本文關(guān)鍵詞： 肝癌細(xì)胞膜抗原 單克隆抗體 雜交瘤技術(shù) 抗原純化　出處：《重慶醫(yī)科大學(xué)》2009年碩士論文　論文類型：學(xué)位論文

【摘要】： 目的:提取肝癌細(xì)胞膜蛋白作為抗原,利用雜交瘤抗體技術(shù)篩選并制備針對(duì)肝癌細(xì)胞膜蛋白的單克隆抗體。 方法:分別利用研磨方法(第一部分)、膜蛋白提取試劑盒(第二部分)、低滲法(第三部分)提取肝癌細(xì)胞HepG2細(xì)胞膜蛋白作為抗原,超速離心法純化抗原。純化后Bradford法進(jìn)行蛋白定量。用提取的抗原每次25ug-50ug劑量免疫Balb/c小鼠;以聚乙二醇法融合免疫小鼠脾細(xì)胞與SP20骨髓瘤細(xì)胞,用HAT、HT選擇培養(yǎng)基篩選雜交瘤;以提取的肝癌細(xì)胞膜蛋白作為包被抗原,用ELISA方法篩選能分泌抗肝癌細(xì)胞膜蛋白單克隆抗體的陽(yáng)性雜交瘤細(xì)胞克隆;擴(kuò)大培養(yǎng)所獲得的單克隆細(xì)胞,制備單克隆抗體,檢測(cè)所獲得單克隆抗體與肝癌細(xì)胞膜蛋白的反應(yīng)性。 結(jié)果:研磨方法提取膜蛋白,免疫四次后,ELISA檢測(cè)免疫小鼠血清抗體效價(jià)達(dá)1:105,尾靜脈沖擊免疫后進(jìn)行細(xì)胞融合,融合四周后,共有10株陽(yáng)性克隆細(xì)胞,擴(kuò)大培養(yǎng)后共有5株克隆生長(zhǎng),但ELISA檢測(cè)均為陰性;膜蛋白提取試劑盒提取膜蛋白,50ug劑量免疫小鼠共四次后,ELISA檢測(cè)免疫小鼠血清抗體效價(jià)均為陰性,用真空干燥法去除有機(jī)溶劑后再次免疫小鼠,小鼠血清抗體效價(jià)仍為陰性,免疫失敗;低滲法提取膜蛋白,免疫小鼠五次后,ELISA檢測(cè)免疫小鼠血清抗體效價(jià)達(dá)1:106,細(xì)胞融合后四周,共有22株陽(yáng)性克隆細(xì)胞,擴(kuò)大培養(yǎng)后共有15株克隆生長(zhǎng),但多次ELISA檢測(cè)細(xì)胞培養(yǎng)上清均為陰性。 結(jié)論:用研磨方法及低滲法提取肝癌細(xì)胞膜蛋白作為抗原,制備針對(duì)肝癌細(xì)胞膜蛋白的單克隆抗體存在一定缺陷。這兩種方法所提取的膜蛋白純度不高、獲得的膜蛋白量小,細(xì)胞膜蛋白抗原成分多,用所提取的膜蛋白制備單克隆抗體相對(duì)比較困難。本實(shí)驗(yàn)?zāi)康氖轻槍?duì)其中各種單一抗原制備單克隆抗體,故研磨方法中以每次25ug劑量免疫小鼠,劑量顯然不足,故在試劑盒方法及低滲法提取膜蛋白時(shí)增加免疫劑量至50ug。用所提取的細(xì)胞膜蛋白免疫小鼠,血清中產(chǎn)生的是多克隆抗體,針對(duì)單一抗原所產(chǎn)生的血清效價(jià)低于針對(duì)細(xì)胞膜蛋白抗原的血清效價(jià),故應(yīng)盡量待血清效價(jià)達(dá)1:106后再進(jìn)行細(xì)胞融合。試劑盒提取膜蛋白純度及定量?jī)?yōu)于研磨方法,但試劑盒中含有的有機(jī)溶劑可能影響了膜蛋白的抗原性,真空干燥法可能不能完全清除有機(jī)溶劑,從而影響免疫小鼠產(chǎn)生抗體的效果。
[Abstract]:Aim: to select and prepare monoclonal antibodies against hepatoma cell membrane proteins by using hybridoma antibody technique. Methods: HepG2 cell membrane proteins were extracted by grinding method (part I), membrane protein extraction kit (part II) and hypoosmotic method (part III). The antigen was purified by ultracentrifugation. The protein was quantified by Bradford method. Balb/c mice were immunized with the extracted antigen at a dose of 25ug-50ug each time. Spleen cells of immunized mice and SP20 myeloma cells were fused with polyethylene glycol method. The ELISA method was used to screen the positive hybridoma cell clones which secreted monoclonal antibodies against hepatoma cell membrane protein. The monoclonal antibody was prepared and the reactivity of the monoclonal antibody to hepatoma cell membrane protein was detected. Results: the membrane protein was extracted by grinding method. The titer of serum antibody of immunized mice was 1: 105 by Elisa after four times immunization. There were 10 positive clones and 5 clones grew after expanded culture, but ELISA was negative. The serum antibody titers of mice immunized with 50ug of membrane protein extraction kit were negative after four times of immunization with Elisa. After removing the organic solvent by vacuum drying method, the mice were immunized again, the antibody titers of the mice were still negative and the immunity failed. The membrane protein was extracted by hypotonic method. The titer of serum antibody of immunized mice was 1: 106 by Elisa after five times. Four weeks after cell fusion, there were 22 positive clone cells. A total of 15 clones grew after expanded culture, but the supernatant of cell culture detected by ELISA was negative. Conclusion: there are some defects in the preparation of monoclonal antibody against hepatoma cell membrane protein by grinding method and hypotonic method. The purity of membrane protein extracted by these two methods is not high. The amount of membrane protein obtained is small, the membrane protein antigen components are many, it is relatively difficult to prepare monoclonal antibody with the extracted membrane protein. The purpose of this experiment is to prepare monoclonal antibody against a variety of single antigens. Therefore, the grinding method to each dose of 25ug immunization mice, the dose is obviously insufficient. Therefore, when the membrane protein was extracted by Kit and hypoosmotic method, the immune dose was increased to 50ug.To immunize mice with the extracted membrane protein, the polyclonal antibody was produced in the serum. The titer of serum produced against single antigen was lower than that against membrane protein antigen. The purity and quantification of membrane protein extracted by the kit was better than the grinding method, but the organic solvent contained in the kit might affect the antigenicity of the membrane protein. Vacuum drying may not completely remove organic solvents, thus affecting the antibody production in immunized mice.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類號(hào)】：R392

【引證文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前1條

1 張任鵬;蓖麻毒素單克隆抗體的制備[D];吉林大學(xué);2013年

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本文編號(hào)：1445950