本文選題：NTHi + 外膜蛋白��； 參考：《北京生物制品研究所》2008年碩士論文

【摘要】： 不可分型的流感嗜血桿菌(NTHi)是一種常定居于鼻咽和后口咽上呼吸道部位的共生菌,在多達(dá)80%的健康兒童以及40%的健康成人的咽部都存在NTHi。NTHi是人類一些疾病的重要病原菌,它們首先寄居在鼻咽部,然后通過咽鼓管到達(dá)中耳,也感染(鼻)竇和/或喉,常導(dǎo)致呼吸道反復(fù)感染,最常引起中耳炎及竇炎。NTHi一直是嬰幼兒和兒童中耳炎的主要病原菌,其引起的中耳炎約占所有中耳炎的1/4～1/3。血清學(xué)研究及抗生素療效調(diào)查提示NTHi是慢性阻塞性肺部疾病(COPD)患者下呼吸道感染的重要原因。最近一些研究提示流感嗜血桿菌也是引起AIDS患者細(xì)菌性肺炎的主要病原菌。過去十年里,NTHi引起的新生兒膿毒癥發(fā)病有所上升,其可導(dǎo)致50%患者死亡,早產(chǎn)兒中死亡率達(dá)90%。因此,中耳炎不論從疾病發(fā)生還是從治療費(fèi)用而言都是一個(gè)國家較大的健康問題。伴隨這些菌耐藥性的增強(qiáng),治療面臨著更大的挑戰(zhàn)。因此,發(fā)展一種安全有效的疫苗預(yù)防NTHi的感染已顯得尤為重要。由于該菌沒有莢膜多糖,故現(xiàn)在尋求一種以NTHi外膜蛋白為基礎(chǔ)的疫苗。 該菌有5個(gè)主要的外膜蛋白,分別為P1(47,000Da)、P2(39,000Da)、P4(30,000Da)、P5(27,000Da)、P6(16,600Da)。這五種外膜蛋白均是殺菌抗體的靶目標(biāo),而P1、P2及P5均受到菌株異源性的限制,P4和P6的抗原性非常穩(wěn)定同時(shí)也高度保守。因此,無論其免疫原性還是菌株保守性,P6都是作為研究預(yù)防不可分型流感嗜血桿菌感染的疫苗成分的非常有潛力的候選者。 本實(shí)驗(yàn)根據(jù)P6的特性來提取,即在buffer B(0.1M Tris,0.5M NaCl,1%SDS,0.1%2-巰基乙醇,PH 8.0)中37℃孵育30min,P6是唯一不溶的蛋白,而在65℃孵育30min,P6即可被溶解下來。首先超聲破碎菌體制備膜制劑,膜制劑在bufferB中37℃孵育30min,高速離心后棄上清,膜制劑繼續(xù)在buffer B中37℃孵育30min,超速離心,如此反復(fù)多次后,即可將膜制劑上的雜蛋白洗去,僅剩下P6,最后在bufferA(0.01 M Tris-0.15 M NaCl,pH 7.4)中65℃孵育30min,P6即可被溶解下來。SDS-PAGE顯示僅一條蛋白帶,影像分析軟件AlphaDigiDoc~(TM)顯示其純度為100%,分子量為16.73kDa,IEF-PAGE電泳后,采用AlphaEaseFC軟件對等電聚焦圖譜進(jìn)行分析,結(jié)果顯示,目的蛋白的等電點(diǎn)為5.49,產(chǎn)率為90.43ug/g濕菌體。以P6不同劑量和針次免疫NIH小鼠,間接ELISA法檢測小鼠血清IgG抗體滴度,結(jié)果顯示:不同劑量免疫均能誘導(dǎo)小鼠產(chǎn)生IgG抗體,其效價(jià)可達(dá)1:10240,說明P6蛋白具有很好的免疫原性。選擇最佳免疫條件免疫小鼠,然后腹腔攻擊NTHi SSI p/225菌株,觀察主動免疫保護(hù)效果,結(jié)果顯示:20LD_(50)攻擊免疫小鼠后,試驗(yàn)組存活率為80%,而陰性對照組存活率為0,表明P6蛋白對成年小鼠有一定的保護(hù)力。
[Abstract]:The indistinguishable Haemophilus influenzae (Haemophilus influenzae) is a symbiotic bacterium that often resides in the upper respiratory tract of nasopharynx and posterior oropharynx. NTHi.NTHi is an important pathogen of human diseases in up to 80% of healthy children and 40% of healthy adults. They first live in the nasopharynx, then reach the middle ear through the eustachian tube, and they also infect the sinus and / or larynx, often leading to repeated respiratory tract infections. The most common cause of otitis media and sinusitis. NTHi has always been the main pathogen of otitis media in infants and children. The resulting otitis media accounts for about a quarter of all otitis media. Serological studies and antibiotic efficacy surveys suggest that NTHi is an important cause of lower respiratory tract infection in patients with chronic obstructive pulmonary disease (COPD). Recent studies have suggested that Haemophilus influenzae is also the leading cause of bacterial pneumonia in AIDS patients. The incidence of neonatal sepsis caused by NTHi has increased in the past decade, which can lead to 50% deaths and 90% deaths among premature infants. Therefore, otitis media is a major health problem in a country both in terms of disease and treatment costs. With the increasing drug resistance of these bacteria, the treatment is facing more challenges. Therefore, it is important to develop a safe and effective vaccine to prevent NTHi infection. Because it has no capsule polysaccharides, a vaccine based on NTHi outer membrane protein is being sought. There are five main outer membrane proteins in this strain, which are P1O47000DaP39000DX, P4O30000DaP5O27000DaP6, 16600 Daanjiao respectively, and the results show that there are five main outer membrane proteins in this strain, which are P1, 47000, P21, 39000, and P4, 300, 000, and P5. These five outer membrane proteins were the target of bactericidal antibodies, while P1P 2 and P 5 were both highly stable and highly conserved by the heterogenous restriction of the strains on the antigenicity of P4 and P6. Therefore, both immunogenicity and conserved strain P6 are very potential candidates for the study of vaccine components for the prevention of non-phenotype Haemophilus influenzae infection. According to the characteristics of P6, the protein was extracted at 37 鈩，

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