枸杞多糖的佐剂效应及流感病毒多表位DNA疫苗的初步研究
发布时间：2018-09-12 09:08
【摘要】：本论文共有三部分组成：(1)流感病毒、流感病毒疫苗及佐剂的简介；(2)枸杞多糖(Lycium barbarum polysaccharide, LBP)对流感灭活疫苗免疫增强作用的研究；(3)流感病毒多表位DNA疫苗的构建及其免疫效力的初步研究。 一、流感病毒、流感病毒疫苗及佐剂的简介。以综述的形式描述了A型流感病毒的基本特征、流感病毒疫苗的研究进展及疫苗佐剂的研究进展等。 二、枸杞多糖(Lycium barbarum polysaccharide, LBP)对流感全病毒灭活疫苗的免疫增强作用的研究。取LBP的不同剂量,分别与0.015μg流感全病毒(A/Vietnam/1194/2004(H5N1))灭活疫苗共同免疫小鼠,阳性对照组为氢氧化铝(100μg)与灭活疫苗共同免疫组(Alum+Vaccine);阴性对照组为灭活疫苗单独免疫组；空白对照组为PBS免疫组。一次免疫后三周收集血清,用ELISA方法测定血清中特异性的IgG抗体滴度及IgG 1和IgG 2a两种分型抗体滴度,并使用HI方法检测中和抗体滴度；一次免疫后四周用致死量(10×LD50)流感病毒(A/Chicken/Henan/12/2004 (H5N1))攻击小鼠,通过观察小鼠的体重丢失率、肺部病毒量、存活率来反映佐剂的免疫增强效果和疫苗的保护作用。结果显示,LBP 800μg剂量组能显著增加血清抗体水平,并提高小鼠抗致死量流感病毒攻击的能力,其免疫增强效果与氢氧化铝相当,且与铝佐剂合用有佐剂协同作用。 三、流感病毒多表位DNA疫苗的构建及其免疫效力的初步研究。多表位DNA疫苗是建立在常规DNA疫苗基础上的一种新型疫苗。它是用表位作免疫原,这样就比较容易在一个表达载体上克隆病原体的多个抗原基因中具有免疫活性的部分。本研究鉴于当前多种亚型流感病毒共存的局面,应用生物信息学理论筛选出H1、H3、H5、H9亚型流感病毒的共同保守的多个优势抗原表位,设计一种针对多个亚型流感病毒的多表位核酸疫苗。主要筛选流感病毒主要抗原(H5HA、H5NA、NP、M、PB)优势抗原表位基因,以生物信息学软件优化其排列结构合成流感多表位基因(MP、ME),以pCAGGSP7为载体,构建了2个重组质粒：1、pCAGGSP7/MP; 2、pCAGGSP7/ME。分别用这2个重组质粒肌注免疫6-8周龄SPF BALB/c小鼠,阴性对照组不免疫。免疫三次,间隔为2周,每次每只小鼠的剂量为100ELISA法检测小鼠血清中的针对四种亚型的流感抗体。第三次免疫后两周以致死量的四种亚型的病毒攻击小鼠,攻毒前进行T淋巴细胞亚类数量的检测。攻毒后第三天取肺检测小鼠肺部病毒含量,并定期观测小鼠的死亡情况。结果发现,免疫小鼠获得了针对H1、H3、H5、H9四种亚型流感病毒的体液和细胞免疫反应；攻毒试验表明,阴性对照组小鼠(每种亚型各10只)分别在攻毒后7-14 d内全部死亡,实验组小鼠都获得了部分保护。上述结果说明,我们构建的多表位DNA疫苗有良好的免疫原性,为最终获得能同时预防多种亚型流感病毒的多价疫苗奠定了基础。
[Abstract]:This thesis consists of three parts: (1) introduction of influenza virus, influenza virus vaccine and adjuvant, (2) study on the immune enhancement effect of Lycium barbarum polysaccharide (Lycium barbarum polysaccharide, LBP) on inactivated influenza vaccine; (3) Construction and immunological efficacy of influenza virus multiepitope DNA vaccine. Introduction of influenza virus vaccine and adjuvant. The basic characteristics of influenza A virus, the research progress of influenza virus vaccine and the research progress of vaccine adjuvant are described in this paper. Second, the immune enhancement effect of Lycium barbarum polysaccharide (Lycium barbarum polysaccharide, LBP) on inactivated influenza vaccine was studied. The mice were immunized with 0.015 渭 g A/Vietnam/1194/2004 (H5N1) inactivated vaccine at different doses of LBP. The positive control group was aluminum hydroxide (100 渭 g) and inactivated vaccine co-immunized group (Alum Vaccine); negative control group was inactivated vaccine alone. The blank control group was immunized with PBS. Serum samples were collected three weeks after the first immunization. The titers of specific IgG antibodies and IgG 1 and IgG 2a antibodies were determined by ELISA method. The neutralization antibody titers were detected by HI method. Four weeks after one immunization, mice were attacked with a lethal dose (10 脳 LD50) of influenza virus (H5N1). The immune enhancement effect of adjuvant and the protective effect of vaccine were reflected by observing the weight loss rate, the amount of lung virus and the survival rate of mice. The results showed that LBP 800 渭 g group could significantly increase the level of serum antibody and enhance the ability of mice to resist the lethal influenza virus attack. The immune enhancement effect of LBP 800 渭 g group was similar to that of aluminum hydroxide, and the adjuvant combined with aluminum adjuvant had synergistic effect. Third, the construction and immune efficacy of influenza virus multiepitope DNA vaccine. Polyepitope DNA vaccine is a new vaccine based on conventional DNA vaccine. It uses epitopes as immunogen, which makes it easier to clone the immunoreactive parts of multiple antigenic genes of pathogens in a single expression vector. In view of the coexistence of many subtypes of influenza viruses, we used bioinformatics theory to screen the common and conserved multiple dominant epitopes of H1H3H3, H5, H9 subtype influenza viruses. A multiepitope nucleic acid vaccine against multiple influenza viruses was designed. The dominant epitope genes of influenza virus major antigen (H _ 5HAH _ 5NAN) were screened, and its arrangement was optimized by bioinformatics software to synthesize influenza polyepitope gene (MP,ME). Using pCAGGSP7 as vector, two recombinant plasmids: 1 pCAGGSP7 / MPP and 2pCAGGSP7 / MEM were constructed. The two recombinant plasmids were injected intramuscularly to SPF BALB/c mice aged 6 to 8 weeks, but the negative control group was not immunized. The mice were immunized three times at intervals of 2 weeks. Each mouse was given 100ELISA assay to detect the influenza antibodies against the four subtypes in the serum of the mice. Two weeks after the third immunization, the mice were attacked with four lethal subtypes of virus, and the number of T lymphocyte subtypes was detected before the attack. The lung was taken from the third day after the attack to detect the lung virus content and the death rate of the mice was observed regularly. The results showed that the humoral and cellular immune responses against the four subtypes of influenza virus H1H3, H5 and H9 were obtained in the immunized mice, and the mice in the negative control group (10 mice in each subtype) died within 7-14 days after the attack, the results showed that all the mice in the negative control group died within 7 to 14 days after the attack. The mice in the experimental group were partially protected. These results suggest that the constructed multiepitope DNA vaccine has good immunogenicity, which lays the foundation for the final production of multivalent vaccine which can prevent multiple subtype influenza viruses simultaneously.
【学位授予单位】：湖南师范大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392
本文编号：2238548
[Abstract]:This thesis consists of three parts: (1) introduction of influenza virus, influenza virus vaccine and adjuvant, (2) study on the immune enhancement effect of Lycium barbarum polysaccharide (Lycium barbarum polysaccharide, LBP) on inactivated influenza vaccine; (3) Construction and immunological efficacy of influenza virus multiepitope DNA vaccine. Introduction of influenza virus vaccine and adjuvant. The basic characteristics of influenza A virus, the research progress of influenza virus vaccine and the research progress of vaccine adjuvant are described in this paper. Second, the immune enhancement effect of Lycium barbarum polysaccharide (Lycium barbarum polysaccharide, LBP) on inactivated influenza vaccine was studied. The mice were immunized with 0.015 渭 g A/Vietnam/1194/2004 (H5N1) inactivated vaccine at different doses of LBP. The positive control group was aluminum hydroxide (100 渭 g) and inactivated vaccine co-immunized group (Alum Vaccine); negative control group was inactivated vaccine alone. The blank control group was immunized with PBS. Serum samples were collected three weeks after the first immunization. The titers of specific IgG antibodies and IgG 1 and IgG 2a antibodies were determined by ELISA method. The neutralization antibody titers were detected by HI method. Four weeks after one immunization, mice were attacked with a lethal dose (10 脳 LD50) of influenza virus (H5N1). The immune enhancement effect of adjuvant and the protective effect of vaccine were reflected by observing the weight loss rate, the amount of lung virus and the survival rate of mice. The results showed that LBP 800 渭 g group could significantly increase the level of serum antibody and enhance the ability of mice to resist the lethal influenza virus attack. The immune enhancement effect of LBP 800 渭 g group was similar to that of aluminum hydroxide, and the adjuvant combined with aluminum adjuvant had synergistic effect. Third, the construction and immune efficacy of influenza virus multiepitope DNA vaccine. Polyepitope DNA vaccine is a new vaccine based on conventional DNA vaccine. It uses epitopes as immunogen, which makes it easier to clone the immunoreactive parts of multiple antigenic genes of pathogens in a single expression vector. In view of the coexistence of many subtypes of influenza viruses, we used bioinformatics theory to screen the common and conserved multiple dominant epitopes of H1H3H3, H5, H9 subtype influenza viruses. A multiepitope nucleic acid vaccine against multiple influenza viruses was designed. The dominant epitope genes of influenza virus major antigen (H _ 5HAH _ 5NAN) were screened, and its arrangement was optimized by bioinformatics software to synthesize influenza polyepitope gene (MP,ME). Using pCAGGSP7 as vector, two recombinant plasmids: 1 pCAGGSP7 / MPP and 2pCAGGSP7 / MEM were constructed. The two recombinant plasmids were injected intramuscularly to SPF BALB/c mice aged 6 to 8 weeks, but the negative control group was not immunized. The mice were immunized three times at intervals of 2 weeks. Each mouse was given 100ELISA assay to detect the influenza antibodies against the four subtypes in the serum of the mice. Two weeks after the third immunization, the mice were attacked with four lethal subtypes of virus, and the number of T lymphocyte subtypes was detected before the attack. The lung was taken from the third day after the attack to detect the lung virus content and the death rate of the mice was observed regularly. The results showed that the humoral and cellular immune responses against the four subtypes of influenza virus H1H3, H5 and H9 were obtained in the immunized mice, and the mice in the negative control group (10 mice in each subtype) died within 7-14 days after the attack, the results showed that all the mice in the negative control group died within 7 to 14 days after the attack. The mice in the experimental group were partially protected. These results suggest that the constructed multiepitope DNA vaccine has good immunogenicity, which lays the foundation for the final production of multivalent vaccine which can prevent multiple subtype influenza viruses simultaneously.
【学位授予单位】：湖南师范大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392
【参考文献】
相关期刊论文 前10条
1 陈庆伟,陈志桃;枸杞多糖药理作用研究进展[J];海峡药学;2005年04期
2 穆冰,刘晓峰,熊进,勾艾莉;枸杞多糖的急性毒性及致突变性[J];环境与职业医学;2002年03期
3 刘彦平,李积东;枸杞多糖对小鼠NK细胞和白细胞活性的免疫调节作用[J];青海医学院学报;2001年01期
4 孙艳,官杰,王琪,陈海洋;枸杞多糖对荷瘤小鼠免疫功能的增强效应及抑瘤作用的研究[J];齐齐哈尔医学院学报;2001年05期
5 汪积慧,李鸿梅;枸杞多糖免疫调节作用的研究[J];齐齐哈尔医学院学报;2002年11期
6 何彦丽,应逸,罗荣敬,王斌,杜标炎,罗惠;枸杞多糖对荷瘤小鼠淋巴细胞亚群及树突状细胞表达的影响[J];广州中医药大学学报;2005年04期
7 周娅,佟书娟,王宁萍,赵建宁,黎雪如;枸杞多糖对小鼠巨噬细胞内酶活性及NO诱生的影响[J];山东中医杂志;2000年06期
8 沈诚诚,刘庆良;破伤风类毒素非磷脂脂质体疫苗的实验研究[J];上海免疫学杂志;2000年01期
9 王玲，，杜守英;枸杞多糖的免疫调研究进展[J];上海免疫学杂志;1995年02期
10 王玲，李俊，李欣，钱玉昆;枸杞多糖2对辐射损伤小鼠免疫功能恢复的影响[J];上海免疫学杂志;1995年04期
本文编号：2238548
本文链接：https://www.wllwen.com/yixuelunwen/shiyanyixue/2238548.html
最近更新
教材专著
