HIF1α特异性人源喉癌噬菌体单链抗体的制备及其对HEP2细胞放射增敏的实验研究

发布时间：2018-04-06 02:01

本文选题：噬菌体单链抗体scFv　切入点：喉癌　出处：《重庆医科大学》2017年硕士论文

【摘要】：目的利用噬菌体肽库技术,制备人源化HIF1α喉癌单链抗体,检测抗体性能,并研究其对HEP2细胞放射敏感性的影响,为喉癌靶向治疗和放射增敏研究提供实验室数据。方法提取喉癌患者癌旁阳性淋巴结总RNA,通过RT-PCR和重叠延伸PCR(SOE-PCR)扩增得到可变区基因片段。将扩增片段重组到噬菌体载体pCANTAB5E中,转化至TG1大肠杆菌,以制备初级抗体库。先后以HEP2细胞及HIF1α纯化抗原对抗体库进行免疫亲和富集,制备HEP2细胞特异性HIF1α单链抗体scFv。SDS-PAGE电泳检测单链抗体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,CCK8检测scFv联合6MV-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细胞放射线辐照存活曲线。结果成功制备HIF1α人喉癌单链抗体scFv;SDS-PAGE电泳证实其可溶性表达且分子量约为34 kDa;Western blot实验表明其能下调HEP2细胞中HIF1α蛋白的表达;ELISA实验检测到该抗体对HIF1α抗原的识别率为79%;细胞免疫化学显示该抗体与HEP2细胞特异性结合。CCK8检测结果显示scFv联合6MV-X线照射后,HEP2细胞存活率较单纯X线照射组明显降低(P0.05);克隆形成实验结果显示scFv干预后,HEP2细胞对放射辐照的增敏比SER为1.89。结论成功构建了人源喉癌噬菌体单链抗体库,并筛选出能与HIF1α蛋白特异性结合的单链抗体,且其能增加HEP2细胞对放射线的敏感性,为喉癌的靶向治疗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 伪 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 伪 purified antigen.Preparation of HEP2 cell specific HIF1 伪 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 伪 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 伪 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 伪 protein in HEP2 cells. Elisa assay showed that the recognition rate of HIF1 伪 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 伪 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.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R739.65

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