靶向EGFR分子的荧光探针设计及其对非小细胞肺癌体内外检测功能的研究

The Generation of a EGFR Targeted scFv Conjugate Fluorochrome Which Detected the Non-small Cell Lung Cancer Invitro and vivo

目的使用单链抗体偶联荧光染料的手段构建一种探针,其可对表皮生长因子受体(EGFR)阳性非小细胞肺癌进行体内外检测。方法通过Overlap PCR(polymerase chain reaction)的方法通过柔性肽G4S将爱必妥(西妥昔单抗)的轻链与重链连接,构建成工程载体,并通过毕赤酵母进行表达纯化,获得靶向EGFR的单链抗体;流式细胞术检测单链抗体对EGFR阳性非小细胞肺癌细胞A549的结合能力;将单链抗体与碱性荧光染料罗丹明B偶联,使用激光共聚焦检测单链抗体在体外对A549细胞的靶向能力;将单链抗体与近红外荧光染料NIRB-NHS偶联,通过CCD照相机实时收集荧光信号检测偶联探针在荷瘤裸鼠体内的靶向能力,并判断其开发为检测试剂的潜力。结果通过基因工程手段和毕赤酵母表达,成功获得抗EGFR单链抗体,经Western blot鉴定结果说明单链抗体Anti-EGFR scFv表达及装配正确。流式细胞术检测Anti-EGFR scFv与非小细胞肺癌细胞A549的结合率为40.3%。体外靶向性实验验证单链抗体Anti-EGFR scFv可以在体外很好地靶向非小细胞肺癌细胞A549,单链抗体组的细胞平均荧光强度为136.38±7.62,明显优于阻断对照组(19.83±2.75)。体内近红外成像实验中,通过荷A549细胞移植瘤裸鼠给药后的荧光信号分析结果显示单链抗体探针可以很好靶向至肿瘤部位,分析热点区域的最大肿瘤/正常组织荧光信号比可知,单链抗体组的信号比为7.54±1.42,明显强于阻断对照组(1.17±0.28)。结论本文采用毕赤酵母表达体系成功构建并表达了单链抗体Anti-EGFR scFv。单链抗体保留了母本抗体的结合能力,并体现了很好的体外和体内的靶向能力,其靶向能力可以应用于EGFR阳性非小细胞肺癌的检测领域,拥有潜在的临床应用前景。

Objective Using the single chain fragment variable （scFv）conjugate fluorochrome to establish a probe, which can target and detect Human Epidermal Growth Factor Receptor type 2 （EGFR）positive non- small cell lung cancer invitro and invivo. Methods The new scFv was obtained by overlapping PCR between variable region of heavy chain from patented Cetuximab, G4S linker and variable region of light chain. And Pichiapastoris was used to express Anti-EGFR scFv. The binding activity of Anti-EGFR scFv to non-small cell lung cancer cell line A549 was detected by flow cytometry. The in-vitro targeting prosperities of Anti- EGFR scFv to non-small cell lung cancer cell line A549 was detected by laser confocal microscope through linking Anti-EGFR scFv with alkaline dye Rhodamine B. A549-beating nude mice model was established to evaluate the targeting activity of Anti-EGFR scFv in vivo. CCD camera was used to image the dynamics of Anti-EGFR scFv conjugated near infrared dye IRB-NHS in vivo. Results The target protein anti-EGFR single chain fragment variable was purified from culture supernatants by nickel affinity chromatograph, followed by analysis on western blot. Furthermore, anti-EGFR scFv maintained binding activity to A549 and the binding ratio was 40.3%. From the invitro targeting experiment anti-EGFR scFv targeted A549 in vitro. And the average fluorescence intensity of anti-EGFR scFv was 136.38±7.62, which was better than that of blocking group（ 19.83- 2.75 ）. As anticipate, scFv maintained effective in vivo targeting using near infrared （NIR） imaging in A549 xenografts. And the tumor to normal tissue ratio of scFv was 7.54±1.42, which was better than blocking group （ 1. 17±0.28 ）. Conclusion We established and express the anti-EGFR scFv, which maintained the binding activity of parent antibody. What＇ s more, anti-EGFR scFv has a desirable targeting activity invitro and invivo. This ability might be used in the detection and has potential clinical application prospect.