本文選題：肺炎鏈球菌 + Ply突變體�。� 參考：《重慶醫(yī)科大學(xué)》2010年碩士論文

【摘要】： 【背景】在所有可通過(guò)疫苗預(yù)防的疾病中,肺炎鏈球菌疾病是導(dǎo)致全球5歲以下兒童死亡的首要病因。據(jù)世界衛(wèi)生組織估計(jì)全球每年約有160萬(wàn)人死于各種肺炎鏈球菌疾病,其中5歲以下兒童占100萬(wàn),90%以上的死亡病例在發(fā)展中國(guó)家。肺炎鏈球菌抗生素耐藥形勢(shì)也日趨嚴(yán)峻。因此,世界衛(wèi)生組織推薦將肺炎鏈球菌疫苗納入各國(guó)家/地區(qū)的免疫規(guī)劃項(xiàng)目中。 【方法】本研究利用基因重組技術(shù)、重組蛋白原核表達(dá)技術(shù)構(gòu)建,制備了肺炎鏈球菌Lpl蛋白以及第146位氨基酸缺失的溶血素(Ply)。通過(guò)體外溶血試驗(yàn)鑒定Ply蛋白及其突變體ΔA146 Ply蛋白的溶血活性;通過(guò)中和試驗(yàn)和肺組織切片分析明確ΔA146 Ply特異性抗體的中和毒素能力和免疫原性。采用Western技術(shù)分析Ply蛋白和Lpl蛋白在不同菌株中的表達(dá)情況以獲得保守性信息。疫苗候選ClpP、Lpl、ΔA146 Ply蛋白單一或聯(lián)合免疫小鼠,利用鼻腔攻毒方式評(píng)價(jià)其減少細(xì)菌肺內(nèi)定植的能力;利用腹腔攻毒實(shí)驗(yàn)評(píng)價(jià)其對(duì)肺炎鏈球菌引起的嚴(yán)重感染敗血癥的抵抗能力。利用抗體吸附試驗(yàn)將產(chǎn)生的特異性抗體消除,并模擬被動(dòng)保護(hù)實(shí)驗(yàn)分析特異抗體在保護(hù)中的作用。 【結(jié)果】本研究成功制備ΔA146 Ply重組蛋白,其溶血活性全部喪失。ΔA146 Ply蛋白誘導(dǎo)產(chǎn)生的多克隆抗體能有效抑制野生Ply的溶血活性。經(jīng)Western分析,Lpl和Ply在本研究所采用的12株不同血清型肺炎鏈球菌中均有表達(dá)。候選分子單一保護(hù)效果試驗(yàn)顯示: Lpl、ΔA146 Ply、ClpP蛋白免疫后,均能使肺炎鏈球菌血清型19F和14的肺內(nèi)定植減少10倍以上(P0.05);使用強(qiáng)毒力的肺炎鏈球D39(血清型2型)腹腔攻毒,ΔA146 Ply和ClpP組小鼠的中位生存期延長(zhǎng)。在使用D39菌株鼻腔攻毒時(shí),ClpP和Lpl蛋白幾乎沒(méi)有保護(hù)效果;而ΔA146 Ply蛋白單一使用的保護(hù)效率為50%,與聯(lián)合Lpl和(或)ClpP保護(hù)效力相當(dāng)。后續(xù)試驗(yàn)中,我們重點(diǎn)評(píng)價(jià)了三種蛋白聯(lián)合使用(IC247)的有效率,使用致死劑量的14、19F、23F鼻腔攻毒,IC247有效率均為100%;對(duì)于致死劑量的血清型6B鼻腔攻毒,有效率為80%;而對(duì)于由4種不同血清型所致的混合感染,IC247的有效率為70%。IC247所提供的保護(hù)效果與目前市場(chǎng)上使用的23價(jià)多糖疫苗和7價(jià)蛋白結(jié)合疫苗效果相當(dāng)。Lpl、ClpP、ΔA146 Ply蛋白特異性IgG抗體缺失的IC247抗血清在被動(dòng)保護(hù)實(shí)驗(yàn)中不能提供保護(hù)作用。 【結(jié)論】三種表面蛋白具有一定的協(xié)同保護(hù)效果,IC247候選疫苗組合能有效抵抗多種血清型肺炎鏈球菌的感染。IC247候選疫苗所提供的保護(hù)與其特異性抗體密切相關(guān)。
[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.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類(lèi)號(hào)】：R392

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本文編號(hào)：1892527