本文關(guān)鍵詞：全人源抗CCR7噬菌體單鏈抗體的篩選及初步鑒定　出處：《重慶醫(yī)科大學(xué)》2010年碩士論文　論文類型：學(xué)位論文

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【摘要】： 目的:從噬菌體抗體庫(kù)中篩選抗CCR7單鏈融合抗體,并對(duì)其性能進(jìn)行初步檢測(cè)。 方法:PCR檢測(cè)大腸桿菌中ScFv基因插入率;1%瓊脂糖凝膠電泳鑒定Sfi I和Not I雙酶切質(zhì)粒的結(jié)果;分別以乳腺癌細(xì)胞MDA-MB-435s細(xì)胞及CCR7多肽片段為靶抗原對(duì)抗體庫(kù)進(jìn)行4輪和3輪篩選富集。將陽(yáng)性克隆轉(zhuǎn)化E.coli HB2151進(jìn)行可溶表達(dá)�？贵w親和層析純化后,Western-blot結(jié)果顯示獲得抗體相對(duì)分子質(zhì)量為34 kd左右。免疫細(xì)胞化學(xué)、免疫組織化學(xué)檢測(cè)與放射免疫顯像均證實(shí)單鏈抗體與表達(dá)CCR7的乳腺癌細(xì)胞特異性結(jié)合。人工基底膜穿透實(shí)驗(yàn)檢測(cè)131I標(biāo)記scFv抗體對(duì)CCR7抗原表達(dá)陽(yáng)性乳腺癌細(xì)胞侵襲能力的影響。 結(jié)果:ScFv基因插入率為90%(18/20),雙酶切鑒定檢測(cè)到目的條帶。經(jīng)4輪細(xì)胞篩選,3輪抗原篩選抗CCR7抗原的噬菌體抗體得到了明顯富集,在E.coli HB2151中實(shí)現(xiàn)可溶表達(dá)。Western-blot結(jié)果顯示獲得抗體相對(duì)分子質(zhì)量為34kd左右。免疫細(xì)胞化學(xué)、免疫組織化學(xué)檢測(cè)與放射免疫顯像均證實(shí)單鏈抗體與表達(dá)CCR7的乳腺癌細(xì)胞特異性結(jié)合。人工基底膜穿透實(shí)驗(yàn)顯示131I標(biāo)記scFv抗體可抑制CCR7抗原表達(dá)陽(yáng)性乳腺癌細(xì)胞的侵襲能力。 結(jié)論:利用乳腺癌細(xì)胞MDA-MB-435s和CCR7多肽片段為靶抗原,從噬菌體抗體庫(kù)中篩選獲得具有較高特異性的抗CCR7單鏈抗體�？贵w在體內(nèi)體外均與腫瘤細(xì)胞表達(dá)抗原特異結(jié)合,并在體外可抑制其侵襲作用。
[Abstract]:Aim: to screen anti CCR7 single chain fusion antibody from phage antibody library and to test its properties. Methods the insertion rate of ScFv gene in Escherichia coli was detected by 10% PCR. 1% agarose gel electrophoresis was used to identify the plasmids digested by Sfi I and Not I. Using breast cancer cell MDA-MB-435s cells and CCR7 polypeptide fragments as target antigens, the antibody library was screened for 4 and 3 rounds, respectively. The positive clones were transformed into E. coli. HB2151 was expressed and purified by affinity chromatography. The results of Western-blot showed that the relative molecular weight of the antibody was about 34kd and immunocytochemistry. Immunohistochemistry and radioimmunoimaging confirmed the specific binding of scFv to breast cancer cells expressing CCR7. Detection of 131I-labeled scFv Antibody against CCR7 by artificial basement membrane Penetration Assay. Effect of prokaryotic expression on invasive ability of breast cancer cells. Results the insertion rate of the 10% scFv gene was 90% and 18 / 20%. The target band was detected by double enzyme digestion and was screened by 4 rounds of cells. The phage antibodies against CCR7 antigens were significantly enriched by three rounds of antigen screening. The immunocytochemistry showed that the relative molecular weight of the antibody was about 34kd. Immunohistochemistry and radioimmunoimaging confirmed the specific binding of scFv to breast cancer cells expressing CCR7. Artificial basement membrane Penetration assay showed that 131I-labeled scFv antibody could inhibit CCR. 7 the invasive ability of breast cancer cells positive for antigen expression. Conclusion: MDA-MB-435s and CCR7 polypeptide fragments were used as target antigens in breast cancer cells. A highly specific anti CCR7 single chain antibody was obtained from phage phage antibody library. In vivo and in vitro, the antibody could bind to tumor cell expression antigen and inhibit its invasion in vitro.
【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】：2010
【分類號(hào)】：R392

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