全人源抗肝癌噬菌体单链抗体的筛
发布时间：2018-11-05 18:50
【摘要】： 目的:从全人源噬菌体单链抗体库中大规模筛选抗肝癌噬菌体单链抗体(scFv),鉴定其特异性;以绿色荧光蛋白(EGFP)为示踪分子构建、表达抗肝癌scFv与EGFP融合蛋白,体外、体内实验研究抗肝癌scFv的靶向特异性,为肝癌的临床诊断和导向治疗奠定基础。 方法:用肝癌细胞系HepG_2细胞和肝细胞系QSG-7701细胞对初级抗体库进行正负淘洗富集以及ELISA筛选,选择与肝癌细胞反应强阳性而与肝细胞反应弱阳性或者阴性的克隆进行免疫组化鉴定并测序。测序后构建阳性克隆SA3基因与EGFP融合基因的原核表达载体EGFP-SA3/pET-25b(+)并转化大肠杆菌BL21(DE3),以IPTG诱导表达;变性条件下纯化的融合蛋白EGFP-SA3用稀释法复性以后与HepG_2细胞经体外孵育后在荧光显微镜下观察单链抗体SA3与肝癌细胞的结合作用;然后通过尾静脉将其注射入荷肝癌裸鼠体内观察EGFP-SA3的体内靶向作用。 结果:对抗体库进行三轮正负淘洗和富集,从第三轮淘洗后的LB平板上随机挑取2798个克隆进行间接法ELISA,得到979个与HepG_2呈阳性反应的克隆,阳性率为35%,其中有3个克隆与HepG_2呈强阳性反应,而与QSG-7701、HUVEC等人正常细胞系呈弱阳性或阴性。选择克隆SA3进行免疫细胞化学染色,结果与ELISA一致。免疫组织化学结果表明SA3与肝癌组织和肝组织阳性率分别为72.1%和8.3%,差别具有统计学意义(P＜0.01)。重组表达载体EGFP-SA3/pET-25b(+)经IPTG诱导表达后SDS-PAGE显示融合蛋白EGFP-SA3分子量大小为58 KD,主要以包涵体的形式存在;在变性的条件下用His-tag磁珠纯化试剂盒纯化并复性的EGFP-SA3与HepG_2细胞孵育之后在荧光显微镜下观察可见细胞膜上有明显的绿色荧光聚积。体内实验表明,注射EGFP-SA3的荷肝癌小鼠的肿瘤部位有绿色荧光发出,而对照组小鼠的肿瘤部位未见荧光,显示EGFP-SA3具有良好的肿瘤特异性。 结论:通过大规模的细胞ELISA筛选从噬菌体单链抗体库中获得3株抗肝癌scFv阳性克隆,免疫组织化学鉴定结果表明阳性克隆SA3具有较好的肝癌特异性。通过分子生物学方法成功构建并表达纯化了SA3与绿色荧光蛋白的融合蛋白EGFP-SA3,动物实验表明EGFP-SA3在荷肝癌裸鼠动物模型体内具有良好的靶向性,能明显的聚积在肿瘤部位。
[Abstract]:Objective: to screen anti-hepatoma phage single chain antibody (scFv),) from human phage scFv library on a large scale to identify its specificity. The fusion protein of scFv and EGFP was constructed with green fluorescent protein (EGFP) as tracer molecule. In vitro, the target specificity of anti-HCC scFv was studied in vivo, which laid a foundation for clinical diagnosis and guiding therapy of HCC. Methods: the primary antibody library was enriched by positive and negative washing and ELISA screening with hepatoma cell line HepG_2 and liver cell line QSG-7701. The clones with strong positive reaction and weak positive or negative reaction with hepatoma cells were identified by immunohistochemistry and sequenced. After sequencing, the prokaryotic expression vector EGFP-SA3/pET-25b () of SA3 gene and EGFP fusion gene was constructed and transformed into Escherichia coli BL21 (DE3) for IPTG induced expression. The fusion protein EGFP-SA3 purified under denaturation condition was renatured by dilution and incubated with HepG_2 cells in vitro to observe the binding effect of single chain antibody (SA3) SA3 to hepatoma cells under fluorescence microscope. Then it was injected into nude mice to observe the in vivo targeting effect of EGFP-SA3. Results: three rounds of positive or negative washing and enrichment were carried out on the antibody library. 2798 clones were randomly selected from the plate of LB after the third round of washing, and 979 clones with positive reaction to HepG_2 were obtained by indirect method. The positive rate was 35%. Three clones were strongly positive for HepG_2 and weakly positive or negative for normal cell lines with QSG-7701,HUVEC et al. SA3 clones were selected for immunocytochemical staining and the results were consistent with those of ELISA. The immunohistochemical results showed that the positive rates of SA3 were 72.1% and 8.3% in HCC and liver tissues, respectively. The difference was statistically significant (P < 0. 01). After the recombinant expression vector EGFP-SA3/pET-25b () was induced by IPTG, SDS-PAGE showed that the molecular weight of the fusion protein EGFP-SA3 was 58 KD, mainly in the form of inclusion body. Under the condition of denaturation, the EGFP-SA3 purified by His-tag magnetic bead purification kit was incubated with HepG_2 cells. The green fluorescence accumulation on the cell membrane was observed under fluorescence microscope. In vivo experiments showed that there was green fluorescence in the tumor site of hepatoma bearing mice injected with EGFP-SA3, but no fluorescence was found in the tumor site of the control group, indicating that EGFP-SA3 had good tumor specificity. Conclusion: three anti-HCC scFv positive clones were obtained from phage scFv library by large-scale cell ELISA screening. The results of immunohistochemical analysis showed that the positive SA3 clones had good HCC specificity. A fusion protein of SA3 and green fluorescent protein (EGFP-SA3,) was successfully constructed and expressed by molecular biological methods. The results of animal experiments showed that EGFP-SA3 has a good targeting ability in nude mice bearing hepatocellular carcinoma. It can obviously accumulate in the tumor area.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392;R735.7
[Abstract]:Objective: to screen anti-hepatoma phage single chain antibody (scFv),) from human phage scFv library on a large scale to identify its specificity. The fusion protein of scFv and EGFP was constructed with green fluorescent protein (EGFP) as tracer molecule. In vitro, the target specificity of anti-HCC scFv was studied in vivo, which laid a foundation for clinical diagnosis and guiding therapy of HCC. Methods: the primary antibody library was enriched by positive and negative washing and ELISA screening with hepatoma cell line HepG_2 and liver cell line QSG-7701. The clones with strong positive reaction and weak positive or negative reaction with hepatoma cells were identified by immunohistochemistry and sequenced. After sequencing, the prokaryotic expression vector EGFP-SA3/pET-25b () of SA3 gene and EGFP fusion gene was constructed and transformed into Escherichia coli BL21 (DE3) for IPTG induced expression. The fusion protein EGFP-SA3 purified under denaturation condition was renatured by dilution and incubated with HepG_2 cells in vitro to observe the binding effect of single chain antibody (SA3) SA3 to hepatoma cells under fluorescence microscope. Then it was injected into nude mice to observe the in vivo targeting effect of EGFP-SA3. Results: three rounds of positive or negative washing and enrichment were carried out on the antibody library. 2798 clones were randomly selected from the plate of LB after the third round of washing, and 979 clones with positive reaction to HepG_2 were obtained by indirect method. The positive rate was 35%. Three clones were strongly positive for HepG_2 and weakly positive or negative for normal cell lines with QSG-7701,HUVEC et al. SA3 clones were selected for immunocytochemical staining and the results were consistent with those of ELISA. The immunohistochemical results showed that the positive rates of SA3 were 72.1% and 8.3% in HCC and liver tissues, respectively. The difference was statistically significant (P < 0. 01). After the recombinant expression vector EGFP-SA3/pET-25b () was induced by IPTG, SDS-PAGE showed that the molecular weight of the fusion protein EGFP-SA3 was 58 KD, mainly in the form of inclusion body. Under the condition of denaturation, the EGFP-SA3 purified by His-tag magnetic bead purification kit was incubated with HepG_2 cells. The green fluorescence accumulation on the cell membrane was observed under fluorescence microscope. In vivo experiments showed that there was green fluorescence in the tumor site of hepatoma bearing mice injected with EGFP-SA3, but no fluorescence was found in the tumor site of the control group, indicating that EGFP-SA3 had good tumor specificity. Conclusion: three anti-HCC scFv positive clones were obtained from phage scFv library by large-scale cell ELISA screening. The results of immunohistochemical analysis showed that the positive SA3 clones had good HCC specificity. A fusion protein of SA3 and green fluorescent protein (EGFP-SA3,) was successfully constructed and expressed by molecular biological methods. The results of animal experiments showed that EGFP-SA3 has a good targeting ability in nude mice bearing hepatocellular carcinoma. It can obviously accumulate in the tumor area.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392;R735.7
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