2002
29) 邱汝先．祛咳喘糖浆中盐酸双氯醇胺的含量测定方法[J]．药物分析杂志．1988，8(1)：29
30) 邱阳生，杨根海，何方洋．沙丁胺醇单克隆抗体的制备及其鉴定[J]．中国兽医科技．2002，32(10)：25～26
31) 萨姆布拉克J，弗里奇EF，曼尼阿蒂斯T．分子克隆实验指南[M]．金冬雁 黎孟枫，侯云德．第2版．北京：科学出版社，1991
32) 邵明洋．兽药残留危害及监控对策[J]．中国兽药杂志．2003，37(2)：1～4
33) 沈倍奋．分子文库[M]．北京．科学出版社．2001
34) 司徒镇强．细胞培养[M]．西安：世界图书出版公司，1996，西安
35) 汪家政，范明．蛋白质技术手册[M]．北京：科学出版社，2001
36) 王树槐，门立强，刘琪．液质联用(LC／MS)定性及定量检测猪视网膜组织中盐酸克伦特罗残留[J]．中国兽药杂志．2003，37(3)：36～38
37) 王琰．基因工程抗体研究进展[J]．中国免疫学杂志．1999，19(5)：193～195
38) 王泽永．兽药残留的分析方法[J]．中国兽药杂志．2002，36(10)：36～38
39) 谢孟峡，刘嫒，蒋敏．固相萃取-气相色谱-质谱分析肉样中盐酸克伦特罗的残留量[J]．分析化学．2002，30(10)：1308～1311
40) 杨安钢，毛积芳，药立波．生物化学与分子生物学实验指导[M]．北京：高等教育出版社，2001．
41) 杨利国．酶免疫测定技术[M] 南京南京大学出版社，1998，南京
42) 杨珊瑛．反相离子对HPLC法测定情安喘定片中微量盐酸克伦特罗[J]．中草药．1999，30(9)：368～370
43) 杨曙明，宋荣，范理．饲料和畜产品中β2肾上腺素激动剂测定方法研究进展[J]．农业质量标准．2003，1(2)
44) 张谷生，容庚培．单克隆抗体在医学上的应用[M]．上海科学技术出版社．1987，91～110
45) 张亮．东方田鼠天然IgG3抗体相关的日本血吸虫抗原基因的研究，中国农业科学院硕士论文[D]．2002
46) 张剑勇．兽药残留监测与动物性食品安全卫生[J]．中国兽药杂志．2001，35(4)：60～61
47) 中国药典[M]2000版
48) 周家驹，王亭．药物设计中的分子模拟化方法[M]．北京．科学出版社．2001
49) 周得刚，闫小峰．动物性产品中的兽药残留监测体系建设迫在眉睫[J]．中国兽药杂志．2002，36(2)：1～3
50) 周业康．盐酸双氯醇胺的比色测定法的改进[J]．药物分析杂志．1984，4(2)：113～114
51) Albert Adam, nicole gercais, avo panoyan. Detection of clenbuterol residues in hair[J]. Analyst. 1994,119(12):2663～2666
52) Alexander Staudt, Reza Mobini, Michael Fu, β_1-adrenoceptor antibodies induce positive inotropic response in isolated cardiomyocytes[J].European Journal of Pharmacology. 2001, 423 (2～3) :115～119
53) Barbara Krebs, Robert Rauchenberger, Silke Reiffert. High-throughput generation and engineering of recombinant human antibodies[J], Journal of Immunological Methods, 2001,254(1) 67～84
54) Clackso. Making antibody fragments using phage display libraries[J]
nature. 1991,345,624～628
55) D.Boyd, P. Shearan, J.p.Hopkins. Application of matrix solid phase dispersion for determination of clenbuterol in liver samples [J].Analytica Chimica Acta. 1993,27(5):221～226
56) Diane Lebesgue, Gerd Wallukat, Alfredo Mijares, An agonist-like monoclonal antibody against the human β 2-adrenoceptor[J],European Journal of Pharmacology. 1998, 348(1):123～133
57) D. J. Smith. Sterochemical composition of Clenbuterol Residues in edible tissue of swine [J]. J Agric food Chem.2000,48:6036～6043
58) D.J. Smith. Total radiactive residues and clenbuterol residues in swine after dietary administration of 14C clenbuterol for seven days and preslaughter withdrawal periods of zero, three, or sevendays[J]. J.Animal Science. 2000,78 (11): 2903～2912
59) Gardner M J, Flint D J. Long-term reductions in GH, insulin-like growth factor-1 and body weight gain in rats treated neonatally with antibodies to rat GH[J], J Endocrinol,1990.124:381～386
60) Gocayne J, Robinson D A, FitzGerald M G, et al. Primary structure of rat cardiac beta-adrenergic and muscarinic cholinergic receptors obtained by automated DNA sequence analysis: further evidence for a multigene family [J].Journal Proc. Natl. Acad. Sci. U.S.A. 1987,84:8296～8300.
61) Goldberg MR, Robertson Dyohimbine.a pharmacololical probe for study of the α2 adrenergic receptor[J] .Pharmacol Rev. 1983,35:143～180.
62) Guy Degand And Anne Bernes Duyckaerts. Determination of agonists in urinr by an enzyme immunoassays based on the use of an antisalbutamol antiserum[J]. Analytica Chimica Avt. 1993,27(5):241～247
63) H. D. Meyer, L. RinkeO. Residue screening for the β agonists Clenbuterol, sakbutmol and cimaterol in urine using enzyme immunoassay and High-Performance Liquid Chromatography[J]. Journal Of Chromatography. 1991,564:551～556
64) helga sauerwein, andreas gleixner and Heinricb H. D. meyer. Detection of the anabolic β adrenoceptor agonist clenbuterol In human scalp hair by HPLC/EIA[J]. clinical chemistry. 1996,42(11): 1869～1870
65) I .M.Reital, Mthanavala, D K Male, et al. Anti-idiotypes as surrogate antigens structure considerations[J] Immunology Today: 1985,6(9)265～267
66) Isabelle Navarro-Teulon, Sylvie Peraldi-Roux,.Expression in Escherichia coli of soluble and M13 phage-displayed forms of a single-chain antibody fragment specific for digoxin: assessment in a novel drug immunoassay[J], Immunotechnology, 1995,1 (1), 41～52
67) Kaita De Wach, Hubert De Brabander And Dirk Courtheyn .LC-MS-MS to detect and identify four beta-agonists and quantify clenbuterol in liver[J] .Analyst. 1998,123,2701～2705
68) Kathlynn C Brown, New approaches for cell-specific targeting:identification of cell-selective peptides from combinatorial libraries[J], Current Opinion in Chemical Biology. 2000, 4(1) 16～21
69) K. Charltonb, W.J. Harrisa and AJ. Porter , The isolation of super-sensitive anti-hapten antibodies from combinatorial antibody libraries derived from sheep[J] Biosensors andBioelectronics, 2001, 16 (9～12): 639～646
70) Knottc ,Browery Ng, Pharmacological characterization of GABAA and GABAB receptors in mamalian cns by recptor binding assay[J], Neuroendocrine Research Methods. 1991,12:699～722
71) Kristia Na, Ew I Vanoosthuyze, Cor J. M. Arrts. Development of a fast and simp le method for determination of agonists in urine by extravction on empore membranes and detection by a test strip immunoassay [J].J Agric food Chem. 1997,45 (8) 3129～3137
72) Kunakar Pou,Huy Ong And Albert AdamKunakar. Combined immunoextraction approach coupled to chemiluminescence enzyme immunoassay for the determination of trace levels of salbutamol and clenbuterol in tissue samples[J].Analyst. 1994,119(12):2659～2662
73) Laurrent Debrauwer And Geoges Bories. Determination of clenbuterol residues by Liquid Chromatography -Electrospay Mass Spectrometry[J]. Analytica Chimica Acta. 1993,27(5):231～239
74) M.A.Bacigalupo, A. Ius, G.Meroni Dovis And E Petruzzlli. Comparison of time-resolved fluroimmunoassay and immmunoenzymometric assay for
Clenbuterol [J].Analyst. 1995,120(8) :2269～2271
75) Martinez-Navarro J. F. Food poisoning related to the consumption of illicit β agonist in liver [J]. Lancet. 1990,346:180
76) Nakada M T, Haskell K M, Ecker D J, Stadel J M.. Genetic regulation of beta-2-adrenergic receptors in 3T3-L1 fibroblasts [J].Journal Biochem. 1989,260:53-59
77) Peter Batjoens, Dirk Courtheyn, Huber F. De Brabander,.Gas Chromatographic-Tandem Mass Spectrometric analysis of clenbuterol residues in faeces[J].Journal Of Chromatography A. 1996,750:133～139
78) P. Gonzalez Gigosos, C.A. Febte Smpayo. Liquid chromatographic analysis of clenbuterol residues in food products of animal origin using diphasic dialysis exatraction [J] .Chromatographia. 1996,43(5/6): 271～274
79) Ph. Delahaut, Dubois, I Pribar et al. Development of a specifc radiimmunoassay for the detection of clenbuterol determination residues in treated cattle[J].Food Additives And Contaminants. 1991,8(1): 43～45
80) Pluce C, Lamaison D, Kieck G, et al. Collective human food poisonings by clenbuterol residue in veal liver[J]. Vet. Hum. Toxicol. 1991,33:480～481
81) Randy M.Wadkinsa, Joel P. Goldena, Leo M. Pritsiolasa.Detection of multiple toxic agents using a planar array immunosensor[J],Biosensors and Bioelectronics. 1998, 13, 639～646
82) R.J.H.Pickett And M.J. Suaer. Determination of clenbuterol in bovine urine by enzyme immunoassay following concentrarion and clean-up by Immunoaffmity Chronatography[J].Analytica Chimica Acta. 1993,27(5)269～273
83) Roger T, Wilson Joseph. Determination of clenbuterolin cattle, sheep,and swine tissues by Electron Ionization Gaschromatography/Mass Spectrometry[J].journal of AOAC international. 1994,77(4):917～924
84) Rupert J L, Monsalve M V, Devine D V. Beta2-adrenergic receptor allele frequencies in the Quechua, a high altitude native population [J]. Journal Ann. Hum. Genet. 2000,64:135～143.
85) Sachdev S.Sidhu. Phage display in pharmaceutical biotechnology[J], Current
Opinion in Biotechnology,2000, 11(6):610～616
86) Scippo M-L; Weerdt C.V.D.;Willemsen P. Detection of illegal growth promoters in biological samples using receptor binding assays[J]Analytica Chimica Acta,2002, 473, 1, 135～141
87) S.D.Bucknall, A.L.Mackenzi, M.J. Sauer, .Determination of clenbuterol in bovine liver by enzyme immunossay[J]. Analytica Chimica Acta. 1993,27(5):227～230
88) T.Visser, M.J. Vredenbregt And A P J M De Jong. Cryotrapping Gas Chromatography -Fourier Transform Infrared Spectrometry: a new technique to confirm the presence of β-agonists in animal material[J] .Analytica Chimica Acta. 1993,27(5): 205～214
89) Ulrik B.Nielsen and James D.Marks, antibodies and targeted cancer therapy: direct selection from phage display libraries[J], Internalizing Pharmaceutical Science and Technology Today, 2000, 3(8) :282～291
90) Vogel M, Miescher S, Kuhn S. Mimicry of human IgE epitopes by anti-idiotypic antibodies[J]. J Mol Biol,2000 May 298:729～35
91) Weilin L Shelver And David J Smith Evaluation of commercial immunoassays for cross-reactivity to clenbuterol stereisomers and bovine metabolites[J].Food Additives And Contaminants.2000,17(10): 837～845
92) xiao rp,lakatta eg.β1 adrenoreceptor stimulation and β2 adrenoreceptor stimulation differ in their effect on contraction cytosolic Ca~(2+) current in single rat ventricular cells[J].Circ Rev. 1993,73:286～300.
93) Yamamoto J, Iwata. K.Enzyme immunoassay for clenbuterol, a β adrenergic stimulant, [J]journal of immunoassay 1982,3,55～171
94) Yang Y.T, And Ecellgott Ma. Mutiple action of adrenergic agonists on skeletal muscle and aclipose tissue[J].Biochemistry. 1989,261:1～10
2) 陈继明，黄日新，陆承平等．酶联免疫吸附测定样品中克伦特罗含量[J]．中国兽药杂志．1999，33(2)：19～22
3) 陈新建，陈梅英，赵会杰．免疫学在植物科学中的应用[M]．中国农业大学出版社
4) 陈勇，周光宏，刘清．猪β2肾上腺素能受体基因的克隆与鉴定[J]．畜牧兽医学报．2002，33(1)：36～39
5) 陈勇，周光宏．猪β2-AR基因重组质粒的快速筛选．生物技术[J]．2002，12(5)：5～6．
6) 陈杖榴，杨桂香，孙永学等．兽药残留毒性与生态毒理学研究进展[J]．华南农业大学学报．2001，22(1)：88～91
7) 程大敦，陶爱群，姚杏苏．喘敌素和克喘素的分析[J]．药物分析杂志．1986，6(3)：153～155
8) 方炳虎，陈杖榴，黄显会．克伦特罗在猪体内的生物利用度及药物动力学研究[J]．畜牧兽医学报．1997，28(3)：233～237
9) 冯振卿，李玉华，仇镇宁等．日本血吸虫单克隆抗独特型抗体NP30诱导抗卵免疫的研究[J]．中国人兽共患病杂志．1999，15(1)：17～20
10) 管晓红，马磊．日本血吸虫单克隆抗独特型抗体NP30的建株与初步鉴定[J]．中国寄生虫学与寄生虫病杂志．1991，9(4)：261～263
11) 黄传书，金伯泉，汪美先等．一种用于制备MCAB的体内和体外联合免疫诱导方法[J]．单克隆抗体通讯，1993，9(1)：54～56
12) 黄永科，刘清．猪抗克伦特罗抗血清的制备及其IGg纯化与鉴定[J]．畜牧与兽医．2001，33(3)：6～9
13) 姜金霞，钟建平，张胜权．HPLC法测定皮肤渗透液中克喘素含量[J]．药物分析杂志．1992，12(2)：118～119
14) 孔科，袁宗辉，范盛先．Development of a screening assay Of Clenbuterol residue in pig liver by ELISA Kit[J]．华中农业大学学报．2000，19(3)：252～255
15) 李明远，肖玉，肖丽英等。抗HEL抗独特型抗体准备与酶功能模拟初探[J]．中华微生物和免疫学杂志．1996，16(4)：292
16) 李述文．实用有机化学手册[M]．上海：上海科技出版社，1981，429-430
17) 罗道栩，邓国东，肖田安．CHARMⅡ检测在兽药残留分析中的应用[J]．中国兽药杂志．2003，37(2)：1～4
18) 吕宝璋，田英．受体学概论[M]．北京：科学出版社，1991．
19) 吕贵霞，刁有祥，许胜勇．建立完善的兽药残留留监测体系势在必行[J]．中国兽药杂志．2002，36(9)：7～9
20) 马颖哲．一次脾内直接注射免疫动物制备单克隆抗体[J]．白求恩医科大学学报．1993，19(4)：409～410
21) 马有福，马春艳．孕马血清促性腺激素单克隆抗体的研制[J]．畜牧兽医学报．1996，27(2)：125～130．
22) 缪晓辉，ELISA技术的某些改进[J]，上海免疫学杂志，1994，14(4)229
23) 农业部公告，105号．允许使用的饲料添加剂管理条例[Z]
24) 农业部公告，168号．关于分布食品动物饲料药物添加剂使用规范[Z]
25) 农业部、卫生部、国家药品监督管理局公告，176号．禁止在饲料和动物饮用水中使用的药物品种目录[Z]
26) 农业部文件，农牧发(2002)1号．关于分布食品动物禁用的兽药及其它化合物清单的通知[Z]
27) 农业部文件，农牧发(1997)3号[Z]
28) 秦路平．生物活性物质的高通量筛选[M]．第二军医大学出版社．2002
29) 邱汝先．祛咳喘糖浆中盐酸双氯醇胺的含量测定方法[J]．药物分析杂志．1988，8(1)：29
30) 邱阳生，杨根海，何方洋．沙丁胺醇单克隆抗体的制备及其鉴定[J]．中国兽医科技．2002，32(10)：25～26
31) 萨姆布拉克J，弗里奇EF，曼尼阿蒂斯T．分子克隆实验指南[M]．金冬雁 黎孟枫，侯云德．第2版．北京：科学出版社，1991
32) 邵明洋．兽药残留危害及监控对策[J]．中国兽药杂志．2003，37(2)：1～4
33) 沈倍奋．分子文库[M]．北京．科学出版社．2001
34) 司徒镇强．细胞培养[M]．西安：世界图书出版公司，1996，西安
35) 汪家政，范明．蛋白质技术手册[M]．北京：科学出版社，2001
36) 王树槐，门立强，刘琪．液质联用(LC／MS)定性及定量检测猪视网膜组织中盐酸克伦特罗残留[J]．中国兽药杂志．2003，37(3)：36～38
37) 王琰．基因工程抗体研究进展[J]．中国免疫学杂志．1999，19(5)：193～195
38) 王泽永．兽药残留的分析方法[J]．中国兽药杂志．2002，36(10)：36～38
39) 谢孟峡，刘嫒，蒋敏．固相萃取-气相色谱-质谱分析肉样中盐酸克伦特罗的残留量[J]．分析化学．2002，30(10)：1308～1311
40) 杨安钢，毛积芳，药立波．生物化学与分子生物学实验指导[M]．北京：高等教育出版社，2001．
41) 杨利国．酶免疫测定技术[M] 南京南京大学出版社，1998，南京
42) 杨珊瑛．反相离子对HPLC法测定情安喘定片中微量盐酸克伦特罗[J]．中草药．1999，30(9)：368～370
43) 杨曙明，宋荣，范理．饲料和畜产品中β2肾上腺素激动剂测定方法研究进展[J]．农业质量标准．2003，1(2)
44) 张谷生，容庚培．单克隆抗体在医学上的应用[M]．上海科学技术出版社．1987，91～110
45) 张亮．东方田鼠天然IgG3抗体相关的日本血吸虫抗原基因的研究，中国农业科学院硕士论文[D]．2002
46) 张剑勇．兽药残留监测与动物性食品安全卫生[J]．中国兽药杂志．2001，35(4)：60～61
47) 中国药典[M]2000版
48) 周家驹，王亭．药物设计中的分子模拟化方法[M]．北京．科学出版社．2001
49) 周得刚，闫小峰．动物性产品中的兽药残留监测体系建设迫在眉睫[J]．中国兽药杂志．2002，36(2)：1～3
50) 周业康．盐酸双氯醇胺的比色测定法的改进[J]．药物分析杂志．1984，4(2)：113～114
51) Albert Adam, nicole gercais, avo panoyan. Detection of clenbuterol residues in hair[J]. Analyst. 1994,119(12):2663～2666
52) Alexander Staudt, Reza Mobini, Michael Fu, β_1-adrenoceptor antibodies induce positive inotropic response in isolated cardiomyocytes[J].European Journal of Pharmacology. 2001, 423 (2～3) :115～119
53) Barbara Krebs, Robert Rauchenberger, Silke Reiffert. High-throughput generation and engineering of recombinant human antibodies[J], Journal of Immunological Methods, 2001,254(1) 67～84
54) Clackso. Making antibody fragments using phage display libraries[J]
nature. 1991,345,624～628
55) D.Boyd, P. Shearan, J.p.Hopkins. Application of matrix solid phase dispersion for determination of clenbuterol in liver samples [J].Analytica Chimica Acta. 1993,27(5):221～226
56) Diane Lebesgue, Gerd Wallukat, Alfredo Mijares, An agonist-like monoclonal antibody against the human β 2-adrenoceptor[J],European Journal of Pharmacology. 1998, 348(1):123～133
57) D. J. Smith. Sterochemical composition of Clenbuterol Residues in edible tissue of swine [J]. J Agric food Chem.2000,48:6036～6043
58) D.J. Smith. Total radiactive residues and clenbuterol residues in swine after dietary administration of 14C clenbuterol for seven days and preslaughter withdrawal periods of zero, three, or sevendays[J]. J.Animal Science. 2000,78 (11): 2903～2912
59) Gardner M J, Flint D J. Long-term reductions in GH, insulin-like growth factor-1 and body weight gain in rats treated neonatally with antibodies to rat GH[J], J Endocrinol,1990.124:381～386
60) Gocayne J, Robinson D A, FitzGerald M G, et al. Primary structure of rat cardiac beta-adrenergic and muscarinic cholinergic receptors obtained by automated DNA sequence analysis: further evidence for a multigene family [J].Journal Proc. Natl. Acad. Sci. U.S.A. 1987,84:8296～8300.
61) Goldberg MR, Robertson Dyohimbine.a pharmacololical probe for study of the α2 adrenergic receptor[J] .Pharmacol Rev. 1983,35:143～180.
62) Guy Degand And Anne Bernes Duyckaerts. Determination of agonists in urinr by an enzyme immunoassays based on the use of an antisalbutamol antiserum[J]. Analytica Chimica Avt. 1993,27(5):241～247
63) H. D. Meyer, L. RinkeO. Residue screening for the β agonists Clenbuterol, sakbutmol and cimaterol in urine using enzyme immunoassay and High-Performance Liquid Chromatography[J]. Journal Of Chromatography. 1991,564:551～556
64) helga sauerwein, andreas gleixner and Heinricb H. D. meyer. Detection of the anabolic β adrenoceptor agonist clenbuterol In human scalp hair by HPLC/EIA[J]. clinical chemistry. 1996,42(11): 1869～1870
65) I .M.Reital, Mthanavala, D K Male, et al. Anti-idiotypes as surrogate antigens structure considerations[J] Immunology Today: 1985,6(9)265～267
66) Isabelle Navarro-Teulon, Sylvie Peraldi-Roux,.Expression in Escherichia coli of soluble and M13 phage-displayed forms of a single-chain antibody fragment specific for digoxin: assessment in a novel drug immunoassay[J], Immunotechnology, 1995,1 (1), 41～52
67) Kaita De Wach, Hubert De Brabander And Dirk Courtheyn .LC-MS-MS to detect and identify four beta-agonists and quantify clenbuterol in liver[J] .Analyst. 1998,123,2701～2705
68) Kathlynn C Brown, New approaches for cell-specific targeting:identification of cell-selective peptides from combinatorial libraries[J], Current Opinion in Chemical Biology. 2000, 4(1) 16～21
69) K. Charltonb, W.J. Harrisa and AJ. Porter , The isolation of super-sensitive anti-hapten antibodies from combinatorial antibody libraries derived from sheep[J] Biosensors andBioelectronics, 2001, 16 (9～12): 639～646
70) Knottc ,Browery Ng, Pharmacological characterization of GABAA and GABAB receptors in mamalian cns by recptor binding assay[J], Neuroendocrine Research Methods. 1991,12:699～722
71) Kristia Na, Ew I Vanoosthuyze, Cor J. M. Arrts. Development of a fast and simp le method for determination of agonists in urine by extravction on empore membranes and detection by a test strip immunoassay [J].J Agric food Chem. 1997,45 (8) 3129～3137
72) Kunakar Pou,Huy Ong And Albert AdamKunakar. Combined immunoextraction approach coupled to chemiluminescence enzyme immunoassay for the determination of trace levels of salbutamol and clenbuterol in tissue samples[J].Analyst. 1994,119(12):2659～2662
73) Laurrent Debrauwer And Geoges Bories. Determination of clenbuterol residues by Liquid Chromatography -Electrospay Mass Spectrometry[J]. Analytica Chimica Acta. 1993,27(5):231～239
74) M.A.Bacigalupo, A. Ius, G.Meroni Dovis And E Petruzzlli. Comparison of time-resolved fluroimmunoassay and immmunoenzymometric assay for
Clenbuterol [J].Analyst. 1995,120(8) :2269～2271
75) Martinez-Navarro J. F. Food poisoning related to the consumption of illicit β agonist in liver [J]. Lancet. 1990,346:180
76) Nakada M T, Haskell K M, Ecker D J, Stadel J M.. Genetic regulation of beta-2-adrenergic receptors in 3T3-L1 fibroblasts [J].Journal Biochem. 1989,260:53-59
77) Peter Batjoens, Dirk Courtheyn, Huber F. De Brabander,.Gas Chromatographic-Tandem Mass Spectrometric analysis of clenbuterol residues in faeces[J].Journal Of Chromatography A. 1996,750:133～139
78) P. Gonzalez Gigosos, C.A. Febte Smpayo. Liquid chromatographic analysis of clenbuterol residues in food products of animal origin using diphasic dialysis exatraction [J] .Chromatographia. 1996,43(5/6): 271～274
79) Ph. Delahaut, Dubois, I Pribar et al. Development of a specifc radiimmunoassay for the detection of clenbuterol determination residues in treated cattle[J].Food Additives And Contaminants. 1991,8(1): 43～45
80) Pluce C, Lamaison D, Kieck G, et al. Collective human food poisonings by clenbuterol residue in veal liver[J]. Vet. Hum. Toxicol. 1991,33:480～481
81) Randy M.Wadkinsa, Joel P. Goldena, Leo M. Pritsiolasa.Detection of multiple toxic agents using a planar array immunosensor[J],Biosensors and Bioelectronics. 1998, 13, 639～646
82) R.J.H.Pickett And M.J. Suaer. Determination of clenbuterol in bovine urine by enzyme immunoassay following concentrarion and clean-up by Immunoaffmity Chronatography[J].Analytica Chimica Acta. 1993,27(5)269～273
83) Roger T, Wilson Joseph. Determination of clenbuterolin cattle, sheep,and swine tissues by Electron Ionization Gaschromatography/Mass Spectrometry[J].journal of AOAC international. 1994,77(4):917～924
84) Rupert J L, Monsalve M V, Devine D V. Beta2-adrenergic receptor allele frequencies in the Quechua, a high altitude native population [J]. Journal Ann. Hum. Genet. 2000,64:135～143.
85) Sachdev S.Sidhu. Phage display in pharmaceutical biotechnology[J], Current
Opinion in Biotechnology,2000, 11(6):610～616
86) Scippo M-L; Weerdt C.V.D.;Willemsen P. Detection of illegal growth promoters in biological samples using receptor binding assays[J]Analytica Chimica Acta,2002, 473, 1, 135～141
87) S.D.Bucknall, A.L.Mackenzi, M.J. Sauer, .Determination of clenbuterol in bovine liver by enzyme immunossay[J]. Analytica Chimica Acta. 1993,27(5):227～230
88) T.Visser, M.J. Vredenbregt And A P J M De Jong. Cryotrapping Gas Chromatography -Fourier Transform Infrared Spectrometry: a new technique to confirm the presence of β-agonists in animal material[J] .Analytica Chimica Acta. 1993,27(5): 205～214
89) Ulrik B.Nielsen and James D.Marks, antibodies and targeted cancer therapy: direct selection from phage display libraries[J], Internalizing Pharmaceutical Science and Technology Today, 2000, 3(8) :282～291
90) Vogel M, Miescher S, Kuhn S. Mimicry of human IgE epitopes by anti-idiotypic antibodies[J]. J Mol Biol,2000 May 298:729～35
91) Weilin L Shelver And David J Smith Evaluation of commercial immunoassays for cross-reactivity to clenbuterol stereisomers and bovine metabolites[J].Food Additives And Contaminants.2000,17(10): 837～845
92) xiao rp,lakatta eg.β1 adrenoreceptor stimulation and β2 adrenoreceptor stimulation differ in their effect on contraction cytosolic Ca~(2+) current in single rat ventricular cells[J].Circ Rev. 1993,73:286～300.
93) Yamamoto J, Iwata. K.Enzyme immunoassay for clenbuterol, a β adrenergic stimulant, [J]journal of immunoassay 1982,3,55～171
94) Yang Y.T, And Ecellgott Ma. Mutiple action of adrenergic agonists on skeletal muscle and aclipose tissue[J].Biochemistry. 1989,261:1～10