香菇多糖联合丙肝核酸疫苗诱导抗原特异性免疫反应的研究

发布时间：2018-05-10 09:59

本文选题：丙型肝炎 + 非结构蛋白3　；参考：《华中科技大学》2008年硕士论文

【摘要】： 第一部分丙型肝炎病毒非结构蛋白3(NS3)DNA疫苗的制备与鉴定目的:大量制备丙型肝炎病毒非结构蛋白3(NS3)DNA疫苗,并进行定量及定性分析,为进一步研究其免疫活性奠定基础。方法:将pcDNA3.1-HCV/NS3转化感受态大肠杆菌HB101,克隆后挑取单个菌落扩增,用超纯质粒大量快速提取试剂盒提取pcDNA3.1-HCV/NS3,提取物分别进行核酸定量检测和琼脂糖凝胶电泳鉴定。结果:本试验成功的转化pcDNA3.1-HCV/NS于大肠杆菌中,铺平皿温育16h后有较多的单菌落生长。扩增提取产物经核酸定量检测,浓度在1.5μg/μl-2μg/μl范围内,纯度均接近1.8,因免疫小鼠所需要的DNA浓度为1μg/μl,所以提取的DNA在浓度和纯度上符合要求,另将扩增提取产物双酶切后经琼脂糖凝胶电泳证实扩增的质粒DNA包含目的基因NS3片段,分子量为1.8Kb。结论:本实验成功制备了用于免疫动物的HCV/ NS3 DNA疫苗,为进一步研究其免疫活性奠定了基础。第二部分香菇多糖作为免疫佐剂对丙肝病毒非结构蛋白3(NS3)体液免疫效果的影响目的:探讨中药香菇多糖(LNT)作为免疫佐剂对HCV/ NS3核酸疫苗体液免疫效果的影响。方法:HCV-NS3核酸疫苗肌注免疫BALB/c小鼠(100μg/只),10天后加强免疫一次,同时灌胃给予高(1mg/ml)、中(0.5mg/ml)、低(0.1mg/ml)三种剂量的香菇多糖,于末次免疫后第1、3、5周分别采集小鼠尾血,分离收集血清冻存。用HCV/NS3抗原多肽包被酶标板,采用间接酶联免疫吸附实验(ELISA)法检测小鼠血清特异性IgG抗体水平。并采用ELISA方法检测血清中细胞因子IL-4和IFN-γ分泌水平。结果:二次免疫后1周各组免疫小鼠血清中均产生了不同水平的抗体,以中剂量LNT为佐剂的免疫组在各时间点产生的特异性IgG水平最为显著,而且随着时间的增加,中剂量LNT为佐剂的免疫组和单独免疫核酸疫苗组的特异性IgG水平呈现出上升的趋势,而以高剂量和低剂量的LNT为佐剂免疫组的抗体水平随时间变化不大;中剂量香菇多糖为佐剂免疫组产生的特异性抗体水平较单独免疫核酸疫苗组在各时间点均有显著性提高(P0.05)。细胞因子分析显示以不同剂量香菇多糖作为佐剂的免疫组较之单独免疫核酸疫苗组IFN-γ的分泌增加,其中中剂量的香菇多糖联合HCV/NS3核酸疫苗组产生IFN-γ的水平较之单独使用核酸疫苗组,差异非常显著(P0.01)。而IL-4水平各组间无明显差异(P0.05)。结论:通过二次肌注免疫HCV/NS3核酸疫苗,小鼠体内产生了抗NS3抗原的特异性IgG抗体,而且随着时间的延长,特异性抗体水平也随之升高。中剂量的香菇多糖为佐剂能够增强HCV/NS3核酸疫苗诱导的特异性体液免疫反应。香菇多糖能够刺激Th1类细胞因子IFN-γ分泌,而对Th2类细胞因子IL-4的分泌未明显影响。第三部分香菇多糖作为佐剂对丙肝病毒非结构蛋白3(NS3)细胞免疫效果的影响目的:探讨中药香菇多糖(LNT)联合丙型肝炎病毒(HCV)非结构蛋白NS3核酸疫苗诱导NS3抗原特异性细胞免疫作用。方法:将不同剂量的香菇多糖与HCV/NS3质粒(100μg/只),采用初次加强免疫法联合免疫BALB/c小鼠。第二次免疫10天后取小鼠脾脏,制备脾细胞悬液,与NS3多肽抗原共同孵育作为效应细胞。另培养NS3抗原致敏的P815细胞作为靶细胞。采用MTT法分别检测NS3抗原特异性T细胞增殖反应和细胞毒T细胞特异性杀伤反应(CTL)。结果:研究发现中剂量和低剂量香菇多糖与NS3质粒联用明显增强NS3抗原特异性T细胞增殖反应(P0.05)。高剂量及中剂量香菇多糖与HCV/NS3核酸疫苗联合应用,在效靶细胞比为50:1和25:1时,可诱导更强NS3抗原特异性CTL反应,与单独免疫NS3质粒相比有显著性差异(P0.05)。而低剂量的香菇多糖佐剂免疫组的CTL杀伤率与单独使用核酸疫苗组相比没有明显差异。总的趋势是随着效靶细胞比值(E/T)的增大,效应细胞对靶细胞的杀伤力也增大。结论:采用初次加强免疫法将中剂量香菇多糖(0.5mg/ml)与HCV/ NS3核酸疫苗联合免疫BALB/c小鼠,可激发明显增强的抗原特异性T细胞增殖和CTL反应,并结合试验第二部分的细胞因子分析结果,提示香菇多糖作为丙肝病毒NS3核酸疫苗的免疫佐剂,可加强抗原特异性免疫应答效应。
[Abstract]:Part one: preparation and identification of hepatitis C virus non structural protein 3 (NS3) DNA vaccine
Objective: to prepare large quantities of hepatitis C virus non structural protein 3 (NS3) DNA vaccine, and to conduct quantitative and qualitative analysis, so as to lay a foundation for further study of its immune activity.
Methods: pcDNA3.1-HCV/NS3 was transformed into HB101 of Escherichia coli. After cloning, single colony was amplified and pcDNA3.1-HCV/NS3 was extracted with a large number of rapid extraction kits of super pure plasmids. The extract was identified by nucleic acid quantitative detection and agarose gel electrophoresis.
Results: the experiment successfully transformed pcDNA3.1-HCV/NS into Escherichia coli, and there were more single colony growth after paving Petri dish for 16h. The extracted product was detected by nucleic acid, the concentration was in the range of 1.5 mu g/ Mu L-2 g/ Mu L, the purity was close to 1.8, and the DNA concentration needed by the immune mice was 1 u g/ Mu L, so the extracted DNA was in the concentration and purity. According to the requirements, the amplified products were digested and confirmed by agarose gel electrophoresis. The plasmid DNA contained the target gene NS3 fragment with a molecular weight of 1.8Kb.
Conclusion: this experiment successfully prepared HCV/ NS3 DNA vaccine for immune animals and laid the foundation for further study of its immune activity. The effect of second part of letinous edodes polysaccharide as an immune adjuvant on the humoral immune effect of non structural protein 3 (NS3) of hepatitis C virus (HCV)
Objective: To investigate the effect of letinous edodes polysaccharide (LNT) as an immune adjuvant on the humoral immunity of HCV/ NS3 nucleic acid vaccine.
Methods: HCV-NS3 nucleic acid vaccine was injected into BALB/c mice (100 mu g/). After 10 days, the immunization was strengthened. At the same time, three doses of letinous edodes polysaccharide were given to high (1mg/ml), middle (0.5mg/ml) and low (0.1mg/ml) doses. After the last immunization, the tail blood of mice was collected respectively, and the serum frozen storage was collected. The enzyme labeled plate was wrapped with HCV/NS3 antigen polypeptide. The serum specific IgG antibody level of mice was detected by indirect enzyme linked immunosorbent assay (ELISA), and the level of cytokine IL-4 and IFN- gamma in serum was detected by ELISA.
Results: different levels of antibodies were produced in the serum of mice immunized at 1 weeks after the two immunization. The specific IgG level produced by the medium dose LNT as the adjuvant at all time points was the most significant, and with the time increasing, the specific IgG level of the medium dose LNT as the adjuvant and the single immune nucleic acid vaccine group showed the specific level. The level of antibody in the adjuvant immunization group with high dose and low dose of LNT changed little with time. The specific antibody level produced by the medium dose letinous edodes polysaccharide as the adjuvant immunization group was significantly higher than that of the individual immune nucleic acid vaccine group at all time points (P0.05). The cytokine analysis showed that the different doses of letinous edodes polysaccharide were taken at different doses. As an adjuvant, the immunization group increased the secretion of IFN- gamma in the individual immune nucleic acid vaccine group, in which the level of IFN- gamma produced by the medium dose of letinous edodes polysaccharide combined with HCV/NS3 nucleic acid vaccine group was significantly higher than that of the individual using nucleic acid vaccine group (P0.01), but there was no significant difference between each group of IL-4 (P0.05).
Conclusion: the specific IgG antibody against NS3 antigen was produced in the mice by two intramuscular injection of HCV/NS3 nucleic acid vaccine, and the specific antibody level increased with the prolongation of time. The medium dose of letinous edodes polysaccharide could enhance the specific humoral immune response induced by the HCV/NS3 nucleic acid vaccine. The letinous edodes polysaccharide could stimulate Th1 Cytokine IFN- gamma secretion was not significantly affected by the secretion of Th2 cytokine IL-4.
The third part is the effect of letinous edodes polysaccharide as adjuvant on cellular immunity of hepatitis C virus non structural protein 3 (NS3).
Objective: To explore the specific cellular immunity of letinous edodes antigen (LNT) combined with hepatitis C virus (HCV) non structural protein NS3 nucleic acid vaccine to induce NS3 antigen.
Methods: the different doses of letinous edodes polysaccharide and HCV/NS3 plasmid (100 mu g/) were used to immunization of BALB/c mice with the first strengthening immunization. The spleen was obtained from the spleen of the mice after second days of immunization. The spleen cell suspension was prepared, and the NS3 polypeptide antigen was incubated as the effector cell. The P815 cells of NS3 antigenic antigen were incubated as the target cells. The MTT method was used respectively. NS3 antigen specific T cell proliferation and cytotoxic T cell specific killing reaction (CTL) were detected.
Results: the study found that the combination of the medium dose and low dose letinous edodes polysaccharide and the NS3 plasmid significantly enhanced the NS3 antigen specific T cell proliferation response (P0.05). The combination of high dose and medium dose of letinous edodes polysaccharide and HCV/NS3 nucleic acid vaccine could induce a stronger NS3 antigen specific CTL reaction when the target cells were 50:1 and 25:1, and the immune NS3 plasmid was immunized alone. There was a significant difference (P0.05). The CTL killing rate of the low dose letinous edodes polysaccharide adjuvant immunization group was not significantly different from that of the single nucleic acid vaccine group. The general trend was that the effect cells increased with the increase of target cell ratio (E/T).
Conclusion: the combined immunization of medium dose letinous edodes polysaccharide (0.5mg/ml) and HCV/ NS3 nucleic acid vaccine in BALB/c mice can stimulate obviously enhanced antigen specific T cell proliferation and CTL reaction, and combined with the results of cytokine analysis in the second part of the test, it suggests that the letinous edodes polysaccharide is used as the immuno adjuvant of the hepatitis C virus NS3 nucleic acid vaccine. It can strengthen the antigenic specific immune response effect.

【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392

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