EGFR靶向单链抗体制备及介导siRNA内化NSCLC细胞的生物学效应
发布时间：2019-06-18 09:08
【摘要】：目的:EGFR-TKIs耐药引起肿瘤进展是当前NSCLC治疗的主要重点和难点,siRNA可沉默耐药相关基因表达但缺乏靶向性。为提高siRNA的靶向性,本研究设计并优化EGFR靶向单链抗体核苷酸序列,在大肠杆菌中表达后,纯化人源性抗EGFR单链抗体(scFv,s-9R)融合蛋白,并对纯化产物的抗原结合活性、细胞内化活性及核算携带能力进行鉴定;研究s-9R携带siRNA在体内、外的抗肿瘤活性。方法:1.根据人源性抗EGFR抗体轻、重链可变区氨基酸序列,设计、合成其单链抗体核酸表达序列,将轻链与重链核酸序列通过G4S连接肽相连并在核酸序列末端加入九聚精氨酸及His.tag表达序列,以构建scFv及s-9R融合基因。将测序正确的融合基因连接入原核表达载体pGEX-4T-1,转化大肠杆菌BL21(DE3),诱导表达后行亲和层析纯化并酶切GST.tag。表达产物经SDS-PAGE和Western blot鉴定后,ELISA分析融合蛋白的抗原结合活性,凝胶迁移阻滞实验检测s-9R与核酸的结合活性,间接免疫荧光和流式细胞术检测检测融合蛋白的内化活性。2.scfv及s-9r与egfr-sirna,met-sirna,kras-sirna及her2-sirna分别混合后,加入h1975,h1993,a549,spc-a1,pc-9及h69细胞培养液中共孵育,同时加入/不加入gefitinib。通过rt-qpcr,westernblot实验分析基因表达水平;通过mtt实验、流式细胞学实验、平板克隆实验分析egfr-tkis细胞药物敏感性。3.使用近红外染料dylight800nhsester对单链抗体融合蛋白进行标记,经鼠尾静脉对h1975细胞荷瘤裸鼠注射标记后的融合蛋白,活体成像观察融合蛋白在肿瘤部位富集及全身代谢过程。使用cy5荧光染料标记sirna,经h1975细胞荷瘤裸鼠鼠尾静脉注射融合蛋白/cy5-sirna,活体成像观察融合蛋白/cy5-sirna在肿瘤部位富集及全身代谢过程。经鼠尾静脉注射融合蛋白/egfr-sirna混合物,并同时gefitinib灌胃,观察裸鼠荷瘤变化;经鼠尾静脉注射融合蛋白/her2-sirna混合物,观察裸鼠荷瘤变化。剥离荷瘤组织后对组织切片进行免疫组化实验,观察egfr、her2、ki67表达情况;对组织切片进行tunel标记,观察细胞凋亡情况。结果:1.pgex-4t-1-scfv及pgex-4t-1-s-9r质粒转化大肠杆菌bl21(de3)后,iptg诱导表达融合蛋白scfv及s-9r,经sds-page及westernblot证实表达成功;elisa分析证实两条单链抗体融合蛋白具有良好的egfr结合活性;凝胶迁移阻滞实验表明s-9r具有核酸结合活性;间接免疫荧光及流式细胞术实验显示两条单链抗体融合蛋白能够特异性结合并内化入egfr阳性的肿瘤细胞中,而不能内化入egfr表达阴性肿瘤细胞中;s-9r可以携带fam-sirna结合并内化入egfr阳性的肿瘤细胞中,而scfv则无此功能。2.s-9r可以携带egfr-sirna,met-sirna,kras-sirna及her2-sirna内化入肿瘤细胞中,并下调细胞中egfr、met、kras、her2基因的表达;联合应用gefitinib后h1975、h1993及a549细胞的凋亡水平较对照组明显提高;s-9r/her2-sirna混合物可以使spc-a1,pc-9增殖能力、克隆形成能力降低,细胞周期出现g1期停滞。3.小动物活体成像显示两条单链抗体融合蛋白本身能够通过循环系统富集于裸鼠荷瘤部位,其中s-9r可以携带cy5/fam荧光素标记的sirna富集于裸鼠荷瘤部位,并内化入瘤细胞中;经鼠尾静脉注射s-9r/egfr-sirna混合物联合gefitinib灌胃,或单独经鼠尾静脉注射s-9R/HER2-siRNA混合物可以使裸鼠荷瘤生长受到明显抑制,病理切片显示肿瘤细胞靶蛋白表达减少,增殖能力下降,凋亡细胞增多,荷瘤血管形成能力降低。实验组裸鼠生存时间延长,移植瘤成瘤率下降,荷瘤体积变小、重量减轻。结论:所设计、构建的EGFR靶向单链抗体scFv及s-9R可以在体内、外特异性的识别结合EGFR表达阳性NSCLC细胞。s-9R可以携带特异性siRNA内化入肿瘤细胞之中抑制靶蛋白表达,发挥siRNA的生物学作用,具有恢复Gefitinib敏感性,抑制肿瘤细胞增殖、促进细胞凋亡的作用。我们的研究为解决当前NSCLC临床治疗中所面对的酪氨酸激酶抑制剂耐药问题,以及治疗以HER2为驱动基因的NSCLC,提供了一条新的方法和思路。
[Abstract]:Objective: EGFR-TKIs resistance-induced tumor progression is the main focus and difficulty in the treatment of NSCLC, and siRNA can be used to silence resistance-related gene expression but lacks targeting. In order to improve the targeting of the siRNA, the present study designs and optimizes the EGFR-targeting single-chain antibody nucleosonic acid sequence, and after the expression in E. coli, the humanized anti-EGFR single-chain antibody (scFv, s-9R) fusion protein is purified, and the antigen binding activity of the purified product is carried out, The cell-internalization activity and the carrying capacity of the cell were identified; and the anti-tumor activity of the siRNA in vivo and in vitro was studied by the study of s-9R. Method:1. according to the human-derived anti-EGFR antibody light and heavy chain variable region amino acid sequence, the single-chain antibody nucleic acid expression sequence is synthesized, the light chain and the heavy-chain nucleic acid sequence are connected through a G4S connection peptide and a 9-polyarginine and a His.tag expression sequence are added at the end of the nucleic acid sequence, In ord to construct that fusion gene of scFv and s-9R. The correct fusion gene was ligated into the prokaryotic expression vector pGEX-4T-1, transformed into E. coli BL21 (DE3), the expression product was purified by affinity chromatography, and the enzyme-digested GST.tag. expression product was identified by SDS-PAGE and Western blot, and the antigen binding activity of the fusion protein was analyzed by ELISA. The binding activity of s-9R and the nucleic acid, the indirect immunofluorescence and flow cytometry were used to detect the internalized active .2.scfv of the fusion protein and s-9r and egfr-sirna, met-sirna, kras-sirna and her2-sirna, respectively, and then added to the culture medium of h1975, h1993, a549, spc-al, pc-9 and h69. At the same time, gefitinib was added/ not added. The expression level of gene was analyzed by rt-qpcr and western blot. The fusion protein of the single-chain antibody fusion protein was labeled by using the near-infrared dye dylight800nhester, and the fusion protein after the injection of the h1975 cell tumor-bearing nude mice was injected via the mouse tail vein, and the fusion protein was concentrated in the tumor site and the whole body metabolism process. Cy5 fluorescent dye was used to mark the sirna, and the fusion protein/ cy5-sirna was injected into the tumor-bearing nude mouse tail vein by the h1975 cell tumor-bearing nude mouse tail, and the fusion protein/ cy5-sirna was observed in the tumor site and the whole body metabolism process. The mice were injected with the fusion protein/ egfr-sirna mixture and gefitinib was given intragastric administration to observe the tumor-bearing changes of the nude mice, and the tumor-bearing changes of the nude mice were observed by the combination of the fusion protein/ her2-sirna mixture in the tail of the mice. The expression of egfr, her2 and ki67 was observed after the tumor-bearing tissue was peeled off and the expression of egfr, her2 and ki67 was observed. Results :1.pgex-4t-1-scfv and pgex-4t-1-s-9r plasmids were transformed into E. coli bl21 (de3), and iptg induced the expression of the fusion protein scfv and s-9r. The expression of the fusion protein scfv and s-9r was confirmed by sds-page and western blot. indirect immunofluorescence and flow cytometry show that two single-chain antibody fusion proteins can be specifically combined and internalized into egfr-positive tumor cells, and can not be internalized into egfr expression-negative tumor cells; s-9r can carry the fam-sirna binding and internalized into egfr-positive tumor cells, scfv does not have such a function. s-9r can carry egfr-sirna, met-sirna, kras-sirna and her2-sirna into the tumor cells and down-regulate the expression of egfr, met, kras, and her2 in the cells, and the level of apoptosis in h1975, h1993 and a549 cells after the combined application of gefitinib is significantly higher than that of the control group; s-9r/ her2-sirna mixtures may enable the proliferation of spc-a1, pc-9, The ability of the clone formation decreased and the cell cycle showed a phase-1 stagnation. in vivo imaging of small animals, two single-chain antibody fusion proteins can be enriched in a tumor-bearing part of a nude mouse through a circulating system, wherein s-9r can carry a cy5/ fam fluorescein-labeled sirna to be enriched in a tumor-bearing part of a nude mouse, And the tumor cell target protein expression is reduced, the proliferation ability is reduced, the apoptosis cell is increased, and the tumor-bearing blood vessel forming capacity is reduced. The survival time of the nude mice in the experimental group was prolonged, the tumor-forming rate of the transplanted tumor was decreased, the tumor-bearing volume was small, and the weight was reduced. Conclusion: The designed and constructed EGFR-targeting single-chain antibody scFv and s-9R can identify and bind EGFR-expressing positive NSCLC cells in vivo and in vitro. S-9R can carry specific siRNA into the tumor cells to inhibit the expression of the target protein, play the biological function of the siRNA, have the function of restoring the sensitivity of the Gefitinib, inhibiting the proliferation of the tumor cells and promoting the apoptosis of the cells. Our study provides a new approach and thought to address the problem of the resistance of tyrosine kinase inhibitors in the clinical treatment of NSCLC and to treat NSCLC with HER2 as a driving gene.
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R734.2
本文编号：2501357
[Abstract]:Objective: EGFR-TKIs resistance-induced tumor progression is the main focus and difficulty in the treatment of NSCLC, and siRNA can be used to silence resistance-related gene expression but lacks targeting. In order to improve the targeting of the siRNA, the present study designs and optimizes the EGFR-targeting single-chain antibody nucleosonic acid sequence, and after the expression in E. coli, the humanized anti-EGFR single-chain antibody (scFv, s-9R) fusion protein is purified, and the antigen binding activity of the purified product is carried out, The cell-internalization activity and the carrying capacity of the cell were identified; and the anti-tumor activity of the siRNA in vivo and in vitro was studied by the study of s-9R. Method:1. according to the human-derived anti-EGFR antibody light and heavy chain variable region amino acid sequence, the single-chain antibody nucleic acid expression sequence is synthesized, the light chain and the heavy-chain nucleic acid sequence are connected through a G4S connection peptide and a 9-polyarginine and a His.tag expression sequence are added at the end of the nucleic acid sequence, In ord to construct that fusion gene of scFv and s-9R. The correct fusion gene was ligated into the prokaryotic expression vector pGEX-4T-1, transformed into E. coli BL21 (DE3), the expression product was purified by affinity chromatography, and the enzyme-digested GST.tag. expression product was identified by SDS-PAGE and Western blot, and the antigen binding activity of the fusion protein was analyzed by ELISA. The binding activity of s-9R and the nucleic acid, the indirect immunofluorescence and flow cytometry were used to detect the internalized active .2.scfv of the fusion protein and s-9r and egfr-sirna, met-sirna, kras-sirna and her2-sirna, respectively, and then added to the culture medium of h1975, h1993, a549, spc-al, pc-9 and h69. At the same time, gefitinib was added/ not added. The expression level of gene was analyzed by rt-qpcr and western blot. The fusion protein of the single-chain antibody fusion protein was labeled by using the near-infrared dye dylight800nhester, and the fusion protein after the injection of the h1975 cell tumor-bearing nude mice was injected via the mouse tail vein, and the fusion protein was concentrated in the tumor site and the whole body metabolism process. Cy5 fluorescent dye was used to mark the sirna, and the fusion protein/ cy5-sirna was injected into the tumor-bearing nude mouse tail vein by the h1975 cell tumor-bearing nude mouse tail, and the fusion protein/ cy5-sirna was observed in the tumor site and the whole body metabolism process. The mice were injected with the fusion protein/ egfr-sirna mixture and gefitinib was given intragastric administration to observe the tumor-bearing changes of the nude mice, and the tumor-bearing changes of the nude mice were observed by the combination of the fusion protein/ her2-sirna mixture in the tail of the mice. The expression of egfr, her2 and ki67 was observed after the tumor-bearing tissue was peeled off and the expression of egfr, her2 and ki67 was observed. Results :1.pgex-4t-1-scfv and pgex-4t-1-s-9r plasmids were transformed into E. coli bl21 (de3), and iptg induced the expression of the fusion protein scfv and s-9r. The expression of the fusion protein scfv and s-9r was confirmed by sds-page and western blot. indirect immunofluorescence and flow cytometry show that two single-chain antibody fusion proteins can be specifically combined and internalized into egfr-positive tumor cells, and can not be internalized into egfr expression-negative tumor cells; s-9r can carry the fam-sirna binding and internalized into egfr-positive tumor cells, scfv does not have such a function. s-9r can carry egfr-sirna, met-sirna, kras-sirna and her2-sirna into the tumor cells and down-regulate the expression of egfr, met, kras, and her2 in the cells, and the level of apoptosis in h1975, h1993 and a549 cells after the combined application of gefitinib is significantly higher than that of the control group; s-9r/ her2-sirna mixtures may enable the proliferation of spc-a1, pc-9, The ability of the clone formation decreased and the cell cycle showed a phase-1 stagnation. in vivo imaging of small animals, two single-chain antibody fusion proteins can be enriched in a tumor-bearing part of a nude mouse through a circulating system, wherein s-9r can carry a cy5/ fam fluorescein-labeled sirna to be enriched in a tumor-bearing part of a nude mouse, And the tumor cell target protein expression is reduced, the proliferation ability is reduced, the apoptosis cell is increased, and the tumor-bearing blood vessel forming capacity is reduced. The survival time of the nude mice in the experimental group was prolonged, the tumor-forming rate of the transplanted tumor was decreased, the tumor-bearing volume was small, and the weight was reduced. Conclusion: The designed and constructed EGFR-targeting single-chain antibody scFv and s-9R can identify and bind EGFR-expressing positive NSCLC cells in vivo and in vitro. S-9R can carry specific siRNA into the tumor cells to inhibit the expression of the target protein, play the biological function of the siRNA, have the function of restoring the sensitivity of the Gefitinib, inhibiting the proliferation of the tumor cells and promoting the apoptosis of the cells. Our study provides a new approach and thought to address the problem of the resistance of tyrosine kinase inhibitors in the clinical treatment of NSCLC and to treat NSCLC with HER2 as a driving gene.
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R734.2
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