吩噻嗪类药物广谱单克隆抗体及阿莫西林单链抗体的制备
发布时间：2019-01-07 07:47
【摘要】：随着我国畜牧业的发展,兽药及药物添加剂的使用量呈逐年上升趋势。例如,有不法企业在动物饲料中添加吩噻嗪类药物以减少畜禽活动量达到增重的目的,或在动物运输过程中使用此类药物以减少动物的失重和死亡率。还有许多养殖企业在养殖过程大量使用青霉素类药物以预防或治疗动物的多种细菌性疾病。这些药物的使用会造成其在动物性食品中的药物残留,对公众健康造成潜在的危害。本研究的目的是制备吩噻嗪类药物的单克隆抗体和阿莫西林的单链抗体,为研究免疫检测方法或检测产品提供物质基础。在实验中,首先对2-氯吩噻嗪(Ch PZ)进行了分子改造,将其用溴乙酸进行衍生化合成半抗原(Ch PZD),然后采用混合酸酐法将Ch PZD分别与牛血清白蛋白(BSA)和卵清蛋白(OVA)偶联制成Ch PZ的免疫原(Ch PZD-BSA)和包被原(Ch PZD-OVA)。用Ch PZD-BSA免疫BALB/C小鼠,利用间接竞争ELISA法测定血清的效价及特异性。选择血清效价高且对Ch PZ有特异性的小鼠脾细胞与骨髓瘤细胞融合制备杂交瘤细胞。以间接ELISA方法筛选阳性杂交瘤细胞,采用有限稀释法经过多次筛选得到两株阳性杂交瘤细胞株,采用体内诱生法使小鼠产生腹水,以饱和硫酸铵法和Protein A亲和层析柱纯化法对抗体进行纯化,最后得到能识别五种吩噻嗪类药物(氯丙嗪、奋乃静、异丙嗪、乙酰丙嗪、氟奋乃静)的广谱单克隆抗体。另一方面,从分泌抗阿莫西林单克隆抗体的杂交瘤细胞中提取总RNA,反转录得到单链c DNA,再通过PCR扩增分别得到编码抗体重链(VH)和轻链(VL)的基因片段,然后将VH和VL拼接成单链抗体的基因片段(Sc Fv)。将Sc Fv和表达载体PET-32a进行连接后得到重组表达载体,并转化表达菌株BL21(DE3)感受态表达产生融合His标签的阿莫西林单链抗体。本研究制备了吩噻嗪类药物的广谱单克隆抗体和阿莫西林的单链抗体,这两种抗体将对监控养殖业中吩噻嗪类药物和青霉素类药物的非法使用和残留,保障动物源性食品安全有重要意义。
[Abstract]:With the development of animal husbandry in China, the use of veterinary drugs and drug additives is increasing year by year. For example, illegal companies add phenothiazine drugs to animal feed to reduce animal activity and gain weight, or use such drugs in animal transport to reduce animal weightlessness and mortality. Many aquaculture companies use penicillin drugs to prevent or treat bacterial diseases in animals. The use of these drugs can cause drug residues in animal foods and pose a potential health hazard to the public. The aim of this study was to prepare monoclonal antibodies against phenothiazines and single chain antibodies of amoxicillin to provide a material basis for the study of immunoassay methods or detection products. In the experiment, 2-chlorophenothiazine (Ch PZ) was firstly modified to synthesize hapten (Ch PZD), by derivatization with bromoacetic acid. Then Ch PZD was coupled with bovine serum albumin (BSA) (BSA) and ovalbumin (OVA) to form the immunogen (Ch PZD-BSA) and the envelope (Ch PZD-OVA) of Ch PZ by mixed anhydride method. BALB/C mice were immunized with Ch PZD-BSA. The titer and specificity of serum were determined by indirect competitive ELISA method. Hybridoma cells were prepared by fusion of murine spleen cells and myeloma cells with high serum titer and specificity to Ch PZ. Indirect ELISA method was used to screen positive hybridoma cells. Two positive hybridoma cell lines were obtained by limited dilution method. Ascites were induced in mice by in vivo induction method. The antibody was purified by saturated ammonium sulfate and Protein A affinity chromatography. At last, a broad-spectrum monoclonal antibody was obtained to recognize five phenothiazines (chlorpromazine, perphenazine, promethazine, acetpromazine, fluphenazine). On the other hand, the reverse transcription of total RNA, was obtained from hybridoma cells secreting monoclonal antibodies against amoxicillin, and the fragments encoding heavy chain (VH) and light chain (VL) were obtained by PCR amplification, respectively. Then VH and VL are spliced together to form a single chain antibody gene fragment, (Sc Fv). The recombinant expression vector was obtained by ligating Sc Fv with the expression vector PET-32a, and the expression strain BL21 (DE3) was transformed into an amoxicillin single chain antibody (scFv) fused with His tag. In this study, broad-spectrum monoclonal antibodies against phenothiazines and single-chain antibodies of amoxicillin were prepared, which will monitor the illegal use and residues of phenothiazine and penicillin drugs in aquaculture. It is of great significance to ensure the safety of animal-derived food.
【学位授予单位】：河北农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S859.84
本文编号：2403407
[Abstract]:With the development of animal husbandry in China, the use of veterinary drugs and drug additives is increasing year by year. For example, illegal companies add phenothiazine drugs to animal feed to reduce animal activity and gain weight, or use such drugs in animal transport to reduce animal weightlessness and mortality. Many aquaculture companies use penicillin drugs to prevent or treat bacterial diseases in animals. The use of these drugs can cause drug residues in animal foods and pose a potential health hazard to the public. The aim of this study was to prepare monoclonal antibodies against phenothiazines and single chain antibodies of amoxicillin to provide a material basis for the study of immunoassay methods or detection products. In the experiment, 2-chlorophenothiazine (Ch PZ) was firstly modified to synthesize hapten (Ch PZD), by derivatization with bromoacetic acid. Then Ch PZD was coupled with bovine serum albumin (BSA) (BSA) and ovalbumin (OVA) to form the immunogen (Ch PZD-BSA) and the envelope (Ch PZD-OVA) of Ch PZ by mixed anhydride method. BALB/C mice were immunized with Ch PZD-BSA. The titer and specificity of serum were determined by indirect competitive ELISA method. Hybridoma cells were prepared by fusion of murine spleen cells and myeloma cells with high serum titer and specificity to Ch PZ. Indirect ELISA method was used to screen positive hybridoma cells. Two positive hybridoma cell lines were obtained by limited dilution method. Ascites were induced in mice by in vivo induction method. The antibody was purified by saturated ammonium sulfate and Protein A affinity chromatography. At last, a broad-spectrum monoclonal antibody was obtained to recognize five phenothiazines (chlorpromazine, perphenazine, promethazine, acetpromazine, fluphenazine). On the other hand, the reverse transcription of total RNA, was obtained from hybridoma cells secreting monoclonal antibodies against amoxicillin, and the fragments encoding heavy chain (VH) and light chain (VL) were obtained by PCR amplification, respectively. Then VH and VL are spliced together to form a single chain antibody gene fragment, (Sc Fv). The recombinant expression vector was obtained by ligating Sc Fv with the expression vector PET-32a, and the expression strain BL21 (DE3) was transformed into an amoxicillin single chain antibody (scFv) fused with His tag. In this study, broad-spectrum monoclonal antibodies against phenothiazines and single-chain antibodies of amoxicillin were prepared, which will monitor the illegal use and residues of phenothiazine and penicillin drugs in aquaculture. It is of great significance to ensure the safety of animal-derived food.
【学位授予单位】：河北农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S859.84
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