人源胸腺素α原和白介素2融合基因的真核表达及免疫保护效果初探

发布时间：2018-01-17 21:02

本文关键词：人源胸腺素α原和白介素2融合基因的真核表达及免疫保护效果初探　出处：《广西师范大学》2008年硕士论文　论文类型：学位论文

【摘要】： 人源胸腺素α原(prothymosinα,proTα)是具有促进细胞增殖和显著的免疫刺激活性的酸性蛋白,主要用于治疗肿瘤、肝炎、免疫系统等疾病。白介素2(interleukin 2,IL-2)是一种糖蛋白,它能增强NK细胞的活性,对肾细胞癌、黑色素瘤、结肠癌、直肠癌有明显疗效,但过量使用IL-2会带来不良反应。ProTα和IL-2存在紧密的免疫刺激协同作用,proTα通过调节IL-2诱导的NK敏感细胞和LAK细胞毒性,使IL-2的有效浓度降低一半。它们协同于自体瘤的效果明显强于单独使用IL-2的效果。ProTα和IL-2联合使用不但能提高抗肿瘤的效果,还可降低单独使用IL-2产生的毒副作用。本研究利用PCR技术从前期构建好的pcDNA3.1-PTI重组质粒中克隆出proTα-linker-IL-2融合基因,并对其进行改造,在5′端加入粒细胞-巨噬细胞集落刺激因子(GM-CSF)的信号肽序列。将改造好的proTα-linker-IL-2融合基因(PI)克隆至真核载体pVAX中构建成重组质粒pVAX-PI,再将重组质粒转化大肠杆菌DH5α,通过菌落PCR和酶切筛选阳性单菌落,提取质粒进行测序鉴定。测序结果显示,改造后的融合基因序列与预期序列一致。提取无内毒素的重组质粒pVAX-PI并定量分析。采用脂质体转染试剂将重组质粒转染哺乳动物细胞COS-7中进行表达,分别收集质粒转染细胞后24h、48h、72h、96h的细胞上清,用ELISA法定量分析细胞上清中IL-2蛋白表达量,结果显示,24h、48h、72h、96h转染上清中的IL-2蛋白含量分别为5.4、7.8、6.1、5.7ug/L。采用CTLL-2依赖细胞株测得转染后24h、48h、72h、96h细胞上清中的IL-2活性分别为43、58、54、49IU/ml。将小鼠原代脾细胞与细胞转染上清一起培养48h后,MTT实验结果表明,转染上清能刺激小鼠脾细胞增殖,表达的融合蛋白具有proTα的活性。最后,通过ECL western blotting分析和鉴定了表达的融合蛋白的分子量约为31kDa。本次研究初步发现pVAX-PI对小鼠黑色素瘤有一定的抑制作用,并且对荷瘤鼠体内细胞因子IFN-γ和IL-4均有一定的诱生作用。pVAX-PI质粒免疫组的小鼠外周血清中IFN-γ和IL-4的含量随时间的推移而逐渐上升。荷瘤小鼠接种pVAX-PI质粒后第5周,ELISA定量分析小鼠外周血清中IFN-γ和IL-4的含量,肌肉注射联合在体电脉冲刺激组分别为222pg/ml和157pg/ml,单纯肌肉注射组分别为129pg/ml和68pg/ml。以上结果说明,pVAX-PI质粒在荷瘤鼠的免疫体系中具有一定调节作用。
[Abstract]:Human thymosin 伪 proosin 伪 (Thymosin 伪 prot 伪) is an acidic protein that promotes cell proliferation and has significant immunostimulatory activity. It is mainly used in the treatment of tumor and hepatitis. Interleukin 2 interleukin 2 (IL 2) is a glycoprotein that enhances the activity of NK cells in renal cell carcinoma, melanoma, and colon cancer. Rectal cancer has obvious curative effect, but excessive use of IL-2 can bring adverse reaction. ProT 伪 and IL-2 have close immune stimulation synergism. ProT 伪 regulates the cytotoxicity of NK sensitive cells and LAK cells induced by IL-2. The effective concentration of IL-2 was reduced by half. They were more effective than those of IL-2 alone. Prot 伪 and IL-2 could not only improve the anti-tumor effect. It also reduces the side effects of IL-2 alone. In this study, proT 伪 -linker-IL-2 fusion gene was cloned from pcDNA3.1-PTI recombinant plasmid constructed by PCR technique and modified. The signal peptide sequence of granulocyte-macrophage colony stimulating factor (GM-CSF) was added to the 5'terminal. The modified proT 伪 -linker-IL-2 fusion gene was modified. The recombinant plasmid pVAX-PI was constructed by cloning into eukaryotic vector pVAX. The recombinant plasmid was transformed into Escherichia coli DH5 伪. The positive single colony was screened by colony PCR and enzyme digestion. The plasmid was sequenced and identified. The modified fusion gene sequence was consistent with the expected sequence. The recombinant plasmid pVAX-PI without endotoxin was extracted and quantitatively analyzed. The recombinant plasmid was transfected into mammalian COS-7 by liposome transfection reagent for expression. The supernatants of the cells were collected at 24 h, 48 h, 72 h and 96 h after transfection, and the expression of IL-2 protein in the supernatant was quantitatively analyzed by ELISA method. The results showed that the expression of IL-2 protein was 24 h after transfection. The content of IL-2 protein in the supernatant of 48h / 72h / 96h was 5.4g / L respectively. 24 hours after transfection with CTLL-2 dependent cell line, the content of IL-2 protein was 5.7U / L, respectively. The activity of IL-2 in the supernatant of mouse primary spleen cells and the supernatant of murine primary spleen cells was cultured for 48 h. The results of MTT assay showed that the transfected supernatant could stimulate the proliferation of mouse spleen cells, and the expressed fusion protein had the activity of proT 伪. The molecular weight of the expressed fusion protein was about 31 kDa by ECL western blotting. In this study, we found that pVAX-PI has a certain inhibitory effect on melanoma in mice. Moreover, the levels of IFN- 纬 and IL-4 in peripheral serum of mice immunized with pVAX-PI plasmid were induced by IFN- 纬 and IL-4 in tumor-bearing mice over time. The tumor-bearing mice were inoculated with pVAX-PI plasmid for 5 weeks. The levels of IFN- 纬 and IL-4 in peripheral serum of mice were determined by ELISA. The results of intramuscular injection combined with electrical pulse stimulation in vivo were 222 PG / ml and 157 PG / ml, respectively. The results showed that pVAX-PI plasmid could regulate the immune system of tumor-bearing mice.
【学位授予单位】：广西师范大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392;Q78

【引证文献】