RDP-p53融合蛋白對(duì)小鼠IgG和炎癥細(xì)胞因子的影響
發(fā)布時(shí)間：2019-05-28 05:20
【摘要】：p53基因是重要的腫瘤抑制基因,其編碼的p53蛋白在細(xì)胞周期調(diào)控、DNA修復(fù)和誘導(dǎo)細(xì)胞凋亡等方面均具有關(guān)鍵性作用,人類50%以上的腫瘤組織中存在p53基因突變,是所檢測(cè)到的最常見的基因突變,有研究人員指出,野生型p53蛋白功能的缺失是腫瘤發(fā)生的必要條件。將p53蛋白導(dǎo)入腫瘤細(xì)胞就可以發(fā)揮較好的的抗腫瘤功效,但是p53蛋白作為大分子物質(zhì)自身難以穿過細(xì)胞膜進(jìn)入腫瘤細(xì)胞,使其應(yīng)用受到了一定限制。 Kumar等研究發(fā)現(xiàn)來源于狂犬病毒衣殼糖蛋白的衍生肽段RDP (rabies virus glycoprotein derived peptide)能攜帶抗病毒的siRNA通過血腦屏障特異性地傳遞到感染了腦炎的小鼠腦中,大大提高患腦炎小鼠的存活率,并且RDP作為載體具有較高的安全性。Kumar等對(duì)RDP的開創(chuàng)性研究為解決大分子難以穿過細(xì)胞膜及血腦屏障入腦的問題提供了很好的基礎(chǔ),我們實(shí)驗(yàn)室前期成功利用RDP攜帶大分子蛋白質(zhì)BDNF'快速特異性進(jìn)入腦內(nèi)。以往研究中一般通過往細(xì)胞穿膜肽(cell-penetrating peptides, CPP)攜帶大分子進(jìn)入細(xì)胞,RDP多肽作為新型腦靶向遞藥載體克服了CPP無組織特異性的缺陷。 本實(shí)驗(yàn)利用生物技術(shù)通過大腸桿菌表達(dá)RDP-p53融合蛋白,以期通過腦靶向遞藥載體RDP攜帶p53穿過血腦屏障,用于開發(fā)治療腦腫瘤比如神經(jīng)膠質(zhì)瘤的新型藥物。生物技術(shù)藥物在體內(nèi)存在的最普遍的問題之一是免疫反應(yīng),它不僅降低藥物療效,而且損害機(jī)體,免疫原性的評(píng)價(jià)成為闡明這些藥物臨床安全性和有效性的關(guān)鍵因素,評(píng)價(jià)非期望出現(xiàn)的免疫原性是生物技術(shù)藥物臨床前和臨床評(píng)價(jià)的重要內(nèi)容。本實(shí)驗(yàn)通過ELISA檢測(cè)血清IgG的水平來評(píng)價(jià)免疫原性的強(qiáng)弱。p53除具有抗腫瘤作用外,還與炎癥介質(zhì)之間存在一定聯(lián)系,p53可以影響炎癥細(xì)胞因子(inflammatory cytokine)的分泌；研究表明機(jī)體釋放的一系列炎癥細(xì)胞因子可以對(duì)藥物代謝酶的表達(dá)和功能產(chǎn)生調(diào)控,機(jī)體對(duì)藥物的處置過程會(huì)發(fā)生顯著改變。因此,炎癥細(xì)胞因子可能會(huì)影響RDP-p53融合蛋白的藥理作用。本實(shí)驗(yàn)通過ELISA檢測(cè)血清IL-1β、IL-6、TNF-α的水平來分析腹腔注射不同劑量的RDP-p53融合蛋白對(duì)小鼠血清炎癥細(xì)胞因子水平的影響,為研究RDP-p53融合蛋白抗神經(jīng)膠質(zhì)瘤的藥理作用奠定基礎(chǔ)。 實(shí)驗(yàn)分兩部分進(jìn)行：第一部分：以質(zhì)粒pET28a-p53為模板擴(kuò)增p53基因,并克隆至原核表達(dá)載體pET28a-RDP中,構(gòu)建重組表達(dá)質(zhì)粒pET28a-RDP-p53,轉(zhuǎn)化至大腸桿菌Rosetta, IPTG誘導(dǎo)表達(dá)蛋白并純化,SDS-PAGE確定該蛋白準(zhǔn)確性。第二部分：將昆明小鼠分為空白對(duì)照組(等容生理鹽水)和RDP-p53融合蛋白高、中、低劑量(4mg/kg、2mg/k、1mg/kg)組,經(jīng)腹腔注射免疫,每隔2d給藥1次,30d后眼球取血,用酶聯(lián)免疫吸附法(ELISA)測(cè)定血清中IgG和炎癥細(xì)胞因子(白細(xì)胞介素1p、白細(xì)胞介素6和腫瘤壞死因子α)的含量。 實(shí)驗(yàn)結(jié)果：1雙酶切及測(cè)序結(jié)果顯示,p53基因已克隆入表達(dá)載體中；RDP-p53融合蛋白在上清和沉淀中均獲得表達(dá)。2ELISA測(cè)定結(jié)果表明：與對(duì)照組比較,低、中劑量組小鼠血清中IgG含量沒有明顯升高(P0.05),高劑量組顯著升高(P0.05)；與對(duì)照組比較,低、中劑量組小鼠血清炎癥因子水平?jīng)]有明顯變化(P0.05),高劑量組的IL-1β和TNF-a含量顯著升高(P0.05)。 實(shí)驗(yàn)結(jié)論：成功表達(dá)并純化RDP-p53融合蛋白,其免疫原性較弱且與給藥劑量相關(guān),1mg/kg和2mg/kg劑量下對(duì)小鼠重復(fù)多次腹腔注射RDP-p53融合蛋白不會(huì)促使炎癥細(xì)胞因子水平顯著升高,為RDP-p53融合蛋白腹腔注射給藥劑量的確定提供了實(shí)驗(yàn)依據(jù),可在此給藥劑量條件下進(jìn)一步通過動(dòng)物實(shí)驗(yàn)來研究RDP-p53融合蛋白抗神經(jīng)膠質(zhì)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.
【學(xué)位授予單位】：西南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：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.
【學(xué)位授予單位】：西南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R965
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