【摘要】：目的胰淀素也稱胰島淀粉樣多肽(IAPP),它與胰島素共同存在于胰島B細(xì)胞的分泌顆粒中,與胰高血糖素、胰島素共同調(diào)節(jié)人體血糖平衡。目前研究表明胰淀素的異常分泌及早期形成的可溶性低聚物在局部堆積對(duì)于B細(xì)胞有毒性作用,可引起B(yǎng)細(xì)胞凋亡,最終導(dǎo)致胰島分泌功能的漸進(jìn)性衰竭。該激素自從1988年被發(fā)現(xiàn)以來(lái),其生理和病理意義愈發(fā)被關(guān)注,然而由于胰淀素的體內(nèi)含量低且分子量較小,其mRNA含量?jī)H為胰島素的0.2-0.3%,目前采用單克隆抗體免疫熒光方法檢測(cè),其方法復(fù)雜,抗體缺乏穩(wěn)定性和特異性,用傳統(tǒng)制備抗體的方法如雜交瘤技術(shù)和抗原免疫的方法制備抗體得到的抗體少,且工作繁瑣。給研究這一激素在體內(nèi)的變化規(guī)律帶來(lái)了困難。因此尋找一種快速和具有高度特異性的方法提取和純化胰淀素抗體是研究工作的關(guān)鍵,我們采用噬菌體表面展示技術(shù)的方法,試圖制備人源化的胰淀素抗體,為體外大規(guī)模生產(chǎn)抗體提供可行的方法。 方法用固相法將抗原胰淀素包被到酶聯(lián)板上,應(yīng)用噬菌體抗體庫(kù)技術(shù),篩選特異性的抗體。收集每輪洗脫的噬菌體并進(jìn)行擴(kuò)增,再用于下一輪篩選。同樣的“吸附一洗脫-擴(kuò)增”的富集反應(yīng)共進(jìn)行了5輪。在第5輪篩選后得到的抗胰淀素噬菌體抗體克隆中,選取數(shù)十個(gè)克隆擴(kuò)增,提取其質(zhì)粒,用Nhe I和SpeⅠ雙酶切,去除gⅢ基因,自身環(huán)化構(gòu)建其可溶性表達(dá)噬菌粒,轉(zhuǎn)化至大腸桿菌BL21(DE3)pLysS中,IPTG誘導(dǎo)高效表達(dá),并用SDS-PAGE鑒定所得蛋白是否為目的蛋白,ELISA鑒定其抗原結(jié)合活性和特異性。 結(jié)果通過(guò)對(duì)人天然Fab段噬菌體抗體庫(kù)的5輪篩選,盡管從第一輪到第五輪洗滌次數(shù)由1次增加到5次,最后增加到10次,從固相洗脫下來(lái)的噬菌體滴度卻顯示了明顯的增加趨勢(shì),回收的噬菌體產(chǎn)出率共增加了約443倍,說(shuō)明特異性抗胰淀素的Fab噬菌體抗體得到了富集。構(gòu)建的可溶性Fab抗體表達(dá)載體,在IPTG的誘導(dǎo)下得到了高效表達(dá),生成含有可溶性的Fab抗體的上清。經(jīng)SDS-PAGE蛋白電泳,在相對(duì)分子質(zhì)量約為47KD處可見(jiàn)一蛋白條帶；轉(zhuǎn)膜后HRP標(biāo)記的羊抗人FabIgG抗體進(jìn)一步顯色,證實(shí)了目的蛋白人源特異性Fab片段的表達(dá)；ELISA鑒定證明有3個(gè)克隆可與胰淀素抗原特異性結(jié)合,且與BSA無(wú)交叉反應(yīng)。 結(jié)論噬菌體抗體庫(kù)技術(shù)的出現(xiàn)為人天然抗體的制備開(kāi)辟了新的途徑,該技術(shù)便捷有效,可不經(jīng)免疫,繞過(guò)雜交瘤技術(shù),直接從抗體庫(kù)中篩選特異性抗體,可解決雜交瘤技術(shù)的低效問(wèn)題,并可在體外改善抗體的性能。本工作特異性抗胰淀素Fab抗體的制備及鑒定方法的建立,為進(jìn)一步的研究胰淀素的生理和病理功能奠定了基礎(chǔ)。
[Abstract]:Objective amylin, also known as islet amyloid polypeptide (IAPP), co-exists with insulin in the secretory granules of islet B cells and regulates the blood glucose balance with glucagon and insulin. Recent studies have shown that the abnormal secretion of amylin and the early accumulation of soluble oligomers have toxic effects on B cells, which can induce apoptosis of B cells and eventually lead to progressive failure of pancreatic islet secretory function. Since it was discovered in 1988, the physiological and pathological significance of the hormone has become more and more concerned. However, because of the low content and molecular weight of amylin in vivo, its mRNA content is only 0.2-0.3 of insulin. At present, monoclonal antibody immunofluorescence method is used, its method is complex, the antibody is lack of stability and specificity, the traditional methods of preparing antibody, such as hybridoma technique and antigen-immunized method, the antibody preparation method is less, and the work is tedious. This makes it difficult to study the changes of the hormone in the body. Therefore, to find a rapid and highly specific method to extract and purify amylin antibody is the key of the research. We use phage display technology to prepare human amylin antibody. To provide a feasible method for mass production of antibodies in vitro. Methods Antigen amylin was coated on enzyme linked plate by solid phase method and specific antibody was screened by phage antibody library technique. The phage eluted in each round was collected and amplified, and then used for the next round of screening. The same "adsorption-elution-amplification" enrichment reaction took place for a total of five rounds. After the fifth round of screening, dozens of clones were selected to amplify and extract their plasmids. The plasmids were digested with Nhe I and Spe 鈪，

本文編號(hào)：2389494