本文選題：融合蛋白 + 構建��； 參考：《華中科技大學》2014年博士論文

【摘要】：第一部分IP10-EGFRvIIIscFv融合蛋白的構建和制備目的通過基因工程技術構建細胞因子融合蛋白IP10-EGFRvIIIscFv,并完成融合蛋白的大量擴增、純化與復性。方法基因工程技術構建真核重組表達質(zhì)粒GV219IP10-EGFRvIIIscFv,兩段目的基因依次克隆于重組質(zhì)粒GV219Pcmv啟動子的下游,二者以編碼(Gly4Ser)3的柔性接頭序列(linker)相連。利用限制性核酸內(nèi)切酶將目的基因IP10-scFv從真核表達載體上酶切下來,加入到高效原核表達載體PET3Oa的T7啟動子下游,在IPTG的誘導下高效表達融合蛋白,通過His6-tag標簽鎳磁珠純化蛋白后進行透析復性。并在透析緩沖液中加入GSSG/GSH氧化還原體系,促進變性融合蛋白的復性。以銀染檢測融合蛋白的純度,western-blotting分析融合蛋白。結果成功構建了真核重組表達質(zhì)粒GV219IP10-EGFRvIIIscFv,通過PET高效原核表達系統(tǒng),獲得大量的融合蛋白(包涵體表達),鎳磁珠連續(xù)分選獲得純度95%的融合蛋白,銀染和western-blotting檢測融合蛋白的分子量為46kDa。 第二部分Angiopep-2、IP10-EGFRvIIIscFv融合蛋白表面修飾納米顆粒的制備及腦穿越實驗 目的通過納米技術制備Angiopep-2、IP10-EGFRvIIIscFv表面修飾納米顆粒,并完成納米顆粒穿越血腦屏障的體內(nèi)、體外研究。方法使用生物納米材料聚乳酸-羥基乙酸共聚物(poly(lactic-co-glycolic acid), PLGA)和PLGA-PEG-Mal制備高分子多聚體納米顆粒,同時以Angiopep-2和/或IP10-scFv修飾納米粒表面,分別制備出實驗組AINPs、對照組ANPAs、INPs和NPs。以香豆素-6和若丹明B(RhoB)作為熒光探針,對AINPs的腦內(nèi)遞藥特性進行體外評價。以香豆素-6標記的熒光納米粒,經(jīng)尾靜脈注射BABL/C/Nude nu,通過激光共聚焦檢測對AINPs的腦內(nèi)遞藥特性進行體外評價。以DiR作為熒光探針標記納米顆粒,尾靜脈注射BABL/C/Nude nu,利用活體成像手段實現(xiàn)對AINPs的腦內(nèi)遞藥特性的體外評價。結果我們制備出的AINPs透射電鏡下可見外觀圓整,大小均一,平均粒徑為143.6±0.7nm, Zeta電位為-25.61±3.53mV,體外實驗中,End.3細胞株和原代大鼠腦毛細血管內(nèi)皮細胞(BCECs)能有效攝取AINPs。體內(nèi)實現(xiàn)中,AINPs能顯著穿越BABL/C/Nude nu進入腦實質(zhì)內(nèi)。通過體內(nèi)體外實驗,說明我們制備的AINPs能有效的透過血腦屏障。 第三部分Angiopep-2, IP1O-EGFRvIIIscFv融合蛋白修飾納米粒聯(lián)合CTL的抗腦惡性膠質(zhì)瘤功能研究 目的評價AINPs體外趨化特性和靶向膠質(zhì)瘤細胞特性,以及聯(lián)合CTL治療腦膠質(zhì)瘤實驗。方法Transwell趨化實驗檢測AINPs的趨化特性,利用免疫熒光染色檢測AINPs抗原抗體結合實驗,體外誘導膠質(zhì)瘤抗原特異性DC細胞,并與CD8+T淋巴細胞混合培養(yǎng),制備出膠質(zhì)瘤特異性的細胞毒T淋巴細胞CTL,并聯(lián)合AINPs經(jīng)尾靜脈注射BABL/C/Nude nu原位腦膠質(zhì)瘤模型,觀察荷瘤小鼠的生存時間及腫瘤體積的大小變化。結果AINPs能有效趨化CD8+T淋巴細胞,也能有效的與U87-EGFRvIII細胞株的特異抗原EGFRvIII特異結合。通過DC混合培養(yǎng)技術成功制備出膠質(zhì)瘤特異性的細胞毒T淋巴細胞CTL,在AINPs的趨化作用下,富集在腫瘤組織周圍,實現(xiàn)對膠質(zhì)瘤細胞的有效殺傷作用。
[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.
【學位授予單位】：華中科技大學
【學位級別】：博士
【學位授予年份】：2014
【分類號】：R739.4

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本文編號：1955528