腸道病毒71型病毒樣顆粒的制備及其免疫原性的研究
發(fā)布時間：2018-07-27 11:18
【摘要】：腸道病毒71型(Enterovirus71, EV71)屬小RNA病毒科(Picornaviridae)腸道病毒屬(Entero virus),是手足口病(Hand-foot-and-mouth Disease, HFMD)的主要病原之一,其感染低齡兒童出現(xiàn)的嚴(yán)重神經(jīng)系統(tǒng)及心肺功能衰竭等綜合癥可引起較高的死亡率。自1974年首次報道以來,EV71已在世界范圍內(nèi)引起多次暴發(fā)與流行。EV71引起的神經(jīng)系統(tǒng)并發(fā)癥較其他腸道病毒多且病情嚴(yán)重,已成為備受關(guān)注的公共衛(wèi)生問題,目前因尚無抗EV71特效的藥物,EV71的持續(xù)爆發(fā)流行使得其疫苗的研發(fā)變得非常迫切。目前研究的疫苗形式有多種,和一些亞單位或DNA疫苗相比,滅活疫苗可誘導(dǎo)產(chǎn)生更高強(qiáng)度的特異性免疫應(yīng)答反應(yīng),但滅活疫苗始終存在著安全隱患,而由呈一定空間結(jié)構(gòu)的結(jié)構(gòu)蛋白組成的病毒樣顆粒(VLPs),其形態(tài)上與天然病毒粒子相似,且不含具有感染性的核酸,因此是一種極具開發(fā)潛力的候選疫苗。近年來,已有臺灣學(xué)者報道使用EV71VLPs聯(lián)合弗氏佐劑經(jīng)皮下注射免疫小鼠可刺激產(chǎn)生一定的中和抗體水平,并對新生小鼠起到一定的保護(hù)作用,這為此類疫苗的開發(fā)提供了一定的依據(jù)。但至今未曾有研究報道EV71VLPs疫苗免疫可對機(jī)體產(chǎn)生哪些具體的保護(hù)作用指標(biāo),因此,結(jié)合我國EV71的流行現(xiàn)狀,本研究以C4亞型毒株作為EV71疫苗的候選株,通過桿狀病毒-昆蟲細(xì)胞表達(dá)系統(tǒng)制備了EV71VLPs,利用小鼠模型以更低的免疫劑量聯(lián)合人用A1(OH)3佐劑進(jìn)行了動物實驗來評價該VLPs疫苗刺激產(chǎn)生的體液免疫和細(xì)胞免疫效果,并且,除了對該疫苗的有效性進(jìn)行了評估之外,本研究還通過組織器官包埋切片、HE染色和免疫組化等方法對小鼠各組織器官進(jìn)行了病理學(xué)和病原學(xué)的分析,更為深入的解釋了評估該候選疫苗的免疫原性和免疫保護(hù)性的意義和價值。結(jié)果表明,該EV71VLPs疫苗可在小鼠病毒感染過程中有效的阻止病毒在動物體內(nèi)的增殖,對小鼠可起到完全的保護(hù)效果,是一種很有開發(fā)前景的疫苗。 第一部分：腸道病毒71型病毒樣顆粒的制備、鑒定與純化 自1981年上海發(fā)現(xiàn)HFMD,其后在全國十幾個省份均有該疾病的報道,相關(guān)流行病學(xué)研究表明我國大陸自1998年以后流行的EV71均為C4亞型,因此本研究結(jié)合EV71流行現(xiàn)狀,選取C4亞型毒株做為EV71疫苗研制的候選株。通過提取病毒RNA, RT-PCR擴(kuò)增法分別獲得EV71P1和3CD蛋白的全長基因片段,并將EV71P1和3CD基因片段克隆入同一桿狀病毒穿梭質(zhì)粒Bacmid中,構(gòu)建出重組桿狀病毒表達(dá)質(zhì)粒Bacmid-P1-3CD;脂質(zhì)體介導(dǎo)其轉(zhuǎn)染Sf9昆蟲細(xì)胞獲得表達(dá)P1和3CD的重組桿狀病毒(AcMNPV-P1-3CD)。使用IFA和Western-blot對表達(dá)產(chǎn)物進(jìn)行鑒定和分析。透射電鏡結(jié)果顯示P1經(jīng)3CD切割成的3個蛋白亞基(VP1、VPO和VP3)自主裝配形成了直徑約為27nm的類球形顆粒(即EV71VLPs)。進(jìn)一步對表達(dá)條件進(jìn)行優(yōu)化,結(jié)果顯示MOI值和時間均可影響目的蛋白的表達(dá),其中時間為主要影響因素。選擇優(yōu)化后條件利用無血清培養(yǎng)基對貼壁Sf9細(xì)胞在多層細(xì)胞培養(yǎng)器中進(jìn)行VLPs的大量表達(dá),密度梯度超速離心法純化后,SDS-PAGE分析可見三條大小約為39kD,34kD和26kD的VP1、VPO和VP3特異性條帶。超速離心純化后,電鏡結(jié)果顯示EV71VLPs顆粒結(jié)構(gòu)完好,可用于后期動物免疫實驗及其免疫效果的進(jìn)一步評價。 第二部分：腸道病毒71型病毒樣顆粒免疫原性的研究 對所制備的EV71VLPs免疫原性及其免疫效果進(jìn)行探討,以4～5周齡雌性ICR小鼠為動物模型,分為EV71VLPs組、滅活EV71病毒組和PBS對照，按5μg/只的抗原劑量配伍A1(OH)3佐劑以肌肉注射的方式在第0天、14天和28天進(jìn)行三次免疫。通過檢測EV71特異性抗體、中和抗體滴度和T細(xì)胞免疫反應(yīng)(ELISPOT法)來探討EV71VLPs刺激機(jī)體所產(chǎn)生的細(xì)胞和體液免疫效果,并通過對免疫后小鼠配種所生產(chǎn)乳鼠病毒攻擊實驗來評價EV71VLPs疫苗對小鼠產(chǎn)生的被動保護(hù)作用。此外,對被動免疫保護(hù)實驗小鼠進(jìn)行組織器官包埋切片,通過HE染色、免疫組化等試驗進(jìn)行進(jìn)一步病理損傷和病原學(xué)分析,以揭示該VLPs疫苗對小鼠產(chǎn)生的保護(hù)作用機(jī)制。 研究結(jié)果顯示,在體液免疫水平方面,EV71VLPs和滅活EV71病毒均能刺激機(jī)體產(chǎn)生基本一致水平的EV71特異性抗體(包括總IgG抗體和粘膜部位sIgA抗體),但對同源病毒的中和能力方面,EV71VLPs免疫組略低于滅活EV71免疫組。在細(xì)胞免疫水平方面,EV71VLPs可刺激小鼠機(jī)體產(chǎn)生較好的細(xì)胞免疫反應(yīng)：刺激機(jī)體產(chǎn)生相同水平的EV71特異性IFN-y分泌T淋巴細(xì)胞免疫反應(yīng),并高于滅活EV71組的EV71特異性IL-4分泌T淋巴細(xì)胞數(shù)目,結(jié)合其誘導(dǎo)機(jī)體產(chǎn)生IgG亞型的檢測結(jié)果可以推測EV71VLPs免疫能同時引起機(jī)體Th1和Th2型細(xì)胞免疫反應(yīng)。對脾臟中特異性sIgA抗體分泌記憶性B細(xì)胞的檢測,也說明EV71VLPs和滅活EV71病毒可誘發(fā)機(jī)體產(chǎn)生相同水平的特異性sIgA抗體分泌記憶性B細(xì)胞免疫反應(yīng)效果。 被動保護(hù)實驗表明與對照組相比,經(jīng)疫苗免疫組小鼠均未出現(xiàn)對照組動物所具有的特征性臨床表現(xiàn),病理學(xué)檢測結(jié)果顯示,除骨骼肌組織呈現(xiàn)出輕度的肌纖維萎縮外,其余易感組織器官(腸,心肌,肺)均無異常；器官病原學(xué)分布與病理表現(xiàn)相一致,除免疫組小鼠除骨骼肌組織可檢測到微量的病毒抗原外,其余器官病原學(xué)分布皆呈陰性。而免疫組小鼠骨骼肌組織中所呈現(xiàn)出伴隨輕度肌纖維再生引起的肌細(xì)胞核線性排列,預(yù)示著小鼠正處于從病毒感染中恢復(fù)的狀態(tài)。以上結(jié)果均說明經(jīng)EV71VLPs誘導(dǎo)機(jī)體產(chǎn)生的免疫應(yīng)答反應(yīng)可明顯抑制病毒在小鼠體內(nèi)一些對病毒易感的組織器官中的增殖,在很大程度上能夠阻止病毒的感染,并促使小鼠在經(jīng)病毒感染后呈現(xiàn)出快速的自愈康復(fù)狀態(tài),可有效的保護(hù)機(jī)體免受病毒感染所引起的病理損害,進(jìn)而對小鼠產(chǎn)生良好的保護(hù)作用。至此,本研究證實了該EV71VLPs疫苗是一種很有開發(fā)前景的候選疫苗,為EV71新型基因工程疫苗的研制提供了重要的實驗依據(jù)和基礎(chǔ)。
[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.
【學(xué)位授予單位】：中國疾病預(yù)防控制中心
【學(xué)位級別】：博士
【學(xué)位授予年份】：2012
【分類號】：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.
【學(xué)位授予單位】：中國疾病預(yù)防控制中心
【學(xué)位級別】：博士
【學(xué)位授予年份】：2012
【分類號】：R392
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