三种肺炎链球菌表面蛋白联合诱导的特异性IgG抗体介导的免疫保护

发布时间：2018-05-15 12:49

本文选题：肺炎链球菌 + Ply突变体　；参考：《重庆医科大学》2010年硕士论文

【摘要】： 【背景】在所有可通过疫苗预防的疾病中,肺炎链球菌疾病是导致全球5岁以下儿童死亡的首要病因。据世界卫生组织估计全球每年约有160万人死于各种肺炎链球菌疾病,其中5岁以下儿童占100万,90%以上的死亡病例在发展中国家。肺炎链球菌抗生素耐药形势也日趋严峻。因此,世界卫生组织推荐将肺炎链球菌疫苗纳入各国家/地区的免疫规划项目中。【方法】本研究利用基因重组技术、重组蛋白原核表达技术构建,制备了肺炎链球菌Lpl蛋白以及第146位氨基酸缺失的溶血素(Ply)。通过体外溶血试验鉴定Ply蛋白及其突变体ΔA146 Ply蛋白的溶血活性;通过中和试验和肺组织切片分析明确ΔA146 Ply特异性抗体的中和毒素能力和免疫原性。采用Western技术分析Ply蛋白和Lpl蛋白在不同菌株中的表达情况以获得保守性信息。疫苗候选ClpP、Lpl、ΔA146 Ply蛋白单一或联合免疫小鼠,利用鼻腔攻毒方式评价其减少细菌肺内定植的能力;利用腹腔攻毒实验评价其对肺炎链球菌引起的严重感染败血症的抵抗能力。利用抗体吸附试验将产生的特异性抗体消除,并模拟被动保护实验分析特异抗体在保护中的作用。【结果】本研究成功制备ΔA146 Ply重组蛋白,其溶血活性全部丧失。ΔA146 Ply蛋白诱导产生的多克隆抗体能有效抑制野生Ply的溶血活性。经Western分析,Lpl和Ply在本研究所采用的12株不同血清型肺炎链球菌中均有表达。候选分子单一保护效果试验显示: Lpl、ΔA146 Ply、ClpP蛋白免疫后,均能使肺炎链球菌血清型19F和14的肺内定植减少10倍以上(P0.05);使用强毒力的肺炎链球D39(血清型2型)腹腔攻毒,ΔA146 Ply和ClpP组小鼠的中位生存期延长。在使用D39菌株鼻腔攻毒时,ClpP和Lpl蛋白几乎没有保护效果;而ΔA146 Ply蛋白单一使用的保护效率为50%,与联合Lpl和(或)ClpP保护效力相当。后续试验中,我们重点评价了三种蛋白联合使用(IC247)的有效率,使用致死剂量的14、19F、23F鼻腔攻毒,IC247有效率均为100%;对于致死剂量的血清型6B鼻腔攻毒,有效率为80%;而对于由4种不同血清型所致的混合感染,IC247的有效率为70%。IC247所提供的保护效果与目前市场上使用的23价多糖疫苗和7价蛋白结合疫苗效果相当。Lpl、ClpP、ΔA146 Ply蛋白特异性IgG抗体缺失的IC247抗血清在被动保护实验中不能提供保护作用。【结论】三种表面蛋白具有一定的协同保护效果,IC247候选疫苗组合能有效抵抗多种血清型肺炎链球菌的感染。IC247候选疫苗所提供的保护与其特异性抗体密切相关。
[Abstract]:Background among all vaccine-preventable diseases, Streptococcus pneumoniae is the leading cause of death in children under 5 years of age worldwide. The World Health Organization estimates that every year about 1.6 million people die from various pneumococcal diseases, of which 1 million or 90 percent of deaths in children under the age of five are in developing countries. Antibiotic resistance of Streptococcus pneumoniae is becoming more and more serious. Therefore, the World Health Organization recommends that streptococcus pneumoniae vaccine be included in immunization programs in various countries. [methods] in this study, Lpl protein of Streptococcus pneumoniae and hemolysin of 146th amino acid deletion were prepared by using gene recombination technique and prokaryotic expression technique of recombinant protein. The hemolytic activity of Ply protein and its mutant 螖 A146 Ply protein was determined by hemolysis test in vitro, and the neutralizing toxin ability and immunogenicity of 螖 A146 Ply specific antibody were confirmed by neutralization test and lung tissue section analysis. Western technique was used to analyze the expression of Ply protein and Lpl protein in different strains to obtain conservative information. Single or combined immunized mice with vaccine candidate ClpPpland 螖 A146 Ply protein were used to evaluate their ability to reduce bacterial colonization in lung by nasal attack, and to evaluate their resistance to severe septicemia caused by Streptococcus pneumoniae by abdominal attack test. The specific antibody was eliminated by antibody adsorption test, and the role of specific antibody in protection was analyzed by passive protection experiment. [results] in this study, 螖 A146 Ply recombinant protein was successfully prepared and its hemolytic activity was completely lost. The polyclonal antibody induced by 螖 A146 Ply protein could effectively inhibit the hemolytic activity of wild Ply. Western analysis showed that LPL and Ply were expressed in 12 strains of Streptococcus pneumoniae. The single protective effect test of candidate molecule showed that: Lpl, 螖 A146 PlynClpP protein was immunized, The intrapulmonary colonization of streptococcus pneumoniae serotype 19F and 14 was reduced by more than 10 times (P0.05A), and the median survival time of mice in 螖 A146 Ply and ClpP groups was prolonged by using virulent streptococcus pneumoniae D39 (serotype 2). When D39 strain was used in nasal cavity, ClpP and Lpl proteins had little protective effect, while 螖 A146 Ply protein alone had a protective efficiency of 50%, which was similar to that of combined Lpl and (or) ClpP. In subsequent tests, we evaluated the effective rate of combined use of three proteins (IC247). The effective rate of nasal attack of IC247 was 100 at the lethal dose of 14 ~ (19) F ~ (23) F in nasal cavity, and 100% for a lethal dose of serotype 6B in nasal cavity. The effective rate is 80%, and the effective rate for IC247, which is caused by four different serotypes, is that 70%.IC247 provides the same protective effect as the 23-valent polysaccharide vaccine and the 7-valent protein-binding vaccine currently used on the market. LplnClpP, 螖 A146 Ply protein The IC247 antiserum without specific IgG antibody does not provide protective effect in passive protection test. [conclusion] the three surface proteins have a synergistic protective effect. The combination of IC247 candidate vaccine can effectively resist the infection of various serotype Streptococcus pneumoniae. The protection provided by IC247 candidate vaccine is closely related to its specific antibody.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392

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