Angiopep-2、IP10-EGFRvⅢscFv融合蛋白修饰纳米颗粒联合CTL治疗恶性胶质瘤研究

发布时间：2018-05-30 13:51

本文选题：融合蛋白 + 构建　；参考：《华中科技大学》2014年博士论文

【摘要】：第一部分IP10-EGFRvIIIscFv融合蛋白的构建和制备目的通过基因工程技术构建细胞因子融合蛋白IP10-EGFRvIIIscFv,并完成融合蛋白的大量扩增、纯化与复性。方法基因工程技术构建真核重组表达质粒GV219IP10-EGFRvIIIscFv,两段目的基因依次克隆于重组质粒GV219Pcmv启动子的下游,二者以编码(Gly4Ser)3的柔性接头序列(linker)相连。利用限制性核酸内切酶将目的基因IP10-scFv从真核表达载体上酶切下来,加入到高效原核表达载体PET3Oa的T7启动子下游,在IPTG的诱导下高效表达融合蛋白,通过His6-tag标签镍磁珠纯化蛋白后进行透析复性。并在透析缓冲液中加入GSSG/GSH氧化还原体系,促进变性融合蛋白的复性。以银染检测融合蛋白的纯度,western-blotting分析融合蛋白。结果成功构建了真核重组表达质粒GV219IP10-EGFRvIIIscFv,通过PET高效原核表达系统,获得大量的融合蛋白(包涵体表达),镍磁珠连续分选获得纯度95%的融合蛋白,银染和western-blotting检测融合蛋白的分子量为46kDa。第二部分Angiopep-2、IP10-EGFRvIIIscFv融合蛋白表面修饰纳米颗粒的制备及脑穿越实验目的通过纳米技术制备Angiopep-2、IP10-EGFRvIIIscFv表面修饰纳米颗粒,并完成纳米颗粒穿越血脑屏障的体内、体外研究。方法使用生物纳米材料聚乳酸-羟基乙酸共聚物(poly(lactic-co-glycolic acid), PLGA)和PLGA-PEG-Mal制备高分子多聚体纳米颗粒,同时以Angiopep-2和/或IP10-scFv修饰纳米粒表面,分别制备出实验组AINPs、对照组ANPAs、INPs和NPs。以香豆素-6和若丹明B(RhoB)作为荧光探针,对AINPs的脑内递药特性进行体外评价。以香豆素-6标记的荧光纳米粒,经尾静脉注射BABL/C/Nude nu,通过激光共聚焦检测对AINPs的脑内递药特性进行体外评价。以DiR作为荧光探针标记纳米颗粒,尾静脉注射BABL/C/Nude nu,利用活体成像手段实现对AINPs的脑内递药特性的体外评价。结果我们制备出的AINPs透射电镜下可见外观圆整,大小均一,平均粒径为143.6±0.7nm, Zeta电位为-25.61±3.53mV,体外实验中,End.3细胞株和原代大鼠脑毛细血管内皮细胞(BCECs)能有效摄取AINPs。体内实现中,AINPs能显著穿越BABL/C/Nude nu进入脑实质内。通过体内体外实验,说明我们制备的AINPs能有效的透过血脑屏障。第三部分Angiopep-2, IP1O-EGFRvIIIscFv融合蛋白修饰纳米粒联合CTL的抗脑恶性胶质瘤功能研究目的评价AINPs体外趋化特性和靶向胶质瘤细胞特性,以及联合CTL治疗脑胶质瘤实验。方法Transwell趋化实验检测AINPs的趋化特性,利用免疫荧光染色检测AINPs抗原抗体结合实验,体外诱导胶质瘤抗原特异性DC细胞,并与CD8+T淋巴细胞混合培养,制备出胶质瘤特异性的细胞毒T淋巴细胞CTL,并联合AINPs经尾静脉注射BABL/C/Nude nu原位脑胶质瘤模型,观察荷瘤小鼠的生存时间及肿瘤体积的大小变化。结果AINPs能有效趋化CD8+T淋巴细胞,也能有效的与U87-EGFRvIII细胞株的特异抗原EGFRvIII特异结合。通过DC混合培养技术成功制备出胶质瘤特异性的细胞毒T淋巴细胞CTL,在AINPs的趋化作用下,富集在肿瘤组织周围,实现对胶质瘤细胞的有效杀伤作用。
[Abstract]:The first part of the construction and preparation of IP10-EGFRvIIIscFv fusion protein, the fusion protein IP10-EGFRvIIIscFv was constructed by genetic engineering technology, and the amplification, purification and renaturation of the fusion protein were completed. The recombinant eukaryotic recombinant expression plasmid GV219IP10-EGFRvIIIscFv was constructed by genetic engineering technology. The two target genes were cloned in turn. In the downstream of the recombinant plasmid GV219Pcmv promoter, the two were connected by the flexible joint sequence (linker) encoding (Gly4Ser) 3. The target gene IP10-scFv was cut from the eukaryotic expression vector by restriction endonuclease, and added to the downstream of T7 promoter, PET3Oa of the high prokaryotic expression vector PET3Oa, to express the fusion protein efficiently under the induction of IPTG. The renaturation was carried out after the His6-tag labeled nickel magnetic beads were purified and the redox system was added to the dialysate buffer solution to promote the renaturation of the modified fusion protein. The purity of the fusion protein was detected by silver staining and the fusion protein was analyzed by Western-blotting. The recombinant eukaryotic recombinant expression plasmid GV219IP10-EGFRvIIIscFv was successfully constructed. A large amount of fusion protein (inclusion body expression) was obtained by PET high effective prokaryotic expression system. The fusion protein of 95% purity was obtained by continuous separation of nickel magnetic beads. The molecular weight of the fusion protein detected by silver staining and Western-blotting was 46kDa.
The second part is the preparation of Angiopep-2, IP10-EGFRvIIIscFv fusion protein surface modified nanoparticles and brain penetration test.
Objective to prepare Angiopep-2, IP10-EGFRvIIIscFv surface modified nanoparticles by nano technology, and to complete the in vivo study of nanoparticles through the blood brain barrier in vitro. Methods using the bionano material polylactic acid hydroxy acetic acid copolymer (lactic-co-glycolic acid), PLGA) and PLGA-PEG-Mal to prepare polymer polymer nanoparticles. The experimental group AINPs was prepared on the surface of Angiopep-2 and / or IP10-scFv nanoparticles, and the control group ANPAs, INPs and NPs. with coumarin -6 and Rhodamine B (RhoB) were used as fluorescence probes to evaluate the drug delivery characteristics in the brain in vitro. The fluorescent nanofilt labeled with coumarin -6 was injected into the tail vein and was injected through the laser. The drug delivery characteristics in the brain of AINPs were evaluated in vitro by focusing detection. DiR was used as a fluorescent probe to mark nanoparticles and BABL/C/Nude Nu was injected into the tail vein. In vitro evaluation of the drug delivery characteristics in the brain of AINPs was achieved by living imaging. The results of AINPs transmission electron microscopy showed that the appearance of the AINPs was round, uniform, and the average particle size was 143.6 + 0.7nm, Zeta potential is -25.61 + 3.53mV. In vitro, the End.3 cell line and the primary rat brain capillary endothelial cell (BCECs) can effectively absorb the AINPs. in vivo. AINPs can penetrate the BABL/C/Nude Nu into the brain parenchyma significantly. Through in vitro and in vitro experiments, it is indicated that the AINPs of our preparation can effectively penetrate the blood brain barrier.
The third part is Angiopep-2, IP1O-EGFRvIIIscFv fusion protein modified nanoparticles combined with CTL in the treatment of malignant glioma.
Objective to evaluate the chemotaxis of AINPs in vitro and the characteristics of targeted glioma cells, as well as the combination of CTL in the treatment of glioma. Methods Transwell chemotactic test was used to detect the chemotaxis of AINPs. Immunofluorescence staining was used to detect AINPs antigen antibody binding experiment. The specific DC cells of glioma antigen were induced in vitro and mixed with CD8+T lymphocytes in vitro. The specific cytotoxic T lymphocyte CTL of glioma was prepared and the BABL/C/Nude nu in situ glioma model was injected into the tail vein of AINPs to observe the survival time and the size of tumor in the tumor bearing mice. The results showed that AINPs could effectively chemotaxis the CD8+T lymphocyte and could be effective with the specific antigen EGFRvI of the U87-EGFRvIII cell line. II specific binding. The specific cytotoxic T lymphocyte CTL of glioma was successfully prepared by DC mixed culture technique, which was enriched around the tumor tissue under the chemotaxis of AINPs to achieve the effective killing effect on glioma cells.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R739.4

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