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吩噻嗪类药物单链抗体的制备及进化

标题: 吩噻嗪类药物单链抗体的制备及进化
英文标题: Preparation and Directional Evolution of the Anti-phenothiazines Single Chain Antibody Fragment
作者: 史芳舒
出版时间: 2018-05-31
所在大学: 河北农业大学
关键词: 吩噻嗪类药物,单克隆抗体,单链抗体,分子对接,定点突变,ELISA
英文关键词: phenothiazines,monoclonal antibody,ScFv,molecular docking,directional mutation,ELISA
论文级别: 硕士
学位: 硕士
导师: 王建平;刘静
专业: 基础兽医学
主题: 吩噻嗪类药物单链抗体的制备及进化
页码: 78
摘要: 吩噻嗪类药物在养殖业中的非法使用会造成此类药物在动物源性食品中的残留,从而危害人类健康。在众多残留分析方法中,酶免疫分析法是最常用的快速筛选法。要建立针对吩噻嗪类药物的多残留酶免疫分析法,制备一种广谱识别性抗体至关重要。本研究以2-氯吩噻嗪为半抗原,制备了一株广谱单克隆抗体。间接竞争ELISA法检测表明,该单克隆抗体可以同时识别氯丙嗪、异丙嗪、乙酰丙嗪、奋乃静和氟奋乃静等5种吩噻嗪类药物,IC_(50)为1.22~500 ng/mL,交叉反应率为0.4~98%。然后,从杂交瘤细胞中提取总RNA,反转录为cDNA,扩增出抗体的重链可变区和轻链可变区基因,拼接成单链抗体基因(ScFv),将其插入到BL21(DE3)菌中表达出ScFv抗体。间接竞争ELISA法检测表明,该ScFv抗体对5种药物的IC_(50)为1.31~56 ng/mL,交叉反应率为2.3~99.2%,与母源单克隆抗体相当。利用YASARA软件将该ScFv抗体与5种药物分别进行对接,结果显示,抗体结合腔主要由重链CDR3和轻链CDR1、CDR2、CDR3区组成,关键接触氨基酸为Arg101、Tyr102、Tyr162、Phe164、Tyr179、Leu180、Trp221,疏水作用力是主要的分子间作用力。然后,利用定点突变技术将轻链CDR2的Phe164突变为Pro164,制备出一种ScFv突变体。间接竞争ELISA法检测表明,该突变体对5种药物亲和力得到较大的提升,IC_(50)为0.4~5.1 ng/mL。利用该抗体建立的ELISA法可以检测肉组织中的这5种吩噻嗪类药物,检测限为0.1~1.8 ng/g,添加回收率为66.4~97.2%。本试验基于基因工程和计算模拟的方法,探究了单链抗体的分子识别机制,并对其定向进化,为研究其他小分子物质的分子识别机制及体外进化奠定了基础,对保障动物源性食品安全具有重要意义。
英文摘要: Phenothiazines are used extensively in animals,and their residues in animal derived food are dangers to the consumers.Among the analytical methods,enzyme linked immunosorbent assay is the commonly used screening method.Therefore,it is very important to produce a broad specific antibody for phenothiazines.In this study,2-chlorophenothiazine was used as the hapten to produce a monoclonal antibody.The indirect ELISA method showed that the monoclonal antibody could simultaneously recognize five phenothiazine drugs,including chlorpromazine,promethazine,acepromazine,perphenazine and triflupromazine.The IC_(50) values were in the range of 1.22-500 ng/mL,and the crossreactivities were in the range of 0.4~98%.Then,the total RNA from the hybridoma cell stain was extracted,which was used to reverse transcrib the cDNA.Then the obtained heavy chain fragment and light chain fragment were linked to construct the ScFv gene fragment.The ScFv gene was transformed into expression competent strain BL21(DE3)cell to generate the the ScFv antibody.The results showed that the IC_(50) values for the five PZs were in the range of 1.31-56 ng/mL,similar to its parent monoclonal antibody.The ScFv antibody was docked with the five drugs respectively by YASARA,and the results indicated that the binding cavity was surrounded by CDR1 of heavy chain and CDR1,CDR2,CDR3 of light chain.The key contact amino acids were Arg101,Tyr102,Tyr162,Phe164,Tyr179,Leu180 and Trp221,and the hydrophobic action was main interaction force.Then the Phe164 in light chain CDR2 was mutated to Pro by use of mutant kit to generate the ScFv mutant.The ELISA showed that the mutant showed highly improved affinity to the five drugs with IC_(50) values in the range of 0.4-5.1 ng/mL.An ELISA method based on this mutant was developed to determine the five drugs in meat.The results showed that the detection limit were in the range of 0.1~1.8 ng/g,and the recoveries were in the range of 66.4~97.2%.In this study,the recognition mechanism of ScFv antibody was studied by use of gene engeneering and computational simulation,and the relative mutant is obtained.The results of this study provide the foundation for production of the ScFv antibodies for other veterinary drugs,and are of great significance on protection of the safety of animal drived foods.