TW201100545A - Method for preparing enterovirus virus-like particle and use thereof - Google Patents

Method for preparing enterovirus virus-like particle and use thereof Download PDF

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TW201100545A
TW201100545A TW098127203A TW98127203A TW201100545A TW 201100545 A TW201100545 A TW 201100545A TW 098127203 A TW098127203 A TW 098127203A TW 98127203 A TW98127203 A TW 98127203A TW 201100545 A TW201100545 A TW 201100545A
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virus
baculovirus
promoter
protein
enterovirus
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TW098127203A
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Chinese (zh)
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Yu-Chen Hu
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Nat Univ Tsing Hua
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Abstract

The present invention is related to a method for preparing enterovirus 71 virus-like particle in the baculovirus/insect cell expression system and use thereof. By modifying the baculovirus vector design, the present invention lowers the expression level of protease 3CD and increases the expression level of the capsid protein precursor P1 so as to increase VLP yield. These VLPs can elicit high antibody titers and neutralization titers responses in mice, demonstrating to be as an enterovirus 71 vaccine.

Description

201100545 六、發明說明: 【發明所屬之技術領域】 本發明係關於應用桿狀病毒重組蛋白表現系統來製備腸病毒71型類病 毒顆粒、製造方法及其應用。 【先前技術】 腸病毒(Enterovirus)在分類上屬於微小核糖核酸病毒科 (Picomaviridae) ’ 為單正股(p0Sitive,singie strand) RNA 病毒’感染途 徑主要經由糞便或呼吸道飛沫傳染,能夠感染人體多種系統器官,甚 至造成器官功能衰竭的現象。傳統上使用中和血清試驗可將腸病毒分 為脊趫灰白質炎病毒型(p〇li〇vjmses , PV),克沙奇病毒a型 (Coxsackieviruses A,CAV),克沙奇病毒 B 型(Coxsackieviruses 2B, CBV) ’人類腸道細胞致病性孤兒病毒(Enteric cytopathogenic human orphan viruses ’即伊科病毒Ech〇viruses),及68_71型腸病毒等。由於 近年來基因定序方法的進步,腸病毒分類轉而分為A_D四種類型,目 前共有超過一百種以上的灰清型。腸病毒71型恤_丨挪71,EV7i) 疋目刖已發現至少1〇〇種血清型腸病毒當中的一種。根據其基因分子 定序的差異,又可分為A、B、C三群基因型,其中又可細分為A bi b5、201100545 VI. Description of the Invention: [Technical Field] The present invention relates to a method for producing an enterovirus 71 type virus particle by using a baculovirus recombinant protein expression system, a method for producing the same, and an application thereof. [Prior Art] Enterovirus is classified as Picomaviridae 'p0Sitive, singie strand RNA virus' infection is mainly transmitted through feces or respiratory droplets, which can infect various systems of the human body. Organs, even the phenomenon of organ failure. Traditionally, neutralizing serum tests can be used to divide enterovirus into poliovirus type (p〇li〇vjmses, PV), Croxackieviruses A (CAV), and kesaki virus type B ( Coxsackieviruses 2B, CBV) 'Enteric cytopathogenic human orphan viruses' (Ek〇viruses), and enterovirus type 68_71. Due to advances in gene sequencing methods in recent years, enterovirus classification has been subdivided into four types of A_D, and there are currently more than one hundred ash-clear types. Enterovirus 71 shirt _ 丨 71 71, EV7i) 疋 刖 has found at least one of the serotypes of enterovirus. According to the differences in the sequencing of their genetic molecules, they can be divided into three groups: A, B, and C, which can be subdivided into A bi b5.

Cl C5 ’不同的亞型與其病毒基因演化及地域性相關。在腸病毒各血清 型中,腸病毒71型的致病力特別高,常伴隨著神經系統的併發症且有 致死的可能。 201100545 腸病毒71型病毒顆粒是直徑約為况7nm的二十面體聚合大分子,但 其詳細組鱗魏尚未清楚研究。目前對腸病毒的了駐要來自脊聽 灰白質炎峨科絲痺財)。_、絲痺病毒的研究 ,腸病毒 基因組王長約7500nt ’包括單—開放讀碼框架_ fr継, ORF約6600m),兩側則分別為5,與3,未轉譯區(她肌齡^邮⑽, UTR)。此0RF可分為三區域:朽,p2及p3,並會被轉譯為單一的多 蛋白(P〇lypr〇tein)。P1 帶有病毒殼蛋白:VP4,W2,VP3 及·。P2 及p3則帶有非結構性蛋白質如水解蛋白(pr〇tease) 2A、3C、與 3CD, 以及病毒聚合梅3DP ’主要負責病毒的複製並產生病毒毒性。以小兒 麻痒病毒為例’腸病毒顆粒理論上是由6Q個聰及Vm59個 及綱,與Μ個W0分子(VP2及未分開前的前趨物)所形成。 VP卜VP2與VP3均位於鱗殼外層,而侧則完全位於内層。對目 剛已研究較多的腸病毒,如小兒麻痺病毒、CAV與CBV,已有許多中 和抗原決定部位(neutralization determinants)被發現主要位於VP1另有 少數位於VP2與VP3。此外’這些結構蛋自質亦與病毒毒性減弱、病 毒熱穩定性、宿主範圍、細胞驅向性等有關。 昆蟲桿狀病毒(baculovims)—般簡稱為桿狀病毒,屬於桿狀病毒科 (Baculoviride),其宿主侷限於無脊椎動物,目前據報導已有6〇〇種以 上昆蟲可被感染’其中又以鱗翅目昆蟲為其主要宿主。根據2004年國 際病毒分類委員會(International Committee on Taxonomy of Virus)的最 新分類’桿狀病毒科可分成兩個屬,分別是顆粒體病毒屬 201100545 (Gnmulovims,GW人反极多苒體病备紙Nuck〇p〇lyhedr〇virus,刪) 組成,差別在於包埋體蛋白質的不同。前者的包埋體較小,約為 0.25-0.5μιη,只包埋一顆病毒粒子;後者較大,約為,主要由 大小約為29kD的多角體蛋白(poiyhecjron)所組成,可以包覆較多的病 毒粒子。NPV以其娜_染週誠基因表财式,可在穩定的細胞 株中培養,使其具有發展為基因表現載體的潛力。目前最常作為桿狀 病毒表現載體的病毒是苜蓿尺蠖峨椋多角體病毒(Aut〇graph cah:f〇mica O nuclear Polyhedrosis Vi⑽;AcMNPV)和家蠶核多角體病毒(及洲加 midear polyhedrosis virus ; BmNPV) ’其主要宿主分別為蛾類和家 蠶。 NPV的遺傳物質為雙股環狀結構的DNA,大小約8〇 2〇〇kb,由長桿狀 蛋白勒(nucleocapsid)包覆在内,外層再由脂蛋白被膜(Ηρ〇_^ envelope)所包覆,形成長約4〇_5〇nm,寬約2〇〇 4〇〇nm的病毒結構。 病毒在感染細胞後可產生出兩種型態的病毒粒子:出芽型病毒粒子 O^ded virion; BV)與封埋型病毒粒子(〇cdusi〇n b〇dy_derived 〇V)。出芽型病毒粒子產生於感染週期的早期,感染後新生的 nucleocapsid會由細胞核膜釋出到細胞質裡,並進一步穿出細胞膜;封 埋型病毒粒子則產生於感染週期的後期,且其會被包埋在大小約如心 的多角體蛋白質或晶形結構的蛋白f中’形成大小約的多角體 (polyhedron)或稱之為封埋體。而實驗室常用的桿狀病毒均為出芽型病 毒粒子。 201100545 桿狀病毒表現細建立於簡年,經過二十多年的改進、發展,目前 已廣泛在異源基因的表現系統上,為—雜快速、方便生產外源 重組蛋白質的系統。因此至今已逾數百種源自病毒、細胞、真菌、原 生物、以及動植物基因利用此系統生產出重組蛋白f。甚至一些較為 複雜的蛋白質結構,例如類病毒粒子,也可在宿主體内表現。 桿狀病毒表現載體系統是-種真核細胞財表現魏,料來基因接 於啟動子(pr_㈣的下游放入桿狀病毒中,再利用此重組桿狀病毒 (recombinant baCul〇virus)感染昆蟲細胞,病毒基因啟動子就可能會驅動 此-外來基因生產出重組蛋白質。而由於重組桿狀病毒所感染的昆蟲 細胞是-種真核細胞,因此終白質可進行各種轉譯後修飾 CpostA—odifieations) ’ 桿狀病毒表現_系統所生 產的真核基因重組蛋白質,在抗原性、免疫性和生物活性上通常與其 天然蛋白質相似。加上—般所使用的啟動子為桿狀病毒基因本身所具 有的強勢啟動子(如:P〇1yhedrin或是_啟動子),所以本系統所產出 的重組蛋白質產量相當大(1-600mg/L)。 除了可進練佳的_祕糾,制桿狀病毒祕纟統肖有以下優 點.(1)扣狀病毒具有相當大的基因組(88 200kbp),可容納相當大的外 來基因’使得同-株桿狀病毒可同時攜帶兩組以上的外源基因組。⑵ 因為重組;^狀病毒的宿域侷限於無脊椎動物,目此在安全性方面比 201100545 起其他基因載體,例如腺相關病毒(AAV)等,更為安全。(3)重組 桿狀病毒本身具有足夠的遺傳訊息來感染細胞並繁殖,所以不需要衛 星細胞或衛星病毒(helper cell lines or helper viruses)的存在,相較之下 在後續的應用上較為廣泛,例如腺相關病毒(AAV)的生產往往需要腺 病毒(Adenovirus)的存在,使得在純化AAV時造成一些瓶頸。重組 桿狀病毒/昆蟲細胞系統因具有可懸浮培養的特性,可大規模地利用反 應器生產,對於之後生產程序上的放大更為方便且快速。 ❽ 現今腸病毒71型疫苗的發展方式主要有下列幾種·· (1) 將病毒以福馬林去活化後製成去活化病毒疫苗。 (2) 中研院何美鄉教授實驗室利用多次的病毒繼代培養及篩選,以選殖 出繁殖力強、毒性弱的新病毒株而),進而希望利用這株新的病 毒株作為減毒病毒疫苗(attenuated vaccine)»這種減毒病毒疫苗在初步 的小鼠動物免疫測試實驗中已有不錯的成果。 〇 (3) 利用基因突變製造毒性減弱的EV71病毒株作為免疫抗原。在猴子 免疫實驗中已發現所產生的免疫血清具有中和不同血清型病毒的能 力,並且由此突變的EV71減毒病毒引發的神經毒性相當輕微。 (4) 中研院何美鄉教授與長庚大學施信如教授實驗室以大腸桿菌大量 表現基因重組VP1,再利用純化後的VP1蛋白質作為次單元疫苗 (subunit vaccine )進行免疫測試。 (5) 何美鄉教授實驗室也將VP1基因轉殖入質體中,製成DNA疫苗 (DNA vaccine),再將DNA疫苗注射入動物體内以表現W1蛋白質, 201100545 希望所表現的VP1可以順利誘發免疫反應。 (6)利用減毒的鼠傷寒桿菌攜帶表現W1蛋白質的質體,在小鼠腸道表 現VP1抗原以誘發強烈免疫反應作為口服活菌疫苗。 ⑺利用基因轉殖鼠,將州蛋白質表現在母鼠的乳汁中,做為小鼠的 口服抗原。 ⑻利用基因轉殖的蕃祐,在蕃蘇知見W1蛋白質作為口服疫苗。 (9)直接使用化學合成之VP1胜肽鏈(VP1 peptide)作為免疫抗原以產 生中和血清。 然而’類似(1)·(3)這些減毒病毒或是去活化病毒做為疫苗的方 法,疫苗的本身都含有完整的病毒基因,耻健有病滅染的安全 性考量’並且去活化病毒疫苗在處理過程也會失去部分誘發免疫能 力而像⑷⑼這些直接使用純化後的ypj蛋白質(或多胜狀)或是用 載體表現VP1蛋白質的次單;^疫苗,其誘發的免疫效果也不理想,整 體的效果仍較去活化病毒疫苗差。這可能是由於聰與VP3亦有中和 抗原蚊雜’肖辦卩分作細蚊雜與雜有關,甚至有些位 於不同蛋白質間氨基酸上,單獨用W1作為次單元疫苗可能會因此喪 失了部分重要的抗原決定部位。因此,若能同時利用別,聰,哪 與vp4作為抗原,並保留它們在野生病毒内的原有結構與形狀,則理 論上可誘發更多與更強的抗體。 類病毒顆粒(virus-like partide,VLP)是病毒外殼蛋白質所組成的殼狀 201100545 結構’不含病毒基因體,因此不會有傳染複製的疑慮,使用上較為安 全。由於_毒赚與真實病毒她,因此進人人雜,可誘發體内 的免疫系統產生特定抗體以抵抗真實病毒的侵犯,達到保護的效果。 目前常用的傳統疫苗包括去活化疫苗及減毒疫苗,因其作法是將真實 病毒是以外力方式使抗原決定位不活化或是降低其毒性,病毒的遺傳 物質仍然存在於疫苗當中,因此無法完全免除施打疫苗後被感染的機 會;相較於前者,類病毒顆粒和真實病毒相近的外殼則能保有和真實 病毒較相近的抗原決定部位,引發良好的免疫反應以抵抗真實病毒的 感染。因此,類病毒顆粒疫苗為極具潛力的一種疫苗形式。類病毒顆 粒疫苗在國外已有相當的研究,且目前多數的類病毒顆粒都以桿狀病 毒/昆蟲細胞表現系統製造,如小兒麻痒病毒,愛滋病毒,人類微小病 毒,華式囊病毒等等。而一些臨床上重要病毒的類病毒顆粒也都被表 現出來,例如SARS冠狀病毒、D型肝炎病毒、人類乳突病毒、禽流感 病毒H5N1與流行性感冒病毒等,以進行更多的病毒疫苗研究。其中人 類乳突病毒(HPV)的類病毒顆粒做為子宮頸癌疫苗,已有2家藥廠生 產,其中Merck生產的Gardasil®是以酵母菌系統 cerevisiae)生產’已在台灣及美國核准上市。另外一個子宮頸癌疫苗產 品Cervarix™是由GlaxoSmithKline利用桿狀病毒/昆蟲細胞表現系統 (baculovirus/insect cell expression system)生查的HPV VLP,也已在全世The different subtypes of Cl C5 ' are related to the evolution and regionality of their viral genes. In the serotypes of enteroviruses, the virulence of EV71 is particularly high, often accompanied by complications of the nervous system and the possibility of death. 201100545 Enterovirus 71 virus particles are icosahedral polymeric macromolecules with a diameter of about 7 nm, but the detailed group scales have not been clearly studied. At present, the resident of the enterovirus comes from the ridge to listen to gray matter and sputum. _, silkworm virus research, enterovirus genome length of about 7500nt 'including single-open reading frame _ fr継, ORF about 6600m), on both sides are 5, and 3, untranslated area (she muscle age ^ Mail (10), UTR). This ORF can be divided into three regions: decay, p2 and p3, and will be translated into a single polyprotein (P〇lypr〇tein). P1 carries viral capsid proteins: VP4, W2, VP3 and . P2 and p3 carry non-structural proteins such as pr〇tease 2A, 3C, and 3CD, as well as virally polymerized plum 3DP', which is primarily responsible for viral replication and virus toxicity. Taking pediatric pruritus virus as an example, enterovirus granules are theoretically formed by 6Q Cong and Vm59, and a W0 molecule (VP2 and a precursor before unseparation). VP VP2 and VP3 are located on the outer layer of the scale, while the side is completely located on the inner layer. Many of the enteroviruses that have been studied, such as poliovirus, CAV and CBV, have been found to have many neutralizing epitopes (mainization determinants) found mainly in VP1 and a few in VP2 and VP3. In addition, the self-quality of these structural eggs is also related to the weakening of viral toxicity, thermal stability of the virus, host range, and cell driveability. Baculovims, generally referred to as baculoviruses, belong to the baculoviride family, whose host is confined to invertebrates. It is reported that more than 6 species of insects have been infected. Lepidoptera is its main host. According to the latest classification of the International Committee on Taxonomy of Virus in 2004, the baculoviridae can be divided into two genera, the genus genus 201100545 (Gnmulovims, GW human anti-polar corpus callosum Nuck) 〇p〇lyhedr〇virus, delete) Composition, the difference lies in the difference in embedded protein. The former has a small embedding body of about 0.25-0.5 μm, and only one virion is embedded; the latter is larger, approximately, consisting mainly of polyhedrin (poiyhecjron) having a size of about 29 kD, which can be coated. More virions. NPV is cultivated in stable cell strains with its Na-Zhong Zhoucheng gene formula, which has the potential to develop into a gene expression vector. At present, the virus most commonly used as a baculovirus expression vector is Aut〇graph cah: f〇mica O nuclear Polyhedrosis Vi (10); AcMNPV and Bombyx mori nuclear polyhedrosis virus (and Chaga midear polyhedrosis virus; BmNPV). ) 'The main hosts are moths and silkworms. The genetic material of NPV is a double-stranded circular structure of about 8〇2〇〇kb, which is covered by a long-chain nucleocapsid, and the outer layer is made up of a lipoprotein envelope (Ηρ〇_^ envelope). The coating forms a viral structure of about 4 〇 5 〇 nm and a width of about 2 〇〇 4 〇〇 nm. The virus can produce two types of virions after infection: the budding virions O^ded virion; BV) and the embedded virions (〇cdusi〇n b〇dy_derived 〇V). The budding virions are produced in the early stage of the infection cycle. After infection, the new nucleocapsid will be released from the nuclear membrane into the cytoplasm and further penetrate the cell membrane; the embedded virions will be produced in the later stage of the infection cycle, and they will be packaged. Buried in a protein f that is about the size of a polyhedral protein or crystal structure, a polyhedron of about size is formed or called an embedding body. The baculovirus commonly used in laboratories is a budding virus particle. 201100545 The performance of baculovirus is based on the improvement and development of baculovirus. After more than 20 years of improvement and development, it has been widely used in the expression system of heterologous genes, and it is a system for rapid and convenient production of exogenous recombinant proteins. Thus, more than hundreds of viruses, cells, fungi, protozoa, and animal and plant genes have been used to produce recombinant protein f using this system. Even some of the more complex protein structures, such as virions, can be expressed in the host. The baculovirus expression vector system is a kind of eukaryotic cell, and it is expected that the gene is ligated into the baculovirus downstream of the promoter (pr_(4), and then the recombinant baculovirus is used to infect the insect cells. The viral gene promoter may drive this-foreign gene to produce a recombinant protein. Since the insect cells infected by the recombinant baculovirus are eukaryotic cells, the white matter can be modified to various post-translational CpostA-odifieations. Baculovirus Expression _ The eukaryotic gene recombinant protein produced by the system is usually similar in antigenicity, immunity and biological activity to its natural protein. In addition, the promoter used in general is the strong promoter of the baculovirus gene itself (such as P〇1yhedrin or _promoter), so the recombinant protein produced by this system is quite large (1-600mg). /L). In addition to the excellent _ secret correction, the baculovirus secret system has the following advantages. (1) The cashew virus has a considerable genome (88 200kbp), which can accommodate a considerable foreign gene 'making the same strain Baculovirus can carry more than two sets of exogenous genomes simultaneously. (2) Because of recombination; the pathogen of the virus is confined to invertebrates, and it is safer than other gene vectors such as adeno-associated virus (AAV) in 201100545. (3) The recombinant baculovirus itself has sufficient genetic information to infect cells and multiply, so there is no need for the presence of helper cell lines or helper viruses, which is more extensive in subsequent applications. For example, the production of adeno-associated virus (AAV) often requires the presence of an adenovirus, causing some bottlenecks in the purification of AAV. The recombinant baculovirus/insect cell system has the characteristics of suspension culture and can be produced on a large scale by using a reactor, which is more convenient and rapid for subsequent amplification in the production process. ❽ Today's enterovirus 71 vaccines are mainly developed in the following ways: (1) The virus is deactivated by fumarin to produce a deactivated virus vaccine. (2) The laboratory of Professor He Meixiang of the Academia Sinica used multiple virus subcultures and screenings to select new strains with strong fertility and weak toxicity, and hoped to use this new strain as attenuated. Attenuated vaccine» This attenuated virus vaccine has had good results in preliminary mouse animal immunoassay experiments. 〇 (3) Using genetic mutations to produce EV71 strains with reduced toxicity as immune antigens. The resulting immune sera have been found to have the ability to neutralize different serotype viruses in monkey immunization experiments, and the neurotoxicity induced by the mutated EV71 attenuated virus is rather mild. (4) Prof. He Meixiang from the Academia Sinica and Professor Shi Xinru from Chang Gung University demonstrated gene recombination VP1 in Escherichia coli and used the purified VP1 protein as a subunit vaccine for immunoassay. (5) Professor He Meixiang also transferred the VP1 gene into the plastid to make a DNA vaccine, and then injected the DNA vaccine into the animal to express the W1 protein. 201100545 Hope that the VP1 can be expressed. Smoothly induce an immune response. (6) The attenuated strain of Salmonella typhimurium carrying the plastid representing the W1 protein, and the VP1 antigen expressed in the intestine of the mouse to induce a strong immune response as an oral live vaccine. (7) Using a genetically-transferred mouse, the state protein is expressed in the milk of the mother mouse as an oral antigen of the mouse. (8) Fanyou, who uses gene transfer, sees W1 protein as an oral vaccine in Fansu. (9) A chemically synthesized VP1 peptide (VP1 peptide) was directly used as an immunizing antigen to produce neutralizing serum. However, 'similar to (1) · (3) these attenuated viruses or deactivated viruses as a vaccine, the vaccine itself contains a complete viral gene, shame and disease safety considerations and deactivate the virus The vaccine also loses part of the immune-inducing ability during the treatment process. Like (4) (9), these directly use the purified ypj protein (or multiple wins) or use the vector to express the VP1 protein; the vaccine, the immune effect is not ideal. The overall effect is still worse than the deactivation of the virus vaccine. This may be due to the fact that Cong and VP3 also have neutralizing antigen mosquitoes, which are related to fine mosquitoes and impurities, and even some are located between different protein amino acids. W1 alone as a subunit vaccine may lose some importance. The epitope. Therefore, if you can use both, Cong, and vp4 as antigens and retain their original structure and shape in the wild virus, it is theoretically possible to induce more and stronger antibodies. The virus-like partide (VLP) is a shell-like structure composed of viral coat proteins. The 201100545 structure does not contain viral genomes, so there is no doubt about infection and replication, and it is safe to use. Because _ poison earns her with the real virus, it can cause the immune system in the body to produce specific antibodies to resist the invasion of real viruses and achieve the effect of protection. The traditional vaccines currently used include deactivated vaccines and attenuated vaccines, because the real virus is an external force to make the epitope unactivated or reduce its toxicity. The genetic material of the virus still exists in the vaccine, so it cannot be completely The opportunity to be infected after the vaccination is eliminated; compared with the former, the virus-like granules and the shells with similar real viruses can maintain antigenic sites close to the real virus, triggering a good immune response against the infection of real viruses. Therefore, the virus-like particle vaccine is a highly promising form of vaccine. Viral particle vaccines have been studied abroad, and most of the current virus-like particles are manufactured by baculovirus/insect cell expression systems, such as poliovirus, HIV, human parvovirus, and Chinese cystic virus. and many more. Some virus-like particles of clinically important viruses have also been shown, such as SARS coronavirus, hepatitis D virus, human papilloma virus, avian influenza virus H5N1 and influenza virus, etc., for more viral vaccine research. . The virus-like particles of human papillomavirus (HPV) are used as cervical cancer vaccines and have been produced by two pharmaceutical companies. Among them, Merck's Gardasil® is produced by the yeast system cerevisiae, which has been approved for marketing in Taiwan and the United States. Another cervical cancer vaccine product, CervarixTM, is a HPV VLP that was diagnosed by GlaxoSmithKline using the baculovirus/insect cell expression system.

界80多個國家被核准上市。除此以外,以類病毒顆粒作為包覆載體以 傳送DNA或是藥物進入細胞的研究亦開始慢慢發展中。文獻中指出, 以類病毒顆粒包覆基因送入細胞的效率遠比利用微脂粒或是裸露DNA 201100545 來得高許多,因此在基因治療上 、 乂類病f顆粒作基因治療載體亦是 相當具有潛力。 在腸病毒高達上百種血清型斜,小絲痺病毒已造成錢多年,並 已有去活化疫苗(沙克)與減毒疫苗(沙f)來防治,但許多其它腸病毒 不會造成嚴重臨床錄,因此細究财《«未械苗防治。尤 其是EV系、列病毒發贿晚,至今其基礎研究她其它腸病毒仍屬不 足然而腸病毒71型近年來陸續在國内及各國爆發重大疫情,因此其 病毒學,病理學與生物學方面的基本研究大量增加,對基因組成與變 異性、致病性質、免疫性料病毒資訊有了更進一步的瞭解,因此對 發展新-代的疫苗與檢賴財相當大崎助。但截至目前為止腸 病毒71型錢馳制’主要奴依靠政觸公共衛生監域制,包含 疾病通報及P讀宣導’因此若要進行有賴預防或治療,疫苗或抗病 毒藥物的開發具有極重要的地位。 【發明内容】 許多文獻都曾魄Hi ’在昆蟲峨/微病絲現祕巾影響重組蛋白 質產量的因素有报多,包括桿狀病毒的基因設計、昆蟲細胞株間對於 桿狀病毒的感受性不同而造成下游蛋白質表現量不同、適當的反應器 參數及培養基能夠有效增加重組蛋白的產量等。針對各個基本參數與 類病毒顆粒產量間的影響進行簡單探討。首先本發明改良桿狀病毒的 基因設計’希望新建構的桿狀病毒(Bac-Pl-C3CD)能藉由降低3CD水 解酶的表現量’而使得昆蟲細胞能有更多資源以表現強勢啟動子 201100545 polyhedrin下游的P1蛋白質。然而在不同細胞株之間有不同的胞外類 病毒顆粒表現量分佈。以Sf-9細胞作為宿主細胞,利用弱勢啟動子驅 動3CD以增加VLP產量的策略’破有其功效,然而在以Ηΐ_5進行生 產時,卻無法提升產量。這是由於Hi-5細胞對於強勢啟動子的感受性 很強,而使得由弱勢啟動子驅動的3CD表現量過低,雖能有效大量表 現P1蛋白,但卻因為沒有足夠量的3CD能將P1完整切割成W1、γρο、 VP3,以致無法提升類病毒顆粒產量。另外,藉由小量試產來討論感染 〇 時的細胞濃度及M〇I對於VLP產量的可能的影響,發現提高細胞濃度 確實能夠大幅增加VLP產量。综合所有實驗結果,目前已可利用 Bac-Pl-C3CD在高細胞濃度(4><1〇6 ceiis/ml)時感染Sf_9細胞並且在培 養液中得到高產量的VLP。在胞外可收成的最大產量超過5〇 mg/1,不 僅是過去缝#(約10 mg/1)的5倍社,更可避站破_,減少後 續純化時的困難度。 〇 本發_提供一種表現病毒蛋白質的桿狀病毒轉置載體,其包含:⑴ 一強勢桿狀病毒啟動子及-段連結於概動子下社與其侧的㈣ 酸序列,其中該核普酸序列可轉譯成病毒之病毒殼蛋白;以及㈦一 弱勢啟動子及-段連結於該啟動子下游且與其作用的核普酸序列,其 中該歸酸序列可轉譯成轉鱗之病毒殼蛋㈣轉蛋自,而其中 強勢桿狀病毒啟動子驅動連結於該啟動子下游的核聽序列所轉譯的 蛋白質產量高於弱勢啟動子驅動連結於該啟動子下游的㈣酸序列所 轉譯的蛋白質產量。 11 201100545 在本發明中所述之病毒係指腸病毒,而最佳實施例係為腸病毒7i l 在本發明中所述之強勢桿狀病毒啟動子係選自於桿狀病毒多角體蛋白 啟動子或P10啟動子,而弱勢啟動子係選自於桿狀病毒立即早期啟^ 子、巨細胞病毒啟動子或其他在桿狀病翻可作用的弱勢啟動子。 本發明亦提供-種重組桿狀病毒’其包含可表現病毒蛋白質的桿狀病 毒轉置載體’該重組桿狀病毒用於感染宿主細胞以製備重組蛋白質, 而重組蛋自f雜腸病毒的病毒殼蛋自及水解蛋白。 本發明進-步提供-種表現赫蛋㈣轉狀絲宿主峨,該桿狀 病毒宿主細胞係受到重組桿狀病毒的感染。 在本發明中所述之桿狀病毒宿主細胞為昆蟲細胞,其係選自於Sf_9, 职細胞株,而最佳實施例係為Sf_9細胞株,此外宿主細胞可進1 將病毒蛋自魅成麵毒_病毒赚鱗放至峨外,而病毒蛋白 質為腸病毒的病毒殼蛋白及水解蛋白。 本發明更進-步提供—鋪備類絲難的方法 可表現Pi 、該重組域·絲錢宿主細胞以形成 了表現病,响-嫩赠後,離 12 201100545 上清液以取得類病毒顆粒,該類病毒顆粒為腸病毒的類病毒顆粒。 本發明進一步提供一種用以預防病毒感染的疫苗,其係選自於包括免 疫有效量之重組桿狀病毒,宿主細胞及該宿主細胞所釋出的類病毒顆 粒所組成的群組’以及醫藥上可接受的免疫佐劑。而病毒係指腸病毒。 【實施方式】 材料 Ο 細胞培養 昆蟲細胞培養 本發明中所使用的兩種昆蟲細胞分別是Sf-9 (Invitrogen,Cat. Να 11496-015)及 Hi-5 (Invitrogen,Cat· Νο_ Β855-02) 〇 Sf-9 細胞株分別以 Sf-900 II (GIBCO,Cat No. 10902-088)及帶有 l〇 %胎牛血清(Fetal bovine serum ’ FBS,GIBCO)和 0_m (w/v) Pluronic F_68 (Sigma)的More than 80 countries have been approved for listing. In addition, research on the use of viroid-like particles as a coating carrier to transport DNA or drugs into cells has begun to develop. It is pointed out in the literature that the efficiency of feeding genes into cells by virus-like particles is much higher than that of using microlipid or naked DNA 201100545. Therefore, in gene therapy, the gene therapy vector for sputum f particles is quite potential. In the case of enterovirus up to hundreds of serotypes, small silk fibrovirus has caused money for many years, and has been deactivated vaccine (Shaq) and attenuated vaccine (sand f) to prevent, but many other enteroviruses will not cause serious Clinical recording, so carefully study the financial "« unprotected seedlings. In particular, the EV system and the listed virus have been bribe late. So far, the other herpesviruses are still insufficient. However, the enterovirus 71 has been experiencing major outbreaks in China and other countries in recent years, so its virology, pathology and biology. The basic research has been greatly increased, and the information on gene composition and variability, pathogenicity, and immune virus information has been further understood. Therefore, it is quite helpful to develop new-generation vaccines and check-ups. However, as of now, the enterovirus 71 type of money has been the main slave to rely on the public health supervision system, including disease notification and P reading. Therefore, if it depends on prevention or treatment, the development of vaccines or antiviral drugs is extremely Important position. SUMMARY OF THE INVENTION Many literatures have reported many factors affecting the production of recombinant protein in insect mites/micro-paths, including the genetic design of baculovirus and the sensitivity of insect cell strains to baculovirus. The difference in the amount of downstream protein expression, the appropriate reactor parameters and the medium can effectively increase the yield of recombinant protein. A brief discussion of the effects between various basic parameters and the production of viroid-like particles. Firstly, the genetic design of the improved baculovirus of the present invention 'hope that the newly constructed baculovirus (Bac-Pl-C3CD) can make the insect cells have more resources to express the strong promoter by reducing the expression of the 3CD hydrolase'. 201100545 P1 protein downstream of polyhedrin. However, there are different distributions of extracellular virions in different cell lines. With Sf-9 cells as host cells, the strategy of using a weak promoter to drive 3CD to increase VLP production has its effect, but when it is produced with Ηΐ_5, it cannot increase yield. This is due to the strong sensitivity of Hi-5 cells to strong promoters, which makes the 3CD expression driven by the weak promoter too low. Although it can effectively express P1 protein in large quantities, it can complete P1 because there is not enough 3CD. Cut into W1, γρο, VP3, so that the production of viroid-like particles could not be improved. In addition, a small amount of trial production was used to discuss the cell concentration at the time of infection and the possible effect of M〇I on VLP production, and it was found that increasing the cell concentration did increase the VLP production significantly. Combining all the experimental results, it has been possible to infect Sf_9 cells at a high cell concentration (4 ><1〇6 ceiis/ml) using Bac-Pl-C3CD and obtain high-yield VLPs in the culture solution. The maximum yield of extracellular harvest is more than 5〇 mg/1, which is not only 5 times that of the past ## (about 10 mg/1), but also avoids the difficulty of the subsequent purification. The present invention provides a baculovirus translocation vector for expressing a viral protein, which comprises: (1) a strong baculovirus promoter and a - segment linked to the (tetra) acid sequence of the promoter and its side, wherein the nucleotide acid The sequence can be translated into a viral viral capsid protein; and (7) a weak promoter and a segment are linked to a nucleotide sequence downstream of the promoter and interacting therewith, wherein the acid sequence can be translated into a scaled virus shell egg (four) Eggs, wherein the strong baculovirus promoter drives a nuclear listener sequence linked downstream of the promoter to produce a higher protein yield than the weak promoter drives the protein translation translated by the (tetra) acid sequence downstream of the promoter. 11 201100545 The virus described in the present invention refers to enterovirus, and the most preferred embodiment is enterovirus 7i l. The strong baculovirus promoter described in the present invention is selected from the baculovirus polyhedrin promoter. The promoter or the P10 promoter, and the weak promoter is selected from the baculovirus immediate early starter, the cytomegalovirus promoter, or other vulnerable promoters that act in a baculopathic disease. The present invention also provides a recombinant baculovirus comprising a baculovirus translocation vector capable of expressing a viral protein which is used to infect a host cell to produce a recombinant protein, and a recombinant egg from a virus of f-intestinal virus Shell egg and hydrolyzed protein. The present invention further provides for the expression of a genus (4) transgenic silk host bacterium which is infected with a recombinant baculovirus. The baculovirus host cell described in the present invention is an insect cell, which is selected from the group consisting of Sf_9, a cell line, and the best embodiment is a Sf_9 cell line, and in addition, the host cell can enter the virus egg. The surface virus _ virus earns scales to the outside, while the viral protein is the viral shell protein and hydrolyzed protein of the enterovirus. The present invention provides a further step-by-step method for expressing silky filaments, which can express Pi, the recombinant domain, and the silk cell host cells to form a disease, and after the ringing, the supernatant is taken from 12 201100545 to obtain virus-like particles. The virus particles are viroid-like particles of enterovirus. The invention further provides a vaccine for preventing viral infection, which is selected from the group consisting of an immunologically effective amount of recombinant baculovirus, a host cell and a virus-like particle released by the host cell, and a pharmaceutical An acceptable immune adjuvant. The virus refers to enterovirus. [Embodiment] Materials Ο Cell culture insect cell culture The two insect cells used in the present invention are Sf-9 (Invitrogen, Cat. 11α 11496-015) and Hi-5 (Invitrogen, Cat· Νο_ Β 855-02), respectively. 〇Sf-9 cell line was treated with Sf-900 II (GIBCO, Cat No. 10902-088) and with 10% fetal bovine serum (Fetal bovine serum ' FBS, GIBCO) and 0_m (w/v) Pluronic F_68 ( Sigma)

TNM-FH (Sigma ’ St. Louis ’ MO)兩種培養基進行培養,並於inner Q flask中以轉速90 rpm懸浮培養於5 % c〇2的27°C恒溫培養箱中 細胞株則以Sf-900 II無血清培養基進行培養,放置於5% c〇2的2;rc 怪/m培養相以轉速90 rpm在spinor懸浮培養。本發明使用 培養基培養之sf_9進行纽桿狀病毒之m病毒放大使肖sf9〇〇II 培養之Sf-9進行腸病毒71型類病毒顆粒的生產。 哺乳動物細胞培養 本發明中所使用的哺乳動物細胞主要為人類胚胎橫紋肌肉瘤細胞 13 201100545 (Human Embryonal rhabdomyosarcoma, RD),RD 細胞以含 ι〇。/。胎牛 血清及 PSN 抗生素之 Alpha minimal Essential medium (α-ΜΕΜ, Sigrim) 培養液進行培養’放置於5% C02之37°C恆溫培養箱於培養盤中貼 附培養。本發明使用RD細胞主要作為腸病毒的宿主細胞,以進行腸病 毒的培養增瘦。 重組桿狀病毒 重組桿狀病奏載艚 本發明使用Bac-To-Bac® (Invitrogen,Carlsbad,CA)桿狀病毒表現系統 來進行病毒製備,並以系統中的pFastBac DUAL質體作為基因骨架, 建構重組桿狀病毒。此質體具有兩個桿狀病毒的後期強勢啟動子,分 別為桿狀病毒多角體蛋白啟動子(polyhedrin prom〇ter )及p 1 〇啟動子, 而在兩個啟動子下游各有一個多重選殖位(Muitipie ci〇ning sjte,mcs I 及MCSII)’可將外來基因殖入表現外來基因。因此根據已發表的腸病 毒株TW/2272/98的序列,本發明將帶有病毒外殼蛋白的ρι基因接至 polyhedrin啟動子下游’將3CD基因接至pl〇啟動子下游,並命名此 一病毒為Bac-Pl-3CD。此外,本發明也將3CD基因接至弱勢啟動子下TNM-FH (Sigma ' St. Louis ' MO) medium was cultured and suspended in an inner Q flask at a rotational speed of 90 rpm in a 5% c〇2 27 °C incubator. The cell line was Sf- The medium was cultured in 900 II serum-free medium and placed in 5% c〇2 of 2; the rc/m culture phase was cultured in suspension at spin at 90 rpm. In the present invention, the sf_9 cultured in the medium is used to amplify the m-virus of the baculovirus, and the Sf-9 cultured in the sf9〇〇II is subjected to the production of the enterovirus 71 type virus particles. Mammalian Cell Culture The mammalian cells used in the present invention are mainly human embryonic rhabdomyosarcoma cells 13 201100545 (Human Embryonal rhabdomyosarcoma, RD), and RD cells are contained in ι. /. The fetal calf serum and the PSN antibiotic were cultured in an Alpha minimal Essential medium (α-ΜΕΜ, Sigrim) medium. The cells were placed in a 5% C02 37 ° C incubator and incubated in a culture dish. The present invention uses RD cells mainly as host cells of enterovirus for the growth and thinning of enterovirus. Recombinant baculovirus recombinant bacillus disease The present invention uses the Bac-To-Bac® (Invitrogen, Carlsbad, CA) baculovirus expression system for virus preparation, and uses the pFastBac DUAL plastid in the system as the gene skeleton. Construction of a recombinant baculovirus. This plastid has two late-stage strong promoters of baculovirus, namely the polyhedrin prom〇ter and the p 1 〇 promoter, and each has a multiple selection downstream of the two promoters. The colony (Muitipie ci〇ning sjte, mcs I and MCSII) can colonize foreign genes into foreign genes. Therefore, according to the sequence of the published enterovirus strain TW/2272/98, the present invention connects the ρι gene carrying the viral coat protein to the downstream of the polyhedrin promoter, and the 3CD gene is ligated downstream of the pl〇 promoter, and the virus is named. For Bac-Pl-3CD. In addition, the present invention also links the 3CD gene to a weak promoter.

游刀別為 IE-1 啟動子(baculovirus immediate early promoter)及 CMV 啟動子(cytomegalovirus)下游,命名為 Bac_P1_I3CD 及 Bac_P1_C3CD (如圖1)。 重經&狀病喜放女 本發明中重轉狀病細放大是使用Sf-9細胞株及有10 %胎牛血清的 14 201100545 ΤΝΜ-FH培養基。放大病毒時,需先將sf_9細胞株培養於旋轉角瓶 (Spinner Flask)中’待細胞濃度達到約lxl〇6 cell/ml時,將細胞以5〇〇xg 的轉速離心10分鐘,抽去9/10的培養基,使細胞回溶在1/1〇原始體 積的培養基中。此時加入病毒感染劑量(multiplicity 〇finfecti〇n,Μ〇Ι) 為 0.05 pfU/cell (溶滅斑形成单位/細胞,piaqUe f〇nr^ng unks per ceu)的 病毒液,於室溫下均勻搖晃一小時後,以新鮮培養基補回至原始體積, 並置入spinner於2rc恆溫培養箱懸浮培養。感染4〜6天後,觀察細 〇 胞存活率,當細胞存活率降至60〜70%時,以3〇〇〇Xg的轉速離心10分 鐘以去除細胞及其碎片,收集上清液(即病毒液)避光保存於冰箱 中,若需長期保存則置於-80°C冰箱中。 .終點稀釋法定量病毒效僧 終點稀釋法首先f先將病魏以養基作1G倍的連續稀釋, 之後再將病麵與濃度為1><1〇5 eell/ml的Sf 9細胞以15() μΐ病毒液 〇 對1350 μ1細胞液的比例混合,加入96孔盤(Coring,Coring,NY)中’ 每個病毒蒲倍树12孔。之碰96减置人Π棘培養箱中培 養I3天,在光學顯微鏡下,觀察細麵感__,最後以統計學方 法什算病毒效價(titer),此病毒濃度稱之為感染效價(响比咖恤), 單位為pfo/ml (溶菌斑形成單位/毫升,咖啊f〇nning恤π milliliter) ° 越也歷病毒71型類病卷海# 15 201100545 腸病毒71型類病奏顆物的决吝 本發明中使用Sf-9及Hi-5兩種細胞株來生產EV71 VLP。當懸浮培養 中的細胞濃度達到約 l-6xl06 cell/ml 時,加入 multiplicity of infecti〇n (MOI)為0.001- 的病毒液,再將spimer放入27〇c恆溫培養 箱。為了決定EV71 VLP最佳的收成時間,於感染後每24小時取樣一 次細胞液,並西方墨點法分析VLP的產量。 生物反應器生產腸病毒71型類病毒顆新 本發明目的在於增加類病毒顆粒的單位體積產率,當確認最佳生產條 件後將嘗試以實驗室及生物反應器生產類病毒顆粒。本發明採用The cleavage knife was downstream of the baculovirus immediate early promoter and the CMV promoter (cytomegalovirus) and was named Bac_P1_I3CD and Bac_P1_C3CD (Fig. 1). Heavy-duty & disease-like females The fine-magnification of the re-transformation disease in the present invention is the use of Sf-9 cell line and 14 201100545 ΤΝΜ-FH medium with 10% fetal calf serum. When amplifying the virus, the sf_9 cell strain should be cultured in a spinner flask (Spinner Flask). When the cell concentration reaches about lxl〇6 cell/ml, the cells are centrifuged at 5 〇〇xg for 10 minutes, and 9 is removed. /10 of the medium was allowed to reconstitute the cells in 1/1 〇 original volume of medium. At this time, add the viral infection dose (multiplicity 〇finfecti〇n, Μ〇Ι) to 0.05 pfU / cell (defect plaque forming unit / cell, piaqUe f〇nr ^ ng unks per ceu) virus solution, uniform at room temperature After shaking for one hour, the medium was replenished with fresh medium and placed in a spinner in a 2rc incubator. After 4 to 6 days of infection, the survival rate of the fine cells was observed. When the cell survival rate was reduced to 60 to 70%, the cells were centrifuged at 3 Torr for 10 minutes to remove the cells and fragments thereof, and the supernatant was collected (ie, The virus solution is stored in the refrigerator in the dark. If it needs to be stored for a long time, it should be placed in a -80 °C refrigerator. End-point dilution method for quantification of viral effect 僧 End-point dilution method First, the disease is serially diluted 1G times with the nutrient, and then the Sf 9 cells with a concentration of 1><1〇5 eell/ml are 15() μ ΐ 〇 〇 〇 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 The collision 96 was reduced in human thorn culture incubator for I3 days. Under the optical microscope, the fine surface sensation was observed __, and finally the statistical method was used to calculate the virus titer. The virus concentration was called infection titer. (loud than t-shirt), the unit is pfo/ml (lytic plaque forming unit / ml, coffee ah f〇nning shirt π milliliter) ° Yue also calendar virus type 71 disease volume sea # 15 201100545 Enterovirus 71 type disease The sputum of the present invention uses the Sf-9 and Hi-5 cell lines to produce the EV71 VLP. When the cell concentration in the suspension culture reached about l-6xl06 cell/ml, the multiplicity of infecti〇n (MOI) was added to the virus solution of 0.001-, and the spiker was placed in a 27 °c incubator. In order to determine the optimal harvest time for the EV71 VLP, the cell fluid was sampled every 24 hours after infection and the VLP yield was analyzed by Western blotting. Bioreactor Production of Enterovirus Type 71 Viruses The present invention aims to increase the unit volume yield of viroid-like particles, and will attempt to produce viroid-like particles in laboratories and bioreactors after confirming optimal production conditions. The invention adopts

Sartorius BIOSTAT® B醱酵槽進行生產。BIOSTAT® B屬於攪拌式生物 反應器,利用無氣泡通氣系統進行通氣。本發明以電子氣體混和裝置 調控氮氣、氧氣及空氣的混和比例,並於槽體上空氣部份以打氣幫浦 通氣來增加溶氧量,將旋轉葉片轉速調控在1〇〇 φΠ1,將溫度控制在27 度。 胞内腸病毒71型類病毒顆敖的萃敌 將感染後72小時的Sf-9細胞離心1〇分鐘(R123Arotor,11000 rpm, 18000 xg ’ CR22,Hitachi)收取細胞’去除培養基後,將細胞回溶至 TNE 緩衝液(40 mM Tris,1〇〇 應 NaQ,2 mM MgCl2,丨 mM EDTA, 卩117.4)中’置入-80(1!冰相,利用重複冰;東/解;東(?1恍2£311(^111&\¥)三次 以將細胞打破。之後加入l/io體積的組織分解溶液(lysis buffer,1〇 % NP-40,40 mM Tris ’ 100 mM NaCl,2 mM MgCl2 ’ 1 mM EDTA,pH 7‘4), 16 201100545 靜置於冰上反應30分鐘。將反應後的細胞液離心30分鐘(RA202 rotor ’ 11000 rpm ’ 18000 xg,CR22 ’ Hitachi),收取上清液,此時所得 之上清液即含有類病毒顆粒蛋白質。 胞外腸病毒71型類病卷顆輪的收隼 將感染後不同時間點(如72或%小時)的Sf-9細胞離心30分鐘(R123A rotoi* ’ ll,〇〇〇ipm,18,000 xg ’ CR22 ’ Hitachi)後收取上清液。 丛蛋白質定景法定量類病卷顆粒的鎞|白澧唐 配置不同濃度的牛血清蛋白(BSA,bovine serum albumin)標準品溶液 (12·5-1.5625 gg/ml)後’本發明將每個濃度之標準品取4〇〇 μ1與1〇〇 μ1 蛋白質染劑(BioRad protein assay ; BioRad,CA)混合均勻,置於室溫 下10分鐘,量測在波長595 nm下的吸光值(〇D),製作出標準品BSA 的檢量線(25 pg/mM.5625 pg/ml)。在同時本發明將經由超高速離心後 純化而得的類病毒顆粒樣品以h2〇倍稀釋後,取4〇〇 μ1與1〇〇叫蛋白 質染劑(BioRad)混合均勻,置於室溫下10分鐘,測量其在〇ε>595之吸 收值,可由BSA標準品的吸光值所繪出的檢量線推算出類病毒顆粒總 蛋白濃度。 腸病卷71刑的纯化 本發明使用腸病毒71型為TW/2272/98 (屬於gen〇type 〇:2)及gen〇type Bh TW/2272/98為1998年台灣首次爆發腸病毒大流行時所分離出的 病毒株,而genotype B5則為2007年至2008年腸病毒流行時的主要病 17 201100545 毋株’經基因疋序後證實為genotype Bs。實驗中利用奶細胞來進行 所有病毒株的培養職,病毒_魏程如下:肋細齡15咖培養 盤内培養至約8成滿時,抽掉原來的培養液並加入15时 Phosphate-buffered saline (PBS)緩衝液以清洗殘餘培養液,清洗完細胞 後將PBS去除。另外本發明以1〇 ml不含胎牛血清之α ΜΕΜ培養液稀 釋腸病毒71翻毒液’將_之病麵加人培養射,之後將培養盤 放置於搖晃板上侧1小時進行病毒感染。經丨小時病毒吸附後,去 除含有病毋之α-ΜΕΜ培養液,接著加入含2%胎牛血清及pSN抗生 素的α-ΜΕΜ培養液,於37°C培養箱中培養。當7〇%細胞產生細胞變 大、脫落等典型之細胞病變效應(cyt〇pathic effect,CpE)後,收集感 染後的培養液離心10分鐘(5000 rpm),離心後收取上清液。此外收集 感染之細胞與培養液,於溶液中添加灿_4〇 (最後濃度1%)以幫助病毒 由細胞破片中釋出,並利用兩次冷束/解凍(freeze md thaw)的方式 將細胞打破。細胞破片經過6000 rpm、10分鐘(4〇〇〇xg, R20A2 rotor) 離心去除後,將含有病毒的溶液儲存於_ 8〇。(:或直接進行純化。進行 純化時,將樣品置於4。(:的環境下並持續攪拌,在持續攪拌的環境下 先緩慢加入氣化鈉至濃度2% (w/v),氣化鈉完全融化後,再緩慢加入 聚乙烯丁一醇8000 (PEG 8000)至濃度14% (w/v)(替代方法:以XNE 缓衝液為溶劑配置60% (w/v) PEG 8000溶液,緩慢加入病毒液中至 PEG 8000的濃度為14%(W/V))。PEG 8000都溶解後,將樣品留置於 4°C的環境下並持續攪拌overnight,利用PEG 8000將使病毒沈澱。 沈殿過程完成後,以6000 rpm離心30分鐘(R20A2 rotor)高速離心將 18 201100545 上清液和沉澱物分離,沈殿物以1/10原始病毒體積的頂緩衝液回 溶。將紐溶於TNE緩衝液中,分別配置成重量百分濃度為Μ %、4〇 % (Wv)的溶液’再以針筒及18號長針頭依紐(濃度25 %、卿。 的順序),將溶液由底部注入容量為n ml的超高速離心管中,體積分 別為5、3 ml ;高濃度的溶液由底部注入時,能夠將低濃度的蔬糖溶液 抬升,可以減少不同濃度間溶液的混和。完成25.的嚴糖梯度後, 娜5 ml的樣品小心加在管中溶液的最上層。將所要離心的離心管都 〇 依上述㈣完成後,再掛上·式離錢(Swinging bueket),以 lOOOOOxg、4〇C 離心 4 小時(P40ST rotor,30000 印瓜,113〇〇〇xg,The Sartorius BIOSTAT® B fermentation tank is produced. BIOSTAT® B is an agitated bioreactor that is vented using a bubble-free ventilation system. The invention adopts an electronic gas mixing device to regulate the mixing ratio of nitrogen, oxygen and air, and increases the dissolved oxygen amount by blowing air in the air portion of the tank body, and regulates the rotating blade speed at 1〇〇φΠ1 to control the temperature. At 27 degrees. The enemies of the intracytoplasmic EV71 virus will be centrifuged for 72 minutes after infection for 72 minutes (R123Arotor, 11000 rpm, 18000 xg 'CR22, Hitachi) to collect the cells. After removing the medium, the cells are returned. Dissolve into TNE buffer (40 mM Tris, 1 〇〇 should be NaQ, 2 mM MgCl2, 丨 mM EDTA, 卩 117.4) in 'Set-80 (1! Ice phase, use repeated ice; East / solution; East (? 1恍2£311(^111&\¥) three times to break the cells. Then add l/io volume of tissue decomposition solution (lysis buffer, 1% NP-40, 40 mM Tris '100 mM NaCl, 2 mM MgCl2) ' 1 mM EDTA, pH 7'4), 16 201100545 The reaction was allowed to stand on ice for 30 minutes. The cell solution after the reaction was centrifuged for 30 minutes (RA202 rotor '11000 rpm '18000 xg, CR22 'Hitachi), and the supernatant was collected. At this time, the supernatant solution contains the virus-like particle protein. The extracorporeal enterovirus type 71 disease rounds are collected and the Sf-9 cells at different time points (such as 72 or % hours) after centrifugation are centrifuged for 30 minutes. (R123A rotoi* 'll, 〇〇〇ipm, 18,000 xg 'CR22 ' Hitachi) After receiving the supernatant.鎞 类 类 鎞 澧 澧 澧 澧 澧 配置 配置 配置 配置 配置 配置 配置 配置 配置 配置 配置 配置 配置 配置 配置 配置 配置 配置 配置 配置 配置 配置 配置 配置 配置 配置 配置 配置 配置 配置 配置 配置 配置 配置 配置 配置 配置 配置 配置 配置 配置 配置 配置 配置 配置〇〇μ1 was mixed with 1〇〇μ1 protein stain (BioRad protein assay; BioRad, CA) and placed at room temperature for 10 minutes. The absorbance at 595 nm (〇D) was measured to prepare a standard. BSA calibration curve (25 pg/mM.5625 pg/ml). At the same time, the virus-like particle sample obtained by ultra-high-speed centrifugation of the present invention is diluted by h2〇, and then taken 4〇〇μ1 and 1〇. The protein dye (BioRad) is uniformly mixed, placed at room temperature for 10 minutes, and its absorption value at 〇ε > 595 is measured. The total number of virus-like particles can be estimated from the calibration curve drawn by the absorbance of the BSA standard. Protein concentration. Purification of enteric disease volume 71 This invention uses enterovirus type 71 as TW/2272/98 ( belonging to gen〇type 〇: 2) and gen〇type Bh TW/2272/98 as the first outbreak of enterovirus in Taiwan in 1998. The virus strain isolated during the pandemic, and genotype B5 is the main cause of enterovirus epidemics from 2007 to 2008 17201100545 no strain 'Cloth after gene sequence confirmed genotype Bs. In the experiment, milk cells were used to carry out the cultivation of all virus strains. The virus was as follows: when the ribs were aged 15 coffee cultures to about 80% full, the original culture solution was removed and 15 hours of Phosphate-buffered saline was added. The (PBS) buffer was used to wash the residual culture solution, and after washing the cells, the PBS was removed. Further, in the present invention, 1 〇 ml of the α ΜΕΜ culture medium containing no fetal bovine serum is diluted with the enterovirus 71 sterilizing solution, and the diseased surface is cultured, and then the culture disk is placed on the side of the shaking plate for 1 hour for virus infection. After adsorption by the sputum virus, the α-ΜΕΜ culture solution containing the diseased sputum was removed, followed by the α-ΜΕΜ culture medium containing 2% fetal calf serum and pSN antibiotic, and cultured in a 37 ° C incubator. When 7〇% of the cells produced a typical cytopathy effect (CpE) such as cell enlargement and shedding, the infected culture solution was collected and centrifuged for 10 minutes (5000 rpm), and the supernatant was collected after centrifugation. In addition, the infected cells and culture medium were collected, and Can_4〇 (final concentration 1%) was added to the solution to help release the virus from the cell fragments, and the cells were removed by two freeze/daw methods. break in. After the cell fragments were removed by centrifugation at 6000 rpm for 10 minutes (4 Torr x R20A2 rotor), the virus-containing solution was stored at -8 Torr. (: Or directly purify. When purifying, place the sample in a 4: (with an environment of stirring), slowly add sodium vaporate to a concentration of 2% (w/v) in a continuous stirring environment, and gasify. After the sodium is completely melted, add polyvinyl butanol 8000 (PEG 8000) to a concentration of 14% (w/v). (Alternative method: 60% (w/v) PEG 8000 solution in XNE buffer as solvent, slow Add the virus solution to the concentration of PEG 8000 (14% (W / V)). After the PEG 8000 is dissolved, leave the sample in an environment of 4 ° C and continue to stir overnight, using PEG 8000 will precipitate the virus. After completion, the 18 201100545 supernatant and the sediment were separated by centrifugation at 6000 rpm for 30 minutes (R20A2 rotor), and the sediment was reconstituted with 1/10 of the original virus volume of the top buffer. The ruthenium was dissolved in TNE buffer. , respectively, configured as a solution with a concentration of Μ%, 4〇% (Wv) by weight, and then with a syringe and a 18-gauge long needle (in the order of 25%, in the order of qing), the solution is injected into the volume from the bottom. In the ultra-high speed centrifuge tube of n ml, the volume is 5, 3 ml respectively; when the high concentration solution is injected from the bottom, it can It is enough to raise the low concentration of the vegetable sugar solution, which can reduce the mixing of the solution between different concentrations. After completing the 25. sugar gradient, the sample of Na 5 ml is carefully added to the uppermost layer of the solution in the tube. After completing the above (4), you can hang up the Swinging bueket and centrifuge at 1000°g for 4 hours (P40ST rotor, 30,000 melon, 113〇〇〇xg,

CP100MX ’ Hitachi)。之後以針筒及長針頭由底部慢慢吸取溶液,以每 0.5 ml為單位收集成一管,並以西方點墨分析πρ於各管中的含量病 毒會位於25%及40%中間的界面,以針頭收集介面的帶狀物質 (band).。分析完後將含有VLP的部分集中,稀釋於約10倍樣品體積 的TE緩衝液中’再以超高速離心3〇〇〇〇rpm、2小時(i〇〇〇〇〇xg P4〇ST 〇 rotor)將腸病毒離心沈澱;除去上清液後加入適當的PBs緩衝液回溶 底部的沈澱物,不溶的部分以450〇xg、10分鐘(Universal 32)的離心方 式將之去除,此上清液即含有純化後的腸病毒。純化後的病毒均根據 終點稀釋法分析其病毒效價。病毒效價是根據已感染細胞的典型細胞 病理變化(CPE)的原理,以終點稀釋法(end-point dilution)測定。所得 病毒效價單位為TCID50。實驗方式是將RD細胞培養於96孔細胞培 養盤,當細胞繁殖至約80%滿時,待測的病毒以含2%胎牛血清及PSN 抗生素的α-ΜΕΜ培養液進行連續10倍率稀釋(l〇_2-l〇_1G),將原本的 201100545 培養液抽取出,取⑽μΐ _釋縣_狐中,每個稀釋倍率至少 感染12個孔。感染完後於37〇c、5% c〇2培養箱中培養3天,感染 結束後觀察紀絲孔巾細朗CPE ’糊終⑽釋法公式進行分析。 實施例一 yi^p特性分析古沐 西方墨點法(Westem blotting) 利用垂直式轉印槽(Mini Trans-Blot Cell ; BioRad,Munich,Germany) 將分離完之SDS-PAGE 白質樣本轉印至墙化纖維膜(〇45μηιCP100MX ’ Hitachi). Then slowly suck the solution from the bottom with a syringe and a long needle, collect a tube every 0.5 ml, and analyze the content of πρ in each tube by Western blotting. The virus will be located at the interface between 25% and 40%. The needle collects the band of the interface. After the analysis, the fraction containing the VLP was concentrated and diluted in about 10 times the sample volume of TE buffer' and then ultracentrifuged at 3 rpm for 2 hours (i〇〇〇〇〇xg P4〇ST 〇rotor) The enterovirus is precipitated by centrifugation; the supernatant is removed, and the precipitate of the bottom portion is added by adding appropriate PBs buffer, and the insoluble portion is removed by centrifugation at 450 〇 x g for 10 minutes (Universal 32). That is, it contains the purified enterovirus. The purified virus was analyzed for virus titer according to the endpoint dilution method. Viral titers are determined by end-point dilution based on the principle of typical cellular pathological changes (CPE) of infected cells. The resulting virus titer unit is TCID50. The experimental method is to culture the RD cells in a 96-well cell culture dish. When the cells multiply to about 80% full, the virus to be tested is serially diluted by 10 times with α-ΜΕΜ culture medium containing 2% fetal bovine serum and PSN antibiotics ( l〇_2-l〇_1G), extract the original 201100545 culture solution, take (10) μΐ _ release county _ fox, infect at least 12 holes per dilution rate. After infection, the cells were cultured for 3 days in a 37 °c, 5% c〇2 incubator. After the infection, the fine CPE _ paste final (10) interpretation formula was observed. Example 1 yi^p characterization analysis Westem blotting Transfer of the separated SDS-PAGE white matter sample to the wall using a vertical transfer tank (Mini Trans-Blot Cell; BioRad, Munich, Germany) Fiber membrane (〇45μηι

Mitroeellulose membmne,MC membmne ; Pall Corporation)。轉印前先 剪-張與凝敎小械的NC membmne ’與雜—起制於轉印緩衝 液(transfer buffer,2.5 mM Tris,192 mM glycine,20 % v/v methanob pH 8.3)五分鐘然後將NC membrane置於膠體之上,並在其上下各放一 張以transfer buffer浸潤過的濾紙,轉印電流為1〇〇v,時間6〇分鐘。 將轉印好的membrane置於固定緩衝液(blockingbuffer,25mMTris, 150 mM NaCl ’ 5 °/〇 nonfat powdered milk ’ pH 7.4)中’室溫一小時或 4 〇c 隔夜。接著以稀釋1500倍的mouse anti-VPl單株抗體為第1級抗體, 辨識膜上的VP1蛋白質,在室溫作用2小時或4。(:作用隔夜,然後以 washing buffer (TBST ’ 0.05 °/〇 Tween-20 in TBS)清洗三次,每次約 10 分鐘’以稀釋 2500 倍的 Alkaline Phosphatase-conjugated goat anti-mouseMitroeellulose membmne, MC membmne; Pall Corporation). Before the transfer, the NC membmne 'and the miscellaneous-cutting buffer (2.5 mM Tris, 192 mM glycine, 20% v/v methanob pH 8.3) were transferred to the transfer buffer (2.5 mM Tris, 192 mM glycine, 20% v/v methanob pH 8.3) for five minutes. The NC membrane was placed on top of the gel and a filter paper infiltrated with transfer buffer was placed on top of each other with a transfer current of 1 〇〇v for 6 〇 minutes. The transferred membrane was placed in a blocking buffer (25 mM Tris, 150 mM NaCl '5 ° / 〇 nonfat powdered milk 'pH 7.4) at room temperature for one hour or 4 〇 c overnight. Next, a 1500-fold dilution of mouse anti-VP1 monoclonal antibody was used as the first-order antibody, and the VP1 protein on the membrane was identified and allowed to act at room temperature for 2 hours or 4. (: function overnight, then wash three times with washing buffer (TBST ' 0.05 ° / 〇 Tween-20 in TBS) for about 10 minutes each time to dilute 2500 times Alkaline Phosphatase-conjugated goat anti-mouse

IgG單株抗體(Chemicon,Temecula,CA)作為第2級抗體,辨識抓取 第1級抗體,室溫作用一小時’接著使用呈色劑5_bromo斗chloroindoxyl 20 201100545 phosphate (BCIP)/ nitro blue tetrazolium (NBT) (Sigma)呈色。 酵素連結免疫吸附分析法(Enzyme-Linked Immunosorbent Assay) ELISA又可分為direct與sandwich兩種方法,本發明採用sandwich ELISA的方式以提高精準性。吸附用抗體(地祕⑽打-乳?多株抗體)以 1:10000稀釋後’分別各取100μ1到ELISA的96孔微量滴定盤(Nunc ELISA/EIA Maxisorp ; Nunc,Roskilde,Denmark)的各個孔中,置於室 q 溫下2個小時或是放於4°C隔夜使抗體吸附到孔中的底部。吸附完成 後將溶液吸出’以 100 μΐ 清洗液 PBS-T (0.05% Tween 20 in PBS buffer) 注入各個孔’於室溫下靜置10分鐘後將清洗液取出,重複清洗的步驟 共4次。最後一次取出清洗液後,每孔加入1〇〇 μ1 blocking buffer (含 1%BSA的PBST溶液)於室溫反應1小時,將孔中底部的所有空隙都 填補上蛋白質,接著以清洗液重複清洗4次。取出清洗液後,加入ι〇〇μι 的稀釋後(樣品稀釋倍率為1:1 〇 〇 )待測樣品(如含類病毒顆粒的細胞液) 〇 或腸病毒71型類病毒顆粒標準品(標準品由0.5pg/ml以兩倍序列稀釋 直到0.015625pg/ml)’標準品為胞内生產之類病毒顆粒經由超高速離心 法純化而得,於室溫下反應2小時後,接著以清洗液重複清洗4次。 取出清洗液後’加入100 μ卜稀釋500倍的EV71單株抗體(mouse igG2b monoclonal antibody,Cat No. 3324 ; Chemicon,Temecula,CA),於室 溫下反應2小時,接著以清洗液重複清洗4次。取出清洗液後,加入 100 μΐ、稀釋 2000 倍的 g〇at anti-mouse IgG conjugated with HRP (Kirkegaard & Perry Laboratories ’ Gaithersburg,MD)二級抗體,於室溫 21 201100545 下反應1.5小時’再以清洗液重複清洗4次。於最後一次清洗時,配製 TMB (3,3’,5,5’-tetramethyl benzidine ; Sigma)呈色劑,將 1 mg TMB 溶 於 1 ml dimethyl sulfoxide (DMSO ; Sigma)中,再加入 9 ml 的 Phosphate-Citrate Buffer (0.1 M NaH2P〇4 和 0.1 M citric acid,以碗酸調 整到PH 5),取出清洗液後,每個孔中加入100 μΐ TMB (Sigma,cat# T0440)進行呈色反應,於37 °C下反應約15分鐘,直到反應試劑呈現 明顯藍色。接著於每個孔中加入5〇 μ卜2 M H2S〇4以中止呈色反應, 再將微 1 滴定盤置於 ELISA reader (MRX; Dynex Technology Inc.,Glen d Allen,VA)中,以波長450 nm讀取其ELISA數值。根據類病毒顆粒標 準品的濃度及所得的吸光值,本發明做出檢量線後,再對待測樣品中 的類病毒顆粒作相對定量。 穿透式電子顯微鏡(Transmission Electron Microscopy,TEM) 本發明將純化後的VLP樣品以超高速離心沈在離心管下方後,以loo μΐ ddK2〇回溶。於室溫下’將1〇μ1 (濃度調整為約50pg/ml)純化後的VLP 樣〇口液滴在 TEM 銅網(support films formvar / carbon 200 mesh copper grids,TEDPellaInc,cat# 〇18〇〇_F)上方,靜置吸附5分鐘後,以濾紙 及乾多餘液滴。取 1〇μ1 的 2% ph〇Sph〇tungStic acid (ρτΑ,Sigma,以 NaOH調整到pH 7,2)滴於銅網上負染3〜5分鐘。最後再以滤紙吸除多 餘染劑’將銅_品面朝上,置於除濕下的冷氣房巾,待隔夜風乾後 即可直接_ f拉電子顯魏⑽aehi,H_75⑻)觀察。 22 201100545 免疫金染色及穿透式電子顯微鏡觀察 在進行免疫金粒子的標定實驗時,本發明先將jjgM銅網置於石蠟封口 膜(parafilm)上,取約10μι (濃度為5〇 μβ/ηι1)的類病毒顆粒樣品滴在鍍 碳銅網上,靜置約5分鐘後以棉紙吸掉多餘的樣品,接著將_的鱗 酸鹽緩衝落液(phosphate buffer saline,PBS)滴在鍍碳銅網上清洗掉 多餘的蛋白質並以棉紙吸掉多餘的PBS,重複清洗的步驟三次。清洗 完後,將anti-VPl抗體以PBS稀釋(1:2〇〇稀釋)後,取_稀釋液滴在 銅網上’靜置1小時使抗體辨識吸附到樣品上。當抗體反應時間結束 後,重複上述清洗方法,以PBST清洗銅網表面三次,接著把二級抗體IgG monoclonal antibody (Chemicon, Temecula, CA) as a second-order antibody, recognizes the capture of the first-order antibody, and acts at room temperature for one hour' followed by the coloring agent 5_bromo chloroindoxyl 20 201100545 phosphate (BCIP) / nitro blue tetrazolium ( NBT) (Sigma) is colored. Enzyme-Linked Immunosorbent Assay ELISA can be divided into two methods: direct and sandwich. The present invention adopts the sandwich ELISA method to improve the accuracy. The antibody for adsorption (mystery (10)-milk-multiple antibody) was diluted 1:10000 to each well of 100 μl to ELISA 96-well microtiter plate (Nunc ELISA/EIA Maxisorp; Nunc, Roskilde, Denmark) The antibody was adsorbed to the bottom of the well by placing it at room temperature for 2 hours or at 4 °C overnight. After the adsorption was completed, the solution was aspirated and poured into each well with 100 μΐ of the washing solution PBS-T (0.05% Tween 20 in PBS buffer). After standing at room temperature for 10 minutes, the washing solution was taken out, and the washing step was repeated 4 times. After the last removal of the cleaning solution, add 1 μl of blocking buffer (PBST solution containing 1% BSA) to each well for 1 hour at room temperature, fill all the voids in the bottom of the well with protein, and then wash repeatedly with washing solution. 4 times. After removing the cleaning solution, add ι〇〇μι after dilution (sample dilution ratio 1:1 〇〇) sample to be tested (such as cell fluid containing virus-like particles) 〇 or enterovirus type 71 virus particle standard (standard The product is diluted from 0.5 pg/ml in two-fold sequence until 0.015625 pg/ml). The standard is obtained by ultra-high-speed centrifugation of viral particles such as intracellular production, and reacted at room temperature for 2 hours, followed by a cleaning solution. Repeat the washing 4 times. After removing the washing solution, '100 μl diluted EV71 monoclonal antibody (mouse igG2b monoclonal antibody, Cat No. 3324; Chemicon, Temecula, CA) was added, and reacted at room temperature for 2 hours, followed by repeated washing with washing solution 4 Times. After removing the washing solution, add 100 μM, diluted 2000-fold g〇at anti-mouse IgG conjugated with HRP (Kirkegaard & Perry Laboratories 'Gaithersburg, MD) secondary antibody, and react at room temperature 21 201100545 for 1.5 hours. The cleaning solution was washed repeatedly 4 times. At the last wash, TMB (3,3',5,5'-tetramethyl benzidine; Sigma) was prepared and 1 mg of TMB was dissolved in 1 ml of dimethyl sulfoxide (DMSO; Sigma), followed by 9 ml. Phosphate-Citrate Buffer (0.1 M NaH2P〇4 and 0.1 M citric acid, adjusted to pH 5 with bowl acid). After removing the washing solution, 100 μM TMB (Sigma, cat# T0440) was added to each well for color reaction. The reaction was carried out at 37 ° C for about 15 minutes until the reaction reagent showed a distinct blue color. Then, 5 μM 2 M H2S〇4 was added to each well to stop the color reaction, and the micro 1 titration plate was placed in an ELISA reader (MRX; Dynex Technology Inc., Glen d Allen, VA) at a wavelength. The ELISA values were read at 450 nm. According to the concentration of the viroid particle standard and the obtained absorbance value, after the calibration curve is prepared by the present invention, the virus-like particles in the sample to be tested are relatively quantified. Transmission Electron Microscopy (TEM) In the present invention, the purified VLP sample is submerged under the centrifuge tube by ultracentrifugation, and then dissolved back by loo μΐ ddK2. VLP-like mouthwash after purification of 1〇μ1 (concentration adjusted to about 50pg/ml) at TEM copper mesh (support films formvar / carbon 200 mesh copper grids,TEDPellaInc,cat# 〇18〇〇 Above _F), after standing for 5 minutes, apply filter paper and dry excess droplets. Take 1〇μ1 of 2% ph〇Sph〇tungStic acid (ρτΑ, Sigma, adjusted to pH 7, 2 with NaOH) and drop it on the copper grid for 3~5 minutes. Finally, filter the excess dyeing agent with filter paper. Place the copper _ face up and place it on the cold air towel under dehumidification. After air drying overnight, you can directly observe the electrons (10) aehi, H_75 (8). 22 201100545 Immunogold staining and transmission electron microscopy observation In the calibration experiment of immunogold particles, the present invention first placed jjgM copper mesh on parafilm, about 10μιη (concentration of 5〇μβ/ηι1) The virus-like particle sample was dropped on a carbon-coated copper mesh, and after standing for about 5 minutes, the excess sample was sucked off with a cotton paper, and then the phosphate buffer saline (PBS) was dropped on the carbon-coated carbon. The excess protein was washed off the copper mesh and excess PBS was blotted with tissue paper, and the washing step was repeated three times. After washing, the anti-VP1 antibody was diluted with PBS (1:2 〇〇 dilution), and the _ diluted droplets were allowed to stand on the copper grid for 1 hour to allow the antibody to be adsorbed onto the sample. After the end of the antibody reaction time, the above washing method was repeated, and the surface of the copper mesh was washed three times with PBST, followed by the secondary antibody.

Cg〇ld_conjugated goat anti-mouse IgG,Sigma)以 1:1000 的比例稀釋於 PBS中’取稀釋後抗體1〇μ1滴在銅網上,二級抗體上連接大小為$脆 的金粒子,能齡電子顯微鏡下_呈現。三級抗體反應1小時後, 以PBST清洗銅網表面三次,清洗完後以棉紙吸乾銅網表面溶液,取 1〇μ1的2〇/〇峨酸鶴溶液滴在銅社,靜置染色三分鐘後,利用棉紙將多 餘的染親乾,置錄針乾_ 12小時,似f赋電子顯微鏡 (H-7500)觀察。 遍查株與細胞株組合師德 從先前實驗室研究得知,桿狀絲/昆蟲細絲現祕共同表現腸 病毒71型的蛋白質Pi (以p〇lyhedrin強啟動子驅動)及3CD (以pi〇強 啟動子驅動)’ P1在表現後可被3CD _丨成綱,赠與州,進一 23 201100545 步在細胞内自發性地組裝形成類病毒顆粒。所製備出的桿狀病毒命名 為Bac-Pl-3CD (見圖1)。為提升類病毒顆粒的產量,本發明猜測少量 的3CD水解酶即能使得P1蛋白被切割並形成類病毒顆粒。因此,本發 明將P10啟動子換成在昆蟲細胞内較弱勢的啟動子(IE-1及CMV啟動 子)’希望能夠藉由降低3CD水解酶的量,使得P1蛋白表現量增加, 進一步提高類病毒顆粒的產量。因此本發明也將3CD基因接至弱勢啟 動子’如正-1啟動子(immediate early promoter)及CMV啟動子 (cytomegalovirus)下游,製備出另外二株病毒,Bac-Pl-I3CD及 Bac-Pl-CBCD (見圖 1)。重組桿狀病毒 Bac-Pl-3CD、Bac-Pl-C3CD、 Bac-Pl-I3CD皆可表現P1及3CD重組蛋白。 建構桿狀病毒株完成後’以懸浮培養法在Spinner Flask培養Sf-9及Hi-5 細胞’在細胞濃度達到約lxl〇6 cell/ml時,分別以Bac_P1_3CD、 Bac-Pl-C3CD及 Bac-Pl-I3CD 以 MOI10 進行感染。於感染後48, 72, 96 小時收取細胞液以l〇〇〇〇Xg離心10分鐘,再以西方墨點法初步偵測 VP1蛋白表現。就sf-9細胞而言,由圖2(a)中,比較三株桿狀病毒的 vpi產量’發現不論在感染後那一天所收取的細胞上清液,Bac_p丨 所表現的vpi蛋白產量都比重組桿狀病毒Bac_pi_C3CD及 Bac-Pl-l3CD 的產量來得低。Bac_pi_C3CE^Bac_pi i3CD 則在感染後 第四天可以達到最高的W1產量,且Bac_pi_C3CD所產生的胞外νρι 產量最高。接著’㈣論上表現量較高的昆蟲細麟职進行類病毒 顆粒的生產,以和Sf-9 _的實驗條件進行病毒感染及細胞上清液的 24 201100545 收成分析。實驗結果經Western blot分析後如圖2(b)。相反地,Cg〇ld_conjugated goat anti-mouse IgG, Sigma) diluted 1:1000 in PBS' Take diluted antibody 1〇μ1 drops on copper net, connect the size of the secondary antibody to $ crispy gold particles, age Under the electron microscope _ presentation. After the tertiary antibody was reacted for 1 hour, the surface of the copper mesh was washed three times with PBST. After washing, the surface solution of the copper mesh was blotted with cotton paper, and a solution of 1〇μ1 of 2〇/capric acid crane was dripped in Tongshe, and the dyeing was allowed to stand. After three minutes, the excess dyed hair was dried with cotton paper and placed for -12 hours, as observed by an electron microscope (H-7500). The combination of the strain and the cell strain was learned from previous laboratory studies that the rod-shaped silk/insect filaments together express the protein Pi of the enterovirus type 71 (driven by the p〇lyhedrin strong promoter) and 3CD (with pi Reluctant promoter drive) 'P1 can be 3CD _ 丨 纲 , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , The baculovirus prepared was named Bac-Pl-3CD (see Figure 1). To enhance the production of viroid-like particles, the present invention contemplates that a small amount of 3CD hydrolase can cause the P1 protein to be cleaved and form viroid-like particles. Therefore, the present invention replaces the P10 promoter with a weaker promoter (IE-1 and CMV promoter) in insect cells. It is desirable to increase the amount of P1 protein by reducing the amount of 3CD hydrolase, and further improve the class. The production of viral particles. Therefore, the present invention also ligates the 3CD gene to a weak promoter such as the immediate early promoter and the CMV promoter (cytomegalovirus) to prepare two other viruses, Bac-Pl-I3CD and Bac-Pl-. CBCD (see Figure 1). Recombinant baculoviruses Bac-Pl-3CD, Bac-Pl-C3CD, and Bac-Pl-I3CD can express P1 and 3CD recombinant proteins. After the construction of the baculovirus strain, 'Sf-9 and Hi-5 cells were cultured in Spinner Flask by suspension culture method', when the cell concentration reached about lxl〇6 cell/ml, Bac_P1_3CD, Bac-Pl-C3CD and Bac-, respectively. Pl-I3CD is infected with MOI10. At 48, 72, and 96 hours after infection, the cell fluid was centrifuged at 10 μg for 10 minutes, and the VP1 protein expression was initially detected by Western blotting. In the case of sf-9 cells, comparing the vpi production of three baculoviruses in Figure 2(a), it was found that regardless of the cell supernatant collected on the day after infection, the Vp protein production by Bac_p丨 was The yield was lower than that of the recombinant baculoviruses Bac_pi_C3CD and Bac-Pl-l3CD. Bac_pi_C3CE^Bac_pi i3CD achieved the highest W1 yield on the fourth day after infection, and Bac_pi_C3CD produced the highest extracellular νρι yield. Then, (4) on the production of viroid-like particles, the insects with higher performance were analyzed, and the virus infection and cell supernatant were analyzed by the experimental conditions of Sf-9 _ 24 201100545. The results of the experiment were analyzed by Western blot as shown in Fig. 2(b). Conversely,

Bac-Pl-C3CD及Bac-Pl-I3CD在感染Hi_5細胞後,VP1蛋白的產量遠 比Bac-Pl-3CD少,且VP1蛋白的表現隨時間逐漸下降。但反觀以 Bac-Pl-3CD感染Ηι·5的VP1蛋白表現則隨著時間而逐漸增加。 為了得知用那種桿狀病毒及昆蟲細胞株,可得到最高的類病毒顆粒產 量。本發明以Western blot結果中表現量較好的幾組實驗組再進一步地After infection of Hi_5 cells, Bac-Pl-C3CD and Bac-Pl-I3CD produced much less VP1 protein than Bac-Pl-3CD, and the expression of VP1 protein gradually decreased with time. However, the expression of VP1 protein infecting Ηι·5 with Bac-Pl-3CD gradually increased with time. In order to know which baculovirus and insect cell strains are used, the highest virus-like particle yield can be obtained. The present invention further improves the number of experimental groups with better expression in Western blot results.

❹進行ELISA分析。將三株病毒株感染Sf_9細胞,或是以Bac_pi 3cD 感染Hi-5細胞,共四組實驗條件,在感染第四天以EUSA分析上清液❹Analysis was performed by ELISA. Three strains of virus were infected with Sf_9 cells, or Hi-5 cells were infected with Bac_pi 3cD for a total of four experimental conditions. The supernatant was analyzed by EUSA on the fourth day of infection.

内類病毒顆粒的表現量。結果如圖2(c),以Bac_pi_3CD及Bac-Pl-I3CD 感染Sf_9細胞的胞外類病毒顆粒的產量分別為1.5 mg/L及7.8 mg/L, 而BaC-P1_C3CD組別的產量最高,可達到12 5 mg/L。若以阳為宿 主細胞時’ Bac-Pl-3CD組別可達12.1 mg/L。此兩組感染細胞及病毒組 合產生的VLP產量較佳且結果相近。因此本發明選定兩個細胞與病毒 Ο 、组合(以Bac-P1_C3CD感染Sf-9細胞及以Bac-Pl-3CD感染Hi-5細胞) 來進行最佳感染細胞濃度(CCI,Cell Concentration at Infection)條件探 討。 量隹感染濃唐摆討 除了改良桿狀病毒的基因設計,亦探討感染時的細胞濃度對於Mp產 量的影響。首先’本發明在Sf-9細胞濃度達到1χ106、2χ1〇6、4χ1〇6及 6xl〇6 Cells/ml 時,以 Μ〇Ι 1〇 的 Bac_pi_C3CD 感染 _ 細胞取感染 後第4天的細胞上清液,以ELISA分析胞外類病毒顆粒產量。如圖3 25 201100545 所示,當細胞濃度增加時,類病毒顆粒的產量亦有所提升,以4χ1〇6 cell/ml進行生產時可有最高產量43·3 mg/L。但過高的細胞漠度(6χ1〇6 cell/ml)則可能因為養分短缺使得細胞存活率快速下降,反而造成類病 毒顆粒產量下降。 在Hi-5部分,由於以SF-900II培養Hi-5細胞在旋轉瓶内之最大細胞濃 度為3xl06cells/ml,因此在細胞濃度達到1χ1〇6及2xl〇6cell/m丨時進行 感染,並且在感染後第4天收取細胞上清液,以ELISA定量分析胞外 ◎ 類病毒顆粒產量。如圖3所示,在固定劑量(M〇I 1〇)條件下,提高感 染時的細胞濃度的確能提升VLP產量,與Sf-9細胞的結果相似。當細 胞濃度達2xl06Cell/ml可以有最高的類病毒顆粒量22 2mg/L。 綜合以上結果,Sf-9細胞在細胞濃度達到4xl06cdl/ml時以M〇I 1〇的 Bac-Pl-C3CD病毒劑量進行感染,在第四天可得最大產量。因此決定 以此條件進行感染,並於第四天收取上清液,做為後續反應器生產類 病毒顆粒之生產參數。也將此感染條件所收取的細胞液以高速離心機 離心30分鐘(18000 rpm)後’收取上清液’再進行初步純化作為後續小 鼠實驗之抗原。 反應器生產類病毒顆粒 在旋轉角瓶中確定類病毒顆粒最佳生產條件後,嘗試以實驗室等級攪 拌式生物反應器(BIOSTAT®B)來生產類病毒顆粒。實驗中調整不同的 溶氧量值(DO 15%、30%、60%),以比較不同的溶氧量對於胞外類病 26 201100545 毒顆粒產量的影響。相同生產條件但於旋轉角瓶所生產之類病毒顆粒 產量則做為正對照組(positive)。ELISA分析結果(圖4)顯示,在反應 器内所生產的類病毒顆粒產量比旋轉角瓶所生產的類病毒顆粒產量來 的高。此外,溶氧量控制在30%的類病毒顆粒表現量最高,在第四天 後的最大產量可達64.3mg/L。因此在反應器溶氧量的部分,選定溶氧 量為30%為反應器生產參數。 類病毒顆粒定性分析 以Bac-Pl-C3CD生產的類病毒顆粒產量較先前以Bac_pi_3CD生產類 病毒顆粒的產量有大幅增加。為確認以Bac-Pl-C3CD生產出來的類病 毒顆粒與Bac-Pl-3CD生產出來的類病毒顆粒有相同基本性質,本發明 以Bac-Pl-C3CD感染Sf-9細胞所生產出來的類病毒顆粒為實驗組,而 以Bac-Pl-;3CD感染Hi-5細胞所生產的類病毒顆粒為正對照組,進行 後續各項定性分析。 離心純化後的類病毒顆粒(包含實驗組及正對照組)以磷鎢酸做負染色 後,利用生物型穿透式電子顯微鏡觀察。由圖5(a)可以發現,本發明 所純化出來的VLP有類似圓形的形狀,顆粒大小約為25 3〇nm,和實 際病毒顆粒(27nm)大小相似,並與先前利用Bac-pi_3CD感染扭5細 胞後,收取齡场·自PEG臟結轉_朗析法純化所得到 的VLP (圖5(b)) ’在外觀及大小上絲太大差異。雜巾間呈現黑色, 可能是顆粒内部被染齡人所造賴,因此__應縣中空的顆 粒球。 27 201100545 為了進一步證明這些顆粒就是腸病毒71型VLP,本發明以anti-VPl 單株抗體辨識並抓取腸病毒VLP上的VP1蛋白質,接著以連接金粒子 (5 nm)的二級抗體辨認抓取VP1單株抗體,如此完成的免疫金標誌以 穿透式電子顯微鏡觀察,結果如圖5(c)所示。圖中可以隱約看出VLP 的分佈,但可能是因為使用較多抗體蛋白質,和單純的負染色結果相 比較,顆粒的外觀變的較為模糊。圖中可以明顯看出5 nm金粒子的小 黑點’這些小黑點絕大部分都包圍在類病毒顆粒附近,因此確認這些 顆粒含有腸病毒VLP蛋白質VP1。同時也將實驗結果與先前實驗室以 Bac-Pl-3CD感染Sf-9細胞後’打破細胞並利用超高速離心法所純化類 病毒顆粒後,以上述同樣的抗體進行免疫金標誌及電子顯微鏡觀察的 結果做比對,如圖5 (d)也發現金粒子都包圍類病毒顆粒附近。 綜合以上結果,以穿透式電子顯微鏡觀察到兩組類病毒顆粒的粒徑大 小與形狀相同,也從免疫金染色當中證明W1位於類病毒顆粒的表 面。在此可以初步結論以Bac-Pl-C3CD感染Sf-9細胞後所生產的類病 毒顆粒與實驗室縣以BaoPUCD _ HU _或是卿細胞所生 產的類病毒顆粒的基本性質是非常近似的。 實施例二 iA免疫實驗分析 小鼠免疫及血清樣本收集 為了瞭解純化後的類病毒顆粒在免疫性質上的表現,本發明以腹腔注 28 201100545 射的方式進行動物實驗。在樣品純化完成後,先將總蛋白質(total protein)濃度調整成 40 pg/ml,存放於-80°C 冰箱中。Female BALB/c mice是由國家動物中心訂購,並送往清大動物房進行檢疫後,在小鼠 週齡為6〜8週時進行腹腔注射。進行腹腔注射前,將樣品與相同體積 的佐劑(complete Freund’s adjuvant for priming injection,Sigma > cat# F5881 ; incomplete Freund’s adjuvant for booster injection , Sigma,cat# F5506)以三向閥進行混合約3分鐘,待樣品混合均勻呈白色乳膠狀e 每隻小鼠以500 μΐ (1〇 total protein/mouse)混合後的乳膠狀樣品進行 腹腔注射,此時標示為第〇週。於第四週時,以相同樣品準備方式及 注射方式(但佐劑為incomplete Freund’s adjuvant)進行加強注射(booster injection)。於第7週時,利用臉部採血法收集小鼠血液,採集得來的 小鼠血液’置於4 C冰箱4小時後,以2〇〇〇xg離心1〇分鐘。所得澄 清的小鼠血清以56°C去活化30分鐘後,再進行後續分析。實驗組別 包括以下五組·(1) PBS組:為了消除佐劑對於實驗數值嗎響,本發 明將PBS齡佐舰進行相縣射,並以此崎收集的血清作為數值 評估的依據。⑵負對照組(rBV):為了瞭解抗體反應是否確實來自類 病毒顆粒,而非其他雜蛋白質所造成的誤差,本發明以—株未帶有任 何外源蛋自基關飯雛病毒· Sf_9細祕,與實驗組相同 的純化程序進行純化,收集相同位置的蛋白質,以此作為負對照組。 ⑶正對照組(EV71) ·以蔗糖不連續梯度離^法所純化而得到的腸病 毒’純化後的腸病毒尚具有感染力,因此以肌加熱Μ分鐘去活化。 ⑷正對照組(BaC-P1-3CD):以Bac孔3CD感染鼠細胞所生產出類 29 201100545 病毒顆粒。(5)實驗組(Bac-Pl-C3CD):以Bac-Pl-C3CD感染Sf-9細 胞所生產出類病毒顆粒。每一組實驗動物隻數為四隻。 抗體效價 首先配置 Coating buffer ;以 ddH20 先配製 0.1 M Na2HP04 (Riedel-de Haen ’ cat# 30414) ( pH 約 9.0)及 0.1 MNaH2P04 (聯工,cat# 90093) (pH 約 4.5)。再取固定量 0.1 MNa2HP04 溶液以 0.1 MNaH2P04 將 pH 調至6.8即為Coating buffer (pH 6.8)。接著將純化而得的腸病毒經由蛋 白質分析(protein assay)定量出總蛋白量,並以Coating buffer稀釋到 總蛋白濃度為5 μθμΐ後,於96孔盤加入1〇〇 μΐ,於37 °C靜置一小時 使抗原吸附到孔中的底部。吸附完成後將溶液吸出,以200 μΐ清洗液 PBS-T (0.05% Tween 20 in PBS buffer)注入各個孔,於室溫下靜置3分 鐘後’將清洗液取出’重複清洗的步驟共3次。最後一次取出清洗液 後,加入 100 μΐ 的 blocking buffer (0.1% BSA in PBST)後於 37 °C 下靜 置反應1小時’將孔中底部的所有空隙都填補上蛋白質,接著以清洗 液重複清洗3次。 取出清洗液後’加入1〇〇μ1稀釋後的小鼠血清(以blocking buffer在 eppendorf中做連續兩倍稀釋,稀釋倍率為25到22Q),於37 °C下靜置 反應1小時。以清洗液重複清洗3次後,加入1〇〇 μΐ稀釋5000倍的goat anti-mouse IgG conjugated with HRP (Kirkegaard & Perry Laboratories > Gaithersburg ’ MD)二級抗體,於37 下靜置反應1小時,再以清洗 液重複清洗3次。接著加入100 μΐ/well TMB (Sigma,cat# T〇440)進行 30 201100545 呈色’於室溫下反應。待呈現明顯藍色後,於每個孔中加入5〇_2M ΗΑ〇4以中止呈色反應,將微量滴定盤置於EusA福过(腦咖⑶ Technology lnc.,Glen Allen,VA)中,以波長mo腿讀取其犯认數值。The amount of expression of the inner type of virus particles. The results are shown in Figure 2(c). The yields of extracellular viroid particles infected with Bac_pi_3CD and Bac-Pl-I3CD were 1.5 mg/L and 7.8 mg/L, respectively, while the BaC-P1_C3CD group had the highest yield. Up to 12 5 mg / L. If yang is the host cell, the Bac-Pl-3CD group can reach 12.1 mg/L. The VLP production by the two groups of infected cells and virus combinations was better and the results were similar. Therefore, the present invention selects two cells and virus 、, combination (infecting Sf-9 cells with Bac-P1_C3CD and infecting Hi-5 cells with Bac-Pl-3CD) for optimal concentration of infected cells (CCI, Cell Concentration at Infection) Conditional discussion. In addition to improving the genetic design of baculovirus, the effect of cell concentration at the time of infection on Mp production was also investigated. First, the present invention infects cells infected with Bac_pi_C3CD at a concentration of 1χ106, 2χ1〇6, 4χ1〇6, and 6xl〇6 Cells/ml, and the cell supernatant on the 4th day after infection is taken. Liquid, extracellular viroid particle yield was analyzed by ELISA. As shown in Figure 3 25 201100545, when the cell concentration increases, the production of viroid-like particles also increases, and the highest yield can be 43. 3 mg/L when produced at 4χ1〇6 cells/ml. However, excessive cell infiltration (6χ1〇6 cell/ml) may result in a rapid decrease in cell viability due to a shortage of nutrients, which in turn may result in a decrease in the production of virulence particles. In the Hi-5 part, since the maximum cell concentration of the Hi-5 cells cultured in SF-900II in the spinner flask is 3x10 cells/ml, the infection is carried out at a cell concentration of 1χ1〇6 and 2xl〇6cell/m丨, and The cell supernatant was collected on the 4th day after infection, and the extracellular ◎ viroid particle production was quantitatively analyzed by ELISA. As shown in Figure 3, increasing the concentration of cells at the time of infection at a fixed dose (M〇I 1〇) did increase VLP production, similar to the results of Sf-9 cells. When the cell concentration reaches 2xl06Cell/ml, the highest amount of virus-like particles can be 22 2mg/L. Taken together, the Sf-9 cells were infected with M〇I 1〇 Bac-Pl-C3CD virus at a cell concentration of 4×10 6 cdl/ml, and the maximum yield was obtained on the fourth day. Therefore, it was decided to infect under this condition, and the supernatant was collected on the fourth day as a production parameter for the production of virus-like particles in the subsequent reactor. The cell fluid collected by this infection condition was also centrifuged in a high-speed centrifuge for 30 minutes (18,000 rpm), and then the supernatant was collected and subjected to preliminary purification as an antigen for subsequent mouse experiments. Reactor production of virus-like particles After determining the optimal production conditions for viroid-like particles in a rotating angle bottle, an attempt was made to produce virus-like particles using a laboratory grade stirred bioreactor (BIOSTAT® B). Different dissolved oxygen values (DO 15%, 30%, 60%) were adjusted in the experiment to compare the effects of different dissolved oxygen levels on the production of extracellular disease 26 201100545 toxic particles. The same production conditions but the production of viral particles produced in a rotating angle bottle were used as a positive control. The results of the ELISA analysis (Fig. 4) showed that the production of viroid-like particles produced in the reactor was higher than that produced by the virulet-like particles produced by the rotary angle bottle. In addition, the amount of dissolved oxygen is controlled at 30% of the virus-like particles, and the maximum yield after the fourth day is 64.3 mg/L. Therefore, in the portion of the reactor dissolved oxygen, the dissolved oxygen content was selected to be 30% of the reactor production parameters. Qualitative analysis of viroid-like particles The production of viroid-like particles produced with Bac-Pl-C3CD was significantly increased compared to the previous production of viruvirus particles produced as Bac_pi_3CD. In order to confirm that the viroid-like particles produced by Bac-Pl-C3CD have the same basic properties as the viroid-like particles produced by Bac-Pl-3CD, the present invention infects Sf-9 cells with Bac-Pl-C3CD to produce viroid-like viruses. The granules were the experimental group, and the viroid-like particles produced by infecting Hi-5 cells with Bac-Pl-;3CD were positive control groups, and subsequent qualitative analysis was performed. The virus-like particles after centrifugation (including the experimental group and the positive control group) were negatively stained with phosphotungstic acid and observed by a biotransmission electron microscope. It can be seen from Fig. 5(a) that the VLP purified by the present invention has a circular shape with a particle size of about 25 Å, which is similar in size to the actual virus particles (27 nm) and is previously infected with Bac-pi_3CD. After twisting 5 cells, the VLP (Fig. 5(b))' obtained from the age field and from the PEG dirty knot _ lang method was too different in appearance and size. The black space between the rags may be caused by the people inside the granules, so __ Ying County's hollow granules. 27 201100545 To further demonstrate that these granules are enterovirus 71 VLPs, the present invention recognizes and captures VP1 protein on enterovirus VLPs with anti-VP1 monoclonal antibodies, and then recognizes secondary antibodies linked to gold particles (5 nm). The VP1 monoclonal antibody was taken, and the immunogold label thus completed was observed by a transmission electron microscope, and the results are shown in Fig. 5(c). The distribution of VLP can be vaguely seen in the figure, but it may be due to the use of more antibody proteins, and the appearance of the particles becomes more blurred compared to the simple negative staining results. It can be clearly seen that the small black dots of 5 nm gold particles are mostly surrounded by viroid-like particles, so it is confirmed that these particles contain the enterovirus VLP protein VP1. At the same time, the experimental results were compared with the previous laboratory infection of Sf-9 cells with Bac-Pl-3CD. After breaking the cells and purifying the virus-like particles by ultracentrifugation, the same antibodies were used for immunogold labeling and electron microscopic observation. The results were compared, as shown in Figure 5(d). Gold particles were also found to surround the viroid-like particles. Based on the above results, the particle size and shape of the two types of virus-like particles were observed by a transmission electron microscope, and it was confirmed from the immunogold staining that W1 was located on the surface of the viroid-like particle. It can be concluded here that the virulence particles produced by infecting Sf-9 cells with Bac-Pl-C3CD are very similar to the basic properties of virulent granules produced by laboratory cells in BaoPUCD _HU _ or qing cells. Example 2 Analysis of iA immunoassay Mouse immunization and serum sample collection In order to understand the immunological properties of purified viroid-like particles, the present invention conducts animal experiments by means of intraperitoneal injection. After the sample was purified, the total protein concentration was adjusted to 40 pg/ml and stored in a -80 °C refrigerator. Female BALB/c mice are ordered by the National Animal Center and sent to the Animal Hall of Qingda for quarantine. The mice are injected intraperitoneally at 6-8 weeks of age. Before the intraperitoneal injection, the sample was mixed with the same volume of adjuvant (complete Freund's adjuvant for priming injection, Sigma >cat#F5881; incomplete Freund's adjuvant for booster injection, Sigma, cat# F5506) in a three-way valve for about 3 minutes. The sample was uniformly mixed in a white latex-like e. Each mouse was intraperitoneally injected with a latex sample of 500 μΐ (1〇total protein/mouse), which was indicated as the third week. At the fourth week, booster injection was performed in the same sample preparation method and injection method (but the adjuvant was incomplete Freund's adjuvant). At the 7th week, the blood of the mice was collected by facial blood collection, and the blood of the collected mice was placed in a 4 C refrigerator for 4 hours, and then centrifuged at 2 Torr x for 1 minute. The obtained clarified mouse serum was deactivated at 56 ° C for 30 minutes, and then subjected to subsequent analysis. The experimental group consisted of the following five groups: (1) PBS group: In order to eliminate the effect of the adjuvant on the experimental values, the present invention carried out the PBS-aged ship, and used the collected serum as the basis for numerical evaluation. (2) Negative control group (rBV): In order to understand whether the antibody reaction is indeed caused by the virus-like particles, rather than the errors caused by other miscellaneous proteins, the present invention does not carry any exogenous egg from the genus of the rice worm virus Sf_9 The secret was purified by the same purification procedure as the experimental group, and the protein at the same position was collected as a negative control group. (3) Positive control group (EV71) - Enteric virus obtained by purification by sucrose discontinuous gradient method The purified enterovirus still has an infectivity, so it is activated by heating the muscle for a minute. (4) Positive control group (BaC-P1-3CD): The virus particles were produced by infecting mouse cells with Bac pore 3CD. (5) Experimental group (Bac-Pl-C3CD): Sf-9 cells were infected with Bac-Pl-C3CD to produce viroid-like particles. The number of experimental animals per group was only four. Antibody titer First, configure the Coating buffer; first prepare 0.1 M Na2HP04 (Riedel-de Haen ’ cat# 30414) (pH about 9.0) and 0.1 MNaH2P04 (Lian Gong, cat# 90093) (pH about 4.5) with ddH20. A fixed amount of 0.1 MNa2HP04 solution was adjusted to pH 6.8 with 0.1 MNaH2P04 to obtain a Coating buffer (pH 6.8). Next, the purified enterovirus was quantified by protein assay, and diluted to a total protein concentration of 5 μθμΐ with a Coating buffer. Then, 1 μμΐ was added to a 96-well plate, and the mixture was incubated at 37 ° C. One hour was allowed to adsorb the antigen to the bottom of the well. After the adsorption was completed, the solution was aspirated, and 200 μL of the washing solution PBS-T (0.05% Tween 20 in PBS buffer) was injected into each well, and after standing at room temperature for 3 minutes, the 'cleaning liquid was taken out'. The repeated washing steps were repeated 3 times. . After the last removal of the cleaning solution, 100 μΐ of blocking buffer (0.1% BSA in PBST) was added and the reaction was allowed to stand at 37 ° C for 1 hour. 'All the voids in the bottom of the well were filled with protein, and then washed repeatedly with a cleaning solution. 3 times. After the washing solution was taken out, the serum of the mouse diluted with 1 μμ1 was added (two-fold serial dilution in eppendorf with a blocking buffer, dilution ratio of 25 to 22Q), and the reaction was allowed to stand at 37 ° C for 1 hour. After repeated washings with washing solution for 3 times, a goat anti-mouse IgG conjugated with HRP (Kirkegaard & Perry Laboratories > Gaithersburg 'MD) secondary antibody diluted 5,000 times was added, and the reaction was allowed to stand at 37 for 1 hour. Then repeat the washing 3 times with the cleaning solution. Then, 100 μΐ/well TMB (Sigma, cat# T〇440) was added for 30 201100545 coloring, and the reaction was carried out at room temperature. After the appearance of a clear blue color, 5〇_2M ΗΑ〇4 was added to each well to stop the color reaction, and the microtiter plate was placed in EusA (Brain (3) Technology lnc., Glen Allen, VA). Read the falsified value with the wave mo leg.

讀取吸光值(OD)後,首先必須先檢查陰性組別(注射pBS組)的〇D 值疋否i於0.2 ’若大於〇.2則應判定為測試無效,需重新檢測。而陽 性組別的判定為比陰性組別大〇 2的〇D值。例如,若陰性組別的吸光 〇 值為〇.2 ’則若實驗組稀釋至216後,吸光值低於Ο.4 (0.2+0.2),則此實 驗組效價判定為15。 中和效價 首先將貼附培養的RD細胞以Trypsin脫附下來後,稀釋成2χ1〇5 cells/ml’並將懸浮細胞量種入96孔盤,置於5% c〇2的37〇c 恆溫培養箱中過夜。接著以含有2 % FBS及1 %腦滿〇价的 α-ΜΕΜ對去活化後的小鼠血清樣品進行2倍的連續稀釋(23〜2ls),並 〇 與相同體積的 2000 TCID50live EV71 (混合後為 1000 TCID50 EV71)均 勾混合後,置入37°C恆溫培養箱中進行中和反應1小時。接著,自37<3C 恆溫培養箱中取出培養隔夜的奶細胞,將培養液抽去後,加入 ΙΟΟμΙ/well已反應1小時的血清病毒混合液,之後置入37〇c恆溫培養 箱培養。3天後,觀察96孔盤中細胞病變現象(CPE)的情形。 根據CPE的情形,能保護5〇 % weu不產生CpE的血清稀釋倍率,該 稀釋倍率即為該血清樣品的中和抗體校價。例如,以後的稀釋倍率 31 201100545 組別中和反應皆停止(出現CPE的well數大於50 °/〇) ’則211即為該血 清樣品的中和抗體校價。 交叉保護效果(cross reaction)分析 本發明利用抗體中和試驗,分析以KV3 strain VLP所誘發的小鼠血 清,對於中和不同strain腸病毒71型感染力的效果。實驗方式與中和 試驗類似’首先將貼附培養的RD細胞以Trypsin脫附下來後,稀釋成 2xl05 cells/ml的濃度,並將懸浮細胞種入96孔盤(ΙΟΟμΙ/well)。接著以 含有2% FBS及1% PSN antibiotic的α-ΜΕΜ對去活化後的小鼠血清進 行2倍的連續稀釋(23〜215),稀釋後的血清混合加入不同血清型的腸病 毒71型(如TW/2272/98 strain及Β5 strain) ’所有使用的病毒劑量均為 100 TCIDsq,混合後的樣品於室溫下反應約丨小時,反應完成後移除 96 well培養盤中的培養液,再將1〇〇μ1小鼠血清與待測病毒的混合液 加入培養孔中(最後病毒濃度100 ,並於37τ培養箱中培 養3天後觀察96孔盤中細胞病變現象的情形,並依據所述之中和效價 判定方法來確定血清之中和效價。 統計分析 數值差異性的分析法,是用至少三次重複實驗的數據,心檢驗 (S一 Mest)進行統計分析。計算出的p值(”-)小於〇 〇5表示 數值間有差異’反之若大於_,則表示數侧沒有明顯差異。 結果 32 201100545 蓮病毒顆粒做為疫苗的免▲疬性y 抗體效價分析 為了確定經過VLP免疫後的小鼠是否能產生mfEVW專一性抗體, 首先將實驗所採集的小鼠血清以56〇c加熱3〇分鐘去活化來去除補體 的干擾,接著進行抗體效價分析。如圖6所示。PBS負對照組不會引 發抗體免疫反應,而rBV則會引發些許的免疫反應,抗體效價值約為 27。但實驗組(Bac_P1_C3CD)及兩組正對照組(EV7i及Bac_pi 3cD)的 ❹ 抗體效價值皆可達到212-213,且在統計上並無顯著的差異㈣〇5)。 也清中和效價(neutralization titer)分析 抗體效價只能分析抗體辨認腸病毒抗原的能力,並無法代表實際感染 時抗體中和真實腸病毒的能力,因此以血清巾和效價分析來評估抗體 中和真實病毒的能力。本發明將小鼠血清做連續2倍稀釋(最低稀釋 倍率為23),並與1〇〇 TCI〇5Q的真實EV71(TW/2272/98 strain)混合反應 Ο 兩小時後’加入96孔盤對RD細胞進行感染,於三天後觀察CPE現象, 並計算中和效價。圖7(a)的結果顯示,負對照組PBS及rBV組及在最 低稀釋倍树即無法對EV71進行巾和反應,正對敝EV71在注射後 第七週的中和效價達27,而正對照組Bac-Pl-3CD及實驗組 Bac-Pl-C3CD則有著良好的中和真實EV71的能力,其中和效價值分別 可逹到21α75及21α5,且兩者之間並無顯著的統計學差異(户>〇 〇5)。推 測去活化腸病毒引發中和效價低於實驗組Bac_pi_C3CD或是正對照組 Bac-Pl-3CD的仙可能是目械化毒的純度不高關係,導致無 33 201100545 法引發报好的免疫反應。 抗體對於不同腸病毒株的交叉保護 前述中和效價實驗中所用的活病毒(TW/2272/98)與本發明生產類病毒 顆粒所用的病雜彻為同—麵,均為溯年流行的病毒株。而在 2008年流行的腸病毒?!型則是屬於。為了確定^所誘 發的抗體是雜巾和㈣基因養職毒71型,本發鶴收集的小鼠 血/月針對genotype B5的病毒株進行中和測試。首先以奶細胞來感染 增殖病毋,再測定各個病毒的濃度,利用病毒的中和測試的方法來測 定類病毒顆粒所誘發的抗體對病毒的中和強度。由圖7⑹的結果發現 與圖7(a)類似的結果,負對照組PBS及麟組無法有效中和腸病毒71 型,而正對照組EV71所引發种和效價為π5,依舊低於正對照組 BaC-Pl-3CD (效價達2心),而由Bae_pi_C3(:D生產的類病毒顆粒所誘 發出的抗體,在中和B5病毒株的最高稀釋倍率能夠達到2出,這代表 了由助strain腸病毒71型類病毒顆粒所誘發的抗體亦可以有效的 降低其他不同腸病毒71型病毒株的感染力。 以現有的數據顯示’以新建立的方式所得到_病毒顆粒與本實驗室 先前的生產方式所得_縣驗齡具有作為腸縣W型疫苗候選 者的良好潛力。 【圖式簡單說明】 基因P1及3CD分別轉殖進入 圖1重組桿狀病毒載體之建構。EV71 34 201100545 pFastBac™ DUAL質體中’經過Bac-to-Bac System所建議之實驗流程 後即可製成病毒(a) Bac-Pl-3CD (以pi〇啟動子驅動3CD表現),(b) Bac-Pl-C3CD (以 CMV 啟動子驅動 3CD 表現),(c) Bac-Pl-I3CD (以 IE-1啟動子驅動3CD表現)。 圖 2 比較以三種病毒株(Bac-Pl-3CD,Bac-Pl-C3CD,Bac-Pl-I3CD)在 不同細胞内生產腸病毒71型類病毒顆粒的產量·》當細胞濃度約達ΐχΐ〇6 〇 cell/ml 時’以 MOI10 感染細胞’感染後 2, 3 或 4 天(days post-infection, dpi) ’收取細胞液’以低速離心將細胞離心下來,收取上清液作為胞外 類病毒顆粒樣品。(a)以西方墨點法分析Sf-9細胞的表現量。(b)以西 方墨點法分析Hi-5細胞的表現量。(c)以ELISA定量不同基因重組桿 狀病毒在感染Sf-9細胞(□) ’及以Bac-Pl-3CD感染Hi-5細胞(_) 後72小時類病毒顆粒的總產量。上述的實驗皆於1〇〇 ml spinner flask 操作,實驗中所使用的培養基皆為SF-900II。實驗結果為三次獨立細胞After reading the absorbance value (OD), first check the 〇D value of the negative group (injected pBS group) 疋 No i at 0.2 ’ If it is greater than 〇.2, it should be judged that the test is invalid and need to be retested. The positive group was judged to be a 〇D value greater than the negative group. For example, if the absorbance 阴性 value of the negative group is 〇.2 ’, then if the experimental group is diluted to 216 and the absorbance is lower than Ο.4 (0.2+0.2), the titer of this experimental group is judged to be 15. Neutralization titer First, the attached RD cells were desorbed with Trypsin, diluted to 2χ1〇5 cells/ml′ and the suspended cells were seeded into a 96-well plate, placed at 37°c of 5% c〇2. In a constant temperature incubator overnight. The deactivated mouse serum samples were then serially diluted 2 times (23~2 ls) with 2% FBS and 1% brain full valence α-ΜΕΜ, and 〇 with the same volume of 2000 TCID50live EV71 (mixed After 1000 TCID50 EV71), the mixture was mixed and placed in a 37 ° C incubator for neutralization for 1 hour. Next, the overnight cultured milk cells were taken out from the 37 <3C incubator, and the culture solution was removed, and then the serum virus mixture which had been reacted for 1 hour with ΙΟΟμΙ/well was added, and then placed in a 37 °C incubator. After 3 days, the condition of cytopathic effect (CPE) in the 96-well plate was observed. According to the case of CPE, it is possible to protect 5 〇 % weu from the serum dilution ratio of CpE, which is the neutralizing antibody valence of the serum sample. For example, the subsequent dilution ratio 31 201100545 group neutralization reaction is stopped (the number of CPE wells is greater than 50 ° / 〇) ' then 211 is the neutralizing antibody calibration of the serum sample. Cross-Reaction Analysis The present invention utilizes an antibody neutralization assay to analyze the effect of mouse sera induced by KV3 strain VLP on neutralizing the infectivity of different strains of enterovirus 71. The experimental method was similar to the neutralization test. First, the attached RD cells were desorbed with Trypsin, diluted to a concentration of 2×10 5 cells/ml, and the suspension cells were seeded into a 96-well plate (ΙΟΟμΙ/well). The deactivated mouse serum was then serially diluted 2 times (23 to 215) with α-ΜΕΜ containing 2% FBS and 1% PSN antibiotic, and the diluted serum was mixed with enterovirus 71 of different serotypes ( For example, TW/2272/98 strain and Β5 strain) 'All virus doses used are 100 TCIDsq, and the mixed sample is reacted at room temperature for about 丨 hours. After the reaction is completed, the culture solution in the 96 well culture plate is removed, and then A mixture of 1 μl mouse serum and the virus to be tested was added to the culture well (final virus concentration was 100, and the cytopathic phenomenon in the 96-well plate was observed after culturing for 3 days in a 37 τ incubator, and according to the The neutralization titer determination method is used to determine the serum neutralization titer. The statistical analysis of the numerical difference analysis method is performed by using at least three repeated experiments, and the heart test (S-Mest) is used for statistical analysis. ("-) is less than 〇〇5 means that there is a difference between the values'. If it is greater than _, it means that there is no significant difference on the number side. Results 32 201100545 Lotus virus particles as a vaccine for 疬 y y antibody titer analysis in order to determine the VLP Immunology Whether the latter mouse can produce mfEVW-specific antibodies, firstly, the mouse serum collected by the experiment was heated at 56 ° C for 3 minutes to activate the interference to remove complement, followed by antibody titer analysis. The PBS negative control group did not elicit an antibody immune response, while rBV elicited a slight immune response with an antibody titer of approximately 27. However, the experimental group (Bac_P1_C3CD) and the two groups of positive control groups (EV7i and Bac_pi 3cD) had anti-antibody antibodies. The value can reach 212-213, and there is no statistical difference (4) 〇 5). Neutralization titer analysis of antibody titer can only analyze the ability of antibodies to recognize enterovirus antigens, and can not represent The ability of the antibody to neutralize the real enterovirus during actual infection, so the ability of the antibody to neutralize the real virus is evaluated by serum towel and titer analysis. The present invention serially dilutes the mouse serum by a 2-fold dilution (minimum dilution ratio is 23), and Mixed reaction with 1 〇〇TCI〇5Q real EV71 (TW/2272/98 strain) Ο Two hours later, RD cells were infected by adding 96-well plates, and CPE phenomenon was observed after three days, and the neutralizing titer was calculated. Figure 7(a) The results showed that the PBS and rBV groups in the negative control group and the lowest dilution tree could not respond to the EV71, and the neutralization titer of the EV71 in the seventh week after injection was 27, while the positive control group Bac-Pl. -3CD and the experimental group Bac-Pl-C3CD have a good ability to neutralize the real EV71, and the synergistic value can reach 21α75 and 21α5, respectively, and there is no statistically significant difference between the two (household > 〇 5). It is estimated that the neutralizing titer caused by deactivated enterovirus is lower than that of the experimental group Bac_pi_C3CD or the positive control group Bac-Pl-3CD may be the purity of the mechanized poisoning, resulting in no immune response caused by the 2011 201100545 method. Cross-protection of antibodies against different enterovirus strains The live virus (TW/2272/98) used in the aforementioned neutralization titer experiment is identical to the disease used in the production of virus-like particles of the present invention, and is all retroactive. Virus strain. And the enterovirus that was popular in 2008? ! The type belongs to. In order to confirm that the antibody induced by the drug is a shawl and (4) a gene-raising virus type 71, the mouse blood/month collected by the crane was neutralized for the strain of genotype B5. First, milk cells are used to infect proliferative diseases, and the concentration of each virus is measured. The neutralization test of the virus is used to determine the neutralizing intensity of the virus induced by the virus-like particles. From the results of Fig. 7 (6), similar results to Fig. 7(a) were found, the negative control group PBS and Lin group could not effectively neutralize enterovirus 71 type, while the positive control group EV71 induced species and titer of π5, still lower than positive. The control group BaC-Pl-3CD (potency up to 2 hearts), and the antibody induced by Bae_pi_C3 (:D-produced virus-like particles, the highest dilution ratio of the neutralized B5 strain can reach 2, which represents Antibodies induced by strains of enterovirus 71 virus can also effectively reduce the infectivity of other different enterovirus 71 strains. The available data show that 'virus particles obtained in a newly established way and this experiment The previous production method of the _ county has a good potential as a candidate for the W-type vaccine in the intestine. [Simplified illustration] The genes P1 and 3CD are separately transferred into the construction of the recombinant baculovirus vector of Fig. 1. EV71 34 201100545 The pFastBacTM DUAL plastid can be made into a virus (a) Bac-Pl-3CD (using the pi〇 promoter to drive 3CD) after the Bac-to-Bac System recommended experimental procedure, (b) Bac-Pl -C3CD (with CMV promoter driving 3CD performance) (c) Bac-Pl-I3CD (expressed by the IE-1 promoter to drive 3CD). Figure 2 Comparison of three strains (Bac-Pl-3CD, Bac-Pl-C3CD, Bac-Pl-I3CD) in different cells Production of Enterovirus 71 virus-like particles · When the cell concentration is about 〇6 〇cell/ml 'Infected cells with MOI10' 2 days, 3 or 4 days (days post-infection, dpi) The liquid was centrifuged at low speed to collect the supernatant as a sample of extracellular viroid particles. (a) Analysis of the expression of Sf-9 cells by Western blotting method. (b) Analysis of Hi- by Western blotting method 5 cell expression. (c) ELISA for quantification of different gene recombinant baculovirus in infected Sf-9 cells (□) ' and 72 hours after infection of Hi-5 cells (_) with Bac-Pl-3CD Total yield. The above experiments were all carried out in 1 〇〇ml spinner flask. The medium used in the experiment was SF-900II. The experimental results were three independent cells.

CI W 培養實驗的平均值。 圖3不同細胞濃度對於腸病毒71型類病毒顆粒產量的影響。當Sf-9細 胞(□)濃度約達 1-,2-,4-或 6xl06cells/ml 時,以 Bac-Pl-C3CD 感染 細胞(MOI 10)。或者當Hi-5細胞()濃度達1-或2xl06 cells/ml時, 以Bac-Pl-3CD感染細胞(MOI 10)。感染後72小時後取樣,將細胞離 心下來,再收取上清液以ELISA定量類病毒顆粒的濃度。上述的實驗 皆於100 ml spinner flask操作,實驗中所使用的培養基皆為SF-900II。 35 201100545 實驗結果為二次獨立細胞培養實驗的平均值。 圖4BIOSTAT® β反應器内獨容氧量對於_毒顆粒產率的影響。當 Sf 9細胞達1 xio ceiis/m時接種入反應器内並以9晒培養。培 養時溶氧量控制在50%,當三天後細胞濃度達4χ1〇6 cdls/mi時以 Bac-Pl-C3CD (MOI 1G)感染細胞,錢後溶氧量分別_為15、% 或60%,以比較不同溶氧量對於譬產量的影響。實驗正對照組 (positive)疋以相同感染條件(4 xi〇6ceus/m丨,Μ。】1〇),但生產於旋轉 角瓶(未鋪溶氧量)醜病毒縣產量n絲為__次獨立細胞培CI 養實驗的平均值。The average of the CI W culture experiments. Figure 3. Effect of different cell concentrations on the production of enterovirus 71 virus-like particles. When the concentration of Sf-9 cells (□) was about 1-, 2-, 4- or 6x106 cells/ml, cells were infected with Bac-Pl-C3CD (MOI 10). Alternatively, when the concentration of Hi-5 cells () reached 1 or 2 x 106 cells/ml, cells were infected with Bac-Pl-3CD (MOI 10). Samples were taken 72 hours after infection, the cells were centrifuged, and the supernatant was collected to quantify the concentration of viroid-like particles by ELISA. All of the above experiments were performed on a 100 ml spinner flask, and the medium used in the experiment was SF-900II. 35 201100545 The experimental results are the average of two independent cell culture experiments. Figure 4 shows the effect of the amount of oxygen in the IOSTAT® beta reactor on the yield of toxic particles. When the Sf 9 cells reached 1 xio ceiis/m, they were inoculated into the reactor and cultured in a 9-bath. The amount of dissolved oxygen in culture was controlled at 50%. When the cell concentration reached 4χ1〇6 cdls/mi after three days, the cells were infected with Bac-Pl-C3CD (MOI 1G), and the dissolved oxygen content after the money was -15, % or 60, respectively. % to compare the effect of different dissolved oxygen levels on the yield of alfalfa. The experimental positive control group (positive) with the same infection conditions (4 xi〇6ceus/m丨, Μ.] 1〇), but produced in the rotating angle bottle (undissolved oxygen) ugly virus county output n silk is __ The average of sub-independent cell culture CI experiments.

圖5類病毒雛之外型、大小及表面蛋自。(a)與⑻穿透式電子顯微 鏡照片Kc)與(d)免疫金染色配合穿透式電子顯微鏡確認類病毒顆粒上 含有VP1蛋白。實驗樣本為以Bac-Pl-C3CD感染Sf-9細胞後,純化所 得到的類病毒顆粒(a與c),或是以Bac_P1_3CD感染High_5細胞,經 純化得到的類病毒顆粒(b與d) °Bar,100nm。 CJ 圖6純化後類病毒顆粒注射於小鼠所引發的anti-EV71 IgG抗體效價分 析。負對照組PBS :以滅菌過的PBS與佐劑混合(1:1)作為注射樣品。 負對照組r-BV :以不帶有任何外源蛋白基因的r_BV感染Sf-9。正對照 級EV71 :以蔗糖不連續梯度離心法所純化得到的腸病毒71型病毒。 正對照組Bac-Pl-3CD :以Bac-Pl-3CD感染Hi-5細胞所生產的類病毒 顆粒。實驗組Bac-Pl-C3CD :以Bac-Pl-C3CD感染Sf-9細胞所生產的 36 201100545 類病毒顆粒。這相無娜合,注射人械雜,並在第 周3進行加脸射。血清在第7週收集,進行序馨釋(最低稀釋倍率 為23),再進行分析。 圖7類病毒顆粒所誘發的抗體對於不同腸病毒株的中和效價測試。⑷ β對於腸病毒株顶2272,98 (genGgiOup⑵的巾和效價。⑹血清對 腸病毒71型gen〇gr〇upB5病毒的中和效價。負對照組pBs:以滅菌過 的PBS與佐劑混合(1:1)作為注射樣品。負對照組r_Bv :以不帶有任何 卜源蛋白基_ t_BV錄Sf 9。衫懷組EV<71 : 4糖不連續梯度 、、法所純化仔到的腸病毒71型病毒。正對照組Bac_pi_3CD :以 Ba〇Pl-3CD感染Hi_5細胞所生產的類病毒顆粒。實驗組 Bac-Pl-C3CD :以Bae_pi C3CD感染sf 9細胞所生產的類病毒顆粒。 廷五組樣品分別與佐劑混合,注射入小鼠腹腔,並在第四周進行加強 注射。血清在第7週收集,進行序列稀釋(最低稀釋倍率為23),再進 行分析。進行分析時,血清的最低稀釋倍率為23。 【主要元件符號說明】 無 37Figure 5 shows the type, size and surface of the virus. (a) and (8) transmission electron micrographs Kc) and (d) immunogold staining combined with transmission electron microscopy confirmed that the viral particles contained VP1 protein. The experimental samples were obtained by infecting Sf-9 cells with Bac-Pl-C3CD, purifying the obtained viroid-like particles (a and c), or infecting High_5 cells with Bac_P1_3CD, and purifying the virus-like particles (b and d). Bar, 100nm. CJ Figure 6. Anti-EV71 IgG antibody titer analysis induced by injection of viroid-like particles into mice. Negative control group PBS: sterilized PBS was mixed with an adjuvant (1:1) as an injection sample. Negative control group r-BV: Sf-9 was infected with r_BV without any foreign protein gene. Positive control grade EV71: Enterovirus 71 virus purified by sucrose discontinuous gradient centrifugation. Positive control group Bac-Pl-3CD: viroid-like particles produced by infecting Hi-5 cells with Bac-Pl-3CD. Experimental group Bac-Pl-C3CD: 36 201100545 virus-like particles produced by infecting Sf-9 cells with Bac-Pl-C3CD. This is inconsistent with the injection of humans, and on the 3rd week, the face is shot. Serum was collected at week 7 and sequenced (minimum dilution ratio of 23) and analyzed. Figure 7. Neutralization potency test of antibodies induced by virus-like particles for different enterovirus strains. (4) β for the enterovirus strain top 2272,98 (genGgiOup (2) towel and titer. (6) serum neutralization titer of enterovirus type 71 gen〇gr〇upB5 virus. Negative control group pBs: with sterilized PBS and adjuvant Mix (1:1) as the injection sample. Negative control group r_Bv: without any source protein base _ t_BV recorded Sf 9. Shirt group EV<71: 4 sugar discontinuous gradient, purified by the method Enterovirus 71 virus. Positive control group Bac_pi_3CD: viroid-like particles produced by infecting Hi_5 cells with Ba〇Pl-3CD. Experimental group Bac-Pl-C3CD: viroid-like particles produced by sf 9 cells infected with Bae_pi C3CD. Five groups of samples were mixed with adjuvant, injected into the peritoneal cavity of mice, and boosted in the fourth week. Serum was collected at week 7 and serially diluted (minimum dilution ratio was 23), and then analyzed. The minimum dilution ratio of serum is 23. [Main component symbol description] No 37

Claims (1)

201100545 七、申請專利範圍·· L -種表現病毒蛋自質的桿狀病毒轉置載體,其包含: a. 強勢才干狀病毒啟動子及一段連結於該啟動子下游的核普酸序列, 其中該核普酸序列可轉譯成病毒之病毒_白;以及 b. -弱勢啟動子及-段連結於該啟動子下游的树酸相,其中該核 普酸序列可轉譯成水解病毒之病毒殼蛋白的水解蛋白, 其中強勢桿狀病毒啟動子驅動連結於級動子下游的核魏序列所轉 譯的蛋白質產量祕弱勢啟動子驅動連結於該啟動子下游的核苷酸序 列所轉譯的蛋白質產量。 如申請專利範圍第1項所述之桿狀病毒轉置載體,其中病毒係指腸病 毒0 如申請專利範圍第2項所述之桿狀病毒轉置載體,其_腸病毒係指腸 病毒71型。 4. 5. 如申請專利翻第1項所述之桿狀病毒轉置載體,其中強勢桿狀病毒 啟動子係選自於桿狀病毒多角體蛋白啟動子或piG啟動子。 如申請專利範圍第1項所述之桿狀病毒轉置載體,其中弱勢啟動子係 選自於桿狀病毒立即早期啟動子、巨細胞病毒啟動子或其他在桿狀病 毒内可作用的弱勢啟動子。 項所述之桿狀病毒轉 種重組桿狀病毒,其包含如申請專利範圍第 置栽體。 如申請專利範圍第6項所述之重組桿狀病毒,其用於感染宿主細胞以 製備重組蛋白質。 38 6. 201100545 8.如__7項所述之重紐样狀病毒,其 病毒的病毒殼蛋白及水解蛋白。 戈白質係指腸 9. -種表現縣蛋_做絲紅_,_ 第6項所述之重組桿狀病毒的感染。 *申請專利範圍 讥如申請專利範圍第9項所述之桿狀病毒宿主細胞, Ο Ο 目於Μ-9或Ηι_5細胞株。 A如申請專利範圍第9項所述之宿主細胞,其進一步將病毒蛋白質组成 該病毒的類病毒顆粒並釋放至細胞外。 錢白質組成 A如申物咖第12細撕_,其中梅 的病毒殼蛋白及水解蛋… 毫毒 14. 一種製備類病毒顆粒的方法,其步驟為W構築如中請專利範圍第】 項Γ之桿狀_蝴;⑻職瓣轉祕以形成如申 軸6籍墙桿輪;及㈦爾晴病毒進 仃感染以形成如申請專利範圍第9項所述之宿主細胞。 ^申請專利範圍第Η項所述之方法,其進一步包含經培養後,離心上 凊液以取得類病毒顆粒。 16.如申請專利範圍第14項所述之方法,其中類病毒顆粒係指腸病毒的類 病毒顆粒。 17. 一種用簡防病毒絲的疫苗,其係選自純括免疫有效量如申請專 1 項所述之重組桿狀鱗,如㈣專利範SI第9項所述之宿 主細胞及該宿主細胞所釋出的類病毒顆粒所組成的群組, 以及醫藥上 39 201100545 可接受的免疫佐劑。 18.如申請專利範圍第17項所述之疫苗,其中病毒係指腸病毒。201100545 VII. Scope of Patent Application·· L-type baculovirus transposition vector showing viral egg self-quality, comprising: a. a strong stem virus promoter and a nucleotide sequence linked downstream of the promoter, wherein The nucleotide sequence can be translated into a viral virus-white; and b. - a weak promoter and a - segment are linked to a stucate phase downstream of the promoter, wherein the nucleotide sequence can be translated into a viral shell protein of the hydrolyzed virus A hydrolyzed protein in which a strong baculovirus promoter drives a nuclear profiling sequence linked to a nuclear Wei sequence downstream of a cascade mover to produce a protein-producing protein that is translated by a nucleotide sequence linked downstream of the promoter. The baculovirus transposition vector according to claim 1, wherein the virus refers to enterovirus 0, such as the baculovirus transposition vector described in claim 2, and the enterovirus refers to enterovirus 71. type. 4. The baculovirus transposition vector of claim 1, wherein the strong baculovirus promoter is selected from the group consisting of a baculovirus polyhedrin promoter or a piG promoter. The baculovirus transposition vector of claim 1, wherein the weak promoter is selected from a baculovirus immediate early promoter, a cytomegalovirus promoter or other weak promoter that acts in a baculovirus. child. The baculovirus of the present invention is a recombinant baculovirus, which comprises a vector according to the scope of the patent application. A recombinant baculovirus as described in claim 6, which is for infecting a host cell to produce a recombinant protein. 38 6. 201100545 8. The heavy-duvirus as described in __7, the viral hull protein and the hydrolyzed protein of the virus. Go white matter refers to the intestine 9. - The type of the county egg _ is made of silk red _, _ the infection of the recombinant baculovirus described in Item 6. * Patent application scope, for example, the baculovirus host cell described in claim 9 of the patent application, Ο 目 Μ Η-9 or Ηι_5 cell line. A host cell according to claim 9, which further comprises the viral protein as a viroid-like particle of the virus and released to the outside of the cell. The composition of the white matter of the money, such as the 12th fine tear of the food, _, the virus shell protein and the hydrolyzed egg of the plum... The poisonous 14. A method for preparing the virus-like particle, the step of which is to construct the patent scope of the patent. The rod-shaped _ butterfly; (8) the lobes are secreted to form a wall rod wheel such as a shaft; and (7) the arsenic virus is infected to form a host cell as described in claim 9 of the patent application. The method of claim 2, further comprising, after culturing, centrifuging the mash to obtain viroid-like particles. 16. The method of claim 14, wherein the viroid-like particle is a virus-like particle of enterovirus. 17. A vaccine for the use of a simple anti-viral filament, which is selected from the group consisting of a recombinant rod-shaped scale as described in the application of claim 1, and the host cell and the host cell as described in (4) Patent No. 9. A group of released virus-like particles, and an immunological adjuvant acceptable for use in medicine 2011 201100545. 18. The vaccine of claim 17, wherein the virus is an enterovirus.
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