TWI432576B - Method for producing hscarb2-tg mice and the use thereof as animal model for enterovirus infections - Google Patents
Method for producing hscarb2-tg mice and the use thereof as animal model for enterovirus infections Download PDFInfo
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本發明係關於腸病毒感染動物模式之製備，更特別地係關於SCARB2基因轉殖鼠之製造，以及其做為手足口病之動物模式上的應用。The present invention relates to the preparation of animal models of enterovirus infection, and more particularly to the manufacture of SCARB2 gene transgenic mice and their use as animal models for hand, foot and mouth disease.
腸病毒71型(EV71)已被證實會引起手足口病(HFMD)、皰疹性咽峽炎，和神經系統的病變如無菌性腦膜炎、腦炎及類小兒麻痺症候群，甚至在嬰幼兒造成致死疾病，並且在全世界爆發嚴重的疫情(AbuBakar et al.,1999,Virus Res 61 (1),1-9;Chang,Huang,and Lin,1998,Lancet 352 (9125),367-8；Bible et al.,2007,Rev Med Virol 17 (6),371-9)。及至今日，透過公共衛生對腸病毒71型疫情的控制與醫療照護仍顯不足。目前並沒有疫苗可供臨床適用。因此，研究腸病毒71型感染的機制，以供開發有效的治療藥物或預防性疫苗實屬重要。Enterovirus 71 (EV71) has been shown to cause hand, foot and mouth disease (HFMD), herpetic angina, and neurological disorders such as aseptic meningitis, encephalitis, and polio-like symptoms, even in infants and young children. Lethal disease, and a serious outbreak in the world (AbuBakar et al., 1999, Virus Res 61 (1), 1-9; Chang, Huang, and Lin, 1998, Lancet 352 (9125), 367-8; Bible Et al., 2007, Rev Med Virol 17 (6), 371-9). And to this day, the control and medical care of the enterovirus 71 epidemic through public health is still insufficient. There are currently no vaccines available for clinical use. Therefore, it is important to study the mechanism of enterovirus 71 infection for the development of effective therapeutic or prophylactic vaccines.
現已知有多種動物模式被發展，欲應用於研究EV71感染之致病原因(參見，例如Chen et al.,2004,J Gen Virol 85 (Pt 1),69-77;Nagata et al.,2004,J Gen Virol 85 (Pt 10),2981-9;Ong et al.,2008,J Neuropathol Exp Neurol 67 (6),532-42;Weng et al.,2010,Infect 12 (7),505-10)。而目前使用的腸病毒動物模式，是利用新生一天大之ICR小鼠，以EV71感染進行人為發展而得，新生的ICR小鼠在EV71感染後會出現後肢麻痺，並且最後在受感染2周內死亡(Wu et al.,2001,Vaccine 20 (5-6),895-904)。然而，由於該ICR小鼠僅適用於，評估小鼠對於腸病毒感染或疫苗引起的免疫反應，無法真正模擬在人類受到感染後所引發的免疫反應。同時，由於該小鼠模式並未呈現腸病毒感染後的HFMD症狀，故利用其所進行之研究觀察只侷限於，腸病毒感染後小鼠的存活率及神經性毒性，無法確實應用在評估藥物或疫苗，對於腸病毒感染的治療及/或預防功效。此外，目前使用的ICR小鼠因為免疫系統發育不全，可能無法充分反應腸病毒EV71的毒性，及瞭解其對於神經系統的影響。A variety of animal models are known to be developed for the study of the cause of EV71 infection (see, for example, Chen et al., 2004, J Gen Virol 85 (Pt 1), 69-77; Nagata et al., 2004 , J Gen Virol 85 (Pt 10), 2981-9; Ong et al., 2008, J Neuropathol Exp Neurol 67 (6), 532-42; Weng et al., 2010, Infect 12 (7), 505-10 ). The currently used enterovirus animal model is based on the newborn ICT mice, which are developed by EV71 infection. The newborn ICR mice will have hind limb paralysis after EV71 infection, and finally within 2 weeks of infection. Death (Wu et al., 2001, Vaccine 20 (5-6), 895-904). However, since this ICR mouse is only suitable for assessing the immune response of a mouse to enterovirus infection or vaccine, it cannot truly simulate the immune response elicited after infection in humans. At the same time, since the mouse model does not exhibit the symptoms of HFMD after enterovirus infection, the research observations using it are limited to the survival rate and neurotoxicity of mice after enterovirus infection, and cannot be applied to the evaluation of drugs. Or a vaccine for the therapeutic and/or prophylactic efficacy of enterovirus infection. In addition, currently used ICR mice may not fully respond to the toxicity of enterovirus EV71 due to the underdeveloped immune system, and understand its effects on the nervous system.
再者，目前使用的ICR小鼠是利用小鼠適應過的腸病毒株(mouse-adapted EV71 strain，參見Wang et al.,2004,J Virol 78 (15),7916-24)進行感染試驗，但由於該病毒並沒有存在於自然界，故無法使受感染之ICR小鼠表現出真正的，天然流行腸病毒株感染後應有的HFMD症狀及神經性毒性(Perez-Velez et al.,2007,Clin Infect Dis 45 (8), 950-7)。且經研究發現，以自然界流行的腸病毒株感染已知的成年ICR、Balb/c、CH3、C57BL/6等實驗用小鼠(彼等皆不表現EV71接受體)後，都完全不會引起HFMD症狀及神經性毒性(Wang,2004，如前述；Chen et al.,2004,J Gen Virol 85 (Pt 1), 69-77)。Furthermore, currently used ICR mice are tested for infection using a mouse-adapted EV71 strain (see Wang et al., 2004, J Virol 78 (15), 7916-24), but Since the virus does not exist in nature, it is impossible to make infected ICR mice exhibit the HFMD symptoms and neurotoxicity of a true, naturally occurring enterovirus strain after infection (Perez-Velez et al., 2007, Clin Infect Dis 45 (8) , 950-7). And it has been found that the experimental adult strains of ICR, Balb/c, CH3, C57BL/6, etc. (all of which do not express EV71 receptors) are infected with the enterovirus strains that are naturally prevalent in nature. HFMD symptoms and neurotoxicity (Wang, 2004, supra; Chen et al., 2004, J Gen Virol 85 (Pt 1) , 69-77).
近來研究發現，人類清道夫接受體B類，膜蛋白質2型(human scavenger receptor class B,member 2;hSCARB2)是腸病毒71型及克沙其病毒16型(Coxsackie A16)進入細胞的接受器(Yamayoshi et al.Nat Med 15(7) : 798-801,2009)，雖然小鼠SCARB2與人類SCARB2(hSCARB2)具有85.8%同源性，但顯示其不作為EV71的受體。本發明遂建立hSCARB2基因轉殖鼠作為腸病毒實驗動物感染模式，以期模擬腸病毒之感染途徑，研究其引起手足口病和神經系統病變的致病機轉，並藉以評估腸病毒疫苗在此動物模式中之免疫保護效力，以及候選治療性藥物或疫苗對於腸病毒感染之防治功效，以增進對腸病毒感染致病機制的了解，以及應用於有效腸病毒疫苗的研發。Recent studies have found that human scavenger receptor class B (member 2; hSCARB2) is a receptor for entry of enterovirus 71 and Coxsackie A16 into cells ( Yamayoshi et al. Nat Med 15(7) : 798-801, 2009), although mouse SCARB2 has 85.8% homology with human SCARB2 (hSCARB2), it is shown to be not a receptor for EV71. The invention establishes the hSCARB2 gene transgenic mouse as an enteric virus experimental animal infection mode, in order to simulate the enterovirus infection pathway, study the pathogenesis of causing hand, foot and mouth disease and nervous system disease, and thereby evaluate the enterovirus vaccine in this animal. The immunoprotective efficacy of the model, as well as the efficacy of candidate therapeutic drugs or vaccines for the control of enterovirus infections, to improve understanding of the pathogenesis of enterovirus infections, and for the development of effective enterovirus vaccines.
本發明係基於發現，人類清道夫接受體B類，膜蛋白質2型(以下簡稱SCARB2)為腸病毒進入細胞的接受器，遂建立SCARB2基因轉殖鼠作為腸病毒71型實驗動物感染模式，應用於研究EV71引起的神經系統病變機轉，及分析EV71疫苗的保護作用，以開發有效的治療藥物或是預防腸病毒感染相關疾病的疫苗。The invention is based on the discovery that the human scavenger accepts the B class, the membrane protein type 2 (hereinafter referred to as SCARB2) is the acceptor of the enterovirus into the cell, and the SCARB2 gene transgenic mouse is established as the enterovirus 71 type experimental animal infection mode, and the application thereof To study the neurological pathogenesis caused by EV71, and to analyze the protective effect of EV71 vaccine to develop effective therapeutic drugs or vaccines against diseases related to enterovirus infection.
於是，本發明之一方面係關於，一種製造SCARB2基因轉殖鼠之方法，其包含：將經由微注射入受Elongation factor-1啟動子(EF-1αpromoter)驅動之人類SCARB2蛋白基因(SEQ ID NO. 1)的小鼠胚胎，轉殖到同品系母鼠中使其發育成鼠；利用PCR分析篩檢出帶有SCARB2蛋白基因型之陽性小鼠，與原生型同品系小鼠互相交配，生成F0小鼠；再次以PCR分析篩選出其基因型為異核型(heterozygous)之陽性F0小鼠，並使呈陽性F0小鼠互相交配生成F1小鼠；利用PCR分析篩選得其基因型為同核型(homozygous)之陽性F1小鼠，而得SCARB2基因轉殖鼠；以及利用RT-PCR分析確認F1基因轉殖鼠在主要器官表達SCARB2蛋白。Accordingly, one aspect of the invention relates to a method of producing a SCARB2 gene-transforming mouse comprising: transfecting a human SCARB2 protein gene (SEQ ID NO) driven by an Elongation factor-1 promoter (EF-1αpromoter) via microinjection 1) The mouse embryos are transferred to the same breed mother to develop them into mice; the positive mice bearing the SCARB2 protein genotype are screened by PCR analysis, and the original homologous mice are mated with each other to generate F0 mice; PCR-analyzed positive F0 mice whose genotypes were heterozygous (heterozygous), and the positive F0 mice were mated to each other to form F1 mice; the genotypes were selected by PCR analysis. The positive F1 mouse of homozygous, and the SCARB2 gene transgenic mouse; and the RT-PCR analysis confirmed that the F1 gene transgenic mouse expressed SCARB2 protein in the main organs.
於本發明之一具體實施例，用於基因轉殖之小鼠係C57BL/6小鼠。於本發明之另一具體實施例，用於基因轉殖之小鼠係FVB小鼠。In a specific embodiment of the invention, the mouse for gene transfer is a C57BL/6 mouse. In another embodiment of the invention, the mouse for gene transfer is a FVB mouse.
本發明之SCARB2基因轉殖鼠於自然界流行的腸病毒株感染後，會呈現手足口病(HFMD)之症狀和神經系統的病變。因此，該SCARB2基因轉殖鼠可應用做為手足口病之動物模式。於本發明之一具體實施例，其係用做為腸病毒71型之感染動物模式。於本發明之另一具體實施例，其係用做為克沙奇病毒16型(Coxsackie A16)之感染動物模式。The SCARB2 gene transgenic mouse of the present invention may exhibit symptoms of hand, foot and mouth disease (HFMD) and pathological changes of the nervous system after infection with a naturally occurring enterovirus strain. Therefore, the SCARB2 gene transgenic mouse can be used as an animal model of hand, foot and mouth disease. In one embodiment of the invention, it is used as an animal model of enterovirus 71 infection. In another embodiment of the invention, it is used as an infected animal model of Coxsackie A16.
於另一方面，本發明係關於一種篩選出可有效預防或治療因腸病毒感染所引起之手足口病的疫苗或藥物之方法。於本發明之一具體實施例，該藥物為一種預防性疫苗。於本發明之另一具體實施例，該藥物為一種抗-腸病毒71型之中和抗體。In another aspect, the invention relates to a method of screening for a vaccine or medicament effective to prevent or treat hand, foot and mouth disease caused by enterovirus infection. In a specific embodiment of the invention, the medicament is a prophylactic vaccine. In another embodiment of the invention, the drug is an anti-enteric virus type 71 neutralizing antibody.
本發明之其他特色及優點將於下列實施範例中被進一步舉例與說明，而該實施範例僅作為輔助說明，並非用於限制本發明之範圍。The other features and advantages of the present invention are further exemplified and illustrated in the following examples, which are intended to be illustrative only and not to limit the scope of the invention.
根據本發明所呈現的各種實施例，下述各種儀器、裝置、方法和其相關結果者，實施例中為了方便讀者閱讀所使用的標題或副標題，並不被限制在本發明的範圍之內。此外，在此所提出和披露的某些理論，但無論他們是對還是錯，只要該創作是根據本發明所實施的，而不需考慮任何特定的理論或行動的計畫，都應被限制在本發明的範圍之內。In view of the various embodiments of the present invention, the various instruments, devices, methods, and related results described below are not intended to be limited to the scope of the present invention. In addition, certain theories presented and disclosed herein, but whether they are right or wrong, should be limited as long as the creation is implemented in accordance with the present invention without regard to any particular theory or action plan. It is within the scope of the invention.
將所合成得具有密碼子最適化之人類SCARB2 基因(SEQ ID NO.1)片段使用EcoRI與BamHI限制酶，***pEF-1α質體(該質體係以pcDNA3.1(-)載體(Invitrogen)為背景經修改後而得之載體，其中原有的巨細胞病毒增效子/啟動子以增長因子1α(EF-1α)之啟動子取代)中，位於啟動子與牛生長激素(BGH)聚A尾端間之多重選殖部位(multiple cloning site)，而得到pEF-1α-hSCARB2構體(圖1)。The human SCARB2 gene (SEQ ID NO. 1) fragment obtained by codon-optimization was inserted into the pEF-1α plastid using EcoRI and BamHI restriction enzyme (the plasmid was pcDNA3.1(-) vector (Invitrogen) A modified vector obtained in which the original cytomegalovirus enhancer/promoter is replaced by a promoter of growth factor 1α (EF-1α), located in the promoter and bovine growth hormone (BGH) poly A The multiple cloning site between the caudal ends yielded the pEF-1α-hSCARB2 construct (Fig. 1).
基因轉殖動物之產生係依照Brinster等人(Brinster et al.,1985)所述的操作步驟來進行。所使用之C57BL/6小鼠係購自國家應用研究實驗室-實驗動物中心(National Applied Research Laboratories-Laboratory Animal Center,台灣)。進行顯微注射前先將質體以限制酵素線形化，並使用凝膠萃取套組(Qiagen,德國)將其從瓊脂糖凝膠(Thermo Fisher Scientific,IL,USA)中回收得。The production of the gene-transforming animal was carried out in accordance with the procedure described by Brinster et al. (Brinster et al., 1985). The C57BL/6 mouse line used was purchased from the National Applied Research Laboratories-Laboratory Animal Center (Taiwan). The plastids were linearized with restriction enzymes prior to microinjection and recovered from agarose gel (Thermo Fisher Scientific, IL, USA) using a gel extraction kit (Qiagen, Germany).
將1 ng線形pEF-1α-hSCARB2構體經由顯微注射，導入處於單細胞階段之已受精的C57BL/6小鼠胚胎中，接著將該胚胎植入到同品系假懷孕之母鼠內使其發育成鼠。利用PCR分析篩檢出帶有hSCARB2蛋白基因型之陽性小鼠，與原生型同品系小鼠互相交配，生成F0小鼠。The 1 ng linear pEF-1α-hSCARB2 construct was introduced into the fertilized C57BL/6 mouse embryo at the single-cell stage by microinjection, and then the embryo was implanted into the same-pregnant pregnant mother. Develop into a mouse. Positive mice bearing the hSCARB2 protein genotype were screened by PCR analysis and mated with native homologous mice to generate F0 mice.
PCR分析方法簡述如下：使用組織與細胞基因組DNA萃取套組(Tissue & Cell genomic DNA extraction kit，Favorgen，台灣)從基因轉殖鼠尾部萃取出基因組DNA。藉由使用前向引子：5’-TGGACCAGAGCATCGAGAA-3’(SEQ ID NO.2)與反向引子：5’-TAGGTGTAGGGGCCCACTT-3’(SEQ ID NO.3)於72℃下2分鐘進行之PCR反應擴增一段長度為175 bp之片段(SEQ ID NO.1之98-272 bp)，以偵測hSCARB2基因是否存在基因組DNA中。用於進行PCR反應之條件設定如下：於95℃下2分鐘；隨後進行40次包括於95℃下30秒、於50℃下30秒與於72℃下10秒之循環週期；之後再於72℃下反應2分鐘。The PCR analysis method is briefly described as follows: Genomic DNA was extracted from the tail of a genetically transformed mouse using a tissue and cell genomic DNA extraction kit (Favorgen, Taiwan). PCR reaction was carried out by using a forward primer: 5'-TGGACCAGAGCATCGAGAA-3' (SEQ ID NO. 2) and a reverse primer: 5'-TAGGTGTAGGGGCCCACTT-3' (SEQ ID NO. 3) at 72 ° C for 2 minutes. A fragment of 175 bp in length (98-272 bp of SEQ ID NO. 1) was amplified to detect the presence of the hSCARB2 gene in genomic DNA. The conditions for performing the PCR reaction were set as follows: 2 minutes at 95 ° C; followed by 40 cycles including 30 seconds at 95 ° C, 30 seconds at 50 ° C and 10 seconds at 72 ° C; The reaction was carried out at ° C for 2 minutes.
結果篩檢出六隻帶有轉殖基因之小鼠-No. 2(雄性,m)、No. 9(雌性，f)、No. 18(m)、No. 27(f)、No. 54(m)及No. 62(f)(參見圖2A)。再次以PCR分析篩選出其基因型為異核型(heterozygous)之陽性F0小鼠，並使呈陽性F0小鼠互相交配生成F1小鼠：利用PCR分析篩選得其基因型為同核型(homozygous)之陽性F1小鼠，而得hSCARB2基因轉殖鼠(結果參見圖2B)。藉由將所得之hSCARB2基因轉殖鼠個體彼此進行交配，獲得F1近親交配種小鼠，以維持該基因轉殖純系。Results Six mice with transgenic genes were screened - No. 2 (male, m), No. 9 (female, f), No. 18 (m), No. 27 (f), No. 54 (m) and No. 62(f) (see Fig. 2A). PCR-analyzed positive F0 mice whose genotypes were heterozygous (heterozygous), and the positive F0 mice were mated to each other to form F1 mice: the genotypes were homonucleated by PCR analysis (homozygous) The positive F1 mouse was obtained, and the hSCARB2 gene-transformed mouse was obtained (see Fig. 2B for the results). F1 inbred mice were obtained by mating the obtained hSCARB2 gene transgenic mouse individuals with each other to maintain the gene transgenic line.
進一步以前述之hSCARB2特異性引子對及購自Roche Universal Probe Library Assay Design Center(Roche,Switzerland)之探針組，進行RT-PCR分析(The LightCycler480 Real-Time PCR system)，來確認及定量人類SCARB2蛋白在F1基因轉殖鼠之主要器官中的表達。PCR產物大小為97 bp。使用不同濃度(5、0.5及0.05 pg/ml)之pEF-1α-hSCARB2質體為標準物。比較用以從各器官擴增hSCARB2基因所需要的循環次數。RT-PCR analysis was further performed using the aforementioned hSCARB2-specific primer pair and the probe set purchased from the Roche Universal Probe Library Assay Design Center (Roche, Switzerland) (The LightCycler 480 Real-Time PCR system) to confirm and quantify the expression of human SCARB2 protein in the main organs of F1 transgenic mice. The PCR product size was 97 bp. Different concentrations (5, 0.5 and 0.05 pg/ml) of pEF-1α-hSCARB2 plastids were used as standards. The number of cycles required to amplify the hSCARB2 gene from each organ was compared.
結果顯示，在取自基因轉殖小鼠之包括腦幹、大腦皮質、延腦與脊髓等中樞神經組織，以及肺、肝、脾、腎、十二指腸、小腸等主要器官及皮膚中，皆發現有表現hSCARB2轉殖基因(圖2C)。The results showed that in the main organs and skins such as brain stem, cerebral cortex, cerebral ventricle and spinal cord, as well as the main organs and skin of the lung, liver, spleen, kidney, duodenum, small intestine, etc., were found in the gene-transferred mice. The hSCARB2 transgenic gene was expressed (Fig. 2C).
將1-天或4-天大的小鼠經皮下注射，接種入腸病毒EV71 E59或5746-tw98分離株(每隻小鼠施予1x106 或1x107 pfu，存在10 μL RPMI培養基中)，以模擬觀察新生小鼠受病毒感染後，腸病毒於小鼠主要器官中之分部情形，及外在所發生的病症。對照組係施給VP-SFM培養基。對於腸病毒EV71之偵測，係將經感染之小鼠於特定天數後將其犧牲，採取血液或器官組織樣本，並針對VP1基因進行專一性的即時(real-time)RT-PCR。1-day or 4-day-old mice were injected subcutaneously and inoculated into enterovirus EV71 E59 or 5746-tw98 isolates (each mouse was administered 1x10 6 or 1x10 7 pfu in 10 μL of RPMI medium). The mice were infected with the virus, the fraction of enterovirus in the main organs of the mice, and the externally occurring diseases. The control group was administered VP-SFM medium. For the detection of enterovirus EV71, the infected mice were sacrificed after a certain number of days, blood or organ tissue samples were taken, and a real-time RT-PCR was performed for the VP1 gene.
將自血液樣本或組織均質化產物萃取得之總體RNA，進行反轉錄反應轉變成cDNA。再將所得之cDNA使用EV71 VP1專一性引子對，前向引子：5’-AGAGAGTCACTTGCTTGGCAGACA-3’(SEQ ID NO.4)與反向引子：5’-ACGACTAGTGCCGGTCGGTTTAAT-3’(SEQ ID NO.5)，進行定量PCR分析來偵測EV71之存在量。The total RNA extracted from the blood sample or tissue homogenization product is converted into cDNA by reverse transcription reaction. The resulting cDNA was further EV71 VP1 specific primer pair, forward primer: 5'-AGAGAGTCACTTGCTTGGCAGACA-3' (SEQ ID NO. 4) and reverse primer: 5'-ACGACTAGTGCCGGTCGGTTTAAT-3' (SEQ ID NO. 5) Quantitative PCR analysis was performed to detect the presence of EV71.
由圖3之結果顯示，於腸病毒EV71感染後1至6天，在SCARB2-Tg B6轉殖基因鼠之中樞神經(腦與脊髓)及肝臟、肺臟、小腸、腎臟、脾臟、皮膚等，偵測到有病毒存在。此外，相對於SCARB2-轉殖基因鼠，於非轉殖基因鼠之神經器官(腦，脊髓)和主要器官(肝、肺、腎、皮膚等)皆偵測不到病毒量分布，表示hSCARB2的表現對於EV71在小鼠中之可感染力而言很重要。The results shown in Figure 3 show that the SCARB2-Tg B6 transgenic mouse central nervous system (brain and spinal cord) and liver, lung, small intestine, kidney, spleen, skin, etc., 1 to 6 days after infection with enterovirus EV71 A virus was detected. In addition, compared with SCARB2-transgenic mouse, the distribution of virus was not detected in the nerve organs (brain, spinal cord) and main organs (liver, lung, kidney, skin, etc.) of non-transgenic mouse, indicating hSCARB2 Performance is important for the infectivity of EV71 in mice.
除了測定病毒量分布，亦於施予病毒感染後，每天觀察於對照組和實驗組小鼠產生的手足口病(HFMD)及神經性毒性(CNS)症狀。病理分析發現，以腸病毒71型E59 B型病毒株感染一天大的SCARB2基因轉殖鼠，會使受感染小鼠產生類似出現在人類受腸病毒EV71-感染幼兒之紅疹，且有嚴重毛髮掉落伴隨皮屑(scurf)產生，該等症狀皆為典型的類手足口病症狀(HFMD-like syndrome)(參見圖4)。而且，在施予感染之轉殖基因鼠亦出現包括前後足麻痺，而導致行走困難之神經系統病症(CNS-like syndrome)。在感染後7天開始，轉殖基因鼠即出現後肢麻痺伴隨行動困難的現象，而非轉殖基因鼠在感染後並未呈現此類似病症，關於在感染後第8天至12天期間，所觀察到之行動困難與後肢麻痺的計分結果列示於圖5。In addition to measuring the amount of virus, the symptoms of hand, foot and mouth disease (HFMD) and neurotoxicity (CNS) produced by the control and experimental mice were observed daily after administration of the virus infection. Pathological analysis revealed that infection of the SCARB2 gene transgenic mice with the enterovirus 71 E59 B virus strain resulted in a rash that appeared in human EV71-infected children and had severe hair. Drops are accompanied by scurf, which is a typical HFMD-like syndrome (see Figure 4). Moreover, in the transgenic mouse that is infected, there is also a neurological disorder (CNS-like syndrome) including anterior and posterior foot paralysis. At 7 days after infection, the transgenic mouse showed signs of difficulty in hindlimb paralysis, and the non-transgenic mouse did not present this similar condition after infection, regarding the 8th to 12th day after infection. The results of the observed difficulty in action and hind limb paralysis are shown in Figure 5.
而且由圖6之病毒分布偵測結果顯示，在施予病毒感染後第12天，於已發生後肢麻痺的小鼠之脊髓中可偵測到，但是在沒有呈現任何行走困難之非轉殖基因鼠，則沒有偵測到病毒，表示所觀察到的CNS病症，係由於脊隨受到腸病毒感染所致。Moreover, the virus distribution detection result of Fig. 6 showed that on the 12th day after the administration of the virus infection, it was detected in the spinal cord of the mouse having hindlimb paralysis, but the non-transgenic gene was not present in any difficulty in walking. In the mouse, no virus was detected, indicating that the observed CNS disorder was caused by the infection of the genus by the enterovirus.
前述之觀察結果均顯示，以腸病毒71型感染新生SCARB2基因轉殖鼠，確實會產生類似於腸病毒感染在人類嬰兒所引起的症狀。亦即，本發明之SCARB2基因轉殖鼠可有效做為，天然存在之腸病毒感染的動物模式。The foregoing observations have shown that infection of the newborn SCARB2 gene with Enterovirus 71 does produce symptoms similar to those caused by enterovirus infection in human infants. That is, the SCARB2 gene-transforming mouse of the present invention can be effectively used as an animal model of naturally occurring enterovirus infection.
本實施例係藉由將已知具有腸病毒中和活性之抗EV-71單株抗體，投藥予預先經天然EV71 5746-TW98腸病分離毒株感染之SCARB2基因轉殖鼠，並檢測該單株抗體對於保護小鼠對抗腸病毒感染之功效，以評估及證明本發明之SCARB2基因轉殖鼠，做為抗腸病毒藥物篩選平台之實用性。In this embodiment, an anti-EV-71 monoclonal antibody known to have enterovirus neutralizing activity is administered to a SCARB2 gene-transferred mouse previously infected with a natural EV71 5746-TW98 enterovirus isolate, and the single is detected. The antibody of the strain is useful for protecting the mouse against enterovirus infection, and to evaluate and prove the utility of the SCARB2 gene transgenic mouse of the present invention as an anti-enteric drug screening platform.
實驗方法簡述如下：將7天大之SCARB2基因轉殖鼠以皮下方式，注射入劑量為3 x104 pfu之EV71 5746-TW98腸病毒株進行感染。接著在注射病毒4小時後進行投藥，將該經過腸病毒株感染的SCARB2基因轉殖鼠以腹膜內注射方式，投藥以200 μg之同型小鼠IgG1抗體(做為對照組)，或抗EV-71單株抗體(已被鑑定具有中和活性，參見Chang,HW et al.,Journal of Virological Methods 7(1) : 62,2011)。The experimental method is briefly described as follows: The 7-day-old SCARB2 gene-transplanted mouse was injected subcutaneously into an EV71 5746-TW98 enterovirus strain at a dose of 3 x 10 4 pfu for infection. Then, 4 hours after the injection of the virus, the SCARB2 gene-transfected mouse infected with the enterovirus strain was intraperitoneally injected with 200 μg of the same type mouse IgG1 antibody (as a control group), or anti-EV- 71 monoclonal antibodies (identified to have neutralizing activity, see Chang, HW et al., Journal of Virological Methods 7(1) : 62, 2011).
圖7列示於同型抗體處理組(□，對照組，總數10隻小鼠)，及抗EV-71抗體處理組(■，實驗組，總數12隻小鼠)所得之存活率觀察結果。於hSCARB2基因轉殖鼠可明顯區分，EV-71抗體與對照組抗體在抑制受感染小鼠之致死性上的功效差異，顯示本發明之hSCARB2基因轉殖鼠可用於評估已知或研發中藥物之抗腸病毒感染功效，或用於評估EV71疫苗在此動物模式中之免疫保護效力。Fig. 7 shows the results of survival observations obtained in the same antibody-treated group (□, control group, total of 10 mice), and anti-EV-71 antibody-treated group (■, experimental group, total of 12 mice). The hSCARB2 gene transgenic mice can clearly distinguish the difference in efficacy between the EV-71 antibody and the control antibody in inhibiting the lethality of infected mice, indicating that the hSCARB2 gene transgenic mouse of the present invention can be used to evaluate known or developed drugs. It is effective against enterovirus infection or is used to assess the immunoprotective efficacy of the EV71 vaccine in this animal model.
本說明書中所揭示之全部特徵可以任何組合方式組合。於是，本說明書中所揭示之各別特徵可由依相同、相等或類似目的之替代特徵取代。因此，除非另行清楚地指示，所揭示之各特徵僅為一系列同等物或類似特徵之實例。All of the features disclosed in this specification can be combined in any combination. Thus, the individual features disclosed in this specification can be replaced by alternative features that are the same, equivalent, or similar. Therefore, the various features disclosed are merely examples of a series of equivalents or similar features, unless otherwise clearly indicated.
從前述之說明，習於該項技藝人士可容易地確定本發明之基本特徵，且在未偏離其範圍下，可進行本發明之各種改變與修飾，以使其適於各種不同用途與狀況。因此，於申請專利範圍內亦包含其他具體態樣。From the foregoing description, those skilled in the art can readily determine the essential features of the invention, and various changes and modifications of the invention can be made to adapt to various different uses and conditions without departing from the scope thereof. Therefore, other specific aspects are included in the scope of patent application.
<110>國家衛生研究院<110>National Institute of Health
<120> hSCARB2基因轉殖鼠之製造及其做為腸病毒感染動物模式之應用<120> Manufacture of hSCARB2 gene transgenic mice and its application as an animal model of enterovirus infection
<160> 5<160> 5
<170> FastSEQ for Windows Version 4.0<170> FastSEQ for Windows Version 4.0
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<212> DNA<212> DNA
<213> 人類<213> Human
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<212> DNA<212> DNA
<213> 人造序列<213> Artificial sequence
<220><220>
<221> 前向引子<221> Forward introduction
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<210> 3<210> 3
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<213> 人造序列<213> Artificial sequence
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<221> 反向引子<221> Reverse primer
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圖1為用於本發明方法之攜帶有人類基因的表現載體pEF-1α-hSCARB2之圖譜。Figure 1 is a map of the expression vector pEF-1α-hSCARB2 carrying the human gene used in the method of the present invention.
圖2顯示藉由使用SCARB2-專一性引子進行PCR反應，篩選出帶有人類基因之founder小鼠(A)，及所獲得F1、F2 Tg小鼠(B)的結果。圖2(C)顯示人類SCARB2在SCARB2-轉殖基因鼠之主要器官中的表現。Figure 2 shows the results of screening for the founder mouse (A) carrying the human gene and the obtained F1, F2 Tg mouse (B) by PCR using the SCARB2-specific primer. Figure 2 (C) shows the performance of human SCARB2 in the major organs of SCARB2-transgenic mouse.
圖3為藉由使用VP1-專一性引子進行RT-PCR反應，測定於腸病毒EV71/E59感染後第1至4天，病毒在SCARB2-Tg B6小鼠中的分布情形。Figure 3 is a graph showing the distribution of virus in SCARB2-Tg B6 mice on days 1 to 4 after infection with enterovirus EV71/E59 by RT-PCR using a VP1-specific primer.
圖4為於施予接種後4至6天，在僅接種培養基(A)、非轉殖基因小鼠(C)及以107 pfu EV71/E59感染之hSCARB2-Tg轉殖基因鼠(B)所觀察之類手足口病(HFMD)，包括掉毛與皮屑產生情形。Figure 4 shows hSCARB2-Tg transgenic mouse (B) inoculated only in medium (A), non-transgenic mouse (C) and infected with 10 7 pfu EV71/E59 4 to 6 days after vaccination. Hand, foot and mouth disease (HFMD) observed, including hair loss and dander production.
圖5為感染後第8天至12天期間，所觀察到之行動困難與後肢麻痺的計分圖。Figure 5 is a scoring diagram of the observed difficulty of movement and hind limb paralysis between the 8th and 12th day after infection.
圖6為以VP-1專一性RT-PCR偵測病毒感染後期(感染後12天)，於小鼠中樞神經系統及主要器官之EV-71病毒分布。Figure 6 shows the distribution of EV-71 virus in the central nervous system and major organs of mice in the late stage of viral infection (12 days after infection) by VP-1 specific RT-PCR.
圖7顯示以同型抗體(□)及以抗EV-71抗體處理(■)之hSCARB2-Tg轉殖基因鼠，在對抗病毒感染EV71 5746-TW98的存活率。Figure 7 shows the survival rate of anti-viral infection EV71 5746-TW98 by homologous antibody (□) and hSCARB2-Tg transgenic mouse treated with anti-EV-71 antibody (■).
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