荧光探针偶联GPC3单链抗体对肝癌的体内外检测研究

Study on the detection of hepatocellular carcinoma in vitro and in vivo by fluorescence probe coupled with GPC3 scFv

目的使用单链抗体偶联荧光染料构建探针进行磷脂酰肌醇蛋白聚糖3(GPC3)阳性肝癌的体内外检测。方法通过Overlap聚合酶链反应(PCR)采用柔性肽G4S将罗氏公司开发的Codrituzumab轻链与重链连接,构建工程载体,并通过毕赤酵母进行表达纯化,获得靶向GPC3的单链抗体;采用流式细胞术检测单链抗体对GPC3阳性肝癌细胞Huh-7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Huh-7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GPC3单链抗体,经WB鉴定结果说明单链抗体AntiGPC3scFv表达及装配正确。流式细胞术检测Anti-GPC3scFv与肝癌细胞Huh-7的结合率为40.3%。体外靶向性试验验证单链抗体Anti-GPC3scFv可在体外很好地靶向肝癌细胞Huh-7,单链抗体组细胞平均荧光强度为218.61±9.03,明显优于对照组的31.74±5.28。体内近红外成像试验中,通过Huh-7细胞移植荷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地靶向至肿瘤部位,分析热点区域的最大肿瘤/正常组织荧光信号比可知,单链抗体组信号比为8.27±2.94,明显强于对照组的1.09±0.63。结论采用毕赤酵母表达体系成功构建并表达了单链抗体Anti-GPC3scFv。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靶向能力,其靶向能力可应用于GPC3阳性肝癌的检测领域,拥有潜在的临床应用前景。

Objective A probe is constructed using single chain antibody coupled with fluorescent dyes,which can be detected in vitro and in vivo for phosphatidylinositol 3（GPC3）positive liver cancer.Methods The Overlap polymerase chain reaction（PCR）method was used to connect the light chain and heavy chain of Codrituzumab developed by Roche company with the flexible peptide G4 S,a engineering carrier was construct,and was expressed and purified by Pichia pastoris,and the single chain antibody of the target GPC3 was obtained;the combining ability of the single chain antibody to the GPC3 positive liver cancer cell Huh-7 was detected by flow cytometry;the single chain antibody and alkaline fluorescent dye Luo Danming B was coupled,and laser confocal microscopy was used to detect the targeting ability of single chain antibody to Huh-7 cells in vitro;the single chain antibody with the near infrared fluorescent dye NIRB-NHS was coupled and the targeting ability of the coupling probe through the real-time collection of fluorescence signals with a CCD camera in the tumor bearing nude mice was detected,and its potential as a detection reagent was determined.Results Through gene engineering and Pichia pastoris expression,the anti GPC3 single chain antibody was successfully obtained.The results of Western Blot identification showed that the expression and assembly of single chain antibody Anti-GPC3 scFv were correct.Flow cytometry showed that the binding rate of AntiGPC3 scFv to Huh-7 was 40.3%.In vitro targeting test,the single chain antibody Anti-GPC3 scFv could target hepatoma cell Huh-7 well in vitro,and the average fluorescence intensity of the single chain antibody group was 218.61±9.03,which was significantly better than 31.74±5.28 of the control group.In the near infrared imaging test,the fluorescence signal analysis of the nude mice bearing Huh-7 cell xenografts showed that the single chain antibody probe could be well targeted to the tumor site.Through the analysis of maximum tumor/normal tissue fluorescence signal,the ratio of the single chain antibody group was 8.27±2.94,which was obviously stronger than 1.09±0.63 of the resistance control group.Conclusion The single chain antibody AntiGPC3 scFv was successfully constructed and expressed in Pichia pastoris expression system.Single chain antibody retains the binding ability of parent antibody,and shows good targeting ability in vitro and in vivo.Its targeting ability can be applied to the detection field of GPC3 positive liver cancer and has potential clinical application prospects.