TWI767433B - Cell strain having increased virus production ability and production method thereof - Google Patents
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本發明係關於具有增加的病毒生產能力之細胞株及其製備方法，更具體地，係關於因CCCH型鋅指蛋白的過度表達而具有增加的病毒生產能力之細胞株及其製備方法。The present invention relates to a cell line with increased virus production ability and a preparation method thereof, and more particularly, to a cell line with increased virus production ability due to overexpression of CCCH-type zinc finger protein and a preparation method thereof.
包括活性減毒疫苗(live-attenuated vaccines)及不活化疫苗(inactivated vaccines)在內的傳統疫苗的疫苗接種已證明是預防和控制病毒性疾病的最有效策略。與傳統疫苗生產方法（例如：流行性感冒病毒的雞胚胎內培養與日本腦炎疫苗的鼠腦培養）相比，基於細胞培養的疫苗生產具有明顯的優勢，包括以下方面：（1）用於生產疫苗的細胞株的來源更容易與方便取得，一旦爆發大流行，可快速生產疫苗；（2）與流感病毒株具有更高的相似性，有可能提高保護作用；（3）更能夠控制生產流程，加快了疫苗生產流程的啟動；（4）避免雞蛋和小鼠腦部疫苗引發嚴重過敏反應的風險。然而，以細胞培養的生產流程可能會降低了病毒產量降低以及成本效益。因此，如何提高病毒產量成為改善細胞培養疫苗的關鍵課題。Vaccination with traditional vaccines, including live-attenuated vaccines and inactivated vaccines, has proven to be the most effective strategy for preventing and controlling viral diseases. Compared with traditional vaccine production methods (eg: chicken embryo culture for influenza virus and mouse brain culture for Japanese encephalitis vaccine), cell culture-based vaccine production has distinct advantages, including the following: (1) for use in The source of the cell line for producing the vaccine is easier and more convenient to obtain, and once a pandemic breaks out, the vaccine can be quickly produced; (2) it has a higher similarity with the influenza virus strain, which may improve the protective effect; (3) can control the production more process, speeding up the start of the vaccine production process; (4) avoiding the risk of severe allergic reactions caused by egg and mouse brain vaccines. However, production processes in cell culture may reduce viral yields as well as cost-effectiveness. Therefore, how to improve the virus yield has become a key issue for improving cell culture vaccines.
CCCH型鋅指蛋白是一種參與RNA代謝途徑（例如RNA剪接，聚腺苷酸化(polyadenylation)和信使RNA (mRNA)降解等）的RNA結合分子。全基因組檢索的結果，在人類中共鑑定出57個CCCH基因，再根據氨基酸序列比對可分為6組(Liang, J et al. PLoS one 2008, 3(8), e2880.)。 CCCH-type zinc finger proteins are RNA-binding molecules involved in RNA metabolic pathways such as RNA splicing, polyadenylation, and messenger RNA (mRNA) degradation. As a result of genome-wide search, a total of 57 CCCH genes were identified in humans, and they could be divided into 6 groups according to the amino acid sequence alignment (Liang, J et al. PLoS one 2008 , 3(8), e2880.).
CCCH型鋅指家族的某些成員，例如ZC3H12A（MCP-1誘導蛋白1）、ZFP36 (Tristetraproline, TTP)、ZC3H2（Zinc Finger CCCH型抗病毒蛋白1，也稱為鋅指抗病毒蛋白(Zinc finger antiviral protein, ZAP) ），可以藉由其CCCH型鋅指模體(motif)直接結合特定的病毒RNA序列，並抑制病毒的複製(Zhou, L et al.
Circ Res 2006, 98, 1–10; Taylor, GA et al.
Immunity 19964, 445–454; Gao, G et al.
Science 2002, 297, 1703–1706.)。
Certain members of the CCCH-type zinc finger family, such as ZC3H12A (MCP-1-inducible protein 1), ZFP36 (Tristetraproline, TTP), ZC3H2 (Zinc Finger CCCH-type
大多數CCCH型鋅指蛋白的功能仍不清楚，與病毒的相互作用及其發揮的作用鮮少被討論。除了抗病毒活性，某些成員也可能具有可以增強病毒複製的前病毒活性(proviral activities)，在使用基於細胞培養的平台生產疫苗時，或可利用來提供更佳的病毒產量及成本效益。The functions of most CCCH-type zinc finger proteins remain unclear, and their interactions with viruses and their roles are rarely discussed. In addition to antiviral activity, some members may also possess proviral activities that enhance viral replication, which may be exploited to provide better viral yield and cost-effectiveness when using cell culture-based platforms for vaccine production.
基於上述目的，本發明演示了過表達ZC3HAV1L (Zinc finger CCCH-type, antiviral 1-like)蛋白的細胞可達成更高產量的病毒生產。Based on the above purpose, the present invention demonstrates that cells overexpressing ZC3HAV1L (Zinc finger CCCH-type, antiviral 1-like) protein can achieve higher yield of virus production.
因此，本發明一方面是關於一種在具有產生病毒能力的細胞中導入 ZC3HAV1L基因的生產病毒之變異細胞，其中該變異細胞藉由過表達該基因的產物而增加產生病毒的能力。 Therefore, one aspect of the present invention relates to a virus-producing mutant cell by introducing the ZC3HAV1L gene into a cell with virus-producing ability, wherein the mutant cell increases the virus-producing ability by overexpressing the product of the gene.
在本發明的一些實施例中，該變異細胞被一病毒感染。In some embodiments of the invention, the mutant cell is infected with a virus.
在本發明的一些實施例中，該病毒為黃病毒屬( Flavivirus)或甲型流感病毒屬 ( Alphainfluenzavirus)。 In some embodiments of the invention, the virus is Flavivirus or Alphainfluenzavirus .
在本發明的一些較佳實施例中，該病毒係選自日本腦炎病毒 (Japanese encephalitis virus, JEV)，登革熱病毒 (Dengue virus, DENV)及甲型流感病毒 (Influenza A virus, IAV)。In some preferred embodiments of the present invention, the virus is selected from Japanese encephalitis virus (JEV), Dengue virus (DENV) and Influenza A virus (IAV).
在本發明的一些較佳實例中，該變異細胞為一動物細胞；優選地，該動物細胞為Vero細胞。In some preferred embodiments of the present invention, the mutant cell is an animal cell; preferably, the animal cell is a Vero cell.
在本發明的另一方面，係關於一種製備生產病毒之變異細胞的方法，該方法包括將 ZC3HAV1L基因導入一生產病毒之細胞。 In another aspect of the present invention, it relates to a method for preparing a virus-producing mutant cell, the method comprising introducing the ZC3HAV1L gene into a virus-producing cell.
在本發明的一些實例中，該方法進一步包括用一病毒感染該變異細胞。In some embodiments of the invention, the method further comprises infecting the mutant cell with a virus.
在本發明的一些實施例中，係使用黃病毒屬( Flavivirus)或甲型流感病毒屬 ( Alphainfluenzavirus)感染該變異細胞。 In some embodiments of the invention, the mutant cells are infected with Flavivirus or Alphainfluenzavirus .
在本發明的一些較佳實施例中，係使用日本腦炎病毒 (Japanese encephalitis virus, JEV)，登革熱病毒 (Dengue virus, DENV)或甲型流感病毒 (Influenza A virus, IAV) 感染該變異細胞。In some preferred embodiments of the present invention, the mutant cells are infected with Japanese encephalitis virus (JEV), Dengue virus (DENV) or Influenza A virus (IAV).
在本發明的一些較佳實例中，被感染的該變異細胞為一動物細胞；優選地，該動物細胞為Vero細胞。In some preferred embodiments of the present invention, the infected mutant cell is an animal cell; preferably, the animal cell is a Vero cell.
在本發明的又一方面則關於一種產生所需病毒的方法，該方法包括以下步驟：（1）以所需病毒感染所述的生產病毒之變異細胞；（2）培養該感染所需病毒的變異細胞，然後收集培養基的上清液以回收所需病毒。In yet another aspect of the present invention, it relates to a method for producing a desired virus, the method comprising the steps of: (1) infecting the virus-producing mutant cells with the desired virus; (2) culturing the infected cells of the desired virus Mutate the cells, and then collect the supernatant of the medium to recover the desired virus.
在本發明的一些實施例中，該變異細胞以0.01至0.1 MOI值的所需病毒感染。In some embodiments of the invention, the mutant cells are infected with the desired virus at a MOI value of 0.01 to 0.1.
在本發明的再一方面則關於一種產生所需病毒的方法，該方法包括以下步驟：（1）以所需病毒感染所述的生產病毒之變異細胞；（2）培養該感染所需病毒的變異細胞，然後收集培養基的上清液以回收所需病毒；（3）將回收的病毒減毒或去活化。In yet another aspect of the present invention, it relates to a method for producing a desired virus, the method comprising the steps of: (1) infecting the virus-producing mutant cells with the desired virus; (2) culturing the infected cells of the desired virus mutate cells, and then collect the supernatant of the medium to recover the desired virus; (3) attenuate or deactivate the recovered virus.
在以下實施例中將進一步舉例說明本發明的其他特徵和優點，這些實施例僅用於說明，並不限制本發明的範圍。Other features and advantages of the present invention are further illustrated in the following examples, which are for illustration only and do not limit the scope of the invention.
病毒、細胞株、化學品及抗體Viruses, cell lines, chemicals and antibodies
將JEV PR-9 病毒株 (GenBank 登錄號: AF014161) (Chen, LK et al. Virology 1996, 223, 79-88.) 或DENV-2 PL046 病毒株 (GenBank 登錄號: KJ734727) (Lin, YL et al. J Virol 1998, 72, 9729-9737.) 在添加5%胎牛血清的RPMI-1640培養基的C6/36蚊子細胞(ATCC, CRL-1660)中培養。JEV-EGFP (RP-9株) 及 DENV-2-EGFP (PL046株)由林宜玲博士所提供。JEV及DENV的定量係藉由測定小倉鼠腎臟細胞(Baby hamster kidney cell line; BHK-21 cells)中的噬菌斑生成，該細胞係培養於添加5%胎牛血清、2 mM L-谷氨酰胺(L-glutamine)及1％青黴素-鏈黴素(penicillin-streptomycin)的RPMI-1640培養基。本研究中使用的甲型/波多黎各流感/8/1934 (Influenza A/Puerto Rico/8/1934 ) (H1N1)（GenBank登錄號：CY009444）在狗腎臟的上皮細胞株( Madin-Darby Canine Kidney cells; MDCK cells)中培養，該細胞在Dulbecco改良的Eagle培養基 (DMEM; Gibico)及含有10％胎牛血清（FBS）中生長。甲型流感病毒的定量藉由在MDCK細胞上的噬菌斑形成測定來進行。人類非小細胞肺癌細胞A549 (ATCC, CCL-185)是在添加了10% FBS，2 mM L-谷氨酰胺和1％ P/S的F-12培養基中培養。人胚胎腎細胞293T/17 (ATCC, CRL-11268)是在添加有10% FBS，2 mM L-谷氨酰胺和1％ P/S的Dulbecco改良Eagle培養基（DMEM）中培養。Lipofectamine 2000試劑購自Invitrogen。抗FLAG小鼠單株抗體購自Sigma-Aldrich。抗ZC3HAV1L小鼠單株抗體(F4): sc-514959購自Santa Cruz Biotechnology。小鼠單株抗JEV NS3抗體先前已描述於Chen, LK et al. Virology 1996, 217, 220-229. JEV PR-9 strain (GenBank accession number: AF014161) (Chen, LK et al. Virology 1996 , 223, 79-88.) or DENV-2 PL046 virus strain (GenBank accession number: KJ734727) (Lin, YL et al. al. J Virol 1998 , 72, 9729-9737.) in C6/36 mosquito cells (ATCC, CRL-1660) in RPMI-1640 medium supplemented with 5% fetal bovine serum. JEV-EGFP (strain RP-9) and DENV-2-EGFP (strain PL046) were provided by Dr. Yiling Lin. JEV and DENV were quantified by measuring plaque formation in baby hamster kidney cells (Baby hamster kidney cell line; BHK-21 cells) cultured in supplemented 5% fetal bovine serum, 2 mM L-glutamine RPMI-1640 medium with L-glutamine and 1% penicillin-streptomycin. Influenza A/Puerto Rico/8/1934 (Influenza A/Puerto Rico/8/1934) (H1N1) (GenBank accession number: CY009444) used in this study was developed in a dog kidney epithelial cell line (Madin-Darby Canine Kidney cells; MDCK cells) grown in Dulbecco's modified Eagle's medium (DMEM; Gibico) containing 10% fetal bovine serum (FBS). Quantification of influenza A virus was performed by plaque formation assay on MDCK cells. Human non-small cell lung cancer cells A549 (ATCC, CCL-185) were cultured in F-12 medium supplemented with 10% FBS, 2 mM L-glutamine and 1% P/S. Human embryonic kidney cells 293T/17 (ATCC, CRL-11268) were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS, 2 mM L-glutamine and 1% P/S. Lipofectamine 2000 reagent was purchased from Invitrogen. Anti-FLAG mouse monoclonal antibody was purchased from Sigma-Aldrich. Anti-ZC3HAV1L mouse monoclonal antibody (F4): sc-514959 was purchased from Santa Cruz Biotechnology. Mouse monoclonal anti-JEV NS3 antibodies have been previously described in Chen, LK et al. Virology 1996 , 217, 220-229.
質粒構建、慢病毒的產生及過表達ZC3HAV1L的穩定細胞株的建立Plasmid construction, lentivirus generation and establishment of stable cell line overexpressing ZC3HAV1L
ZC3HAV1L(NM_080660) Human Tagged ORF Clone (RC203208)購自OriGene Technologies。將人類 ZC3HAV1L的cDNA與FLAG-tag ZC3HAV1LHuman Tagged ORF Clone併合，並次選殖(subcloning)至自滅活慢病毒載體（pSIN），其中***基因的表現是由脾臟病灶形成病毒(Spleen focus-forming virus, SFFV)啟動子控制(Godfrey, A et al. Blood 2005, 105, 2510-2518.)。慢病毒的製備方式是，先將人類胚胎腎臟細胞293 T/17與慢病毒表達構建體(pSIN)和兩個輔助質體pMD.G及pCMVΔR8.91用Lipofectamine 2000試劑（Invitrogen）共轉染，再將轉染的細胞在37°C下培養4 -5小時，然後更換新鮮培養基。接著在轉染後24-60小時收集含有病毒顆粒的上清液，並保存在-80°C。過表達ZC3HAV1L蛋白的Vero細胞株製備方式是，用表現FLAG標記的ZC3HAV1L蛋白或對照組EGFP蛋白的慢病毒載體轉導入A549細胞72小時。 ZC3HAV1L (NM_080660) Human Tagged ORF Clone (RC203208) was purchased from OriGene Technologies. The human ZC3HAV1L cDNA was combined with the FLAG-tag ZC3HAV1L Human Tagged ORF Clone, and subcloned into a self-inactivating lentiviral vector (pSIN), in which the inserted gene was expressed by a spleen focus-forming virus (Spleen focus-forming virus). , SFFV) promoter control (Godfrey, A et al. Blood 2005 , 105, 2510-2518.). The lentivirus was prepared by first co-transfecting human embryonic kidney cells 293 T/17 with the lentiviral expression construct (pSIN) and two helper plastids pMD.G and pCMVΔR8.91 with Lipofectamine 2000 reagent (Invitrogen). Transfected cells were incubated at 37°C for an additional 4-5 hours, then replaced with fresh medium. Viral particle-containing supernatants were then collected 24-60 hours post-transfection and stored at -80°C. The Vero cell line overexpressing ZC3HAV1L protein was prepared by transfecting A549 cells with a lentiviral vector expressing FLAG-tagged ZC3HAV1L protein or control EGFP protein for 72 hours.
西方墨點法免疫印跡分析Western blot analysis by western blot
用表現FLAG標記ZC3HAV1L蛋白或表現對照mcherry蛋白的慢病毒載體轉導A549細胞72小時，接著以JEV-EGFP（MOI = 0.01、0.1或1）感染16小時。以含有蛋白酶抑制劑的十二烷基硫酸鈉(SDS)樣品緩衝液（62.5 mM Tris-HCl [pH 6.8], 2％SDS, 10％甘油, 50 mM 二硫蘇糖醇, 0.1％ 溴酚藍）裂解該細胞。等量的蛋白質經SDS-聚丙烯酰胺凝膠電泳分離後，將其轉移到硝酸纖維素膜(Hybond-C Super, Amersham)上。在添加一抗之前，先以添加5％脫脂牛奶的TBS-T (25 mM Tris, 0.8％NaCl, 2.68 mM KCl [pH 7.4], and 0.1％Tween 20)阻隔非特異性抗體結合位點。然後用帶有辣根過氧化物酶(horseradish peroxidase)的二抗（Amersham）處理印跡，並用ECL系統（Amersham）顯影。A549 cells were transduced with lentiviral vectors expressing FLAG-tagged ZC3HAV1L protein or control mcherry protein for 72 hours, followed by infection with JEV-EGFP (MOI = 0.01, 0.1 or 1) for 16 hours. Sodium dodecyl sulfate (SDS) sample buffer (62.5 mM Tris-HCl [pH 6.8], 2% SDS, 10% glycerol, 50 mM dithiothreitol, 0.1% bromophenol blue) containing protease inhibitors ) to lyse the cell. Equal amounts of proteins were separated by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes (Hybond-C Super, Amersham). Nonspecific antibody binding sites were blocked with TBS-T (25 mM Tris, 0.8% NaCl, 2.68 mM KCl [pH 7.4], and 0.1% Tween 20) supplemented with 5% nonfat milk before adding the primary antibody. Blots were then treated with a secondary antibody (Amersham) with horseradish peroxidase and developed with the ECL system (Amersham).
免疫螢光測定Immunofluorescence assay
用表現FLAG標記ZC3HAV1L蛋白或表現對照mcherry蛋白的慢病毒載體轉導A549細胞72小時，然後用JEV-EGFP (MOI = 0.01或0.1)感染指定的時間，在螢光顯微鏡下觀察EGFP蛋白的表現。A549 cells were transduced with lentiviral vectors expressing FLAG-tagged ZC3HAV1L protein or control mcherry protein for 72 hours, then infected with JEV-EGFP (MOI = 0.01 or 0.1) for the indicated times, and the expression of EGFP protein was observed under a fluorescent microscope.
病毒生長曲線測定Virus growth curve assay
關於JEV和DENV-2感染，將12孔盤中生長的細胞單層以指定的病毒感染劑量 (MOI)在37°C下吸附病毒1小時。吸附1小時後，以RPMI 1640培養基溫和洗滌，從細胞中去除未結合的病毒。在指定的感染後時間點(post-infection, p.i.)，收集培養基以用於BHK-21細胞中的噬菌斑形成測定。For JEV and DENV-2 infection, cell monolayers grown in 12-well dishes were adsorbed with virus at the indicated virus infectious dose (MOI) for 1 hour at 37°C. After 1 hour of adsorption, unbound virus was removed from the cells by gentle washing with RPMI 1640 medium. At the indicated post-infection time points (post-infection, p.i.), media was collected for plaque formation assays in BHK-21 cells.
關於IAV(Influenza A virus)感染，將12孔盤中生長的細胞單層以指定的病毒感染劑量 (MOI = 0.01)在37°C下吸附1小時。吸附1小時後，以PBS溫和洗滌，從細胞中去除未結合的病毒。在指定的感染後時間點，收集上清液以用於噬菌斑形成測定。簡言之，將MDCK細胞以6.0 × 10 5細胞/孔布置於6孔盤中，然後在37°C以病毒感染24小時。欲定量的病毒在37℃下於6孔板中進行10倍序列稀釋。感染結束後1小時，將細胞用1 mL PBS洗滌兩次，並用2 mL添加有0.3%瓊脂糖的DMEM/HG培養基覆蓋細胞。在37°C下培養48小時後，將細胞在10%福馬林 (formalin)中固定至少1小時，然後進行結晶紫染色。病毒定量以每毫升噬菌斑形成單位(PFU)計算。 For IAV (Influenza A virus) infection, cell monolayers grown in 12-well dishes were adsorbed at the indicated virus infection dose (MOI = 0.01) for 1 hour at 37°C. After 1 hour of adsorption, unbound virus was removed from the cells by gentle washing with PBS. At the indicated post-infection time points, supernatants were collected for plaque formation assays. Briefly, MDCK cells were plated at 6.0 x 105 cells/well in 6 -well dishes and then infected with virus for 24 hours at 37°C. Viruses to be quantified were serially diluted 10-fold in 6-well plates at 37°C. One hour after the infection ended, cells were washed twice with 1 mL of PBS and overlaid with 2 mL of DMEM/HG medium supplemented with 0.3% agarose. After 48 hours of incubation at 37°C, cells were fixed in 10% formalin for at least 1 hour before crystal violet staining. Virus quantification was calculated as plaque forming units (PFU) per milliliter.
實施例1 Zc3hav1L蛋白在細胞中的過表現Example 1 Overexpression of Zc3hav1L protein in cells
pSIN載體用於構建ZC3HAV1L-Myc-Flag/pSIN質粒。以慢病毒轉導建立過表達FLAG標記ZC3HAV1L蛋白的A549細胞 (ATCC, CCL-185)。西方墨點法顯示了FLAG標記ZC3HAV1L蛋白或對照組EGFP蛋白在細胞中的表達（第1圖）。以抗FLAG及抗ZC3HAV1L單株抗體檢測，蛋白質大小測定約為34 kDa。The pSIN vector was used to construct the ZC3HAV1L-Myc-Flag/pSIN plasmid. A549 cells (ATCC, CCL-185) overexpressing FLAG-tagged ZC3HAV1L protein were established by lentiviral transduction. Western blotting showed the expression of FLAG-tagged ZC3HAV1L protein or control EGFP protein in cells (Figure 1). Detected with anti-FLAG and anti-ZC3HAV1L monoclonal antibodies, the protein size was about 34 kDa.
實施例2 過表現Zc3hav1L蛋白對日本腦炎病毒 (JEV)感染的影響Example 2 Effect of overexpression of Zc3hav1L protein on Japanese encephalitis virus (JEV) infection
用表現FLAG標記ZC3HAV1L蛋白或對照組表現mcherry蛋白的慢病毒載體轉導A549細胞，然後用JEV-EGFP感染。如第2圖所示，在JEV-EGFP感染過表現ZC3HAV1L的細胞6小時 (MOI = 0.01)後，以及mcherry對照組感染24小時後，可觀察到GFP表現。在較高MOI (0.1)的JEV-EGFP感染中，亦須到16小時，在對照組中才觀察到明顯的GFP螢光（第3圖）。A549 cells were transduced with lentiviral vectors expressing FLAG-tagged ZC3HAV1L protein or control group expressing mcherry protein, and then infected with JEV-EGFP. As shown in Figure 2, GFP expression was observed 6 hours after JEV-EGFP infection of ZC3HAV1L-overexpressing cells (MOI = 0.01) and 24 hours after infection of mcherry controls. In the JEV-EGFP infection at higher MOI (0.1), it was not until 16 hours before obvious GFP fluorescence was observed in the control group (Fig. 3).
實施例3 JEV蛋白NS3的西方墨點法分析Example 3 Western blot analysis of JEV protein NS3
用表現FLAG標記ZC3HAV1L蛋白或對照組表現mcherry蛋白的慢病毒載體轉導A549細胞，然後用JEV-EGFP (MOI = 0.01, 0.1, 1)感染16小時。NS3是JEV產生的一種非結構蛋白，通過西方墨點法進行了測定，證明了JEV的複製可以在過表現ZC3HAV1L蛋白的細胞中有效地進行（第4圖）。A549 cells were transduced with lentiviral vectors expressing FLAG-tagged ZC3HAV1L protein or control group expressing mcherry protein, and then infected with JEV-EGFP (MOI = 0.01, 0.1, 1) for 16 hours. NS3, a nonstructural protein produced by JEV, was assayed by Western blotting, demonstrating that JEV replication can be efficiently performed in cells overexpressing the ZC3HAV1L protein (Figure 4).
實施例4 過表達Zc3hav1L蛋白的穩定細胞株的建立Example 4 Establishment of stable cell lines overexpressing Zc3hav1L protein
用表現FLAG標記ZC3HAV1L蛋白的慢病毒載體轉導Vero非洲綠猴腎細胞72小時。將製備的轉導細胞與親代細胞用於間接免疫螢光，並使用小鼠抗FLAG抗體和抗小鼠IgG抗體（Alexa-488）分別作為第一抗體和第二抗體（第5圖）。與親代細胞相比，幾乎所有過表現FLAG標記ZC3HAV1L的細胞都可以被染色。Vero African green monkey kidney cells were transduced with a lentiviral vector expressing the FLAG-tagged ZC3HAV1L protein for 72 hours. The prepared transduced cells and parental cells were used for indirect immunofluorescence, and mouse anti-FLAG antibody and anti-mouse IgG antibody (Alexa-488) were used as primary and secondary antibodies, respectively (Figure 5). Almost all cells overexpressing the FLAG-tagged ZC3HAV1L could be stained compared to the parental cells.
實施例5Example 5 JEVJEV 感染Zc3hav1L蛋白過表現細胞中的病毒生長Virus growth in infected Zc3hav1L protein-overexpressing cells
用JEV (MOI = 0.01)感染具有或不具有ZC3HAV1L過表現的Vero細胞，並分析病毒複製量。使用噬菌斑測定法確定感染後24、48、60、72小時的病毒量（第6圖）。與親代細胞以及GFP過表達細胞相比，過表達ZC3HAV1L蛋白的細胞中的病毒產量增加了近100倍。Vero cells with or without ZC3HAV1L overexpression were infected with JEV (MOI = 0.01) and the amount of virus replication was analyzed. Plaque assays were used to determine viral loads at 24, 48, 60, and 72 hours post-infection (Figure 6). Virus production was increased nearly 100-fold in cells overexpressing the ZC3HAV1L protein compared to parental cells as well as GFP-overexpressing cells.
實施例6Example 6 DENV-2DENV-2 感染Zc3hav1L蛋白過表現細胞中的病毒生長Virus growth in infected Zc3hav1L protein-overexpressing cells
用DENV-2 (MOI = 0.01)感染具有或不具有ZC3HAV1L過表現的Vero細胞，並分析病毒複製量。使用噬菌斑測定法確定感染後24、48、72、96小時的病毒量（第7圖）。與親代細胞以及GFP過表達細胞相比，過表達ZC3HAV1L蛋白的細胞中的病毒產量亦增加了近100倍。Vero cells with or without ZC3HAV1L overexpression were infected with DENV-2 (MOI = 0.01) and the amount of virus replication was analyzed. Plaque assays were used to determine viral loads at 24, 48, 72, and 96 hours post-infection (Figure 7). Virus production in cells overexpressing the ZC3HAV1L protein was also increased by nearly 100-fold compared to parental cells as well as GFP-overexpressing cells.
實施例7 IAV感染Zc3hav1L蛋白過表現細胞中的病毒生長Example 7 Virus growth in IAV infected Zc3hav1L protein overexpressing cells
用IAV (MOI = 0.01)感染具有或不具有ZC3HAV1L蛋白過表現的Vero細胞，並分析病毒複製量。使用噬菌斑測定法確定感染後24、48、64、80小時的病毒量（第8圖）。與親代細胞以及GFP過表達細胞相比，過表達ZC3HAV1L蛋白的細胞中的病毒產量增加了近10倍。Vero cells with or without ZC3HAV1L protein overexpression were infected with IAV (MOI = 0.01) and the amount of viral replication was analyzed. Plaque assays were used to determine viral loads at 24, 48, 64, and 80 hours post-infection (Figure 8). Virus production in cells overexpressing the ZC3HAV1L protein increased nearly 10-fold compared to parental cells as well as GFP-overexpressing cells.
如上述實施例所示，ZC3HAV1L做為新穎的宿主細胞因子，可顯著提高病毒產量。與親代細胞相比，過表達ZC3HAV1L蛋白的細胞導致JEV及DENV的病毒產量增加大約100倍以上，IAV的病毒產量增加超過10倍。與本領域中基於細胞培養的疫苗平台相比，本發明在減毒和去活化疫苗的生產中提供了更高的病毒產量和更好的成本效益。As shown in the above examples, ZC3HAV1L, as a novel host cytokine, can significantly increase virus yield. Compared with parental cells, cells overexpressing ZC3HAV1L protein resulted in approximately a 100-fold increase in viral production of JEV and DENV and a more than 10-fold increase in IAV virus production. Compared to cell culture-based vaccine platforms in the art, the present invention provides higher virus yields and better cost-effectiveness in the production of attenuated and deactivated vaccines.
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第1圖 以西方墨點法分析用慢病毒轉導的A549細胞中FLAG標記ZC3HAV1L蛋白的表現。Figure 1. Western blot analysis of the expression of FLAG-tagged ZC3HAV1L protein in A549 cells transduced with lentivirus.
第2圖 使用JEV-GFP（MOI ＝ 0.01）來分析在過表達ZC3HAV1L蛋白的A549細胞中JEV的複製。Figure 2 JEV-GFP (MOI = 0.01) was used to analyze JEV replication in A549 cells overexpressing ZC3HAV1L protein.
第3圖 使用JEV-GFP（MOI ＝ 0.1）來分析在過表達ZC3HAV1L蛋白的A549細胞中JEV的複製。Figure 3. JEV-GFP (MOI = 0.1) was used to analyze JEV replication in A549 cells overexpressing ZC3HAV1L protein.
第4圖 在Vero細胞中，過表達ZC3HAV1L-FLAG之穩定細胞株的免疫螢光。Figure 4 Immunofluorescence of a stable cell line overexpressing ZC3HAV1L-FLAG in Vero cells.
第5圖 以西方墨點法分析被JEV-EGFP感染的細胞中非結構病毒蛋白NS3的生成。Figure 5. Production of the nonstructural viral protein NS3 in cells infected with JEV-EGFP by Western blot analysis.
第6圖 JEV感染的細胞中的病毒生長曲線。Figure 6 Virus growth curve in JEV-infected cells.
第7圖 DENV-2感染的細胞中病毒的生長曲線。Figure 7 Virus growth curve in DENV-2 infected cells.
第8圖 IAV感染的細胞中病毒的生長曲線。Figure 8 Virus growth curve in IAV-infected cells.
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| 期刊 Baoge Zhang1 et. al., "Zinc Finger CCCH-Type Antiviral Protein 1 Restricts the Viral Replication by Positively Regulating Type I Interferon Response" vol. 11, Article 1912, Front Microbiol.Published online 2020 Aug 14, p.1-14; * |
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