盐酸克伦特罗完全抗原的合成及其偶联工艺的优化

2009 年 4 月 Journal of Chemical Engineering of Chinese Universities Apr. 2009文章编号：1003-9015(2009)02-0355-05

盐酸克伦特罗完全抗原的合成及其偶联工艺的优化

杨香瑜, 黄鹤

(天津大学化工学院生物化工系系统生物工程教育部重点实验室, 天津 300072)

摘要：为获取用于快速免疫分析的免疫抗原和包被抗原，本实验采用重氮化反应制备盐酸克伦特罗(Clenbuterol

hydrochloride, CL)完全抗原，在强酸条件下CL先经重氮化，再与鸡卵清白蛋白(OV A)发生共价取代制备了盐酸克伦特

罗包被抗原(OV A-CL)，并以正交实验确定了共价取代反应的关键影响因素，进一步采用单因素实验确定了反应所需的

最佳条件，并通过紫外扫描对制得的包被抗原进行了鉴定。结果表明，投料比例是盐酸克伦特罗完全抗原合成的关键

因素，搅拌转速和反应时间也起着至关重要的作用。而且偶联反应在1 h内基本完成，延长反应时间对提高产物偶联

比影响不大。当投料比例为30:1，pH值约为8，低速搅拌状况下，包被抗原和免疫抗原的偶联比分别高达29:1和48:1，盐酸克伦特罗完全抗原合成条件的优化筛选取得了预期的效果。

关键词：盐酸克伦特罗；重氮化反应；完全抗原；偶联比

中图分类号：O625.14 文献标识码：A

Synthesis of Clenbuterol Hydrochloride Complete Antigen and Optimization of its

Conjugation Conditions

YANG Xiang-yu, HUANG He

(Key Laboratory of Systems Bioengineering, Ministry of Education, Department of Biochemical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China)

Abstract: In order to synthesize the immunogen and coatigen used for the rapid immunoassay, the diazotization was adopted to prepare the Clenbuterol hydrochloride (CL) complete antigen. During which, the CL was firstly activated under strongly acidic conditions, and the activated CL was then conjugated with ovalbumin (OV A) to form coatigen OV A-CL. The important factors affecting the conjugating reaction were determined by orthogonal experiments, and the optimum value of those important factors was confirmed by single factor experiments. The prepared OV A-CL was characterized by UV scan. It was found that the CL/OV A dropping proportion is the key factor for preparing the complete antigen, while the stirring speed and reaction time are important too. The conjugating reaction can be completed within one hour and prolonging the reaction time has little effect on the increase of coupling ratio. When the CL/OV A dropping proportion (molar ratio) is 30:1, the pH value is 8 and at a low stirring dispersion speed (<250 r⋅min−1), the coupling ratio of coatigen and immunogen can be up to 29:1 and 48:1, respectively. It indicates that the optimization of the conjugation conditions of Clenbuterol hydrochloride complete antigen achieves the expected results.

Key words: Clenbuterol hydrochloride; diazotization; complete antigen; coupling ratio

1前言

盐酸克伦特罗是一种β2-肾上腺受体激动剂 [1]。大量用其作为饲料添加剂，可显著提高畜禽生长速率，瘦肉相对增加[2]。但该药易在牲畜体内沉积，人食用含有该药残留的动物产品后可引起食物中毒，严重

收稿日期：2008-01-29；修订日期：2008-06-02。

基金项目：天津市自然科学基金资助项目(06YFJMJC13200)。

作者简介：杨香瑜(1983-)，女，河北省沧州人，天津大学硕士生。通讯联系人：黄鹤，E-mail：huang@