發(fā)布時(shí)間：2018-05-08 13:52

  本文選題：噬菌體抗體庫(kù)技術(shù) + 伊維菌素��； 參考：《揚(yáng)州大學(xué)》2010年碩士論文

【摘要】： 伊維菌素是由阿維鏈霉菌發(fā)酵合成的一種大環(huán)內(nèi)酯類多組分抗生素,被有效用作農(nóng)藥、獸藥及人抗寄生蟲(chóng)藥物。它能打開(kāi)谷氨酸控制的Cl-通道,導(dǎo)致膜超極化,使肌肉細(xì)胞喪失收縮能力,從而導(dǎo)致蟲(chóng)體死亡。長(zhǎng)期以來(lái),伊維菌素被認(rèn)為是安全的抗寄生蟲(chóng)藥物,但是近年的研究結(jié)果表明,它的殘留能降低牧草產(chǎn)量,影響水生群落,對(duì)環(huán)境平衡有重大威脅。伊維菌素的廣泛應(yīng)用和潛在危害的并存,使得建立一種簡(jiǎn)單高效的伊維菌素殘留檢測(cè)方法很有必要。 目前建立的伊維菌素檢測(cè)方法主要有中空纖維支撐液膜萃取法和液相色譜質(zhì)譜-質(zhì)譜法。這些方法需要昂貴的儀器及繁瑣的前處理步驟,相比之下,免疫檢測(cè)法較為快速、經(jīng)濟(jì)�，F(xiàn)已有關(guān)于伊維菌素單克隆和多克隆抗體的報(bào)道,還未見(jiàn)其單鏈抗體的報(bào)道。本研究在參照國(guó)內(nèi)外相關(guān)研究的基礎(chǔ)上,以期從人源噬菌體抗體庫(kù)中篩選出抗伊維菌素的單鏈抗體,并對(duì)其進(jìn)行可溶性表達(dá)、蛋白結(jié)構(gòu)功能分析,為環(huán)境及農(nóng)產(chǎn)品中伊維菌素的安全監(jiān)控提供材料,為研究抗原抗體反應(yīng)提供理論基礎(chǔ)。噬菌體展示技術(shù)是一種制備高親和力抗體的有力工具,抗體庫(kù)技術(shù)則為篩選高親和力抗體提供了良好的平臺(tái)。展示抗體的基因型與表現(xiàn)型的有效統(tǒng)一,使其不僅在農(nóng)藥殘留檢測(cè)及抗體制備等方面具有廣泛應(yīng)用前景,還能有效地應(yīng)用于基因工程改造。 研究目的和意義:應(yīng)用噬菌體抗體庫(kù)技術(shù),篩選人源抗伊維菌素單鏈抗體,以期應(yīng)用于伊維菌素的安全監(jiān)控。對(duì)篩選出的抗體進(jìn)行可溶性表達(dá)、純化后鑒定其免疫活性。對(duì)抗體的結(jié)構(gòu)功能進(jìn)行分析,為研究抗原抗體結(jié)合反應(yīng)提供理論基礎(chǔ)。 研究方法:以伊維菌素-牛血清白蛋白為包被抗原,應(yīng)用噬菌體抗體庫(kù)技術(shù),從庫(kù)容約為108的人源噬菌體抗體庫(kù)中篩選出抗伊維菌素單鏈抗體。采用固相消減篩選法,共進(jìn)行4輪“吸附-洗脫-富集”。對(duì)結(jié)合活性較強(qiáng)的陽(yáng)性克隆,制備噬菌體上清,轉(zhuǎn)化E.coli HB2151,以IPTG進(jìn)行誘導(dǎo),表達(dá)的可溶性抗體過(guò)鎳親和層析柱純化,建立競(jìng)爭(zhēng)抑制ELISA檢測(cè)方法,進(jìn)行靈敏度測(cè)定。應(yīng)用計(jì)算機(jī)軟件及生物信息學(xué)站點(diǎn)對(duì)抗體的結(jié)構(gòu)功能進(jìn)行分析。 研究結(jié)果:經(jīng)過(guò)四輪富集篩選,從人源抗體庫(kù)中篩選出7株不同的陽(yáng)性克隆,hsIVM8展示了最高的親和力。IPTG成功誘導(dǎo)表達(dá)hsIVM8,SDS-PAGE顯示其分子量約為28 kD,伊維菌素對(duì)純化抗體的抑制中濃度IC50為4.11μg/mL,線性檢測(cè)范圍為0.1-5μg/mL。 研究成果:從人源噬菌體抗體庫(kù)中成功篩選到抗伊維菌素的單鏈抗體,有望應(yīng)用于環(huán)境和農(nóng)產(chǎn)品中伊維菌素的安全監(jiān)控。對(duì)抗體蛋白進(jìn)行的結(jié)構(gòu)功能分析為研究抗體性質(zhì)及與抗原反應(yīng)機(jī)理提供理論基礎(chǔ)。
[Abstract]:Ivermectin is a macrolide multicomponent antibiotic synthesized by Streptomyces avelicus. It is effectively used as a pesticide, veterinary drug and human antiparasitic drug. It can open the Cl- channel controlled by glutamate, lead to membrane hyperpolarization, make muscle cells lose the ability of contraction, and lead to the death of insect body. For a long time, ivermectin has been considered as a safe antiparasitic drug. However, recent studies have shown that Ivermectin residues can reduce forage yield, affect aquatic communities and pose a serious threat to environmental balance. The extensive application of ivermectin and the coexistence of potential hazards make it necessary to establish a simple and efficient method for the detection of Ivermectin residues. At present, Ivermectin detection methods are mainly hollow fiber supported liquid membrane extraction and liquid chromatography-mass spectrometry. These methods require expensive instruments and cumbersome pre-processing procedures, whereas immunoassay is faster and more economical. The monoclonal and polyclonal antibodies against ivermectin have been reported, but no single chain antibodies have been reported. The aim of this study was to screen the single chain antibody against ivermectin from human phage antibody library and analyze its soluble expression and protein structure and function. It provides materials for the safety monitoring of Ivermectin in environment and agricultural products, and provides a theoretical basis for the study of antigen-antibody reaction. Phage display is a powerful tool for preparing high affinity antibodies, and antibody library provides a good platform for screening high affinity antibodies. The effective unification of genotypes and phenotypes of antibodies not only has a wide application prospect in pesticide residue detection and antibody preparation, but also can be effectively used in genetic engineering. Objective and significance: to screen human single chain antibody against ivermectin by phage antibody library technology, and to apply it to the safety monitoring of ivermectin. The antibody was expressed in a soluble way and its immunological activity was identified after purification. The analysis of the structure and function of antibody provides a theoretical basis for the study of antigen-antibody binding reaction. Methods: Ivermecin-bovine serum albumin (BSA) was used as coating antigen and phage antibody library technique was used to screen Ivermectin single chain antibody from human phage antibody library containing about 108. Four rounds of "adsorption-elution-enrichment" were carried out by solid phase subtractive screening. The phage supernatant was prepared and transformed into E.coli HB2151. The expressed soluble antibody was purified by nickel affinity chromatography. A competitive inhibition ELISA detection method was established and the sensitivity was determined. The structure and function of antibody were analyzed by computer software and bioinformatics site. Results: after four rounds of enrichment screening, Seven different positive clones of hsIVM8 were screened from the human antibody library and showed the highest affinity. IPTG successfully induced the expression of hsIVM8 SDS-PAGE showed that its molecular weight was about 28 kD. the median inhibitory concentration of ivermectin on purified antibody was 4.11 渭 g / mL, and the linear detection range was 0.1-5 渭 g / mL. Results: single chain antibodies against Ivermectin were successfully screened from human phage antibody library, which is expected to be applied to the safety monitoring of Ivermectin in environment and agricultural products. The analysis of the structure and function of antibody protein provides a theoretical basis for the study of antibody properties and reaction mechanism with antigens.
【學(xué)位授予單位】：揚(yáng)州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R392

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本文編號(hào)：1861664