蛋白質(zhì)-DNA復(fù)合納米顆�？鼓[瘤疫苗的初步研究
發(fā)布時(shí)間：2019-06-05 08:44
【摘要】： 基因疫苗是近年來剛剛發(fā)展起來的一類新型疫苗,由于能誘導(dǎo)細(xì)胞和體液雙重免疫反應(yīng),同時(shí)具有產(chǎn)品穩(wěn)定、運(yùn)輸方便、制造成本低廉的優(yōu)點(diǎn),因而具有巨大的開發(fā)應(yīng)用潛力。但目前國(guó)內(nèi)外研究的基因疫苗通常免疫效力較低,激發(fā)的免疫應(yīng)答達(dá)不到免疫保護(hù)要求,成為其進(jìn)一步產(chǎn)業(yè)化開發(fā)和應(yīng)用的巨大障礙。 本研究中,我們提出了一種新的基因疫苗增效策略:納米顆粒疫苗模式。將蛋白質(zhì)疫苗和基因疫苗,通過電荷作用結(jié)合在一起,形成納米顆粒,同時(shí)發(fā)揮兩種疫苗形式分別在誘導(dǎo)體液免疫和誘導(dǎo)細(xì)胞免疫反應(yīng)方面的優(yōu)勢(shì),并通過形成納米顆粒,增強(qiáng)抗原遞呈細(xì)胞對(duì)抗原的吞噬、加工和遞呈功能,最終提高基因疫苗的免疫效力。 首先,本研究以腫瘤特異性抗原凋亡抑制蛋白survivin和人絨毛膜促性腺激素hCG CTP37為靶抗原,通過基因工程的方法,原核表達(dá)和純化出一系列可負(fù)載DNA疫苗的特異性復(fù)合腫瘤抗原,即:SLC-survivin-IM,單倍體(survivin)1-IM,雙倍體(survivin)2-IM以及CTP37-IM。在上述復(fù)合抗原蛋白質(zhì)中,融合了富含陽離子氨基酸的魚精蛋白短肽,在生理pH下帶正電荷,通過靜電吸附可以濃縮帶負(fù)電的DNA。同時(shí)蛋白質(zhì)疫苗中還含有穿膜肽和DNA保護(hù)肽,能夠增加外源基因向細(xì)胞內(nèi)傳遞并避免其降解。 然后,將重組腫瘤抗原蛋白復(fù)合物與含有同一腫瘤抗原基因的腫瘤治療性DNA疫苗,靜電耦合成為復(fù)合納米顆粒,實(shí)現(xiàn)基因疫苗的納米顆�；２⑼ㄟ^瓊脂糖凝膠電泳阻滯實(shí)驗(yàn)證明,蛋白質(zhì)可以通過電荷作用與DNA結(jié)合,中和了DNA的電荷,從而阻滯了DNA質(zhì)粒在電泳中移動(dòng);用Dnase消化核酸的保護(hù)實(shí)驗(yàn)證明,納米顆�？杀Ｗo(hù)DNA質(zhì)粒不被降解;通過原子力顯微鏡觀察證實(shí),蛋白質(zhì)與DNA質(zhì)粒形成的納米顆粒的大小約為50-500nm,呈大小不均一的顆粒化。而這種大小的顆粒,適合抗原遞呈細(xì)胞的吞噬和抗原加工、遞呈。最后,通過體外瞬時(shí)轉(zhuǎn)染293T細(xì)胞研究表明,蛋白質(zhì)與DNA質(zhì)粒形成納米顆粒后,并不影響基因在細(xì)胞中的高效表達(dá),這保證了基因疫苗可以正常發(fā)揮作用。 總之,該復(fù)合納米顆粒疫苗,是基因疫苗有效投遞并發(fā)揮免疫保護(hù)作用的新模式,可能具備安全、緩釋和增強(qiáng)免疫激活等優(yōu)點(diǎn)。其應(yīng)用價(jià)值和潛力,有待于進(jìn)一步研究驗(yàn)證。
[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.
【學(xué)位授予單位】：中國(guó)人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R392;R730.5
本文編號(hào)：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.
【學(xué)位授予單位】：中國(guó)人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R392;R730.5
【參考文獻(xiàn)】
相關(guān)期刊論文 前1條
1 王浩;賈銳;閻瑾琦;宋曉國(guó);王宇;于繼云;;凋亡抑制蛋白生存蛋白-2B在大腸桿菌中的表達(dá)、純化及鑒定[J];軍事醫(yī)學(xué)科學(xué)院院刊;2008年01期
，本文編號(hào)：2493396
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