霍乱弧菌O139的LPS-CTB结合疫苗的研究

发布时间：2018-11-16 13:52

【摘要】： 霍乱是一种烈性肠道传染病,是由革兰氏阴性霍乱弧菌引起的严重水样腹泻疾病。人类是霍乱弧菌的天然宿主,通过食入被霍乱弧菌污染的水或食物而感染。其感染的特点是严重腹泻和呕吐,由于水和电解质的流失导致在数小时内造成血容量减少性休克,代谢性酸中毒和钾的缺乏。并发症包括肾衰竭、动脉闭塞、肺水肿、孕妇流产和儿童癫痫。在感染人群中的病死率可达20%以上,而全球每年约有12万人死于霍乱。霍乱的流行特点是爆发式,历史上曾经爆发过7次世界大流行,开始在数个不同地点同时发生,然后迅速传播流行可累及许多国家并持续多年,尤其在非洲、亚洲和拉丁美洲等一些发展中国家较为严重。而控制它的有效途径之一就是免疫预防接种,因此寻求研制一种安全有效适用于各种人群的霍乱疫苗极其重要。已证实霍乱弧菌细胞壁上的脂多糖(LPS)既是毒力因子又是重要的保护性抗原,能诱导机体产生杀弧菌抗体。但是LPS分子量较小,免疫原性弱,且再次免疫后不能产生免疫加强效应。并且多糖类抗原不能诱导婴幼儿体内产生具保护水平的抗体,但此年龄组又是细菌性传染病发病的高危人群。霍乱毒素B亚单位(CTB)是霍乱毒素无毒的部分,具有良好的免疫原性,能产生重要的抗毒抗体。并且已经证实CTB是一种很好的粘膜佐剂,能够诱导机体产生粘膜免疫反应。本研究将CTB作为载体蛋白与LPS共价结合制备成结合疫苗,给多糖成分赋予“载体效应”,既能增强LPS的免疫原性,使其由T细胞非依赖抗原成为T细胞依赖抗原,又使诱导的免疫反应具有免疫记忆和加强效应,产生大量的杀菌抗体;而CTB本身又能产生高效价的抗毒抗体,这正是抗霍乱疫苗应该包括的两个方面。目的是制备一种安全有效的能针对各种人群的口服霍乱疫苗。本研究首先采用热酚水法从霍乱弧菌O139中提取纯化LPS,最终获得纯度良好的LPS干粉。然后用1-氰-4-二甲基氨基吡啶四氟硼酸(CDAP)将提取的LPS活化后与载体蛋白CTB共价偶联,产物经过Superdex 200凝胶过滤层析柱进行纯化,最终制备成高纯度的LPS-CTB共价结合物。用制备的LPS-CTB结合物免疫小鼠,取小鼠血清和粪便进行ELISA检测。采用给以盐水的小鼠做阴性对照,同时进行单独给LPS、单独给CTB以及给rBS-WC疫苗的对照实验。实验结果证明,本研究制备的LPS-CTB结合物不仅能有效诱导小鼠机体产生血清抗LPS和抗CTB的IgG和IgA抗体,其中,抗LPS的IgG和IgA的抗体滴度分别达到1:970和1:1690,抗CTB的IgG和IgA抗体滴度更是高达1:163840。并且该结合物能很好的诱导小鼠的肠粘膜免疫系统产生对霍乱免疫极为重要的分泌型IgA抗体。另外在多次免疫后都能产生免疫加强效应。说明该结合物具有很好的免疫原性。本研究初步制备的这种霍乱结合疫苗为霍乱及其他细菌性疾病的免疫预防提供了良好的基础和思路。
[Abstract]:Cholera is a severe intestinal infectious disease caused by Gram-negative Vibrio cholerae. Humans are natural hosts of Vibrio cholerae, infected by ingestion of water or food contaminated by Vibrio cholerae. Its infection is characterized by severe diarrhea and vomiting, resulting in reduced blood volume shock, metabolic acidosis and potassium deficiency within hours due to the loss of water and electrolyte. Complications include renal failure, artery occlusion, pulmonary edema, miscarriage and childhood epilepsy. The fatality rate among infected populations can be more than 20%, and cholera kills about 120000 people worldwide every year. Cholera is characterized by explosive outbreaks, which have occurred seven times in the history of the world, began at the same time in several different locations, and then spread rapidly in many countries and for many years, especially in Africa, Some developing countries, such as Asia and Latin America, are more serious. One of the effective ways to control it is immunization, so it is very important to develop a safe and effective cholera vaccine for all kinds of people. It has been proved that lipopolysaccharide (LPS) on the cell wall of Vibrio cholerae is not only a virulence factor but also an important protective antigen, which can induce the body to produce vibrio antibody. However, the molecular weight of LPS is small and the immunogenicity is weak. And the polycarbohydrate antigen can not induce the infant to produce the protective level antibody, but this age group is the high risk population of bacterial infectious disease. Cholera toxin B subunit (CTB) is a nontoxic part of cholera toxin. It has good immunogenicity and can produce important anti-virus antibodies. And it has been proved that CTB is a good mucosal adjuvant, which can induce mucosal immune response. In this study, CTB was covalently combined with LPS as a carrier protein to prepare a conjugated vaccine. The conjugated vaccine was endowed with "carrier effect" to the polysaccharide component, which could enhance the immunogenicity of LPS and make it become T-cell dependent antigen from T-cell independent antigen. The induced immune response has immune memory and enhancement effect and produces a large number of bactericidal antibodies. CTB itself can produce high-titer anti-virus antibodies, which are two aspects of anti-cholera vaccine. The aim is to prepare a safe and effective oral cholera vaccine for various populations. In this study, LPS, was extracted and purified from Vibrio cholerae O139 by thermophenol water method. Finally, LPS dry powder with good purity was obtained. The extracted LPS was covalently coupled with the carrier protein CTB after activation with 1-cyano-4-dimethylpyridine tetrafluoroboric acid (CDAP). The product was purified by Superdex 200 gel filtration chromatography. Finally, high purity LPS-CTB covalent conjugate was prepared. Mice were immunized with the prepared LPS-CTB conjugate. Serum and feces were collected for ELISA detection. Mice treated with saline were used as negative control, and CTB and rBS-WC vaccine were given to LPS, alone. The results showed that the LPS-CTB conjugate could not only induce the production of IgG and IgA antibodies against LPS and CTB, but also the titers of IgG and IgA against LPS were 1: 970 and 1: 1690, respectively, in which the titers of IgG and IgA against LPS were 1: 970 and 1: 1690, respectively. The titers of IgG and IgA antibodies against CTB were as high as 1: 163840. The conjugate can well induce the intestinal mucosal immune system of mice to produce secretory IgA antibodies which are very important for cholera immunity. In addition, the immune enhancement effect can be produced after multiple immunization. It shows that the conjugate has good immunogenicity. The cholera conjugate vaccine which was prepared in this study provides a good basis and thought for the immune prevention of cholera and other bacterial diseases.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392

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