蛋白质-DNA复合纳米颗粒抗肿瘤疫苗的初步研究
发布时间：2019-06-05 08:44
【摘要】： 基因疫苗是近年来刚刚发展起来的一类新型疫苗,由于能诱导细胞和体液双重免疫反应,同时具有产品稳定、运输方便、制造成本低廉的优点,因而具有巨大的开发应用潜力。但目前国内外研究的基因疫苗通常免疫效力较低,激发的免疫应答达不到免疫保护要求,成为其进一步产业化开发和应用的巨大障碍。 本研究中,我们提出了一种新的基因疫苗增效策略:纳米颗粒疫苗模式。将蛋白质疫苗和基因疫苗,通过电荷作用结合在一起,形成纳米颗粒,同时发挥两种疫苗形式分别在诱导体液免疫和诱导细胞免疫反应方面的优势,并通过形成纳米颗粒,增强抗原递呈细胞对抗原的吞噬、加工和递呈功能,最终提高基因疫苗的免疫效力。 首先,本研究以肿瘤特异性抗原凋亡抑制蛋白survivin和人绒毛膜促性腺激素hCG CTP37为靶抗原,通过基因工程的方法,原核表达和纯化出一系列可负载DNA疫苗的特异性复合肿瘤抗原,即:SLC-survivin-IM,单倍体(survivin)1-IM,双倍体(survivin)2-IM以及CTP37-IM。在上述复合抗原蛋白质中,融合了富含阳离子氨基酸的鱼精蛋白短肽,在生理pH下带正电荷,通过静电吸附可以浓缩带负电的DNA。同时蛋白质疫苗中还含有穿膜肽和DNA保护肽,能够增加外源基因向细胞内传递并避免其降解。 然后,将重组肿瘤抗原蛋白复合物与含有同一肿瘤抗原基因的肿瘤治疗性DNA疫苗,静电耦合成为复合纳米颗粒,实现基因疫苗的纳米颗粒化。并通过琼脂糖凝胶电泳阻滞实验证明,蛋白质可以通过电荷作用与DNA结合,中和了DNA的电荷,从而阻滞了DNA质粒在电泳中移动;用Dnase消化核酸的保护实验证明,纳米颗粒可保护DNA质粒不被降解;通过原子力显微镜观察证实,蛋白质与DNA质粒形成的纳米颗粒的大小约为50-500nm,呈大小不均一的颗粒化。而这种大小的颗粒,适合抗原递呈细胞的吞噬和抗原加工、递呈。最后,通过体外瞬时转染293T细胞研究表明,蛋白质与DNA质粒形成纳米颗粒后,并不影响基因在细胞中的高效表达,这保证了基因疫苗可以正常发挥作用。 总之,该复合纳米颗粒疫苗,是基因疫苗有效投递并发挥免疫保护作用的新模式,可能具备安全、缓释和增强免疫激活等优点。其应用价值和潜力,有待于进一步研究验证。
[Abstract]:Gene vaccine is a new type of vaccine just developed in recent years. Because it can induce double immune response of cell and body fluid, at the same time, it has the advantages of stable product, convenient transportation and low manufacturing cost, so it has great potential for development and application. However, the immune efficacy of gene vaccines studied at home and abroad is usually low, and the stimulated immune response can not meet the requirements of immune protection, which has become a great obstacle to its further industrial development and application. In this study, we proposed a new gene vaccine synergy strategy: nanoparticles vaccine model. Protein vaccine and gene vaccine are combined to form nanoparticles through charge action, and at the same time, the advantages of the two vaccine forms in inducing humoral immunity and inducing cellular immune response are brought into play, and by forming nanoparticles, To enhance the phagocytosis, processing and presentation of antigen presenting cells, and finally to improve the immune efficacy of gene vaccine. Firstly, using tumor specific antigen apoptosis inhibitor survivin and human chorionic gonadotropin hCG CTP37 as target antigens, a series of specific complex tumor antigens loaded with DNA vaccine were expressed and purified by genetic engineering. That is, SLC-survivin-IM, haploid (survivin) 1 / I, diploid (survivin) 2-IM and CTP37-IM. In the above complex antigen protein, protamine short peptide rich in cationic amino acids was fusion, which was positively charged under physiological pH, and the negatively charged DNA. could be concentrated by electrostatic adsorption. At the same time, the protein vaccine also contains transmembrane peptides and DNA protective peptides, which can increase the transmission of foreign genes to cells and avoid their degradation. Then, the recombinant tumor antigen protein complex and the tumor therapeutic DNA vaccine containing the same tumor antigen gene were electrostatic coupled into composite nanoparticles to realize the nano-granulation of the gene vaccine. The results of agarose gel electrophoresis showed that the protein could bind to DNA through charge interaction and neutralize the charge of DNA, thus blocking the movement of DNA plasmid in electrophoresis. The protective experiment of digesting nucleic acid by Dnase showed that nanoparticles could protect DNA plasmid from degradation, and the size of nanoparticles formed by protein and DNA plasmid was about 50 nm and 500 nm, which was uneven in size by atomic force microscope (atomic force microscope). This size of particles, suitable for antigen presenting cells phagocytosis and antigen processing, presentation. Finally, the transient transfection of 293T cells in vitro showed that the formation of nanoparticles between protein and DNA plasmid did not affect the high expression of genes in cells, which ensured that the gene vaccine could play a normal role. In a word, the composite nanoparticles vaccine is a new model of gene vaccine delivery and immune protection, which may have the advantages of safety, sustained release and enhanced immune activation. Its application value and potential need to be further studied and verified.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392;R730.5
本文编号：2493396
[Abstract]:Gene vaccine is a new type of vaccine just developed in recent years. Because it can induce double immune response of cell and body fluid, at the same time, it has the advantages of stable product, convenient transportation and low manufacturing cost, so it has great potential for development and application. However, the immune efficacy of gene vaccines studied at home and abroad is usually low, and the stimulated immune response can not meet the requirements of immune protection, which has become a great obstacle to its further industrial development and application. In this study, we proposed a new gene vaccine synergy strategy: nanoparticles vaccine model. Protein vaccine and gene vaccine are combined to form nanoparticles through charge action, and at the same time, the advantages of the two vaccine forms in inducing humoral immunity and inducing cellular immune response are brought into play, and by forming nanoparticles, To enhance the phagocytosis, processing and presentation of antigen presenting cells, and finally to improve the immune efficacy of gene vaccine. Firstly, using tumor specific antigen apoptosis inhibitor survivin and human chorionic gonadotropin hCG CTP37 as target antigens, a series of specific complex tumor antigens loaded with DNA vaccine were expressed and purified by genetic engineering. That is, SLC-survivin-IM, haploid (survivin) 1 / I, diploid (survivin) 2-IM and CTP37-IM. In the above complex antigen protein, protamine short peptide rich in cationic amino acids was fusion, which was positively charged under physiological pH, and the negatively charged DNA. could be concentrated by electrostatic adsorption. At the same time, the protein vaccine also contains transmembrane peptides and DNA protective peptides, which can increase the transmission of foreign genes to cells and avoid their degradation. Then, the recombinant tumor antigen protein complex and the tumor therapeutic DNA vaccine containing the same tumor antigen gene were electrostatic coupled into composite nanoparticles to realize the nano-granulation of the gene vaccine. The results of agarose gel electrophoresis showed that the protein could bind to DNA through charge interaction and neutralize the charge of DNA, thus blocking the movement of DNA plasmid in electrophoresis. The protective experiment of digesting nucleic acid by Dnase showed that nanoparticles could protect DNA plasmid from degradation, and the size of nanoparticles formed by protein and DNA plasmid was about 50 nm and 500 nm, which was uneven in size by atomic force microscope (atomic force microscope). This size of particles, suitable for antigen presenting cells phagocytosis and antigen processing, presentation. Finally, the transient transfection of 293T cells in vitro showed that the formation of nanoparticles between protein and DNA plasmid did not affect the high expression of genes in cells, which ensured that the gene vaccine could play a normal role. In a word, the composite nanoparticles vaccine is a new model of gene vaccine delivery and immune protection, which may have the advantages of safety, sustained release and enhanced immune activation. Its application value and potential need to be further studied and verified.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392;R730.5
【参考文献】
相关期刊论文 前1条
1 王浩;贾锐;阎瑾琦;宋晓国;王宇;于继云;;凋亡抑制蛋白生存蛋白-2B在大肠杆菌中的表达、纯化及鉴定[J];军事医学科学院院刊;2008年01期
，本文编号：2493396
本文链接：https://www.wllwen.com/yixuelunwen/shiyanyixue/2493396.html
最近更新
教材专著
