甲型流感病毒DNA疫苗双表达载体的构建及免疫效果的研究

发布时间：2018-06-10 18:31

本文选题：流感病毒 + DNA疫苗　；参考：《吉林大学》2007年博士论文

【摘要】： 本研究以pVAXI载体为骨架,构建出含有微小病毒内部核糖体进入位点(IRES)基因的DNA疫苗双表达载体pVI。为验证该载体的表达效果,又将增强型绿色荧光蛋白(EGFP)基因和新霉素磷酸转移酶(neor)基因作为报告基因连接到pVI载体的上下游多克隆位点处,构建出真核表达载体pEIN。将其转染COS-7细胞,得到具有G418抗性的细胞克隆,同时镜下观察到绿色荧光,表明该载体可成功表达两个外源基因。在此基础上,将甲型流感病毒New Caledonia/20/99(H1N1)株接种于鸡胚的尿囊液中,Trizol法提取流感病毒总RNA,以其为模板,RT-PCR法分离出甲型流感病毒M2基因,同时将GM-CSF基因做为佐剂基因连接到pVI载体上,构建出流感病毒DNA疫苗双表达载体pMIG,测序后,将pMIG质粒转染COS7细胞,Western blot结果表明表达了甲型流感病毒M2蛋白。碱裂解法大提质粒pMIG,用Sepharose-CL 4B柱层析纯化质粒,紫外分光光度计测A260和A280比值,测定纯度和浓度后,琼脂糖电泳检测DNA的完整性。将Balb/C小鼠随机分成5组,每组10只,分组如下:流感疫苗对照组、空载体pVAXI对照组、pVAX/GM-CSF对照组、pVAX/M2实验组、pMIG实验组。将质粒注射于小鼠后腿胫前肌,注射剂量100ug/100ul,流感裂解疫苗对照组4ug抗原/只的剂量。分0、2、4周免疫接种3次,三次免疫剂量与注射部位均相同,初免后第1周开始每周定期对小鼠眼眶采血,检测其免疫效果。经方差分析,流感裂解疫苗对照组免后第二周即开始产生特异性血凝素抗体,到第四周达到峰值。在免后14周抗体还没有消失。而其余各组均未检测到抗体。流式细胞术双标法检测小鼠T-cell表面标记CD4+、CD8+水平及CD4+/CD8+水平,T淋巴细胞表面标记检测表明,流感DNA疫苗组与流感裂解疫苗对照组相比,CD4+/CD8++比例,以及细胞因子(IL-4、IFNγ)水平上均有显著的增强作用。攻毒实验表明,流感DNA疫苗比流感裂解疫苗保护效力高出近一倍。
[Abstract]:In this study, pVAXI vector was used as the skeleton to construct a DNA vaccine double expression vector pVII containing the gene of internal ribosomal entry site (IRES) of parvovirus. To verify the expression effect of the vector, the enhanced green fluorescent protein (EGFP) gene and neomycin phosphotransferase (neo) gene were linked to the upstream and downstream polyclonal sites of pVI vector, and the eukaryotic expression vector pEINwas constructed. It was transfected into COS-7 cells to obtain G418 resistant cell clones. Green fluorescence was observed under microscope, which indicated that the vector could express two foreign genes successfully. Influenza A virus strain New Caledonia / 20 / 99H1 N1 was inoculated into chicken embryo allantoic fluid to extract total RNA of influenza virus by Trizol method. The M2 gene of influenza A virus was isolated by RT-PCR, and the GM-CSF gene was linked to PVI vector as adjuvant gene. The double expression vector pMIG of influenza virus DNA vaccine was constructed. After sequencing, the plasmid pMIG was transfected into COS7 cells by Western blot. The results showed that the plasmid pMIG was extracted by alkaline lysis method and purified by Sepharose-CL4B column chromatography. The ratio of A260 to A280 was measured by ultraviolet spectrophotometer, and the purity and concentration were measured. The DNA integrity was detected by agarose electrophoresis. Balb / C mice were randomly divided into 5 groups, 10 in each group, as follows: influenza vaccine control group. Empty vector pVAXI control group pVAX / GM-CSF control group pVAX / M2 experimental group was injected into the anterior tibial muscle of the hind leg of mice at the dose of 100ug-100ul. the dose of 4ug antigen / mouse in the influenza lytic vaccine control group. The immune dose was the same as that of the injection site. The blood samples were collected from the orbit of mice at the first week after the first immunization, and the immunological effects were tested by ANOVA. Specific hemagglutinin antibodies began to be produced in the influenza lytic vaccine control group at the second week after immunization and reached a peak at the fourth week. The antibody did not disappear at 14 weeks after immunization. However, no antibody was detected in other groups. The detection of T-cell surface labeled CD4 / CD8 and CD4 / CD8 level by flow cytometry showed that the ratio of CD 4 / CD 8 in the influenza DNA vaccine group was higher than that in the influenza lytic vaccine group, and the ratio of CD 4 / CD 8 in the influenza DNA vaccine group was higher than that in the control group. The level of cytokine IL-4 and IFN- 纬 was significantly enhanced. The results showed that the protective efficacy of influenza DNA vaccine was nearly twice as high as that of influenza cleavage vaccine.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R392.1

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