不可分型流感嗜血杆菌外膜蛋白P6的纯化及鉴定

发布时间：2018-06-20 16:24

  本文选题：NTHi + 外膜蛋白　； 参考：《北京生物制品研究所》2008年硕士论文

【摘要】： 不可分型的流感嗜血杆菌(NTHi)是一种常定居于鼻咽和后口咽上呼吸道部位的共生菌,在多达80%的健康儿童以及40%的健康成人的咽部都存在NTHi。NTHi是人类一些疾病的重要病原菌,它们首先寄居在鼻咽部,然后通过咽鼓管到达中耳,也感染(鼻)窦和/或喉,常导致呼吸道反复感染,最常引起中耳炎及窦炎。NTHi一直是婴幼儿和儿童中耳炎的主要病原菌,其引起的中耳炎约占所有中耳炎的1/4～1/3。血清学研究及抗生素疗效调查提示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杆菌也是引起AIDS患者细菌性肺炎的主要病原菌。过去十年里,NTHi引起的新生儿脓毒症发病有所上升,其可导致50%患者死亡,早产儿中死亡率达90%。因此,中耳炎不论从疾病发生还是从治疗费用而言都是一个国家较大的健康问题。伴随这些菌耐药性的增强,治疗面临着更大的挑战。因此,发展一种安全有效的疫苗预防NTHi的感染已显得尤为重要。由于该菌没有荚膜多糖,故现在寻求一种以NTHi外膜蛋白为基础的疫苗。 该菌有5个主要的外膜蛋白,分别为P1(47,000Da)、P2(39,000Da)、P4(30,000Da)、P5(27,000Da)、P6(16,600Da)。这五种外膜蛋白均是杀菌抗体的靶目标,而P1、P2及P5均受到菌株异源性的限制,P4和P6的抗原性非常稳定同时也高度保守。因此,无论其免疫原性还是菌株保守性,P6都是作为研究预防不可分型流感嗜血杆菌感染的疫苗成分的非常有潜力的候选者。 本实验根据P6的特性来提取,即在buffer B(0.1M Tris,0.5M NaCl,1%SDS,0.1%2-巯基乙醇,PH 8.0)中37℃孵育30min,P6是唯一不溶的蛋白,而在65℃孵育30min,P6即可被溶解下来。首先超声破碎菌体制备膜制剂,膜制剂在bufferB中37℃孵育30min,高速离心后弃上清,膜制剂继续在buffer B中37℃孵育30min,超速离心,如此反复多次后,即可将膜制剂上的杂蛋白洗去,仅剩下P6,最后在bufferA(0.01 M Tris-0.15 M NaCl,pH 7.4)中65℃孵育30min,P6即可被溶解下来。SDS-PAGE显示仅一条蛋白带,影像分析软件AlphaDigiDoc~(TM)显示其纯度为100%,分子量为16.73kDa,IEF-PAGE电泳后,采用AlphaEaseFC软件对等电聚焦图谱进行分析,结果显示,目的蛋白的等电点为5.49,产率为90.43ug/g湿菌体。以P6不同剂量和针次免疫NIH小鼠,间接ELISA法检测小鼠血清IgG抗体滴度,结果显示:不同剂量免疫均能诱导小鼠产生IgG抗体,其效价可达1:10240,说明P6蛋白具有很好的免疫原性。选择最佳免疫条件免疫小鼠,然后腹腔攻击NTHi SSI p/225菌株,观察主动免疫保护效果,结果显示:20LD_(50)攻击免疫小鼠后,试验组存活率为80%,而阴性对照组存活率为0,表明P6蛋白对成年小鼠有一定的保护力。
[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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