全人源抗CCR7噬菌体单链抗体的筛选及初步鉴定

发布时间：2018-01-15 14:27

本文关键词：全人源抗CCR7噬菌体单链抗体的筛选及初步鉴定　出处：《重庆医科大学》2010年硕士论文　论文类型：学位论文

【摘要】： 目的:从噬菌体抗体库中筛选抗CCR7单链融合抗体,并对其性能进行初步检测。方法:PCR检测大肠杆菌中ScFv基因插入率;1%琼脂糖凝胶电泳鉴定Sfi I和Not I双酶切质粒的结果;分别以乳腺癌细胞MDA-MB-435s细胞及CCR7多肽片段为靶抗原对抗体库进行4轮和3轮筛选富集。将阳性克隆转化E.coli HB2151进行可溶表达。抗体亲和层析纯化后,Western-blot结果显示获得抗体相对分子质量为34 kd左右。免疫细胞化学、免疫组织化学检测与放射免疫显像均证实单链抗体与表达CCR7的乳腺癌细胞特异性结合。人工基底膜穿透实验检测131I标记scFv抗体对CCR7抗原表达阳性乳腺癌细胞侵袭能力的影响。结果:ScFv基因插入率为90%(18/20),双酶切鉴定检测到目的条带。经4轮细胞筛选,3轮抗原筛选抗CCR7抗原的噬菌体抗体得到了明显富集,在E.coli HB2151中实现可溶表达。Western-blot结果显示获得抗体相对分子质量为34kd左右。免疫细胞化学、免疫组织化学检测与放射免疫显像均证实单链抗体与表达CCR7的乳腺癌细胞特异性结合。人工基底膜穿透实验显示131I标记scFv抗体可抑制CCR7抗原表达阳性乳腺癌细胞的侵袭能力。结论:利用乳腺癌细胞MDA-MB-435s和CCR7多肽片段为靶抗原,从噬菌体抗体库中筛选获得具有较高特异性的抗CCR7单链抗体。抗体在体内体外均与肿瘤细胞表达抗原特异结合,并在体外可抑制其侵袭作用。
[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.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392

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