本文選題：噬菌體單鏈抗體scFv　切入點：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,檢測抗體性能,并研究其對HEP2細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驗室數(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)擴增得到可變區(qū)基因片段。將擴增片段重組到噬菌體載體pCANTAB5E中,轉(zhuǎn)化至TG1大腸桿菌,以制備初級抗體庫。先后以HEP2細胞及HIF1α純化抗原對抗體庫進行免疫親和富集,制備HEP2細胞特異性HIF1α單鏈抗體scFv。SDS-PAGE電泳檢測單鏈抗體scFv的可溶性表達,Western blot檢測其對HIF1α蛋白表達的影響,ELISA和細胞免疫化學(xué)鑒定其特異性,CCK8檢測scFv聯(lián)合6MV-X線照射后HEP2細胞的存活率,克隆形成實驗分析scFv處理后HEP2細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電泳證實其可溶性表達且分子量約為34 kDa;Western blot實驗表明其能下調(diào)HEP2細胞中HIF1α蛋白的表達;ELISA實驗檢測到該抗體對HIF1α抗原的識別率為79%;細胞免疫化學(xué)顯示該抗體與HEP2細胞特異性結(jié)合。CCK8檢測結(jié)果顯示scFv聯(lián)合6MV-X線照射后,HEP2細胞存活率較單純X線照射組明顯降低(P0.05);克隆形成實驗結(jié)果顯示scFv干預(yù)后,HEP2細胞對放射輻照的增敏比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細胞對放射線的敏感性,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)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R739.65

【參考文獻】

相關(guān)期刊論文 前3條

1 羅弋;龐華;李淑杰;曹輝;李少林;樊春波;王潔;;人源抗PeroxiredoxinⅠ肺腺癌噬菌體抗體的制備及鑒定[J];南方醫(yī)科大學(xué)學(xué)報;2010年01期

2 王潔;李建國;龐華;彭志平;李少林;;抗HIF-1α肺腺癌人源單鏈抗體的篩選和初步鑒定[J];第三軍醫(yī)大學(xué)學(xué)報;2009年23期

3 楊家和;雙功能單克隆抗體的基礎(chǔ)與臨床研究進展[J];國外醫(yī)學(xué)(臨床生物化學(xué)與檢驗學(xué)分冊);1998年04期

，

本文編號：1717448