本文選題：噬菌體單鏈抗體scFv　切入點(diǎn)：喉癌　出處：《重慶醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的利用噬菌體肽庫技術(shù),制備人源化HIF1α喉癌單鏈抗體,檢測(cè)抗體性能,并研究其對(duì)HEP2細(xì)胞放射敏感性的影響,為喉癌靶向治療和放射增敏研究提供實(shí)驗(yàn)室數(shù)據(jù)。方法提取喉癌患者癌旁陽性淋巴結(jié)總RNA,通過RT-PCR和重疊延伸PCR(SOE-PCR)擴(kuò)增得到可變區(qū)基因片段。將擴(kuò)增片段重組到噬菌體載體pCANTAB5E中,轉(zhuǎn)化至TG1大腸桿菌,以制備初級(jí)抗體庫。先后以HEP2細(xì)胞及HIF1α純化抗原對(duì)抗體庫進(jìn)行免疫親和富集,制備HEP2細(xì)胞特異性HIF1α單鏈抗體scFv。SDS-PAGE電泳檢測(cè)單鏈抗體scFv的可溶性表達(dá),Western blot檢測(cè)其對(duì)HIF1α蛋白表達(dá)的影響,ELISA和細(xì)胞免疫化學(xué)鑒定其特異性,CCK8檢測(cè)scFv聯(lián)合6MV-X線照射后HEP2細(xì)胞的存活率,克隆形成實(shí)驗(yàn)分析scFv處理后HEP2細(xì)胞放射線輻照存活曲線。結(jié)果成功制備HIF1α人喉癌單鏈抗體scFv;SDS-PAGE電泳證實(shí)其可溶性表達(dá)且分子量約為34 kDa;Western blot實(shí)驗(yàn)表明其能下調(diào)HEP2細(xì)胞中HIF1α蛋白的表達(dá);ELISA實(shí)驗(yàn)檢測(cè)到該抗體對(duì)HIF1α抗原的識(shí)別率為79%;細(xì)胞免疫化學(xué)顯示該抗體與HEP2細(xì)胞特異性結(jié)合。CCK8檢測(cè)結(jié)果顯示scFv聯(lián)合6MV-X線照射后,HEP2細(xì)胞存活率較單純X線照射組明顯降低(P0.05);克隆形成實(shí)驗(yàn)結(jié)果顯示scFv干預(yù)后,HEP2細(xì)胞對(duì)放射輻照的增敏比SER為1.89。結(jié)論成功構(gòu)建了人源喉癌噬菌體單鏈抗體庫,并篩選出能與HIF1α蛋白特異性結(jié)合的單鏈抗體,且其能增加HEP2細(xì)胞對(duì)放射線的敏感性,為喉癌的靶向治療和放射增敏研究提供了新的思路。
[Abstract]:Objective to prepare single chain antibody (scFv) of human HIF1 偽 laryngeal carcinoma by phage phage peptide library, and to study its effect on radiosensitivity of HEP2 cells, and to provide laboratory data for targeted therapy and radiosensitization of laryngeal carcinoma.Methods Total RNAs were extracted from paracancerous lymph nodes of laryngeal cancer patients. The variable region gene fragments were amplified by RT-PCR and overlapping extension PCRSOE-PCR.The amplified fragment was recombined into phage vector pCANTAB5E and transformed into TG1 Escherichia coli to prepare the primary antibody library.The antibody library was enriched with HEP2 cells and HIF1 偽 purified antigen.Preparation of HEP2 cell specific HIF1 偽 single chain antibody scFv.SDS-PAGE electrophoresis to detect the soluble expression of single chain antibody scFv the effect of Western blot on the expression of HIF1 偽 protein; Elisa and immunocytochemistry were used to detect the survival rate of HEP2 cells after scFv combined with 6MV-X line irradiation.Clone formation assay was used to analyze the survival curve of HEP2 cells treated with scFv.Results HIF1 偽 was successfully prepared from human laryngeal carcinoma by SDS-PAGE. The soluble expression and molecular weight of the antibody were about 34kDa. Western blot assay showed that the antibody could down-regulate the expression of HIF1 偽 protein in HEP2 cells. Elisa assay showed that the recognition rate of HIF1 偽 antigen by this antibody was as follows: 1.The cell immunocytochemistry showed that the specific binding of the antibody to HEP2 cells. CCK8 assay showed that the survival rate of HEP2 cells after scFv combined with 6MV-X irradiation was significantly lower than that of simple X-ray irradiation group, and the clone formation test showed that the survival rate of Hep 2 cells after scFv intervention was significantly lower than that of X ray irradiation group.The sensitizing ratio (SER) of cells to radiation was 1.89.Conclusion the phage single chain antibody library of human laryngeal carcinoma was successfully constructed, and the single chain antibody which could specifically bind to HIF1 偽 protein was screened, and it could increase the radiosensitivity of HEP2 cells to radiation.It provides a new idea for the targeted therapy and radiosensitization of laryngeal carcinoma.
【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】：2017
【分類號(hào)】：R739.65

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