肠道病毒71型病毒样颗粒的制备及其免疫原性的研究
发布时间：2018-07-27 11:18
【摘要】：肠道病毒71型(Enterovirus71, EV71)属小RNA病毒科(Picornaviridae)肠道病毒属(Entero virus),是手足口病(Hand-foot-and-mouth Disease, HFMD)的主要病原之一,其感染低龄儿童出现的严重神经系统及心肺功能衰竭等综合症可引起较高的死亡率。自1974年首次报道以来,EV71已在世界范围内引起多次暴发与流行。EV71引起的神经系统并发症较其他肠道病毒多且病情严重,已成为备受关注的公共卫生问题,目前因尚无抗EV71特效的药物,EV71的持续爆发流行使得其疫苗的研发变得非常迫切。目前研究的疫苗形式有多种,和一些亚单位或DNA疫苗相比,灭活疫苗可诱导产生更高强度的特异性免疫应答反应,但灭活疫苗始终存在着安全隐患,而由呈一定空间结构的结构蛋白组成的病毒样颗粒(VLPs),其形态上与天然病毒粒子相似,且不含具有感染性的核酸,因此是一种极具开发潜力的候选疫苗。近年来,已有台湾学者报道使用EV71VLPs联合弗氏佐剂经皮下注射免疫小鼠可刺激产生一定的中和抗体水平,并对新生小鼠起到一定的保护作用,这为此类疫苗的开发提供了一定的依据。但至今未曾有研究报道EV71VLPs疫苗免疫可对机体产生哪些具体的保护作用指标,因此,结合我国EV71的流行现状,本研究以C4亚型毒株作为EV71疫苗的候选株,通过杆状病毒-昆虫细胞表达系统制备了EV71VLPs,利用小鼠模型以更低的免疫剂量联合人用A1(OH)3佐剂进行了动物实验来评价该VLPs疫苗刺激产生的体液免疫和细胞免疫效果,并且,除了对该疫苗的有效性进行了评估之外,本研究还通过组织器官包埋切片、HE染色和免疫组化等方法对小鼠各组织器官进行了病理学和病原学的分析,更为深入的解释了评估该候选疫苗的免疫原性和免疫保护性的意义和价值。结果表明,该EV71VLPs疫苗可在小鼠病毒感染过程中有效的阻止病毒在动物体内的增殖,对小鼠可起到完全的保护效果,是一种很有开发前景的疫苗。 第一部分：肠道病毒71型病毒样颗粒的制备、鉴定与纯化 自1981年上海发现HFMD,其后在全国十几个省份均有该疾病的报道,相关流行病学研究表明我国大陆自1998年以后流行的EV71均为C4亚型,因此本研究结合EV71流行现状,选取C4亚型毒株做为EV71疫苗研制的候选株。通过提取病毒RNA, RT-PCR扩增法分别获得EV71P1和3CD蛋白的全长基因片段,并将EV71P1和3CD基因片段克隆入同一杆状病毒穿梭质粒Bacmid中,构建出重组杆状病毒表达质粒Bacmid-P1-3CD;脂质体介导其转染Sf9昆虫细胞获得表达P1和3CD的重组杆状病毒(AcMNPV-P1-3CD)。使用IFA和Western-blot对表达产物进行鉴定和分析。透射电镜结果显示P1经3CD切割成的3个蛋白亚基(VP1、VPO和VP3)自主装配形成了直径约为27nm的类球形颗粒(即EV71VLPs)。进一步对表达条件进行优化,结果显示MOI值和时间均可影响目的蛋白的表达,其中时间为主要影响因素。选择优化后条件利用无血清培养基对贴壁Sf9细胞在多层细胞培养器中进行VLPs的大量表达,密度梯度超速离心法纯化后,SDS-PAGE分析可见三条大小约为39kD,34kD和26kD的VP1、VPO和VP3特异性条带。超速离心纯化后,电镜结果显示EV71VLPs颗粒结构完好,可用于后期动物免疫实验及其免疫效果的进一步评价。 第二部分：肠道病毒71型病毒样颗粒免疫原性的研究 对所制备的EV71VLPs免疫原性及其免疫效果进行探讨,以4～5周龄雌性ICR小鼠为动物模型,分为EV71VLPs组、灭活EV71病毒组和PBS对照，按5μg/只的抗原剂量配伍A1(OH)3佐剂以肌肉注射的方式在第0天、14天和28天进行三次免疫。通过检测EV71特异性抗体、中和抗体滴度和T细胞免疫反应(ELISPOT法)来探讨EV71VLPs刺激机体所产生的细胞和体液免疫效果,并通过对免疫后小鼠配种所生产乳鼠病毒攻击实验来评价EV71VLPs疫苗对小鼠产生的被动保护作用。此外,对被动免疫保护实验小鼠进行组织器官包埋切片,通过HE染色、免疫组化等试验进行进一步病理损伤和病原学分析,以揭示该VLPs疫苗对小鼠产生的保护作用机制。 研究结果显示,在体液免疫水平方面,EV71VLPs和灭活EV71病毒均能刺激机体产生基本一致水平的EV71特异性抗体(包括总IgG抗体和粘膜部位sIgA抗体),但对同源病毒的中和能力方面,EV71VLPs免疫组略低于灭活EV71免疫组。在细胞免疫水平方面,EV71VLPs可刺激小鼠机体产生较好的细胞免疫反应：刺激机体产生相同水平的EV71特异性IFN-y分泌T淋巴细胞免疫反应,并高于灭活EV71组的EV71特异性IL-4分泌T淋巴细胞数目,结合其诱导机体产生IgG亚型的检测结果可以推测EV71VLPs免疫能同时引起机体Th1和Th2型细胞免疫反应。对脾脏中特异性sIgA抗体分泌记忆性B细胞的检测,也说明EV71VLPs和灭活EV71病毒可诱发机体产生相同水平的特异性sIgA抗体分泌记忆性B细胞免疫反应效果。 被动保护实验表明与对照组相比,经疫苗免疫组小鼠均未出现对照组动物所具有的特征性临床表现,病理学检测结果显示,除骨骼肌组织呈现出轻度的肌纤维萎缩外,其余易感组织器官(肠,心肌,肺)均无异常；器官病原学分布与病理表现相一致,除免疫组小鼠除骨骼肌组织可检测到微量的病毒抗原外,其余器官病原学分布皆呈阴性。而免疫组小鼠骨骼肌组织中所呈现出伴随轻度肌纤维再生引起的肌细胞核线性排列,预示着小鼠正处于从病毒感染中恢复的状态。以上结果均说明经EV71VLPs诱导机体产生的免疫应答反应可明显抑制病毒在小鼠体内一些对病毒易感的组织器官中的增殖,在很大程度上能够阻止病毒的感染,并促使小鼠在经病毒感染后呈现出快速的自愈康复状态,可有效的保护机体免受病毒感染所引起的病理损害,进而对小鼠产生良好的保护作用。至此,本研究证实了该EV71VLPs疫苗是一种很有开发前景的候选疫苗,为EV71新型基因工程疫苗的研制提供了重要的实验依据和基础。
[Abstract]:Enterovirus 71 (Enterovirus71, EV71) belongs to the small RNA virus family (Picornaviridae) enterovirus (Entero virus) and is one of the main pathogens of hand foot and mouth disease (Hand-foot-and-mouth Disease, HFMD). The severe nervous system and cardiopulmonary failure syndrome in the infected children of low age children can cause high mortality. Since 1974, it has been the first time. Since the report, EV71 has caused many outbreak and epidemic of.EV71 caused by the epidemic of nerve system more than other enterovirus and serious disease. It has become a public health problem which has attracted much attention. At present, there is no special drug against EV71. The continuous outbreak of EV71 has made the research and development of the vaccine become very urgent. Compared with some subunits or DNA vaccines, the inactivated vaccine can induce a higher specific immune response, but the inactivated vaccine always has a safety hidden danger, and the virus like particle (VLPs), which is composed of structural protein of a certain spatial structure, is similar to that of the natural virus particles. In recent years, Taiwan scholars have reported that the use of EV71VLPs combined with Freund's adjuvant to immunize mice can stimulate a certain level of neutralization antibody and protect the newborn mice, which provides a new vaccine for the development of this kind of vaccine. But it has not been reported that EV71VLPs vaccine immunization can produce specific protective effects on the body. Therefore, combined with the current status of EV71 in our country, this study takes C4 subtype as a candidate for EV71 vaccine, and has prepared EV71VLPs by baculovirus insect cell expression system, and using mouse model to lower EV71VLPs. The immunization dose combined with human A1 (OH) 3 adjuvant was carried out to evaluate the humoral and cellular immune effects of the VLPs vaccine. Besides, in addition to the evaluation of the effectiveness of the vaccine, the tissues and organs of the mice were carried out by tissue and organ embedding, HE staining and immunohistochemistry. The analysis of pathology and etiology has explained the significance and value of the immunogenicity and immunogenicity of the candidate vaccine. The results show that the EV71VLPs vaccine can effectively prevent the proliferation of the virus in the animal in the process of virus infection in mice, and it can be completely protected in mice. It is a kind of development. The foreground vaccine.
Part one: preparation, identification and purification of enterovirus 71 virus like particles
Since the discovery of HFMD in Shanghai in 1981 and subsequent reports of the disease in more than a dozen provinces throughout the country, related epidemiological studies have shown that EV71 is C4 subtype in mainland China since 1998. Therefore, this study combines the status of EV71 epidemic and selects the C4 subtype as a candidate for EV71 vaccine research. By extracting virus RNA, RT-PCR amplification method The full length gene fragment of EV71P1 and 3CD protein was obtained, and the EV71P1 and 3CD gene fragments were cloned into the same baculovirus shuttle plasmid Bacmid to construct the recombinant baculovirus expression plasmid Bacmid-P1-3CD. Liposomes mediate the transfection of Sf9 insect cells to obtain a heavy group of baculovirus expressing P1 and 3CD (AcMNPV-P1-3CD). IFA and Western were used. -blot was used to identify and analyze the expression products. The transmission electron microscope showed that the 3 protein subunits (VP1, VPO and VP3) made by 3CD were assembled to form spherical particles (i.e. EV71VLPs) with a diameter of about 27nm, and the expression conditions were optimized. The results showed that the expression of the target protein could be affected by the MOI value and time. The main influencing factors. After choosing the optimized condition, the VLPs was expressed in a serum-free medium for the adherent Sf9 cells in the multi-layer cell culture. After the density gradient centrifugation was purified, the SDS-PAGE analysis showed that the VP1, VPO and VP3 specific bands of three sizes were about 39kD, 34kD and 26kD. After the ultra speed centrifugation, the electron microscope results showed E. The structure of V71VLPs particles is intact, which can be used for further animal immune experiments and further evaluation of immune effects.
The second part: immunogenicity of enterovirus 71 virus like particles.
The immunogenicity and immune effect of the prepared EV71VLPs were discussed. 4~5 weeks old female ICR mice were used as animal models, divided into EV71VLPs group, inactivated EV71 virus group and PBS control. Three doses of A1 (OH) 3 adjuvant were combined with 5 mu g/ only with A1 (OH) 3 adjuvant. The specificity of EV71 specificity was detected by the detection of EV71 specificity. The antibody, neutralizing antibody titer and T cell immune response (ELISPOT method) were used to investigate the cellular and humoral immune effects of EV71VLPs stimulated by the body, and to evaluate the passive protective effect of the EV71VLPs vaccine on the mice by the attack experiment on the mouse virus produced by the immunized mice. In order to reveal the protective mechanism of the VLPs vaccine on mice, the tissue and organ embedded slices were further studied by HE staining and immunohistochemistry.
The results show that both EV71VLPs and inactivated EV71 virus can stimulate the basic level of EV71 specific antibodies (including the total IgG antibody and the sIgA antibody of the mucous membrane), but the EV71VLPs immune group is slightly lower than the inactivated EV71 immune group, E, E, and E. V71VLPs stimulates a better cellular immune response in the mice: stimulating the body to produce the same level of EV71 specific IFN-y secreting T lymphocyte immune response, and higher than the EV71 specific IL-4 secreting the number of T lymphocytes in the inactivated EV71 group. Combined with the results of the detection of the IgG subtype induced by the organism, the immunization of EV71VLPs can be conjectured. Th1 and Th2 type cell immune responses were induced in the body. The detection of memory B cells secreted by specific sIgA antibodies in the spleen also indicated that EV71VLPs and inactivated EV71 virus could induce the same level of specific sIgA antibody to secrete memory B cell immune response of the body.
The passive protection experiment showed that compared with the control group, all the mice in the immunized group did not have the characteristic clinical manifestations of the control group, and the pathological examination showed that the other susceptible tissues (intestines, heart muscles and lungs) were not abnormality except that the skeletal muscle tissue showed mild muscle fiber atrophy; the distribution of organ pathogeny and pathological table were found. In addition, the distribution of the other organs in the immune group was negative, except in the immune group, except in the skeletal muscle tissue. In the immune group, the muscle cells in the skeletal muscle of the immune group showed the linear arrangement of the muscle nuclei associated with the regeneration of the mild muscle fibers, indicating that the rat was in the state of recovering from the virus infection. The results show that the immune response induced by EV71VLPs can obviously inhibit the proliferation of some virus susceptible tissues and organs in mice, to a great extent, prevent the virus infection, and promote the rapid self healing state of the mice after the virus infection, which can effectively protect the body from the body. The pathological damage caused by virus infection and the good protective effect on mice. This study confirms that the EV71VLPs vaccine is a promising candidate vaccine and provides important experimental basis and basis for the development of EV71 new genetic engineering vaccine.
【学位授予单位】：中国疾病预防控制中心
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R392
[Abstract]:Enterovirus 71 (Enterovirus71, EV71) belongs to the small RNA virus family (Picornaviridae) enterovirus (Entero virus) and is one of the main pathogens of hand foot and mouth disease (Hand-foot-and-mouth Disease, HFMD). The severe nervous system and cardiopulmonary failure syndrome in the infected children of low age children can cause high mortality. Since 1974, it has been the first time. Since the report, EV71 has caused many outbreak and epidemic of.EV71 caused by the epidemic of nerve system more than other enterovirus and serious disease. It has become a public health problem which has attracted much attention. At present, there is no special drug against EV71. The continuous outbreak of EV71 has made the research and development of the vaccine become very urgent. Compared with some subunits or DNA vaccines, the inactivated vaccine can induce a higher specific immune response, but the inactivated vaccine always has a safety hidden danger, and the virus like particle (VLPs), which is composed of structural protein of a certain spatial structure, is similar to that of the natural virus particles. In recent years, Taiwan scholars have reported that the use of EV71VLPs combined with Freund's adjuvant to immunize mice can stimulate a certain level of neutralization antibody and protect the newborn mice, which provides a new vaccine for the development of this kind of vaccine. But it has not been reported that EV71VLPs vaccine immunization can produce specific protective effects on the body. Therefore, combined with the current status of EV71 in our country, this study takes C4 subtype as a candidate for EV71 vaccine, and has prepared EV71VLPs by baculovirus insect cell expression system, and using mouse model to lower EV71VLPs. The immunization dose combined with human A1 (OH) 3 adjuvant was carried out to evaluate the humoral and cellular immune effects of the VLPs vaccine. Besides, in addition to the evaluation of the effectiveness of the vaccine, the tissues and organs of the mice were carried out by tissue and organ embedding, HE staining and immunohistochemistry. The analysis of pathology and etiology has explained the significance and value of the immunogenicity and immunogenicity of the candidate vaccine. The results show that the EV71VLPs vaccine can effectively prevent the proliferation of the virus in the animal in the process of virus infection in mice, and it can be completely protected in mice. It is a kind of development. The foreground vaccine.
Part one: preparation, identification and purification of enterovirus 71 virus like particles
Since the discovery of HFMD in Shanghai in 1981 and subsequent reports of the disease in more than a dozen provinces throughout the country, related epidemiological studies have shown that EV71 is C4 subtype in mainland China since 1998. Therefore, this study combines the status of EV71 epidemic and selects the C4 subtype as a candidate for EV71 vaccine research. By extracting virus RNA, RT-PCR amplification method The full length gene fragment of EV71P1 and 3CD protein was obtained, and the EV71P1 and 3CD gene fragments were cloned into the same baculovirus shuttle plasmid Bacmid to construct the recombinant baculovirus expression plasmid Bacmid-P1-3CD. Liposomes mediate the transfection of Sf9 insect cells to obtain a heavy group of baculovirus expressing P1 and 3CD (AcMNPV-P1-3CD). IFA and Western were used. -blot was used to identify and analyze the expression products. The transmission electron microscope showed that the 3 protein subunits (VP1, VPO and VP3) made by 3CD were assembled to form spherical particles (i.e. EV71VLPs) with a diameter of about 27nm, and the expression conditions were optimized. The results showed that the expression of the target protein could be affected by the MOI value and time. The main influencing factors. After choosing the optimized condition, the VLPs was expressed in a serum-free medium for the adherent Sf9 cells in the multi-layer cell culture. After the density gradient centrifugation was purified, the SDS-PAGE analysis showed that the VP1, VPO and VP3 specific bands of three sizes were about 39kD, 34kD and 26kD. After the ultra speed centrifugation, the electron microscope results showed E. The structure of V71VLPs particles is intact, which can be used for further animal immune experiments and further evaluation of immune effects.
The second part: immunogenicity of enterovirus 71 virus like particles.
The immunogenicity and immune effect of the prepared EV71VLPs were discussed. 4~5 weeks old female ICR mice were used as animal models, divided into EV71VLPs group, inactivated EV71 virus group and PBS control. Three doses of A1 (OH) 3 adjuvant were combined with 5 mu g/ only with A1 (OH) 3 adjuvant. The specificity of EV71 specificity was detected by the detection of EV71 specificity. The antibody, neutralizing antibody titer and T cell immune response (ELISPOT method) were used to investigate the cellular and humoral immune effects of EV71VLPs stimulated by the body, and to evaluate the passive protective effect of the EV71VLPs vaccine on the mice by the attack experiment on the mouse virus produced by the immunized mice. In order to reveal the protective mechanism of the VLPs vaccine on mice, the tissue and organ embedded slices were further studied by HE staining and immunohistochemistry.
The results show that both EV71VLPs and inactivated EV71 virus can stimulate the basic level of EV71 specific antibodies (including the total IgG antibody and the sIgA antibody of the mucous membrane), but the EV71VLPs immune group is slightly lower than the inactivated EV71 immune group, E, E, and E. V71VLPs stimulates a better cellular immune response in the mice: stimulating the body to produce the same level of EV71 specific IFN-y secreting T lymphocyte immune response, and higher than the EV71 specific IL-4 secreting the number of T lymphocytes in the inactivated EV71 group. Combined with the results of the detection of the IgG subtype induced by the organism, the immunization of EV71VLPs can be conjectured. Th1 and Th2 type cell immune responses were induced in the body. The detection of memory B cells secreted by specific sIgA antibodies in the spleen also indicated that EV71VLPs and inactivated EV71 virus could induce the same level of specific sIgA antibody to secrete memory B cell immune response of the body.
The passive protection experiment showed that compared with the control group, all the mice in the immunized group did not have the characteristic clinical manifestations of the control group, and the pathological examination showed that the other susceptible tissues (intestines, heart muscles and lungs) were not abnormality except that the skeletal muscle tissue showed mild muscle fiber atrophy; the distribution of organ pathogeny and pathological table were found. In addition, the distribution of the other organs in the immune group was negative, except in the immune group, except in the skeletal muscle tissue. In the immune group, the muscle cells in the skeletal muscle of the immune group showed the linear arrangement of the muscle nuclei associated with the regeneration of the mild muscle fibers, indicating that the rat was in the state of recovering from the virus infection. The results show that the immune response induced by EV71VLPs can obviously inhibit the proliferation of some virus susceptible tissues and organs in mice, to a great extent, prevent the virus infection, and promote the rapid self healing state of the mice after the virus infection, which can effectively protect the body from the body. The pathological damage caused by virus infection and the good protective effect on mice. This study confirms that the EV71VLPs vaccine is a promising candidate vaccine and provides important experimental basis and basis for the development of EV71 new genetic engineering vaccine.
【学位授予单位】：中国疾病预防控制中心
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R392
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