吩噻嗪類藥物廣譜單克隆抗體及阿莫西林單鏈抗體的制備
發(fā)布時(shí)間：2019-01-07 07:47
【摘要】：隨著我國(guó)畜牧業(yè)的發(fā)展,獸藥及藥物添加劑的使用量呈逐年上升趨勢(shì)。例如,有不法企業(yè)在動(dòng)物飼料中添加吩噻嗪類藥物以減少畜禽活動(dòng)量達(dá)到增重的目的,或在動(dòng)物運(yùn)輸過(guò)程中使用此類藥物以減少動(dòng)物的失重和死亡率。還有許多養(yǎng)殖企業(yè)在養(yǎng)殖過(guò)程大量使用青霉素類藥物以預(yù)防或治療動(dòng)物的多種細(xì)菌性疾病。這些藥物的使用會(huì)造成其在動(dòng)物性食品中的藥物殘留,對(duì)公眾健康造成潛在的危害。本研究的目的是制備吩噻嗪類藥物的單克隆抗體和阿莫西林的單鏈抗體,為研究免疫檢測(cè)方法或檢測(cè)產(chǎn)品提供物質(zhì)基礎(chǔ)。在實(shí)驗(yàn)中,首先對(duì)2-氯吩噻嗪(Ch PZ)進(jìn)行了分子改造,將其用溴乙酸進(jìn)行衍生化合成半抗原(Ch PZD),然后采用混合酸酐法將Ch PZD分別與牛血清白蛋白(BSA)和卵清蛋白(OVA)偶聯(lián)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,利用間接競(jìng)爭(zhēng)ELISA法測(cè)定血清的效價(jià)及特異性。選擇血清效價(jià)高且對(duì)Ch PZ有特異性的小鼠脾細(xì)胞與骨髓瘤細(xì)胞融合制備雜交瘤細(xì)胞。以間接ELISA方法篩選陽(yáng)性雜交瘤細(xì)胞,采用有限稀釋法經(jīng)過(guò)多次篩選得到兩株陽(yáng)性雜交瘤細(xì)胞株,采用體內(nèi)誘生法使小鼠產(chǎn)生腹水,以飽和硫酸銨法和Protein A親和層析柱純化法對(duì)抗體進(jìn)行純化,最后得到能識(shí)別五種吩噻嗪類藥物(氯丙嗪、奮乃靜、異丙嗪、乙酰丙嗪、氟奮乃靜)的廣譜單克隆抗體。另一方面,從分泌抗阿莫西林單克隆抗體的雜交瘤細(xì)胞中提取總RNA,反轉(zhuǎn)錄得到單鏈c DNA,再通過(guò)PCR擴(kuò)增分別得到編碼抗體重鏈(VH)和輕鏈(VL)的基因片段,然后將VH和VL拼接成單鏈抗體的基因片段(Sc Fv)。將Sc Fv和表達(dá)載體PET-32a進(jìn)行連接后得到重組表達(dá)載體,并轉(zhuǎn)化表達(dá)菌株BL21(DE3)感受態(tài)表達(dá)產(chǎn)生融合His標(biāo)簽的阿莫西林單鏈抗體。本研究制備了吩噻嗪類藥物的廣譜單克隆抗體和阿莫西林的單鏈抗體,這兩種抗體將對(duì)監(jiān)控養(yǎng)殖業(yè)中吩噻嗪類藥物和青霉素類藥物的非法使用和殘留,保障動(dòng)物源性食品安全有重要意義。
[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.
【學(xué)位授予單位】：河北農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S859.84
本文編號(hào)：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.
【學(xué)位授予單位】：河北農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S859.84
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相關(guān)期刊論文 前2條
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