HIF1α特异性人源喉癌噬菌体单链抗体的制备及其对放疗增敏的实验研究

发布时间：2018-04-15 05:05

本文选题：噬菌体单链抗体scFv + 喉癌　；参考：《第三军医大学学报》2017年16期

【摘要】：目的利用噬菌体肽库技术,构建HIF1α人源喉癌单链抗体(single-chain variable fragment,scFv),研究其对放疗敏感性的影响。方法提取喉癌患者癌旁阳性淋巴结总RNA,通过RTPCR和重叠延伸PCR扩增得到可变区基因scFv,将其重组到载体pCANTAB5E中,转化至TG1大肠杆菌,以制备初级抗体库。以HEP2细胞及HIF1α纯化抗原对抗体库进行淘选。SDS-PAGE电泳检测scFv的可溶性表达,Western blot检测其对HIF1α蛋白表达的影响,ELISA和细胞免疫化学鉴定其特异性,CCK-8检测scFv联合X线照射后HEP2细胞的存活率,克隆形成实验分析scFv处理后HEP2细胞放疗存活曲线。结果成功制备HIF1α人源喉癌单链抗体scFv,SDS-PAGE电泳证实其可溶性表达且相对分子质量约为34×10~3,Western blot表明其能下调HIF1α蛋白的表达。ELISA检测到该抗体对HIF1α抗原的识别率高达79%,细胞免疫化学显示该抗体与HEP2细胞特异性结合。CCK-8检测结果显示scFv联合X线照射后,较单纯X线照射组明显降低了HEP2细胞的存活率(P0.05),克隆形成实验结果显示scFv对放射的增敏比为1.89。结论成功构建了HIF1α人源喉癌单链抗体,且其能增加HEP2细胞对放疗的敏感性。
[Abstract]:Objective to construct single-chain variable fragment scFvn of HIF1 伪 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 伪 purified antigens were used to detect the soluble expression of scFv by SDS-PAGE electrophoresis. The effect of scFv on the expression of HIF1 伪 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 伪 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 伪 protein. The recognition rate of HIF1 伪 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 伪 human laryngeal carcinoma was successfully constructed, and it could increase the sensitivity of HEP2 cells to radiotherapy.
【作者单位】：重庆医科大学附属第一医院肿瘤科;重庆医科大学附属第一医院分子肿瘤及表观遗传学重庆市重点实验室;重庆医科大学附属第一医院放射医学与肿瘤研究室;
【基金】：重庆市卫生和计生委中医药科技项目(ZY20150245) 肿瘤科国家临床重点专科建设项目[国卫办医函(2013)544号]~~
【分类号】：R739.65

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