以HBC和HPV16L1为载体的流感通用疫苗研究
发布时间：2018-11-05 12:25
【摘要】： 背景知识: 流感(Influenza)是由流行性感冒病毒(Influenza virus)感染引起的急性呼吸道传染病。流感病毒属于正粘病毒科,可感染人和多种动物。流感病毒抗原性易发生变异,抗原的漂移和转换给流感的防治带来了较大困难。接种疫苗仍然是预防和控制流感的主要手段。目前使用的疫苗包括全病毒疫苗、裂解疫苗和减毒活疫苗,但都需要每年更换疫苗株接种。因此,发展流感通用疫苗,实现几年接种免疫一次是流感疫苗研究发展的方向。 目的: 本实验构建以乙肝核心蛋白(HBC)和人乳头瘤病毒主要衣壳蛋白(HPV16 L1)为载体的甲型流感病毒M2基因胞外区(M2e)通用疫苗杆粒,利用昆虫细胞杆状病毒表达系统,进行初步的蛋白表达,为发展安全、有效、稳定的流感通用疫苗奠定基础。 方法与结果: 1.将甲型流感病毒M2e基因序列设计在引物上,利用PCR技术分别与HBC和HPV16 L1相连。将M2e-HBC和M2e-HPV16 L1序列分别克隆到供体质粒pFastBac HTA上,构建两个重组质粒,分别为M2e-HBC-pFastBac HTA和M2e-HPV16 L1-pFastBac HTA。 2.用E.coli DH10Bac中的穿梭载体Bacmid与重组质粒转座,使含有M2e-HBC和M2e-HPV16 L1基因的表达盒插入Bacmid的attTn7的接受位点,获得重组Bacmid。 3.将挑选的阳性重组子DNA转染sf9细胞,取上清和细胞,上清继续扩增病毒,用空斑试验测定病毒效价;免疫荧光法检测蛋白表达;细胞做破胞处理后,将上清和裂解液进行SDS-PAGE分析;将感染病毒的细胞裂解后透射电镜观察。结果证明,M2e-HBC和M2e-HPV16 L1基因有蛋白表达,而对照呈现阴性;电镜可见细胞内病毒样颗粒(VLP)形成。 结论: 构建了含有M2e-HBC和M2e-HPV16 L1的杆粒。利用Bac-to-Bac杆状病毒表达系统,将构建好的杆粒转染sf9昆虫细胞,得到含有重组杆状病毒的上清,扩增病毒,感染细胞后,表达出M2e-HBC和M2e-HPV16 L1目的蛋白。流感病毒样颗粒抗原M2e-HBC和M2e-HPV16 L1高效表达,为流感病毒通用疫苗的研制奠定了基础。
[Abstract]:Background: influenza (Influenza) is an acute respiratory infection caused by influenza virus (Influenza virus) infection. Influenza viruses belong to the orthomyxovirus family and infect humans and many animals. The antigenicity of influenza viruses is easy to mutate, and the drift and transformation of antigens bring great difficulties to the prevention and treatment of influenza. Vaccination remains the primary means of influenza prevention and control. Vaccines currently in use include whole-virus vaccines, lytic vaccines and live attenuated vaccines, but require annual replacement of the vaccine strain. Therefore, it is the direction of influenza vaccine research and development to develop a universal influenza vaccine and to vaccinate once in a few years. Objective: to construct a universal vaccine of influenza A virus M2 gene extracellular region (M2e) using hepatitis B core protein (HBC) and human papillomavirus main capsid protein (HPV16 L1) as vectors. The baculovirus expression system of insect cells was used to carry out the preliminary protein expression, which laid the foundation for the development of a safe, effective and stable universal influenza vaccine. Methods and results: 1. The M2e gene sequence of influenza A virus was designed on primer and linked to HBC and HPV16 L1 by PCR technique. M2e-HBC and M2e-HPV16 L1 sequences were cloned into donor plasmid pFastBac HTA, and two recombinant plasmids, M2e-HBC-pFastBac HTA and M2e-HPV16 L1-pFastBac HTA., were constructed. 2. By transposing the shuttle vector Bacmid in E.coli DH10Bac with the recombinant plasmid, the expression box containing M2e-HBC and M2e-HPV16 L1 gene was inserted into the acceptance site of Bacmid attTn7, and the recombinant Bacmid. was obtained. 3. The positive recombinant DNA was transfected into sf9 cells. The supernatant and the supernatant were used to amplify the virus, the titer of the virus was determined by plaque test, and the protein expression was detected by immunofluorescence assay. The supernatant and the lytic fluid were analyzed by SDS-PAGE, and the infected cells were observed by transmission electron microscope. The results showed that M2e-HBC and M2e-HPV16 L1 gene had protein expression, but the control showed negative expression, and the virus like (VLP) was found in the cells under electron microscope. Conclusion: rods containing M2e-HBC and M2e-HPV16 L1 were constructed. The recombinant baculovirus supernatant was obtained by transfection of sf9 insect cells with Bac-to-Bac baculovirus expression system. The recombinant baculovirus supernatant was amplified and the target proteins of M2e-HBC and M2e-HPV16 L1 were expressed after infection. The high expression of influenza virus-like particle antigen M2e-HBC and M2e-HPV16 L1 lays a foundation for the development of influenza virus vaccine.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392
本文编号：2312076
[Abstract]:Background: influenza (Influenza) is an acute respiratory infection caused by influenza virus (Influenza virus) infection. Influenza viruses belong to the orthomyxovirus family and infect humans and many animals. The antigenicity of influenza viruses is easy to mutate, and the drift and transformation of antigens bring great difficulties to the prevention and treatment of influenza. Vaccination remains the primary means of influenza prevention and control. Vaccines currently in use include whole-virus vaccines, lytic vaccines and live attenuated vaccines, but require annual replacement of the vaccine strain. Therefore, it is the direction of influenza vaccine research and development to develop a universal influenza vaccine and to vaccinate once in a few years. Objective: to construct a universal vaccine of influenza A virus M2 gene extracellular region (M2e) using hepatitis B core protein (HBC) and human papillomavirus main capsid protein (HPV16 L1) as vectors. The baculovirus expression system of insect cells was used to carry out the preliminary protein expression, which laid the foundation for the development of a safe, effective and stable universal influenza vaccine. Methods and results: 1. The M2e gene sequence of influenza A virus was designed on primer and linked to HBC and HPV16 L1 by PCR technique. M2e-HBC and M2e-HPV16 L1 sequences were cloned into donor plasmid pFastBac HTA, and two recombinant plasmids, M2e-HBC-pFastBac HTA and M2e-HPV16 L1-pFastBac HTA., were constructed. 2. By transposing the shuttle vector Bacmid in E.coli DH10Bac with the recombinant plasmid, the expression box containing M2e-HBC and M2e-HPV16 L1 gene was inserted into the acceptance site of Bacmid attTn7, and the recombinant Bacmid. was obtained. 3. The positive recombinant DNA was transfected into sf9 cells. The supernatant and the supernatant were used to amplify the virus, the titer of the virus was determined by plaque test, and the protein expression was detected by immunofluorescence assay. The supernatant and the lytic fluid were analyzed by SDS-PAGE, and the infected cells were observed by transmission electron microscope. The results showed that M2e-HBC and M2e-HPV16 L1 gene had protein expression, but the control showed negative expression, and the virus like (VLP) was found in the cells under electron microscope. Conclusion: rods containing M2e-HBC and M2e-HPV16 L1 were constructed. The recombinant baculovirus supernatant was obtained by transfection of sf9 insect cells with Bac-to-Bac baculovirus expression system. The recombinant baculovirus supernatant was amplified and the target proteins of M2e-HBC and M2e-HPV16 L1 were expressed after infection. The high expression of influenza virus-like particle antigen M2e-HBC and M2e-HPV16 L1 lays a foundation for the development of influenza virus vaccine.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392
【参考文献】
相关期刊论文 前1条
1 朱建国;A型流感病毒基质蛋白研究进展[J];动物医学进展;2004年02期
，本文编号：2312076
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