纳米乳剂及中草药提取物流感病毒疫苗佐剂研究

发布时间：2018-06-27 01:43

本文选题：流感 + 疫苗　；参考：《中国协和医科大学》2010年硕士论文

【摘要】： 流行性感冒简称流感,是由流感病毒(influenza virus)引起的急性呼吸系统疾病,具有传播迅速,可反复感染的特点。预防流感大流行爆发的最有效手段是接种流感疫苗。传统流感病毒疫苗目前还存在很多需要改进的地方,使用佐剂是改进方法之一。在流感疫苗中使用佐剂能够增强其免疫原性,并且能够减少单剂流感疫苗的用量,对于开发能够预防潜在的大流行流感的疫苗以及在大流行流感爆发时候迅速生产足够剂量的疫苗等均具有现实意义。第一部分纳米乳剂作为H3N2灭活流感病毒疫苗佐剂的研究乳剂作为流感疫苗佐剂已经具有一定的应用,纳米乳剂是粒径小于100nm的乳剂。本实验首先寻找具有安全性的,能够形成纳米乳剂的表面活性剂、助表面活性剂、油相和水相,之后通过三元相图法对各相的成分及比例进行研究,确定EL和Span-80质量比为1：1的混合物作为表面活性剂,配方为EL-Span-80-1,2丙二醇-注射用大豆油。将得到的优化纳米乳剂包裹H3N2灭活流感病毒疫苗接种昆明种小鼠。纳米乳剂组小鼠的血凝抑制效价在42d时达到1920,中和抗体滴度达到447,均超过铝佐剂组及疫苗组4倍以上,并且该组小鼠体重增长正常,证明纳米乳剂作为流感疫苗佐剂同时具有安全性及有效性,是较好的流感疫苗佐剂候选。第二部分中草药提取物作为H3N2灭活流感病毒疫苗佐剂的研究我国具有中草药提取和应用的传统优势。一些中草药在应对禽流感以及甲型H1N1流感的过程中扮演了重要角色,某些中草药提取物能够增强免疫反应也是研究人员的共识。本实验采用40种中草药提取物作为流感疫苗的佐剂候选,在动物体内进行试验,从中筛选有效的佐剂,并研究佐剂与疫苗的剂量的最佳配比。首先将40种中草药提取物分别与H3N2灭活流感病毒疫苗混合,接种小鼠,初免后每隔7d尾部动脉采血,21d时加免一剂,采血后分离血清,测定各中草药提取物佐剂组及对照组对H3N2病毒的血凝抑制效价并进行对比,初步筛选出锁阳、天冬及鳖甲三种能够明显提高血凝抑制抗体的佐剂候选。之后对初步筛选中效果最好的锁阳提取物设计流感疫苗及锁阳剂量的二次旋转回归正交试验,证明2.5μg血凝素含量和0.8mg锁阳含量是最优化比例,数据拟合为1778,与实验数据1824基本相符,证明该方法合理,数据可靠。锁阳血凝抑制效价可达1824,中和抗体滴度达562,均高于铝佐剂组及疫苗组4倍以上。并且各组小鼠体重增长超过空白对照组,证明锁阳作为佐剂具有安全性及有效性。
[Abstract]:Influenza is an acute respiratory disease caused by influenza virus (influenza virus). It is characterized by rapid transmission and repeated infection. The most effective way to prevent an outbreak of influenza pandemic is to get vaccinated against it. Traditional influenza virus vaccines still need to be improved, and adjuvant is one of the methods. The use of adjuvants in flu vaccines enhances their immunogenicity and reduces the amount of a single dose of flu vaccine. It is of practical significance to develop a vaccine that can prevent potential pandemic influenza and to produce enough vaccine rapidly in the event of pandemic influenza outbreak. The first part is the study on the use of nano-emulsion as adjuvant of inactivated influenza virus vaccine of H _ 3N _ 2. The emulsion has been used as adjuvant of influenza vaccine to some extent. Nanometer emulsion is an emulsion with particle size smaller than 100nm. In this experiment, we first looked for the surfactant, cosurfactant, oil phase and water phase with safety, which can form nanometer emulsion, and then studied the composition and proportion of each phase by ternary phase diagram method. The mixture of El and Span-80 was determined as surfactant with the mass ratio of 1:1. The formula was EL-Span-80-1 + 2-propanediol and soybean oil for injection. The optimized nano-emulsion encapsulated H 3 N 2 inactivated influenza virus vaccine was inoculated into Kunming mice. The hemagglutination inhibition titer of nano-emulsion group reached 1920 and neutralizing antibody titer reached 447 at 42 days, which was more than 4 times higher than that of aluminum adjuvant group and vaccine group, and the weight of the group was normal. It is proved that nano-emulsion is a safe and effective adjuvant for influenza vaccine and is a good candidate for influenza vaccine adjuvant. The second part is the study of Chinese herbal medicine extract as adjuvant of inactivated influenza virus vaccine of H _ 3N _ 2. China has the traditional advantages of Chinese herbal medicine extraction and application. Some Chinese herbal medicines have played an important role in dealing with avian flu and influenza A (H1N1), and some Chinese herbal extracts have also been agreed by researchers to enhance the immune response. In this experiment, 40 kinds of Chinese herbal extracts were used as adjuvant candidates for influenza vaccine, and the effective adjuvant was screened in vivo, and the optimal proportion of adjuvant and vaccine was studied. First, 40 kinds of Chinese herbal medicine extracts were mixed with H3N2 inactivated influenza virus vaccine and inoculated into mice. After the first immunization, a dose was added every 7 days to collect blood from caudal artery for 21 days, and the serum was isolated after blood collection. The hemagglutination inhibition titers of H3N2 virus in the adjuvant group and the control group were determined and compared. Three adjuvant candidates which could significantly improve the hemagglutination inhibition antibody were selected. After that, we designed the influenza vaccine and the second rotation regression orthogonal test of the dose of lock positive for the best result of the preliminary screening. It was proved that 2.5 渭 g hemagglutinin and 0.8mg concentration were the optimal proportion. The fitting of the data is 17788.It is basically consistent with the experimental data 1824, which proves that the method is reasonable and the data is reliable. The inhibitory titers of hemagglutination and neutralizing antibody titers were 1824 and 562 respectively, which were 4 times higher than those in aluminum adjuvant group and vaccine group. The weight gain of each group was higher than that of the blank control group, which proved the safety and efficacy of the adjuvant.
【学位授予单位】：中国协和医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392

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