全人源抗肝癌噬菌體單鏈抗體的篩
發(fā)布時(shí)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(gòu)建、表達(dá)抗肝癌scFv與EGFP融合蛋白,體外、體內(nèi)實(shí)驗(yàn)研究抗肝癌scFv的靶向特異性,為肝癌的臨床診斷和導(dǎo)向治療奠定基礎(chǔ)。 方法:用肝癌細(xì)胞系HepG_2細(xì)胞和肝細(xì)胞系QSG-7701細(xì)胞對初級抗體庫進(jìn)行正負(fù)淘洗富集以及ELISA篩選,選擇與肝癌細(xì)胞反應(yīng)強(qiáng)陽性而與肝細(xì)胞反應(yīng)弱陽性或者陰性的克隆進(jìn)行免疫組化鑒定并測序。測序后構(gòu)建陽性克隆SA3基因與EGFP融合基因的原核表達(dá)載體EGFP-SA3/pET-25b(+)并轉(zhuǎn)化大腸桿菌BL21(DE3),以IPTG誘導(dǎo)表達(dá);變性條件下純化的融合蛋白EGFP-SA3用稀釋法復(fù)性以后與HepG_2細(xì)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(xì)胞的結(jié)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(jié)果:對抗體庫進(jìn)行三輪正負(fù)淘洗和富集,從第三輪淘洗后的LB平板上隨機(jī)挑取2798個(gè)克隆進(jìn)行間接法ELISA,得到979個(gè)與HepG_2呈陽性反應(yīng)的克隆,陽性率為35%,其中有3個(gè)克隆與HepG_2呈強(qiáng)陽性反應(yīng),而與QSG-7701、HUVEC等人正常細(xì)胞系呈弱陽性或陰性。選擇克隆SA3進(jìn)行免疫細(xì)胞化學(xué)染色,結(jié)果與ELISA一致。免疫組織化學(xué)結(jié)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(jì)學(xué)意義(P＜0.01)。重組表達(dá)載體EGFP-SA3/pET-25b(+)經(jīng)IPTG誘導(dǎo)表達(dá)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(fù)性的EGFP-SA3與HepG_2細(xì)胞孵育之后在熒光顯微鏡下觀察可見細(xì)胞膜上有明顯的綠色熒光聚積。體內(nèi)實(shí)驗(yàn)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(jié)論:通過大規(guī)模的細(xì)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(xué)鑒定結(jié)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(xué)方法成功構(gòu)建并表達(dá)純化了SA3與綠色熒光蛋白的融合蛋白EGFP-SA3,動(dòng)物實(shí)驗(yàn)表明EGFP-SA3在荷肝癌裸鼠動(dòng)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(xué)位授予單位】：中南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：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(xué)位授予單位】：中南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R392;R735.7
【參考文獻(xiàn)】
相關(guān)期刊論文 前8條
1 何小鵑,李官成,朱建高,李躍輝,周國華;鼻咽癌人源抗獨(dú)特型單鏈抗體的制備及篩選[J];癌癥;2004年02期
2 朱建高,胡錦躍,李官成,李躍輝,周國華,李小玲,孫去病;抗大腸癌噬菌體單鏈抗體的篩選及初步鑒定[J];中國生物化學(xué)與分子生物學(xué)報(bào);2001年01期
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