RDP-p53融合蛋白对小鼠IgG和炎症细胞因子的影响
发布时间：2019-05-28 05:20
【摘要】：p53基因是重要的肿瘤抑制基因,其编码的p53蛋白在细胞周期调控、DNA修复和诱导细胞凋亡等方面均具有关键性作用,人类50%以上的肿瘤组织中存在p53基因突变,是所检测到的最常见的基因突变,有研究人员指出,野生型p53蛋白功能的缺失是肿瘤发生的必要条件。将p53蛋白导入肿瘤细胞就可以发挥较好的的抗肿瘤功效,但是p53蛋白作为大分子物质自身难以穿过细胞膜进入肿瘤细胞,使其应用受到了一定限制。 Kumar等研究发现来源于狂犬病毒衣壳糖蛋白的衍生肽段RDP (rabies virus glycoprotein derived peptide)能携带抗病毒的siRNA通过血脑屏障特异性地传递到感染了脑炎的小鼠脑中,大大提高患脑炎小鼠的存活率,并且RDP作为载体具有较高的安全性。Kumar等对RDP的开创性研究为解决大分子难以穿过细胞膜及血脑屏障入脑的问题提供了很好的基础,我们实验室前期成功利用RDP携带大分子蛋白质BDNF'快速特异性进入脑内。以往研究中一般通过往细胞穿膜肽(cell-penetrating peptides, CPP)携带大分子进入细胞,RDP多肽作为新型脑靶向递药载体克服了CPP无组织特异性的缺陷。 本实验利用生物技术通过大肠杆菌表达RDP-p53融合蛋白,以期通过脑靶向递药载体RDP携带p53穿过血脑屏障,用于开发治疗脑肿瘤比如神经胶质瘤的新型药物。生物技术药物在体内存在的最普遍的问题之一是免疫反应,它不仅降低药物疗效,而且损害机体,免疫原性的评价成为阐明这些药物临床安全性和有效性的关键因素,评价非期望出现的免疫原性是生物技术药物临床前和临床评价的重要内容。本实验通过ELISA检测血清IgG的水平来评价免疫原性的强弱。p53除具有抗肿瘤作用外,还与炎症介质之间存在一定联系,p53可以影响炎症细胞因子(inflammatory cytokine)的分泌；研究表明机体释放的一系列炎症细胞因子可以对药物代谢酶的表达和功能产生调控,机体对药物的处置过程会发生显著改变。因此,炎症细胞因子可能会影响RDP-p53融合蛋白的药理作用。本实验通过ELISA检测血清IL-1β、IL-6、TNF-α的水平来分析腹腔注射不同剂量的RDP-p53融合蛋白对小鼠血清炎症细胞因子水平的影响,为研究RDP-p53融合蛋白抗神经胶质瘤的药理作用奠定基础。 实验分两部分进行：第一部分：以质粒pET28a-p53为模板扩增p53基因,并克隆至原核表达载体pET28a-RDP中,构建重组表达质粒pET28a-RDP-p53,转化至大肠杆菌Rosetta, IPTG诱导表达蛋白并纯化,SDS-PAGE确定该蛋白准确性。第二部分：将昆明小鼠分为空白对照组(等容生理盐水)和RDP-p53融合蛋白高、中、低剂量(4mg/kg、2mg/k、1mg/kg)组,经腹腔注射免疫,每隔2d给药1次,30d后眼球取血,用酶联免疫吸附法(ELISA)测定血清中IgG和炎症细胞因子(白细胞介素1p、白细胞介素6和肿瘤坏死因子α)的含量。 实验结果：1双酶切及测序结果显示,p53基因已克隆入表达载体中；RDP-p53融合蛋白在上清和沉淀中均获得表达。2ELISA测定结果表明：与对照组比较,低、中剂量组小鼠血清中IgG含量没有明显升高(P0.05),高剂量组显著升高(P0.05)；与对照组比较,低、中剂量组小鼠血清炎症因子水平没有明显变化(P0.05),高剂量组的IL-1β和TNF-a含量显著升高(P0.05)。 实验结论：成功表达并纯化RDP-p53融合蛋白,其免疫原性较弱且与给药剂量相关,1mg/kg和2mg/kg剂量下对小鼠重复多次腹腔注射RDP-p53融合蛋白不会促使炎症细胞因子水平显著升高,为RDP-p53融合蛋白腹腔注射给药剂量的确定提供了实验依据,可在此给药剂量条件下进一步通过动物实验来研究RDP-p53融合蛋白抗神经胶质瘤效果。
[Abstract]:The p53 gene is an important tumor suppressor gene, and the encoded p53 protein has a key role in the aspects of cell cycle regulation, DNA repair and induction of cell apoptosis, and the p53 gene mutation in the tumor tissue of more than 50% is the most common gene mutation detected, It was noted that the deletion of the wild-type p53 protein function was a necessary condition for tumorigenesis. The introduction of the p53 protein into the tumor cell can play a better anti-tumor effect, but the p53 protein is difficult to enter the tumor cell through the cell membrane as a macromolecular substance, so that the application of the p53 protein is limited. Kumar et al. found that the derived peptide (RDP) derived from the rabies virus capsid glycoprotein can carry antiviral siRNA to the brain of mice infected with encephalitis through the blood-brain barrier, thus greatly improving the survival of the mice with encephalitis Rate, and RDP as a carrier with higher security The groundbreaking of the RDP by Kumar et al provides a good basis for solving the problem of the difficulty of the macromolecules to penetrate the cell membrane and to the brain of the blood-brain barrier In the early stage of our lab, we successfully used RDP to carry macromolecular protein BDNF 'into the brain. In the past, the cell-specific peptide (CPP) was used to carry macromolecules into the brain. The novel brain-targeting drug delivery vector overcomes the defect of no tissue specificity of the CPP as a novel brain-targeting drug delivery vehicle. In this experiment, the expression of RDP-p53 fusion protein was expressed in E. coli by using biological technology, with a view to carrying p53 through the blood through the brain-targeted drug delivery vector RDP. A brain barrier for the development of new drugs for the treatment of brain tumors, such as glioma. One of the most common problems in the body of biotechnology is the immune response, which not only reduces the efficacy of the drug, but also The evaluation of the harm to the organism and the immunogenicity has become the key to clarify the clinical safety and effectiveness of these drugs. The key factors and the non-expected immunogenicity were the important contents of the preclinical and clinical evaluation of biotechnological drugs. The level of serum IgG was detected by ELISA in this experiment to evaluate the immunogenicity. It is suggested that a series of inflammatory cytokines released by the body can regulate the expression and function of the drug metabolizing enzyme, and the body has a significant change in the treatment of the drug. In this study, the levels of IL-1, IL-6 and TNF-1 in the serum of mice were detected by ELISA, and the levels of IL-1, IL-6, and TNF-1 in the serum of mice were detected by ELISA. To study the effect of RDP-p53 fusion protein on the anti-glioma. The first part is to amplify the p53 gene with the plasmid pET28a-p53 and to be cloned into the prokaryotic expression vector pET28a-RDP to construct the recombinant expression plasmid pET28a-RDP-p53 and to transform to E. coli Ro. Setta, induced by IPTG and purified, and the accuracy of the protein was determined by SDS-PAGE. The second part: Kunming mice were divided into blank control group (isovolume normal saline) and RDP-p53 fusion protein with high, medium and low dose (4 mg/ kg,2 mg/ k,1 mg/ kg). D. After 1 and 30 days of administration, the blood was taken and the serum IgG and inflammatory cytokines (interleukin1, interleukin6 and tumor necrosis factor) were determined by enzyme-linked immunosorbent assay (ELISA). The results of the two-enzyme digestion and sequencing showed that the p53 gene was already in the form of a double-enzyme digestion and sequencing. The results showed that the content of IgG in serum of mice with low and medium dose was not significantly higher than that of the control group (P0.05), and the high-dose group was significantly higher (P0.05). The levels of IL-1 and TNF-a in the high-dose group were significantly higher than those in the control group (P0.05). (P0.05). Experimental conclusion: The RDP-p53 fusion protein was successfully expressed and purified, and its immunogenicity was higher. The low and dose-related,1-mg/ kg and 2-mg/ kg-dose administration of RDP-p53 fusion protein in mice at a dose of 1 mg/ kg and 2 mg/ kg did not cause a significant increase in the level of inflammatory cytokines, which was administered to the intraperitoneal injection of the RDP-p53 fusion protein. It is determined that the experimental basis is provided and the RDP-p53 fusion egg can be further studied by animal experiments under this administration dose.
【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R965
[Abstract]:The p53 gene is an important tumor suppressor gene, and the encoded p53 protein has a key role in the aspects of cell cycle regulation, DNA repair and induction of cell apoptosis, and the p53 gene mutation in the tumor tissue of more than 50% is the most common gene mutation detected, It was noted that the deletion of the wild-type p53 protein function was a necessary condition for tumorigenesis. The introduction of the p53 protein into the tumor cell can play a better anti-tumor effect, but the p53 protein is difficult to enter the tumor cell through the cell membrane as a macromolecular substance, so that the application of the p53 protein is limited. Kumar et al. found that the derived peptide (RDP) derived from the rabies virus capsid glycoprotein can carry antiviral siRNA to the brain of mice infected with encephalitis through the blood-brain barrier, thus greatly improving the survival of the mice with encephalitis Rate, and RDP as a carrier with higher security The groundbreaking of the RDP by Kumar et al provides a good basis for solving the problem of the difficulty of the macromolecules to penetrate the cell membrane and to the brain of the blood-brain barrier In the early stage of our lab, we successfully used RDP to carry macromolecular protein BDNF 'into the brain. In the past, the cell-specific peptide (CPP) was used to carry macromolecules into the brain. The novel brain-targeting drug delivery vector overcomes the defect of no tissue specificity of the CPP as a novel brain-targeting drug delivery vehicle. In this experiment, the expression of RDP-p53 fusion protein was expressed in E. coli by using biological technology, with a view to carrying p53 through the blood through the brain-targeted drug delivery vector RDP. A brain barrier for the development of new drugs for the treatment of brain tumors, such as glioma. One of the most common problems in the body of biotechnology is the immune response, which not only reduces the efficacy of the drug, but also The evaluation of the harm to the organism and the immunogenicity has become the key to clarify the clinical safety and effectiveness of these drugs. The key factors and the non-expected immunogenicity were the important contents of the preclinical and clinical evaluation of biotechnological drugs. The level of serum IgG was detected by ELISA in this experiment to evaluate the immunogenicity. It is suggested that a series of inflammatory cytokines released by the body can regulate the expression and function of the drug metabolizing enzyme, and the body has a significant change in the treatment of the drug. In this study, the levels of IL-1, IL-6 and TNF-1 in the serum of mice were detected by ELISA, and the levels of IL-1, IL-6, and TNF-1 in the serum of mice were detected by ELISA. To study the effect of RDP-p53 fusion protein on the anti-glioma. The first part is to amplify the p53 gene with the plasmid pET28a-p53 and to be cloned into the prokaryotic expression vector pET28a-RDP to construct the recombinant expression plasmid pET28a-RDP-p53 and to transform to E. coli Ro. Setta, induced by IPTG and purified, and the accuracy of the protein was determined by SDS-PAGE. The second part: Kunming mice were divided into blank control group (isovolume normal saline) and RDP-p53 fusion protein with high, medium and low dose (4 mg/ kg,2 mg/ k,1 mg/ kg). D. After 1 and 30 days of administration, the blood was taken and the serum IgG and inflammatory cytokines (interleukin1, interleukin6 and tumor necrosis factor) were determined by enzyme-linked immunosorbent assay (ELISA). The results of the two-enzyme digestion and sequencing showed that the p53 gene was already in the form of a double-enzyme digestion and sequencing. The results showed that the content of IgG in serum of mice with low and medium dose was not significantly higher than that of the control group (P0.05), and the high-dose group was significantly higher (P0.05). The levels of IL-1 and TNF-a in the high-dose group were significantly higher than those in the control group (P0.05). (P0.05). Experimental conclusion: The RDP-p53 fusion protein was successfully expressed and purified, and its immunogenicity was higher. The low and dose-related,1-mg/ kg and 2-mg/ kg-dose administration of RDP-p53 fusion protein in mice at a dose of 1 mg/ kg and 2 mg/ kg did not cause a significant increase in the level of inflammatory cytokines, which was administered to the intraperitoneal injection of the RDP-p53 fusion protein. It is determined that the experimental basis is provided and the RDP-p53 fusion egg can be further studied by animal experiments under this administration dose.
【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R965
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