本文關(guān)鍵詞：人源性抗bFGF單鏈抗體及雙價(jià)抗體在畢赤酵母中的表達(dá)、純化及鑒定　出處：《暨南大學(xué)》2012年碩士論文　論文類(lèi)型：學(xué)位論文

  更多相關(guān)文章： bFGF 抗體片段 畢赤酵母 肺癌

【摘要】：目的：為了提高人源性抗bFGF抗體的表達(dá)量，構(gòu)建抗bFGF單鏈抗體ScFv、雙價(jià)抗體Diabody的酵母表達(dá)載體，利用畢赤酵母表達(dá)系統(tǒng)表達(dá)人源性的抗bFGF ScFv及Diabody并研究其生物學(xué)活性。 方法：從噬菌體抗體庫(kù)篩選出的人源性抗bFGF抗體基因中亞克隆單鏈抗體ScFv基因；以抗bFGF單鏈抗體為模板，設(shè)計(jì)引物通過(guò)PCR的方法分別擴(kuò)增出VH、VL，并通過(guò)重疊PCR引入中間連接肽（G4S）將其連接構(gòu)建Diabody基因；將測(cè)序正確的ScFv、Diabody基因分別克隆入酵母表達(dá)載體pPICZαA中，構(gòu)建酵母表達(dá)工程菌，SDS-PAGE和Western Blot鑒定其表達(dá)，間接ELISA檢驗(yàn)其特異性；經(jīng)鎳離子親和層析、陰離子交換層析兩步法純化目的蛋白，將純化的抗bFGF ScFv及Diabody抗體作用于人肺腺癌細(xì)胞株A549，CCK8法檢測(cè)其增值抑制率。Hoechst33258熒光染色及流式細(xì)胞術(shù)檢驗(yàn)Diabody誘導(dǎo)A549的凋亡。 結(jié)果：成功構(gòu)建人源性抗bFGF ScFv及Diabody酵母表達(dá)載體，，獲得3株高表達(dá)ScFv及2株高表達(dá)Diabody的酵母工程菌，經(jīng)1%甲醇誘導(dǎo)約48h表達(dá)量即可恒定，SDS-PAGE及Western Blot檢測(cè)結(jié)果顯示目的蛋白約36kDa、34kDa，經(jīng)兩步純化抗體的純度可達(dá)90%以上，間接ELISA檢驗(yàn)?zāi)康牡鞍拙膳cbFGF特異性結(jié)合，Diabody的親和力要優(yōu)于ScFv，均可抑制人肺腺癌細(xì)胞株A549的增殖，并呈劑量依賴(lài)性。Hoechst33258熒光染色及流式細(xì)胞術(shù)結(jié)果顯示Diabody能夠誘導(dǎo)A549的凋亡。 結(jié)論：bFGF作為關(guān)鍵的促血管生成因子，與腫瘤的發(fā)生、發(fā)展密切相關(guān)，通過(guò)構(gòu)建并表達(dá)了抗bFGF單鏈抗體ScFv、雙價(jià)抗體Diabody，體外研究表明其均可抑制人肺腺癌細(xì)胞株A549的增殖，并呈劑量依賴(lài)性，并且Diabody能夠誘導(dǎo)A549的凋亡，從而為進(jìn)一步研究抗bFGF抗體的生物學(xué)特性、結(jié)構(gòu)與功能的關(guān)系等奠定了基礎(chǔ)。
[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 偽 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.
【學(xué)位授予單位】：暨南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類(lèi)號(hào)】：R392

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本文編號(hào)：1429574