本文關(guān)鍵詞： 不可分型流感嗜血桿菌(nontypeable Haemophilus influenzae NTHi) 外膜蛋白P6(outer membrane protein P6 P6) 核酸疫苗 質(zhì)粒 免疫　出處：《河北北方學(xué)院》2011年碩士論文　論文類型：學(xué)位論文

【摘要】：不可分型流感嗜血桿菌(nontypeable Haemophilus influenzae, NTHi)是一種在人群中攜帶率很高的病原菌，該菌能夠引起急性中耳炎，鼻竇炎，結(jié)膜炎，慢性支氣管炎的反復(fù)發(fā)作及惡化等嚴(yán)重感染性疾病，對(duì)人類尤其是嬰幼兒及老年人的健康威脅極大，因此目前亟待研究一種新型、安全有效的疫苗來(lái)預(yù)防NTHi的感染。研究證明，在所有流感嗜血桿菌菌株中(含不可分型流感嗜血桿菌)高度保守的外膜蛋白P6(outer membrane protein P6, P6)可以誘導(dǎo)機(jī)體產(chǎn)生保護(hù)性的體液免疫、細(xì)胞免疫及黏膜免疫，但是天然P6蛋白含量少且提取過(guò)程極為繁瑣。近年來(lái)核酸免疫的發(fā)展為我們研制疫苗提供了新的思路，本文就是通過(guò)構(gòu)建真核表達(dá)重組質(zhì)粒pcDNA3.1/His A-P6，并將其免疫動(dòng)物觀察其免疫效果，以確定P6在NTHi疫苗研制中的應(yīng)用價(jià)值。 以NTHi全基因組為模板通過(guò)PCR擴(kuò)增獲得P6目的基因，構(gòu)建真核質(zhì)粒pcDNA3.1/His A-P6，，通過(guò)菌落PCR、酶切、基因測(cè)序的方法確定重組質(zhì)粒構(gòu)建成功，并將重組質(zhì)粒轉(zhuǎn)染至HeLa細(xì)胞，以間接免疫熒光蛋白法檢測(cè)其表達(dá)產(chǎn)物；動(dòng)物實(shí)驗(yàn)中，將45只8w齡BALB/c小鼠隨機(jī)分為3組：PBS組、pcDNA3.1/His A空質(zhì)粒組、pcDNA3.1/His A-P6重組質(zhì)粒組，每組每只小鼠分別經(jīng)股四頭肌注射免疫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共進(jìn)行3次免疫。末次免疫后2w，每組取10只小鼠CCK-8法分析脾淋巴細(xì)胞增殖情況；ELISA法分析其脾淋巴細(xì)胞IFN-γ和IL-4產(chǎn)生水平；末次免疫后3w，每組其余5只小鼠鼻內(nèi)接種1×108 CFU/只NTHi，于第3d、7d將100μL PBS灌洗鼻腔檢測(cè)NTHi清除率；接種NTHi后1w處死小鼠，取其鼻粘膜組織，HE染色法分析鼻粘膜病理變化。 成功構(gòu)建了真核載體pcDNA3.1/His A-P6，經(jīng)PCR、酶切、測(cè)序證實(shí)插入的基因片段為P6蛋白編碼基因,而且該重組質(zhì)粒能夠在HeLa細(xì)胞中表達(dá)目的蛋白。動(dòng)物實(shí)驗(yàn)中，pcDNA3.1/His A-P6免疫組組小鼠特異性脾淋巴細(xì)胞刺激指數(shù)及其所產(chǎn)生的IFN-γ水平均高于pcDNA3.1/His A、PBS對(duì)照組(P0.01)，而IL-4水平各組無(wú)差異；pcDNA3.1/HisA-P6組在NTHi感染后第3d、7d對(duì)其清除率分別為80%、90%，顯著高于pcDNA3.1/His A、PBS對(duì)照組(P0.01)；HE染色顯示目的基因組小鼠鼻粘膜組織結(jié)構(gòu)基本正常，而對(duì)照組鼻粘膜紊亂、脫落。 該研究成功構(gòu)建了真核表達(dá)質(zhì)粒pcDNA3.1/His A-P6，將其免疫BALB/c小鼠后可誘導(dǎo)小鼠產(chǎn)生明顯的抗NTHi保護(hù)效應(yīng)，提示pcDNA3.1/His A-P6核酸疫苗具有潛在的疫苗研究與開(kāi)發(fā)價(jià)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- 緯 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- 緯 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.
【學(xué)位授予單位】：河北北方學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R378.4

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