【作者】 杨恩成；

【导师】 迟宝荣； 金宁一；

【作者基本信息】 吉林大学 ， 内科学， 2010， 博士

【摘要】 本研究利用分子生物学和免疫学等技术和手段,探索了利用多结构域嵌合蛋白提高裸质粒DNA转导效率的问题。首先,在PCR方法分别克隆了GAL4/147、NLS-GAL4/147、Tat-GAL4/147和Tat-NLS-GAL4/147序列,并将其克隆于酵母表达载体pPIC9K,成功构建了酵母表达质粒pPI-G、pPI-NG、pPI-TG和pPI-TNG,并在酵母菌株GS115内对目的蛋白进行了表达,通过对表达时间、温度、转速、甲醇浓度等条件的优化,最终获得了目的蛋白G、NG、TG和TNG。基于GAL4特异性识别／结合序列UAS、EGFP基因和Apoptin基因,成功构建了能够被融合蛋白特异性识别／结合的真核表达质粒DNA,并在体外对融合蛋白与UAS质粒的结合效率和条件进行了探索。通过MTT染色、AO/EB染色、流式细胞术、AnnexinV染色、DAPI染色、Caspase活性检测等方法探讨了融合蛋白介导的重组质粒对人肝癌细胞HepG-2的转导和抑制作用。实验结果表明,融合蛋白能够有效介导重组质粒对人肝癌细胞HepG-2的转导,并能够实现对人肝癌细胞HepG-2生长的抑制。本研究复制了C57BL／6小鼠荷小鼠肝癌(H22)肿瘤模型,通过瘤内注射融合蛋白-重组质粒复合物的方式,检测抑瘤率、生存期等指标,探讨了融合蛋白介导的重组质粒在动物体内的转导作用和对体内实体肿瘤的抑制作用。实验结果表明,融合蛋白在动物体内也可有效介导重组质粒的转导,并能够实现对C57BL／6小鼠荷小鼠H22肿瘤模型实体肿瘤的抑制。上述研究为研制安全、高效、低毒的裸质粒DNA转导辅助系统,拓展裸质粒DNA的应用空间奠定了基础。更多还原

【Abstract】 The Expression of the Specific Chimeric Multidomain DNA Transfer Protein in Pichia pastoris and its Transduction in vivo and in vitroGene therapy is based on the specific introduction of therapeutic genes into individual cells to treat diseases, and successful gene therapy depends on the development of efficient delivery systems. Current DNA delivery approaches can be divided into viral and non-viral platforms. Viral DNA vectors are widely used because of their high level of uptake and foreign gene expression. There are disadvantages with viral systems, however, including infectivity, a restriction in the size of incorporated DNA, and immunogenicity. Non-viral systems have generally included cationic polymers, peptides and liposomes. Non-viral receptor-mediated gene delivery strategies afford more flexibility in design and construction, in which multi-domain recombinant chimeric proteins as well as peptide-protein conjugates have been widely investigated.The GAL4 protein is a transcriptional activator that binds to the galactose UAS sequence in the yeast genome and activates the transcription of genes encoding enzymes of the galactose metabolic pathway. The N-terminal portion (residues 1-147) of GAL4, which has high affinity for a specific 17 bp oligonucleotide sequence (17 mer), mediates DNA recognition and binding and NMR analysis has demonstrated that the core DNA binding domain of GAL4 contains a Cys-X2-Cys-X6-Cys-X6-Cys-X2-Cys-X6-Cys motif in which the six cysteines coordinate two zinc ions, forming a bimetal-thiolate cluster. Preliminary studies have demonstrated that GAL4 may enhance DNA transfection, which supported the potential application of GAL4 in non-viral DNA transfer vectors.A series of small protein domains, termed cell-penetrating peptides (CPPs) or protein transduction domains (PTDs), have been described and are thought to penetrate the plasma membrane directly from the cell surface. The Tat protein, a transcriptional activator from HIV type 1 (HIV-1), was first reported to cross the plasma membrane after exogenous delivery. It is remarkable that the Tat basic domain also mediates efficient cellular internalization and cellular transduction activities when attached to large protein cargos. These properties have made Tat an attractive component of synthetic gene delivery vehicle for plasmid DNA, proteins, nanoparticles, and viral vectors.In this study, we generated a novel genetically engineered chimeric protein, TG, in which one domain (GAL4) acts as a carrier for plasmid DNA uptake, while another (Tat) enables the compounds (TG/plasmid or TNG/plasmid) to be endocytosed. Specifically, we purified and assessed the ability of the purified TG or TNG recombinant proteins to bind a reporter plasmid pUAS-EGFP containing the GAL417 bp oligonucleotide recognition sequence and EGFP gene and to mediate transduction of HepG-2 cells. Our studies determined that the TG or TNG chimeric protein is an efficient carrier of plasmid DNA, and this strategy may offer a new approach for enhancement of clinical gene therapy.To evaluate the TG-or TNG mediated cell transfection, the HepG-2 human hepatoma cell line was analyzed by fluorescence. HepG-2 cells were seeded the day before the assay and incubated with the complexes of TG/pUAS-EGFP, TNG/pUAS-EGFP, G/pUAS-EGFP or NG/pUAS-EGFP, respectively, for 48 h at 37℃. The HepG-2 cells incubated with TG/pUAS-EGFP or TNG/pUAS-EGFP complex exhibited strong green fluorescence. In contrast, no fluorescence was visualized in the cells incubated with G/pUAS-EGFP or NG/pUAS-EGFP complex under the same conditions. These results demonstrated that the Tat peptide fusion in the TG or TNG protein mediated efficient gene transfer and resulted in significant expression of the exogenous gene.The cell transduction and anti-tumor effects of TG and TNG in HepG-2 cells were identified by MTT. The results showed that, except for TG/pUAS-Apoptin or TNG/pUAS-Apoptin, other treatments (G/pUAS-Apoptin, NG/pUAS-Apoptin, pUAS-Apoptin alone, G protein alone, NG protein alone, TG protein alone, TNG protein alone) did not kill HepG-2 cells in varying degrees. While, there was no significant difference between the anti-tumor effects of TG/pUAS-Apoptin and the anti-tumor effects of TNG/pUAS-Apoptin. In the certain treatment doses, the anti-tumor effects of TG/pUAS-Apoptin or TNG/pUAS-Apoptin were heightened with the extending of treatment time, and resulted in some time-effect relationship. It is presumed that the suppression rates of TG/pUAS-Apoptin or TNG/pUAS-Apoptin were concerned with treatment doses.By using AO/EB assay, DAPI assay, Annexin V assay and Caspses activity assay, we analyzed the mechanism of the anti-tumor effects mediated by TG/pUAS-Apoptin or TNG/pUAS-Apoptin. The results showed that the TG/pUAS-Apoptin or TNG/pUAS-Apoptin complex can induce apoptosis of HepG-2 cells. While, there was no significant difference between the apoptosis induce function of TG/pUAS-Apoptin and the poptosis induce function of TNG/pUAS-Apoptin.We also constrcted the C57BL/6 mice model bearing H22 by transplanting H22 cells into the right hind limb of the mice. And then, the in vivo anti-tumor effects of TG/pUAS-Apoptin or TNG/pUAS-Apoptin were observed through the mice model. The results showed that TG/pUAS-Apoptin and TNG/pUAS-Apoptin treatment groups demonstrate varying degree anti-tumor effects. The suppression rates of TG/pUAS-Apoptin and TNG/pUAS-Apoptin were 27% and 28.6%, respectively, which significant higher than the other groups (Saline, pUAS, pUAS-Apoptin, TG, TNG, TG/pUAS and TNG/pUAS). While, there was no significant difference between the suppression rate of TG/pUAS-Apoptin and the suppression rate of TNG/pUAS-Apoptin. We, then, observed the mean survival of the mice model. The results showed that the mean survival rates of both TG/pUAS-Apoptin and TNG/pUAS-Apoptin treatment groups were 66.7%, and much higher that Saline, pUAS, pUAS-Apoptin, TG, TNG, TG/pUAS or TNG/pUAS treatment groups. To approach the effects of TG/pUAS-Apoptin and TNG/pUAS-Apoptin on immune system, the contents of IL-2, IFN-y, IL-4 and IL-10 were identified. The results showed that TG/pUAS-Apoptin, TNG/pUAS-Apoptin and the other recombinant proteins (G and NG) did not affect the concentration of IL-2, IFN-y, IL-4 and IL-10.In summary, the recombinant protein TG and TNG basing on GAL4/147 and Tat peptid in this study, which has both UAS specific recognition and membrane penetration abilities, can transduct plasmid containing UAS sequences, and suppress HepG-2 cells and inhibit solid tumor in animal model by mediating Apoptin containing plasmid, effectively. The study provide a novel subsystem for the expanding use of nacked plasmid DNA.更多还原