一株针对人EGFR的单链抗体克隆与哺乳细胞表达

Cloning and Expression of Single Chain Antibody Against Human EGFR

目的：制备特异性抗人表皮生长因子受体（EGFR）的单链抗体（sc Fv）,鉴定其生物学活性,为进一步研究基于单链抗体的免疫治疗奠定基础。方法：从分泌抗人EGFR单克隆抗体的杂交瘤细胞系提取总RNA,利用5’RACE技术扩增轻链和重链可变区（VL、VH）基因,构建具有VL、VH基因的单链抗体基因,并将构建的单链抗体基因克隆到真核细胞表达载体pc DNA3.1中进行表达和鉴定。ELISA鉴定单链抗体对抗原的特异性;Fortebio检测抗原抗体间的亲和力,流式细胞术检测单链抗体结合肺癌细胞系天然EGFR的功能活性。结果：获得唯一的轻重链可变区序列VL、VH,成功构建EGFR-sc Fv,特异性与天然EGFR蛋白结合,亲和力达3.22×10-9mol/L。结论：成功构建了抗人EGFR单链抗体,为肺癌免疫导向治疗研究奠定了基础。

Objective： Epidermal growth factor receptor（ EGFR） is overexpressed in tumors and is associated with metastasis,invasion and poor prognosis of tumor cells. Therefore,EGFR has become an attractive target for the treatment of cancer. Several small molecule compounds that specifically inhibit EGFR have been clinically developed. Preparation of specific antibody to human EGFR with an identified biological activity is essential for this target-related immunotherapeutic study,such as a construction of single chain antibody fragment（ sc Fv）. Methods： Total RNA was extracted from the hybridoma cell line specific to EGFR. The light and heavy chain variable region genes VLand VHwere obtained by 5’ RACE technology. The constructed sc Fv antibody gene was cloned into the eukaryotic expression vector pc DNA3. 1. ELISA was used to detect the specificity of expressed single chain antibody to human EGFR protein. The affinity between antigen and antibody was detected by Fortebio. Flow cytometry was used to detect the functional activity of single chain antibody by which binding to natural EGFR on lung cancer cell lines. Results： The VLand VHsequences of light and heavy chain variable regions were obtained,and assembled EGFR-sc Fv was successfully expressed. The EGFR-sc Fv was specifically bound to natural EGFR protein with an affinity of 3. 22 × 10-9 mol/L,and the sc Fv was further indentified based on its binding to EGFR that expressed by lung cancer cell line H1975. Conclusion： The anti-human EGFR single chain antibody was successfully constructed,which lays the foundation for development of EGFR-targeted immunotherapy.