【摘要】： 流感是一個(gè)古老且第一個(gè)實(shí)行全球性監(jiān)測的病毒性急性呼吸道傳染病。流感危害極為嚴(yán)重，流行時(shí)波及面廣，有很高的超額死亡率，對人群健康、甚至對社會穩(wěn)定產(chǎn)生重大影響，對經(jīng)濟(jì)造成難以估計(jì)的損失。 流感病毒屬于正粘病毒科(Orthomyxovirdae)，是一種負(fù)鏈RNA病毒，病毒基因組為單股8節(jié)段負(fù)鏈RNA。流感病毒最大的特點(diǎn)是容易變異，以逃避人體的免疫力，這也是流感不斷發(fā)生流行而不能根除的原因。疫苗免疫仍是當(dāng)今預(yù)防流感最有效的手段。 近年來，由于DNA疫苗能誘導(dǎo)出較好的體液免疫和細(xì)胞免疫而日益受到重視，被譽(yù)為疫苗的第三次革命。本研究選用H1N1流感病毒A／PR／8／34株表面的兩個(gè)糖蛋白血凝素HA、神經(jīng)氨酸酶NA和病毒核殼體內(nèi)部的NP蛋白的編碼基因?yàn)槟康幕蜻M(jìn)行流感DNA疫苗的研究。提取病毒RNA，反轉(zhuǎn)錄成cDNA，以cDNA為模板擴(kuò)增出HA、NA和NP片段，成功構(gòu)建重組質(zhì)粒pVAX1／HA、pVAX1／NA和pVAX1／NP。利用重疊延伸PCR的方法，在成功構(gòu)建的pVAX1／HA、pVAX1／NA DNA疫苗基礎(chǔ)上，，將HA1及NA胞外區(qū)通過柔性氨基酸接頭連接起來，構(gòu)建成融合表達(dá)的雙分子DNA疫苗。重組質(zhì)粒轉(zhuǎn)染COS-7細(xì)胞，免疫組化方法和Western blot方法檢測目的基因在體外培養(yǎng)的哺乳動(dòng)物細(xì)胞中的表達(dá)。 將重組質(zhì)粒以100μg／只肌肉注射免疫BALB／c小鼠，隔周免疫一次，加強(qiáng)免疫3次，檢測體液免疫和細(xì)胞免疫的指標(biāo)。結(jié)果顯示，單分子的HA、NA和NP DNA疫苗初次免疫后能檢測到血清總的IgG抗體水平，加強(qiáng)免疫后抗體水平顯著升高，抗體亞型及免疫小鼠脾淋巴細(xì)胞流式細(xì)胞檢測的結(jié)果顯示，三個(gè)分子的DNA疫苗誘導(dǎo)Th1型細(xì)胞免疫應(yīng)答。免疫小鼠攻毒實(shí)驗(yàn)進(jìn)一步證明我們所構(gòu)建的DNA疫苗具有較好的免疫保護(hù)性。 ELISA檢測雙分子DNA疫苗誘導(dǎo)產(chǎn)生的IgG抗體、IgG抗體亞類，流式細(xì)胞儀分析免疫小鼠的脾淋巴細(xì)胞，結(jié)果證明雙分子DNA疫苗能夠刺激小鼠產(chǎn)生特異的體液免疫應(yīng)答，也能夠誘導(dǎo)Th1型細(xì)胞免疫應(yīng)答，但其抗體水平及CD4~+T／CD8~+T的值均基本相當(dāng)于NA DNA疫苗，并沒有體現(xiàn)出抗原性的增強(qiáng)，其原因還有待于進(jìn)一步的研究。 利用以asd為基礎(chǔ)的宿主-載體平衡系統(tǒng)，構(gòu)建了以減毒沙門氏菌運(yùn)送的HA DNA及NADNA活載體疫苗。重組菌具有良好的安全性和穩(wěn)定性，口服免疫小鼠后，可以刺激機(jī)體產(chǎn)生體液免疫和粘膜免疫，攻毒實(shí)驗(yàn)顯示了其好的免疫保護(hù)性。以上研究為后續(xù)的流感基因疫苗研究奠定了基礎(chǔ)。
[Abstract]:Influenza is an ancient and the first viral acute respiratory disease to be monitored globally. Influenza is extremely serious, spread widely during the epidemic, has a high excess mortality rate, has a significant impact on the health of the population and even on social stability, and causes incalculable losses to the economy. Influenza virus belongs to Orthomyxoviridae (Orthomyxovirdae), is a negative-stranded RNA virus, the virus genome is single-stranded 8-segment negative-stranded RNA.. The biggest feature of influenza viruses is that they mutate easily to escape the body's immunity, which is why influenza continues to spread and cannot be eradicated. Vaccination is still the most effective way to prevent influenza today. In recent years, more and more attention has been paid to DNA vaccine because of its ability to induce better humoral and cellular immunity, which is regarded as the third revolution of vaccine. In this study, two glycoprotein hemagglutinin (HA,) neuraminidase NA on the surface of H1N1 influenza virus A/PR/8/34 strain and the encoding gene of NP protein in the nuclear shell of the virus were used as target genes to study the influenza DNA vaccine. The recombinant plasmids pVAX1/HA,pVAX1/NA and pVAX1/NP. were successfully constructed by extracting the viral RNA, reverse transcription into cDNA, and using cDNA as template to amplify the HA,NA and NP fragments. On the basis of successfully constructed pVAX1/HA,pVAX1/NA DNA vaccine, the HA1 and NA extracellular regions were connected by flexible amino acid junction to form a fusion expressed bimolecular DNA vaccine using overlapping extension PCR method. The recombinant plasmid was transfected into COS-7 cells. The expression of the target gene in mammalian cells in vitro was detected by immunohistochemistry and Western blot methods. The recombinant plasmid was injected intramuscularly into BALB/c mice at a dose of 100 渭 g per mouse. The mice were immunized once every other week, and 3 times by intensified immunization. The indexes of humoral immunity and cellular immunity were detected. The results showed that the total serum IgG antibody level could be detected after primary immunization with single molecule HA,NA and NP DNA vaccine, and the antibody level was significantly increased after enhanced immunization. The results of antibody subtype and flow cytometry analysis of spleen lymphocytes in immunized mice showed that the total IgG antibody level was detected after primary immunization. Three molecules of DNA vaccine induce Th1 type cellular immune response. The DNA vaccine was further proved to be immune protective by immunizing mice. ELISA was used to detect the IgG antibody subclass induced by bimolecular DNA vaccine. The spleen lymphocytes of mice immunized with bimolecular DNA vaccine were analyzed by flow cytometry. The results showed that bimolecular DNA vaccine could stimulate specific humoral immune response and induce Th1 type cellular immune response in mice. However, the antibody level and the value of CD4~ T / CD8T were similar to those of NA DNA vaccine, and did not reflect the enhancement of antigenicity. HA DNA and NADNA live vector vaccines transported by attenuated Salmonella were constructed by using the host-vector equilibrium system based on asd. The recombinant bacteria have good safety and stability. After oral immunization, the recombinant bacteria can stimulate humoral immunity and mucosal immunity. The above studies have laid the foundation for the further study of influenza gene vaccine.
【學(xué)位授予單位】：中國人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2006
【分類號】：R392

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相關(guān)期刊論文 前2條

1 李彥鋒,靳鳳爍,何畏,江軍,王洛夫,靳文生;鼠精子表面蛋白cyritestin在沙門氏菌平衡致死系統(tǒng)中的高效表達(dá)[J];免疫學(xué)雜志;2001年05期

2 姚碧濤;李鵬;焦新安;周麗麗;劉秀梵;王希良;;重組流感病毒H1N1活菌疫苗的構(gòu)建及其免疫效果的初步研究[J];免疫學(xué)雜志;2006年06期

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