以HBc颗粒为载体甲型流感通用疫苗的初步研究
发布时间：2018-09-07 21:31
【摘要】： 【研究背景】 流感病毒(Influenza virus )是一种能够引起严重性呼吸道传播疾病的正粘科属病毒。根据其内部核蛋白(NP)和基质蛋白(M)的抗原性不同,可分为甲(A)、乙(B)、丙(C)三种类型。其中,甲型流感病毒曾引起多次大范围的流行,上世纪共出现四次大流行,最为严重的是1918年的大流感,造成了全世界5千多万人死亡。2009年世界范围内暴发的猪源性甲型流感病毒流行,传染性强,波及范围广,再次突显了甲型流感病毒感染的严重性及其对人类社会造成的巨大经济负担。 接种疫苗是预防流感发生与传播的最有效方式。目前市场应用的流感疫苗主要有三种:全病毒灭活疫苗、裂解疫苗、亚单位疫苗。这些疫苗对同亚型流感病毒感染预防有效,但在不同亚型病毒之间的保护效果较弱。此外,流感病毒的变异速度非常快,流感疫苗每年都要更换,给疫苗的生产制备造成了诸多不便。世界卫生组织是依据当年全世界范围流感病毒变化情况来预测并推荐下一年流感疫苗生产用组分的,预测的准确度将会直接影响疫苗的保护效率,如预测失败将造成流感爆发流行的潜在威胁。因此,研制出一种具有广泛保护作用的通用型疫苗对于应对流感病毒突变后的突然爆发,迅速建立人群免疫屏障,阻断流感大流行蔓延,降低其危害性方面具有极其重要的实际意义。 甲型流感通用疫苗的研发主要基于流感病毒基质蛋白胞外功能区M2e (Ectodomain of matrix protein 2),其在各种甲型流感病毒中具有极高的保守性,且可诱导特异性保护抗体产生。此外还有高度保守的流感病毒核蛋白NP(Nucleoprotein),其表面具有多个细胞毒性T淋巴细胞(CTL)识别表位,可诱导细胞免疫。但M2e和NP表位的分子量小、免疫原性低、易降解,在体内难以产生足够的免疫反应。所以,使用结构复杂病毒蛋白作为免疫原性放大载体成为表位疫苗研发的热点。其中,乙肝病毒核心蛋白HBc(Hepatitis B virus core protein , HBc)蛋白具有天然的颗粒组装能力,能特异性激发针对外源表位的体液、细胞免疫。近年来,HBc颗粒的应用方面已经有了较成熟研究结果。疫苗研究中以HBc作为载体的疫苗抗原有很多,如乙肝病毒表面抗原、人乳头瘤病毒E7蛋白、B群链球菌CPS糖蛋白等,在临床前实验研究中已观察到肯定的疗效。因此,发展以HBc颗粒为载体的流感病毒表位疫苗研究具有很好的前景和应用价值。 【目的】 本研究的目的是构建以乙肝病毒核心蛋白(HBc)为载体的基于M2e蛋白的甲型流感通用疫苗抗原,利用杆状病毒表达系统及大肠杆菌表达系统表达、纯化和鉴定,并进行疫苗免疫效果初步评价,为发展安全、有效、具有广泛保护作用的甲型流感通用疫苗奠定基础。 【方法与结果】 第一部分sf9昆虫细胞表达甲型流感M2e通用疫苗抗原的构建、表达、纯化及鉴定通过基因工程方法,构建含M2e重复片段的3M2e-HBc-pFastBacHTA重组质粒。将该重组质粒与DH10Bac中的穿梭载体Bacmid同源重组,获得3M2e-HBc-Bacmid重组杆粒。提取杆粒DNA转染sf9昆虫细胞,得到含有重组杆状病毒。继续扩增病毒,感染细胞后,免疫荧光检测成功表达出3M2e-HBc目的蛋白。Western blot检测该蛋白具有免疫活性。利用pFastBacHTA载体上带有的组氨酸标签,进行亲和层析,纯化获得3M2e-HBc重组蛋白。电镜结果显示,3M2e-HBc目的基因可以通过杆状病毒sf9昆虫细胞表达系统表达并正确装配出病毒样颗粒。 第二部分大肠杆菌表达甲型流感M2e-NP通用疫苗抗原的构建、表达、纯化及鉴定通过基因工程方法,构建并鉴定获得3M2e-NP-HBc-pET 21a重组表达质粒,并转化大肠杆菌细胞,IPTG诱导表达首先获得了包涵体形式表达的重组蛋白。经变性及复性纯化处理后,包涵体蛋白形成的重组病毒颗粒的状态不理想。经进一步优化诱导表达条件获得了部分可溶性表达的重组蛋白,Western blot检测证明该蛋白具有免疫活性。采用层析方法获得纯化的3M2e-NP-HBc重组蛋白。电镜观察结果显示,大肠杆菌表达的可溶性重组蛋白能够自动装配成大小约为30nm的病毒样颗粒。 第三部分甲型流感通用疫苗免疫效果初步评价 通过动物免疫实验,初步评价甲型流感通用疫苗的免疫应答效果及交叉保护作用。采用间接ELISA法、流式细胞术、ELISOPT技术,对疫苗在Balb/C小鼠体内的免疫应答效果进行了评估。在对大肠杆菌表达的流感通用疫苗评价的结果中显示,疫苗以滴鼻和腹腔注射两种途径免疫小鼠,均可以诱导产生针对不同亚型流感病毒的特异性体液及细胞免疫应答,并且滴鼻免疫能够激发更高的黏膜局部免疫应答,说明采用滴鼻免疫这种更接近于流感病毒自然感染途径的方式免疫对提高流感疫苗的保护效果具有重要意义。同时,攻毒试验结果显示,以A/Beijing/501和A/PR/8/34两株代表性的甲型流感病毒攻毒后,疫苗免疫组相比对照组明显降低小鼠的死亡率,表明大肠杆菌表达的以HBc为载体的甲型流感通用疫苗具有良好的免疫原性交叉保护作用。 【结论】 本研究利用Bac-to-Bac杆状病毒sf9昆虫细胞表达系统和大肠杆菌原核表达系统,分别成功制备了两种的以HBc为载体的基于流感病毒M2e蛋白的流感通用疫苗,并主要对大肠杆菌表达的流感通用疫苗进行了动物免疫效果评价。实验结果显示,该疫苗具有很好的免疫原性,可以刺激机体产生系统免疫应答和黏膜局部免疫应答,并且能产生有效的交叉保护作用,为甲型流感通用疫苗的深入研究提供了理论依据。
[Abstract]:[background]
Influenza virus (Influenza virus) is a kind of Orthomyxovirus which can cause severe respiratory diseases. According to the antigenicity of nucleoprotein (NP) and matrix protein (M), Influenza virus can be divided into three types: A (A), B (B) and C (C). Influenza A virus has caused a wide range of pandemics, and there were four major outbreaks in the last century. What is most serious is the large influenza in 1918, which resulted in the death of more than 5 million people worldwide. The outbreak of swine influenza A * virus in the world in.2009 was epidemic, infectious and spread widely. It once again highlighted the seriousness of influenza A virus infection and its enormous economic burden on human society.
Vaccination is the most effective way to prevent the occurrence and spread of influenza. At present, there are three kinds of influenza vaccines in the market: whole virus inactivated vaccine, lysis vaccine and subunit vaccine. The World Health Organization (WHO) predicts and recommends the components for the next year's influenza vaccine production based on changes in influenza viruses worldwide. The accuracy of the prediction will directly affect the efficiency of vaccine protection, such as the failure of the prediction. Therefore, it is of great practical significance to develop a universal vaccine with extensive protective effect in dealing with the sudden outbreak of influenza virus mutation, rapidly establishing a population immune barrier, blocking the spread of influenza pandemic and reducing its harmfulness.
The development of universal influenza A vaccine is mainly based on the extracellular domain M2e (Ectodomain of matrix protein 2) of influenza virus, which is highly conserved among influenza viruses and can induce the production of specific protective antibodies. Multiple cytotoxic T lymphocyte (CTL) epitopes can induce cellular immunity. However, M2e and NP epitopes are small in molecular weight, low in immunogenicity, easy to degrade, and difficult to produce sufficient immune response in vivo. Therefore, the use of complex viral proteins as immunogenic amplification vectors has become a hot topic in epitope vaccine research and development. Hepatitis B virus core protein (HBc) has a natural ability to assemble particles, which can specifically stimulate humoral and cellular immunity against exogenous epitopes. In recent years, there have been more mature research results in the application of HBc particles. Pro-HPV E7 protein, group B streptococcal CPS glycoprotein and so on have been observed to be effective in preclinical studies. Therefore, the development of influenza virus epitope vaccine with HBc particles as carriers has a good prospect and application value.
[Objective]
The aim of this study is to construct a universal influenza A vaccine antigen based on M2e protein with hepatitis B virus core protein (HBc) as carrier, express, purify and identify the influenza A vaccine antigen by baculovirus expression system and E. coli expression system, and preliminarily evaluate the immune effect of the vaccine, so as to develop a safe, effective and widely protective influenza A vaccine. The universal vaccine is the foundation.
[methods and results]
The first part is the construction, expression, purification and characterization of the M2e vaccine antigen expressed by Sf9 insect cells. The 3M2e-HBc-pFastBacHTA recombinant plasmid containing M2e repeat fragment was constructed by genetic engineering. The recombinant plasmid was homologously recombined with the shuttle vector Bacmid in DH10Bac to obtain 3M2e-HBc-Bacmid recombinant plasmid. The recombinant protein 3M2e-HBc was successfully expressed by immunofluorescence assay. Western blot assay showed that the protein was immunocompetent. The recombinant protein 3M2e-HBc was purified by affinity chromatography with the histidine tag on the pFastBacHTA vector. The results showed that the target gene of 3M2e-HBc could be expressed in baculovirus Sf9 insect cell expression system and assembled into virus-like particles correctly.
In the second part, the recombinant expression plasmid 3M2e-NP-HBc-pET 21a was constructed and identified by genetic engineering and transformed into E.coli cells. The recombinant protein expressed in the form of inclusion body was first obtained by IPTG induction. The recombinant protein of 3M2e-NP-HBc was purified by chromatography. Electron microscopy showed that the recombinant protein of 3M2e-NP-HBc was purified from the colon stem. The soluble recombinant protein expressed by the bacteria can be automatically assembled into a virus like particle size of about 30nm.
The third part is a preliminary evaluation of the immune effect of universal influenza A vaccine.
The immune response and cross-protection of the universal influenza A vaccine in Balb/C mice were preliminarily evaluated by animal immune test. The immune response of the vaccine in Balb/C mice was evaluated by indirect ELISA, flow cytometry and ELISOPT techniques. Both intranasal and intraperitoneal immunization can induce specific humoral and cellular immune responses to different subtypes of influenza viruses, and intranasal immunization can stimulate higher local mucosal immune responses, suggesting that intranasal immunization is a more natural route for influenza virus infection. The protective effect of high influenza vaccine is very important. Meanwhile, the results of virus attack test showed that the mortality of mice in the vaccine immunized group was significantly lower than that in the control group after two typical strains of influenza A virus (A/Beijing/501 and A/PR/8/34) were attacked. Immunogenic cross protection.
[Conclusion]
In this study, Bac-to-Bac baculovirus Sf9 insect cell expression system and E. The vaccine has good immunogenicity, can stimulate the body to produce systemic immune response and local mucosal immune response, and can produce effective cross-protection, which provides a theoretical basis for the further study of influenza A 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】：2010
【分类号】：R392.1
本文编号：2229479
[Abstract]:[background]
Influenza virus (Influenza virus) is a kind of Orthomyxovirus which can cause severe respiratory diseases. According to the antigenicity of nucleoprotein (NP) and matrix protein (M), Influenza virus can be divided into three types: A (A), B (B) and C (C). Influenza A virus has caused a wide range of pandemics, and there were four major outbreaks in the last century. What is most serious is the large influenza in 1918, which resulted in the death of more than 5 million people worldwide. The outbreak of swine influenza A * virus in the world in.2009 was epidemic, infectious and spread widely. It once again highlighted the seriousness of influenza A virus infection and its enormous economic burden on human society.
Vaccination is the most effective way to prevent the occurrence and spread of influenza. At present, there are three kinds of influenza vaccines in the market: whole virus inactivated vaccine, lysis vaccine and subunit vaccine. The World Health Organization (WHO) predicts and recommends the components for the next year's influenza vaccine production based on changes in influenza viruses worldwide. The accuracy of the prediction will directly affect the efficiency of vaccine protection, such as the failure of the prediction. Therefore, it is of great practical significance to develop a universal vaccine with extensive protective effect in dealing with the sudden outbreak of influenza virus mutation, rapidly establishing a population immune barrier, blocking the spread of influenza pandemic and reducing its harmfulness.
The development of universal influenza A vaccine is mainly based on the extracellular domain M2e (Ectodomain of matrix protein 2) of influenza virus, which is highly conserved among influenza viruses and can induce the production of specific protective antibodies. Multiple cytotoxic T lymphocyte (CTL) epitopes can induce cellular immunity. However, M2e and NP epitopes are small in molecular weight, low in immunogenicity, easy to degrade, and difficult to produce sufficient immune response in vivo. Therefore, the use of complex viral proteins as immunogenic amplification vectors has become a hot topic in epitope vaccine research and development. Hepatitis B virus core protein (HBc) has a natural ability to assemble particles, which can specifically stimulate humoral and cellular immunity against exogenous epitopes. In recent years, there have been more mature research results in the application of HBc particles. Pro-HPV E7 protein, group B streptococcal CPS glycoprotein and so on have been observed to be effective in preclinical studies. Therefore, the development of influenza virus epitope vaccine with HBc particles as carriers has a good prospect and application value.
[Objective]
The aim of this study is to construct a universal influenza A vaccine antigen based on M2e protein with hepatitis B virus core protein (HBc) as carrier, express, purify and identify the influenza A vaccine antigen by baculovirus expression system and E. coli expression system, and preliminarily evaluate the immune effect of the vaccine, so as to develop a safe, effective and widely protective influenza A vaccine. The universal vaccine is the foundation.
[methods and results]
The first part is the construction, expression, purification and characterization of the M2e vaccine antigen expressed by Sf9 insect cells. The 3M2e-HBc-pFastBacHTA recombinant plasmid containing M2e repeat fragment was constructed by genetic engineering. The recombinant plasmid was homologously recombined with the shuttle vector Bacmid in DH10Bac to obtain 3M2e-HBc-Bacmid recombinant plasmid. The recombinant protein 3M2e-HBc was successfully expressed by immunofluorescence assay. Western blot assay showed that the protein was immunocompetent. The recombinant protein 3M2e-HBc was purified by affinity chromatography with the histidine tag on the pFastBacHTA vector. The results showed that the target gene of 3M2e-HBc could be expressed in baculovirus Sf9 insect cell expression system and assembled into virus-like particles correctly.
In the second part, the recombinant expression plasmid 3M2e-NP-HBc-pET 21a was constructed and identified by genetic engineering and transformed into E.coli cells. The recombinant protein expressed in the form of inclusion body was first obtained by IPTG induction. The recombinant protein of 3M2e-NP-HBc was purified by chromatography. Electron microscopy showed that the recombinant protein of 3M2e-NP-HBc was purified from the colon stem. The soluble recombinant protein expressed by the bacteria can be automatically assembled into a virus like particle size of about 30nm.
The third part is a preliminary evaluation of the immune effect of universal influenza A vaccine.
The immune response and cross-protection of the universal influenza A vaccine in Balb/C mice were preliminarily evaluated by animal immune test. The immune response of the vaccine in Balb/C mice was evaluated by indirect ELISA, flow cytometry and ELISOPT techniques. Both intranasal and intraperitoneal immunization can induce specific humoral and cellular immune responses to different subtypes of influenza viruses, and intranasal immunization can stimulate higher local mucosal immune responses, suggesting that intranasal immunization is a more natural route for influenza virus infection. The protective effect of high influenza vaccine is very important. Meanwhile, the results of virus attack test showed that the mortality of mice in the vaccine immunized group was significantly lower than that in the control group after two typical strains of influenza A virus (A/Beijing/501 and A/PR/8/34) were attacked. Immunogenic cross protection.
[Conclusion]
In this study, Bac-to-Bac baculovirus Sf9 insect cell expression system and E. The vaccine has good immunogenicity, can stimulate the body to produce systemic immune response and local mucosal immune response, and can produce effective cross-protection, which provides a theoretical basis for the further study of influenza A 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】：2010
【分类号】：R392.1
【共引文献】
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