本文選題：噬菌體單鏈抗體scFv + 喉癌��； 參考：《第三軍醫(yī)大學(xué)學(xué)報(bào)》2017年16期

【摘要】：目的利用噬菌體肽庫技術(shù),構(gòu)建HIF1α人源喉癌單鏈抗體(single-chain variable fragment,scFv),研究其對(duì)放療敏感性的影響。方法提取喉癌患者癌旁陽性淋巴結(jié)總RNA,通過RTPCR和重疊延伸PCR擴(kuò)增得到可變區(qū)基因scFv,將其重組到載體pCANTAB5E中,轉(zhuǎn)化至TG1大腸桿菌,以制備初級(jí)抗體庫。以HEP2細(xì)胞及HIF1α純化抗原對(duì)抗體庫進(jìn)行淘選。SDS-PAGE電泳檢測scFv的可溶性表達(dá),Western blot檢測其對(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,CCK-8檢測scFv聯(lián)合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)胞放療存活曲線。結(jié)果成功制備HIF1α人源喉癌單鏈抗體scFv,SDS-PAGE電泳證實(shí)其可溶性表達(dá)且相對(duì)分子質(zhì)量約為34×10~3,Western blot表明其能下調(diào)HIF1α蛋白的表達(dá)。ELISA檢測到該抗體對(duì)HIF1α抗原的識(shí)別率高達(dá)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。CCK-8檢測結(jié)果顯示scFv聯(lián)合X線照射后,較單純X線照射組明顯降低了HEP2細(xì)胞的存活率(P0.05),克隆形成實(shí)驗(yàn)結(jié)果顯示scFv對(duì)放射的增敏比為1.89。結(jié)論成功構(gòu)建了HIF1α人源喉癌單鏈抗體,且其能增加HEP2細(xì)胞對(duì)放療的敏感性。
[Abstract]:Objective to construct single-chain variable fragment scFvn of HIF1 偽 human laryngeal carcinoma by phage peptide library and to study its effect on radiosensitivity.Methods the total RNAs of lymph nodes adjacent to larynx carcinoma were extracted, and the variable region gene scFvwas amplified by RTPCR and overlapping extension PCR. The gene was recombined into vector pCANTAB5E and transformed into TG1 Escherichia coli to prepare the primary antibody library.HEP2 cells and HIF1 偽 purified antigens were used to detect the soluble expression of scFv by SDS-PAGE electrophoresis. The effect of scFv on the expression of HIF1 偽 protein was detected by Elisa and immunocytochemistry. CCK-8 was used to detect the survival rate of HEP2 cells after scFv combined with X ray irradiation.The survival curve of HEP2 cells treated with scFv was analyzed by clone formation assay.Results HIF1 偽 was successfully prepared from human laryngeal carcinoma by SDS-PAGE and its soluble expression was confirmed by SDS-PAGE, and the relative molecular weight was about 34 脳 10 ~ (-3) blot. Elisa showed that the antibody could down-regulate the expression of HIF1 偽 protein. The recognition rate of HIF1 偽 antigen was as high as 79% by Elisa, and the cell immunity was obtained.The specific binding of the antibody to HEP2 cells. CCK-8 assay showed that scFv combined with X ray irradiation,The survival rate of HEP2 cells was significantly lower than that of X-ray irradiation group, and the clone formation assay showed that the enhancement ratio of scFv to radiation was 1.89.Conclusion the single chain antibody (scFv) of HIF1 偽 human laryngeal carcinoma was successfully constructed, and it could increase the sensitivity of HEP2 cells to radiotherapy.
【作者單位】： 重慶醫(yī)科大學(xué)附屬第一醫(yī)院腫瘤科;重慶醫(yī)科大學(xué)附屬第一醫(yī)院分子腫瘤及表觀遺傳學(xué)重慶市重點(diǎn)實(shí)驗(yàn)室;重慶醫(yī)科大學(xué)附屬第一醫(yī)院放射醫(yī)學(xué)與腫瘤研究室;
【基金】：重慶市衛(wèi)生和計(jì)生委中醫(yī)藥科技項(xiàng)目(ZY20150245) 腫瘤科國家臨床重點(diǎn)�？平ㄔO(shè)項(xiàng)目[國衛(wèi)辦醫(yī)函(2013)544號(hào)]~~
【分類號(hào)】：R739.65

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