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

目的 利用噬菌体肽库技术,构建HIF1 α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RT-PCR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34×103,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(P＜0.05),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.

Preparation of human phage displayed single-chain antibody against hypoxia-inducible factor 1 alpha of laryngeal carcinoma and its effect on radiotherapy sensitization

Objective To construct a phage-displayed library and identify phage-displayed singlechain variable fragment (scFv) antibody against hypoxia-inducible factor 1 alpha (HIF1α) of laryngeal carcinoma.Methods Total RNA of positive lymph node tissues adjacent to laryngeal carcinoma was extracted.ScFv gene fragments were amplified by RT-PCR and splicing-overlap-extension (SOE) PCR,then linked into the pCANTAB5E phagemid vector.The recombinant plasmids were transformed into E.coli TG1 cells in order to create the primary phage-displayed scFv library.The primary scFv library was performed by biopanning against HEP2 cells and HIF1 α antigen.The properties of the product were identified by Western blotting,and its specificity was detected by ELISA and immunocytochemical assay.CCK8 assay was used to detect its effect on cell viability,and cell clone assay was used to detect its effect on survival fraction.Results A phage-displayed scFv library targeting HIF1α antigen and HEP2 cells was successfully constructed.The prepared scFv antibody had an apparent molecular weight of 34 × 103 and down-regulated the expression of HIF1 α protein in HEP2 cells.ELISA showed that the obtained scFv antibody had a positive recognition rate of 79％ to HIF1α antigen.Immunocytochemical assay indicated that it could specifically bind to HEP2 cells.The obtained scFv antibody decreased the viability of HEP2 cells after X-ray radiation when compared with the cells treated by radiation alone (P ＜ 0.05).Colony formation test showed that the sensitization enhancement ratio (SER) of the antibody to radiation was 1.89.Conclusion A human antiHIF1α scFv antibody of laryngeal carcinoma is successfully prepared,and it can increase the radiation sensitivity of HEP2 cells.