狂犬病浓缩纯化灭活疫苗的实验研究

发布时间：2018-11-08 07:42

【摘要】： 据我国CDC统计,2008年我国人狂犬病发病数已突破3000例,正在形成第三次流行高峰。流行病学调查表明,95%以上的人狂犬病病例是因为与患病或带毒的犬和猫直接或间接接触引起的。因此,有效遏制人狂犬病,必须控制动物狂犬病,研制安全有效的动物狂犬疫苗,对狂犬病流行地区的犬、猫等动物施行强制免疫接种。而疫苗的质量,即疫苗的免疫原性和安全性是预防狂犬病的关键。为此,我们开展了狂犬病浓缩纯化灭活疫苗的实验研究。本研究首先对犬五联弱毒疫苗中的狂犬病毒ERA株第15代Vero细胞培养毒Rb/E3-15进行了鉴定。鉴定后的Rb/E3-15株,用Vero细胞大量增殖,经醋酸锌沉淀浓缩, Sepharose 4FF层析纯化;通过BPL(β-丙内酯)和甲醛的灭活比较实验,选择了低浓度BPL灭活抗原,配以合适比例的脂质体和Al(OH)3佐剂,制备了狂犬病浓缩纯化灭活佐剂疫苗。为了找到一种对狂犬疫苗抗体检测的理想方法,我们分别用本实验室建立的荧光抗体病毒中和试验(FAVN)、快速荧光灶抑制试验和犬抗体检测试剂盒(ELISA法)对实验室收集的狂犬疫苗免疫后的犬血清进行测定比较,结果显示FAVN与ELISA法、RFFIT法具有相似的敏感性与特异性。用制备的浓缩纯化灭活佐剂疫苗免疫小鼠和犬,FAVN法测定免疫后抗体,结果显示该浓缩纯化灭活疫苗可诱导小鼠和犬产生高水平中和抗体,小鼠攻毒实验使小鼠获得完全的免疫保护;在犬体内诱导的中和抗体水平明显优于浓缩纯化前疫苗,犬的外周血淋巴细胞转试验检测表明,脂质体佐剂疫苗的细胞免疫效果要优于Al(OH)3佐剂疫苗。该浓缩纯化灭活疫苗接种受试动物后无不良反应,疫苗安全性好。
[Abstract]:According to CDC statistics, the number of rabies cases in China has exceeded 3000 cases in 2008, which is forming the third epidemic peak. Epidemiological survey showed that more than 95% of human rabies cases were caused by direct or indirect contact with sick or infected dogs and cats. Therefore, it is necessary to control animal rabies, develop a safe and effective animal rabies vaccine, and enforce vaccination against dogs and cats in rabies endemic areas. The quality of the vaccine, that is, the immunogenicity and safety of the vaccine, is the key to the prevention of rabies. Therefore, we carried out an experimental study on rabies concentrated and purified inactivated vaccine. In this study, the virus Rb/E3-15 of rabies virus ERA strain cultured in the 15th passage of Vero cells was first identified. The identified Rb/E3-15 strain was proliferated by Vero cells and concentrated by zinc acetate precipitation and purified by Sepharose 4FF chromatography. By comparing the inactivation of BPL (尾 -propionolactone) and formaldehyde, the inactivated antigen of low concentration BPL was selected, and the adjuvant of liposome and Al (OH) 3 was used to prepare the concentrated and purified adjuvant vaccine of rabies. In order to find an ideal method for detection of rabies vaccine antibody, we used fluorescent antibody neutralization test (FAVN),) established in our laboratory. The rapid fluorescent foci inhibition test and canine antibody test kit (ELISA) were used to detect and compare the serum of rabies vaccine immunized in laboratory. The results showed that FAVN had similar sensitivity and specificity to ELISA method and RFFIT method. Mice and dogs were immunized with concentrated and purified inactivated adjuvant vaccine. The antibody was determined by FAVN method. The results showed that the concentrated and purified inactivated vaccine could induce high level neutralizing antibody in mice and dogs. The mice were immunized completely by the virus attack test. The level of neutralizing antibody induced in dogs was significantly better than that of pre-concentrated and purified vaccines. The peripheral blood lymphocyte transposition test of dogs showed that the cellular immune effect of liposome adjuvant vaccine was better than that of Al (OH) _ 3 adjuvant vaccine. There was no adverse reaction after inoculation of the concentrated and purified inactivated vaccine, and the vaccine was safe.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392

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