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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(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。
OBJECTIVE: To construct single-chain variable fragment (scFv) of human HIF-1α human laryngeal squamous cell carcinoma using phage peptide library technology and study its effect on radiosensitivity. Methods Total RNA was extracted from para-cancerous lymph nodes in patients with laryngeal cancer. The variable region gene scFv was amplified by RTPCR and overlap extension PCR. The scFv was recombined into vector pCANTAB5E and transformed into E. coli TG1 to prepare primary antibody library. Antibody pools were panned with HEP2 cells and HIF1α purified antigen. SDS-PAGE was used to detect the soluble expression of scFv. The expression of HIF1α protein was detected by Western blot. The specificity of HIF1α protein was detected by ELISA and immunocytochemistry. The survival rate of HEP2 cells was detected by CCK-8 after scFv combined with X-ray. The radiotherapy survival curves of HEP2 cells after scFv treatment were analyzed. Results The single chain Fv scFv of HIF1α human laryngeal carcinoma was successfully prepared and its soluble expression was confirmed by SDS-PAGE. The relative molecular mass was about 34 × 10-3. Western blot showed that it can down-regulate the expression of HIF1α protein. ELISA detection of the antibody recognition rate of HIF1α antigen as high as 79%, immunocytochemistry showed that the antibody and HEP2 cells specific binding. The results of CCK-8 showed that the scFv combined with X-ray irradiation significantly reduced the survival rate of HEP2 cells (P <0.05) as compared with X-ray irradiation alone. The results of colony formation assay showed that the scFv sensitization ratio to radiation was 1.89. Conclusion The single chain Fv antibody of HIF1α human laryngeal carcinoma was successfully constructed and it can increase the sensitivity of HEP2 cells to radiotherapy.