人源性抗bFGF单链抗体及双价抗体在毕赤酵母中的表达、纯化及鉴定

发布时间：2018-01-15 18:33

本文关键词：人源性抗bFGF单链抗体及双价抗体在毕赤酵母中的表达、纯化及鉴定　出处：《暨南大学》2012年硕士论文　论文类型：学位论文

【摘要】：目的：为了提高人源性抗bFGF抗体的表达量，构建抗bFGF单链抗体ScFv、双价抗体Diabody的酵母表达载体，利用毕赤酵母表达系统表达人源性的抗bFGF ScFv及Diabody并研究其生物学活性。方法：从噬菌体抗体库筛选出的人源性抗bFGF抗体基因中亚克隆单链抗体ScFv基因；以抗bFGF单链抗体为模板，设计引物通过PCR的方法分别扩增出VH、VL，并通过重叠PCR引入中间连接肽（G4S）将其连接构建Diabody基因；将测序正确的ScFv、Diabody基因分别克隆入酵母表达载体pPICZαA中，构建酵母表达工程菌，SDS-PAGE和Western Blot鉴定其表达，间接ELISA检验其特异性；经镍离子亲和层析、阴离子交换层析两步法纯化目的蛋白，将纯化的抗bFGF ScFv及Diabody抗体作用于人肺腺癌细胞株A549，CCK8法检测其增值抑制率。Hoechst33258荧光染色及流式细胞术检验Diabody诱导A549的凋亡。结果：成功构建人源性抗bFGF ScFv及Diabody酵母表达载体，，获得3株高表达ScFv及2株高表达Diabody的酵母工程菌，经1%甲醇诱导约48h表达量即可恒定，SDS-PAGE及Western Blot检测结果显示目的蛋白约36kDa、34kDa，经两步纯化抗体的纯度可达90%以上，间接ELISA检验目的蛋白均可与bFGF特异性结合，Diabody的亲和力要优于ScFv，均可抑制人肺腺癌细胞株A549的增殖，并呈剂量依赖性。Hoechst33258荧光染色及流式细胞术结果显示Diabody能够诱导A549的凋亡。结论：bFGF作为关键的促血管生成因子，与肿瘤的发生、发展密切相关，通过构建并表达了抗bFGF单链抗体ScFv、双价抗体Diabody，体外研究表明其均可抑制人肺腺癌细胞株A549的增殖，并呈剂量依赖性，并且Diabody能够诱导A549的凋亡，从而为进一步研究抗bFGF抗体的生物学特性、结构与功能的关系等奠定了基础。
[Abstract]:Objective: to improve the expression of human anti bFGF antibody and construct the yeast expression vector of anti bFGF single chain antibody scFv and bivalent antibody Diabody. Human anti bFGF ScFv and Diabody were expressed by Pichia pastoris expression system and their biological activities were studied. Methods: single chain antibody (ScFv) gene was cloned from human anti bFGF antibody gene selected from phage antibody library. Using bFGF single chain antibody as template, the primers were designed to amplify VL by PCR. The Diabody gene was constructed by introducing the intermediate ligation peptide (G4S) into the Diabody gene by overlapping PCR. The correctly sequenced ScFvn Diabody gene was cloned into yeast expression vector pPICZ 伪 A to construct yeast expression engineering bacteria. SDS-PAGE and Western Blot were used to identify its expression, and indirect ELISA was used to detect its specificity. The target protein was purified by nickel ion affinity chromatography and anion exchange chromatography. The purified anti bFGF ScFv and Diabody antibodies were applied to human lung adenocarcinoma cell line A549. The proliferation inhibition rate of A549 was detected by CCK8 assay. Hoechst33258 fluorescence staining and flow cytometry were used to detect the apoptosis of A549 induced by Diabody. Results: the yeast expression vectors against bFGF ScFv and Diabody were successfully constructed, and three strains of high expression ScFv and two strains of yeast engineering bacteria with high expression of Diabody were obtained. The results of SDS-PAGE and Western Blot analysis showed that the target protein was about 36kDa ~ 34kDa. The purity of the antibody can reach more than 90% after two-step purification, and the affinity of the target protein to bFGF by indirect ELISA detection is superior to that of ScFv. Can inhibit the proliferation of human lung adenocarcinoma cell line A549. The results of fluorescence staining and flow cytometry showed that Diabody could induce apoptosis of A549 in a dose-dependent manner. Conclusion as a key angiogenic factor, the bFGF is closely related to the development and development of the tumor. The anti bFGF scFv, bivalent antibody Diabody was constructed and expressed. In vitro studies showed that it could inhibit the proliferation of human lung adenocarcinoma cell line A549 in a dose-dependent manner and Diabody could induce apoptosis of A549. The results provide a basis for further study on the biological characteristics, the relationship between structure and function of anti-bFGF antibodies.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392

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