枸杞多糖的佐劑效應(yīng)及流感病毒多表位DNA疫苗的初步研究
發(fā)布時間：2018-09-12 09:08
【摘要】：本論文共有三部分組成：(1)流感病毒、流感病毒疫苗及佐劑的簡介；(2)枸杞多糖(Lycium barbarum polysaccharide, LBP)對流感滅活疫苗免疫增強(qiáng)作用的研究；(3)流感病毒多表位DNA疫苗的構(gòu)建及其免疫效力的初步研究。 一、流感病毒、流感病毒疫苗及佐劑的簡介。以綜述的形式描述了A型流感病毒的基本特征、流感病毒疫苗的研究進(jìn)展及疫苗佐劑的研究進(jìn)展等。 二、枸杞多糖(Lycium barbarum polysaccharide, LBP)對流感全病毒滅活疫苗的免疫增強(qiáng)作用的研究。取LBP的不同劑量,分別與0.015μg流感全病毒(A/Vietnam/1194/2004(H5N1))滅活疫苗共同免疫小鼠,陽性對照組為氫氧化鋁(100μg)與滅活疫苗共同免疫組(Alum+Vaccine);陰性對照組為滅活疫苗單獨免疫組；空白對照組為PBS免疫組。一次免疫后三周收集血清,用ELISA方法測定血清中特異性的IgG抗體滴度及IgG 1和IgG 2a兩種分型抗體滴度,并使用HI方法檢測中和抗體滴度；一次免疫后四周用致死量(10×LD50)流感病毒(A/Chicken/Henan/12/2004 (H5N1))攻擊小鼠,通過觀察小鼠的體重丟失率、肺部病毒量、存活率來反映佐劑的免疫增強(qiáng)效果和疫苗的保護(hù)作用。結(jié)果顯示,LBP 800μg劑量組能顯著增加血清抗體水平,并提高小鼠抗致死量流感病毒攻擊的能力,其免疫增強(qiáng)效果與氫氧化鋁相當(dāng),且與鋁佐劑合用有佐劑協(xié)同作用。 三、流感病毒多表位DNA疫苗的構(gòu)建及其免疫效力的初步研究。多表位DNA疫苗是建立在常規(guī)DNA疫苗基礎(chǔ)上的一種新型疫苗。它是用表位作免疫原,這樣就比較容易在一個表達(dá)載體上克隆病原體的多個抗原基因中具有免疫活性的部分。本研究鑒于當(dāng)前多種亞型流感病毒共存的局面,應(yīng)用生物信息學(xué)理論篩選出H1、H3、H5、H9亞型流感病毒的共同保守的多個優(yōu)勢抗原表位,設(shè)計一種針對多個亞型流感病毒的多表位核酸疫苗。主要篩選流感病毒主要抗原(H5HA、H5NA、NP、M、PB)優(yōu)勢抗原表位基因,以生物信息學(xué)軟件優(yōu)化其排列結(jié)構(gòu)合成流感多表位基因(MP、ME),以pCAGGSP7為載體,構(gòu)建了2個重組質(zhì)粒：1、pCAGGSP7/MP; 2、pCAGGSP7/ME。分別用這2個重組質(zhì)粒肌注免疫6-8周齡SPF BALB/c小鼠,陰性對照組不免疫。免疫三次,間隔為2周,每次每只小鼠的劑量為100ELISA法檢測小鼠血清中的針對四種亞型的流感抗體。第三次免疫后兩周以致死量的四種亞型的病毒攻擊小鼠,攻毒前進(jìn)行T淋巴細(xì)胞亞類數(shù)量的檢測。攻毒后第三天取肺檢測小鼠肺部病毒含量,并定期觀測小鼠的死亡情況。結(jié)果發(fā)現(xiàn),免疫小鼠獲得了針對H1、H3、H5、H9四種亞型流感病毒的體液和細(xì)胞免疫反應(yīng)；攻毒試驗表明,陰性對照組小鼠(每種亞型各10只)分別在攻毒后7-14 d內(nèi)全部死亡,實驗組小鼠都獲得了部分保護(hù)。上述結(jié)果說明,我們構(gòu)建的多表位DNA疫苗有良好的免疫原性,為最終獲得能同時預(yù)防多種亞型流感病毒的多價疫苗奠定了基礎(chǔ)。
[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.
【學(xué)位授予單位】：湖南師范大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R392
本文編號：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.
【學(xué)位授予單位】：湖南師范大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R392
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