全人源抗肝癌噬菌體單鏈抗體的篩
發(fā)布時間：2018-11-05 18:50
【摘要】： 目的:從全人源噬菌體單鏈抗體庫中大規(guī)模篩選抗肝癌噬菌體單鏈抗體(scFv),鑒定其特異性;以綠色熒光蛋白(EGFP)為示蹤分子構建、表達抗肝癌scFv與EGFP融合蛋白,體外、體內(nèi)實驗研究抗肝癌scFv的靶向特異性,為肝癌的臨床診斷和導向治療奠定基礎。 方法:用肝癌細胞系HepG_2細胞和肝細胞系QSG-7701細胞對初級抗體庫進行正負淘洗富集以及ELISA篩選,選擇與肝癌細胞反應強陽性而與肝細胞反應弱陽性或者陰性的克隆進行免疫組化鑒定并測序。測序后構建陽性克隆SA3基因與EGFP融合基因的原核表達載體EGFP-SA3/pET-25b(+)并轉化大腸桿菌BL21(DE3),以IPTG誘導表達;變性條件下純化的融合蛋白EGFP-SA3用稀釋法復性以后與HepG_2細胞經(jīng)體外孵育后在熒光顯微鏡下觀察單鏈抗體SA3與肝癌細胞的結合作用;然后通過尾靜脈將其注射入荷肝癌裸鼠體內(nèi)觀察EGFP-SA3的體內(nèi)靶向作用。 結果:對抗體庫進行三輪正負淘洗和富集,從第三輪淘洗后的LB平板上隨機挑取2798個克隆進行間接法ELISA,得到979個與HepG_2呈陽性反應的克隆,陽性率為35%,其中有3個克隆與HepG_2呈強陽性反應,而與QSG-7701、HUVEC等人正常細胞系呈弱陽性或陰性。選擇克隆SA3進行免疫細胞化學染色,結果與ELISA一致。免疫組織化學結果表明SA3與肝癌組織和肝組織陽性率分別為72.1%和8.3%,差別具有統(tǒng)計學意義(P＜0.01)。重組表達載體EGFP-SA3/pET-25b(+)經(jīng)IPTG誘導表達后SDS-PAGE顯示融合蛋白EGFP-SA3分子量大小為58 KD,主要以包涵體的形式存在;在變性的條件下用His-tag磁珠純化試劑盒純化并復性的EGFP-SA3與HepG_2細胞孵育之后在熒光顯微鏡下觀察可見細胞膜上有明顯的綠色熒光聚積。體內(nèi)實驗表明,注射EGFP-SA3的荷肝癌小鼠的腫瘤部位有綠色熒光發(fā)出,而對照組小鼠的腫瘤部位未見熒光,顯示EGFP-SA3具有良好的腫瘤特異性。 結論:通過大規(guī)模的細胞ELISA篩選從噬菌體單鏈抗體庫中獲得3株抗肝癌scFv陽性克隆,免疫組織化學鑒定結果表明陽性克隆SA3具有較好的肝癌特異性。通過分子生物學方法成功構建并表達純化了SA3與綠色熒光蛋白的融合蛋白EGFP-SA3,動物實驗表明EGFP-SA3在荷肝癌裸鼠動物模型體內(nèi)具有良好的靶向性,能明顯的聚積在腫瘤部位。
[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.
【學位授予單位】：中南大學
【學位級別】：碩士
【學位授予年份】：2008
【分類號】：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.
【學位授予單位】：中南大學
【學位級別】：碩士
【學位授予年份】：2008
【分類號】：R392;R735.7
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相關期刊論文 前8條
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