流感病毒DNA疫苗的构建及免疫效果的初步研究
发布时间：2018-08-27 15:16
【摘要】： 流感是一个古老且第一个实行全球性监测的病毒性急性呼吸道传染病。流感危害极为严重，流行时波及面广，有很高的超额死亡率，对人群健康、甚至对社会稳定产生重大影响，对经济造成难以估计的损失。 流感病毒属于正粘病毒科(Orthomyxovirdae)，是一种负链RNA病毒，病毒基因组为单股8节段负链RNA。流感病毒最大的特点是容易变异，以逃避人体的免疫力，这也是流感不断发生流行而不能根除的原因。疫苗免疫仍是当今预防流感最有效的手段。 近年来，由于DNA疫苗能诱导出较好的体液免疫和细胞免疫而日益受到重视，被誉为疫苗的第三次革命。本研究选用H1N1流感病毒A／PR／8／34株表面的两个糖蛋白血凝素HA、神经氨酸酶NA和病毒核壳体内部的NP蛋白的编码基因为目的基因进行流感DNA疫苗的研究。提取病毒RNA，反转录成cDNA，以cDNA为模板扩增出HA、NA和NP片段，成功构建重组质粒pVAX1／HA、pVAX1／NA和pVAX1／NP。利用重叠延伸PCR的方法，在成功构建的pVAX1／HA、pVAX1／NA DNA疫苗基础上，，将HA1及NA胞外区通过柔性氨基酸接头连接起来，构建成融合表达的双分子DNA疫苗。重组质粒转染COS-7细胞，免疫组化方法和Western blot方法检测目的基因在体外培养的哺乳动物细胞中的表达。 将重组质粒以100μg／只肌肉注射免疫BALB／c小鼠，隔周免疫一次，加强免疫3次，检测体液免疫和细胞免疫的指标。结果显示，单分子的HA、NA和NP DNA疫苗初次免疫后能检测到血清总的IgG抗体水平，加强免疫后抗体水平显著升高，抗体亚型及免疫小鼠脾淋巴细胞流式细胞检测的结果显示，三个分子的DNA疫苗诱导Th1型细胞免疫应答。免疫小鼠攻毒实验进一步证明我们所构建的DNA疫苗具有较好的免疫保护性。 ELISA检测双分子DNA疫苗诱导产生的IgG抗体、IgG抗体亚类，流式细胞仪分析免疫小鼠的脾淋巴细胞，结果证明双分子DNA疫苗能够刺激小鼠产生特异的体液免疫应答，也能够诱导Th1型细胞免疫应答，但其抗体水平及CD4~+T／CD8~+T的值均基本相当于NA DNA疫苗，并没有体现出抗原性的增强，其原因还有待于进一步的研究。 利用以asd为基础的宿主-载体平衡系统，构建了以减毒沙门氏菌运送的HA DNA及NADNA活载体疫苗。重组菌具有良好的安全性和稳定性，口服免疫小鼠后，可以刺激机体产生体液免疫和粘膜免疫，攻毒实验显示了其好的免疫保护性。以上研究为后续的流感基因疫苗研究奠定了基础。
[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.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R392
本文编号：2207666
[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.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R392
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相关期刊论文 前2条
1 李彦锋,靳凤烁,何畏,江军,王洛夫,靳文生;鼠精子表面蛋白cyritestin在沙门氏菌平衡致死系统中的高效表达[J];免疫学杂志;2001年05期
2 姚碧涛;李鹏;焦新安;周丽丽;刘秀梵;王希良;;重组流感病毒H1N1活菌疫苗的构建及其免疫效果的初步研究[J];免疫学杂志;2006年06期
本文编号：2207666
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