不可分型流感嗜血杆菌外膜蛋白P6基因真核表达质粒的构建及其免疫原性分析

发布时间：2018-02-26 10:04

本文关键词： 不可分型流感嗜血杆菌(nontypeable Haemophilus influenzae NTHi) 外膜蛋白P6(outer membrane protein P6 P6) 核酸疫苗质粒免疫　出处：《河北北方学院》2011年硕士论文　论文类型：学位论文

【摘要】：不可分型流感嗜血杆菌(nontypeable Haemophilus influenzae, NTHi)是一种在人群中携带率很高的病原菌，该菌能够引起急性中耳炎，鼻窦炎，结膜炎，慢性支气管炎的反复发作及恶化等严重感染性疾病，对人类尤其是婴幼儿及老年人的健康威胁极大，因此目前亟待研究一种新型、安全有效的疫苗来预防NTHi的感染。研究证明，在所有流感嗜血杆菌菌株中(含不可分型流感嗜血杆菌)高度保守的外膜蛋白P6(outer membrane protein P6, P6)可以诱导机体产生保护性的体液免疫、细胞免疫及黏膜免疫，但是天然P6蛋白含量少且提取过程极为繁琐。近年来核酸免疫的发展为我们研制疫苗提供了新的思路，本文就是通过构建真核表达重组质粒pcDNA3.1/His A-P6，并将其免疫动物观察其免疫效果，以确定P6在NTHi疫苗研制中的应用价值。以NTHi全基因组为模板通过PCR扩增获得P6目的基因，构建真核质粒pcDNA3.1/His A-P6，，通过菌落PCR、酶切、基因测序的方法确定重组质粒构建成功，并将重组质粒转染至HeLa细胞，以间接免疫荧光蛋白法检测其表达产物；动物实验中，将45只8w龄BALB/c小鼠随机分为3组：PBS组、pcDNA3.1/His A空质粒组、pcDNA3.1/His A-P6重组质粒组，每组每只小鼠分别经股四头肌注射免疫100μL PBS，100μL含100μg pcDNA3.1/His A的PBS，100μL含100μg pcDNA3.1/HisA-P6的PBS，于第0d，14d和28d共进行3次免疫。末次免疫后2w，每组取10只小鼠CCK-8法分析脾淋巴细胞增殖情况；ELISA法分析其脾淋巴细胞IFN-γ和IL-4产生水平；末次免疫后3w，每组其余5只小鼠鼻内接种1×108 CFU/只NTHi，于第3d、7d将100μL PBS灌洗鼻腔检测NTHi清除率；接种NTHi后1w处死小鼠，取其鼻粘膜组织，HE染色法分析鼻粘膜病理变化。成功构建了真核载体pcDNA3.1/His A-P6，经PCR、酶切、测序证实插入的基因片段为P6蛋白编码基因,而且该重组质粒能够在HeLa细胞中表达目的蛋白。动物实验中，pcDNA3.1/His A-P6免疫组组小鼠特异性脾淋巴细胞刺激指数及其所产生的IFN-γ水平均高于pcDNA3.1/His A、PBS对照组(P0.01)，而IL-4水平各组无差异；pcDNA3.1/HisA-P6组在NTHi感染后第3d、7d对其清除率分别为80%、90%，显著高于pcDNA3.1/His A、PBS对照组(P0.01)；HE染色显示目的基因组小鼠鼻粘膜组织结构基本正常，而对照组鼻粘膜紊乱、脱落。该研究成功构建了真核表达质粒pcDNA3.1/His A-P6，将其免疫BALB/c小鼠后可诱导小鼠产生明显的抗NTHi保护效应，提示pcDNA3.1/His A-P6核酸疫苗具有潜在的疫苗研究与开发价值。
[Abstract]:Haemophilus influenzae nontypeable Haemophilus influenzae (NTHiae) is a pathogen with high carrying rate in the population. It can cause severe infectious diseases such as acute otitis media, sinusitis, conjunctivitis, chronic bronchitis and other serious infectious diseases. There is a great threat to human health, especially to infants and the elderly. Therefore, it is urgent to study a new, safe and effective vaccine to prevent NTHi infection. In all Haemophilus influenzae strains (including Haemophilus influenzae), highly conserved outer membrane protein P6outer membrane protein P6 (P6) can induce protective humoral, cellular and mucosal immunity. However, the natural P6 protein content is low and the extraction process is very complicated. In recent years, the development of nucleic acid immunization has provided us with a new way of thinking for the development of vaccine. In this paper, the eukaryotic expression plasmid pcDNA3.1/His A-P6 was constructed, and its immune effect was observed in order to determine the application value of P6 in the development of NTHi vaccine. The P6 target gene was obtained by PCR amplification from the whole NTHi genome, and the eukaryotic plasmid pcDNA3.1/His A-P6 was constructed. The recombinant plasmid was successfully constructed by colony PCR, restriction endonuclease digestion and gene sequencing, and the recombinant plasmid was transfected into HeLa cells. In animal experiments, 45 8-week-old BALB/c mice were randomly divided into 3 groups: pcDNA3.1 / his A empty plasmid group and pcDNA3.1 / his A-P6 recombinant plasmid group. Each mouse in each group was immunized with 100 渭 L PBS100 渭 L PBS100 渭 L containing 100 渭 g pcDNA3.1/HisA through the quadriceps femoris. The mice were immunized for 3 times on the 14th and 28th days after the last immunization. After the last immunization, 10 mice in each group were immunized with CCK-8 method to analyze the proliferation of splenic lymphocytes by Elisa. The levels of IFN- 纬 and IL-4 in splenic lymphocytes were analyzed by Elisa. Three weeks after the last immunization, the other 5 mice in each group were inoculated with 1 脳 108CFU / NTHi. 100 渭 L PBS was perfused into the nasal cavity on the 3rd day to detect the clearance rate of NTHi, and the mice were killed at 1 week after inoculation, and the nasal mucosa tissues were stained with HE staining to analyze the pathological changes of the nasal mucosa. The eukaryotic vector pcDNA3.1/His A-P6 was successfully constructed. The inserted gene fragment was confirmed to be P6 protein encoding gene by PCR, restriction endonuclease digestion and sequencing. Moreover, the recombinant plasmid could express the target protein in HeLa cells. In animal experiment, the specific spleen lymphocyte stimulating index and IFN- 纬 level of mice immunized with pcDNA3.1% his A-P6 were higher than that of pcDNA3.1/His Agna PBS control group (P0.01), while the IL-4 level in each group was higher than that in pcDNA3.1/His Agna PBS control group. The clearance rates of pcDNA3.1 / HisA-P6 group on the 3rd day after NTHi infection were 80 and 90 respectively, which were significantly higher than that of pcDNA3.1/HisA control group (P 0.01). In the control group, the nasal mucosa was disordered and shedding. In this study, the eukaryotic expression plasmid pcDNA3.1/His A-P6 was successfully constructed. After immunizing BALB/c mice, it could induce the mice to produce obvious anti-#en2# protective effect, suggesting that the pcDNA3.1/His A-P6 nucleic acid vaccine has potential value in the research and development of the vaccine.
【学位授予单位】：河北北方学院
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R378.4

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