TW202229556A - 針對SARS-CoV-2之改良型DNA疫苗 - Google Patents
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Abstract
本發明提供一種DNA,其係編碼冠狀病毒(SARS CoV-2)之刺突蛋白或其片段者,且上述DNA之序列中所含之一部分或全部密碼子就人類之表現得到最佳化。
Description
本發明係關於一種用於在人類中誘導針對SARS-CoV-2(Severe acute respiratory syndrome coronavirus 2,嚴重急性呼吸道症候群冠狀病毒2)之免疫之DNA疫苗。
由細菌或病毒引起之感染症不限於局部地區之感染,係具有會於社會上廣泛蔓延之潛在風險之疾病。根據其感染力或患者所產生之症狀,有時亦會成為社會性問題。亦有不少感染症甚至引發了歷史上之重要事件,例如天花、鼠疫、流行性感冒(西班牙流行性感冒)等。該等感染症隨著化學治療劑、抗生素、疫苗、治療用藥等之開發/普及、以及衛生環境之改善而被克服,或者其等之威脅已得到減小。然而,新興、再興感染症目前依然存在。
疫苗係有效之感染症對策。曾因高致死率而令人畏懼之天花藉由在全球範圍內實施根絕措施而得到根絕,在此過程中天花疫苗所發揮之作用非常大。關於嚴重之感染症,亦有法律規定必須接受疫苗接種(在日本有白喉、百日咳、破傷風、急性脊髓灰質炎),以在維持公共衛生之方面有效地發揮功能。
作為傳統之疫苗,已知有病原體之自然減毒株或人工減毒株、及藉由化學處理、物理處理而滅活之病原體。例如,流行性感冒疫苗係以使用雞蛋大規模地製造之流行性感冒病毒作為原料所製造。
近年來,對以藉由基因工程方法所製造之病原體之特定成分、例如蛋白質作為有效成分之疫苗(組分疫苗,例如專利文獻1)、以及編碼上述蛋白質之核酸本身(DNA疫苗,例如專利文獻2;RNA疫苗,例如專利文獻3)、搭載有上述核酸之病毒載體(病毒載體疫苗,例如專利文獻4)之研究不斷推進。
如上所述,不大量製造病原體本身而製造疫苗之技術正在不斷開發之中。但是,為了創製具備足以實用之性能之疫苗,而需要反覆試誤並根據對象病原體或其成分來設計疫苗。
[先前技術文獻]
[專利文獻]
[專利文獻1]國際公開第2018/074558號說明書
[專利文獻2]國際公開第2014/034735號說明書
[專利文獻3]國際公開第2018/075980號說明書
[專利文獻4]國際公開第2018/189522號說明書
[發明所欲解決之問題]
本發明之目的在於提供一種旨在預防、治療作為新興感染症之SARS-CoV-2感染症之DNA疫苗。
[解決問題之技術手段]
本發明人等對在人類中能夠誘導針對具有高感染力之SARS-CoV-2之免疫的DNA疫苗之製造反覆進行了努力研究,發現,藉由適當地修飾編碼上述病毒之刺突蛋白之核酸之密碼子,會使細胞內之刺突蛋白之表現效率顯著提昇,從而完成本發明。
即,本發明提供:
[1]一種DNA,其係編碼冠狀病毒(SARS CoV-2)之刺突蛋白或其片段者,且所含之一部分或全部密碼子就人類之表現得到最佳化;
[2]如[1]所記載之DNA,其中SARS CoV-2基因組RNA之編碼刺突蛋白或其片段之鹼基序列中所含之密碼子的約50%以上被替換為其他密碼子;
[3]如[1]所記載之DNA,其編碼刺突蛋白之全長;
[4]如[1]所記載之DNA,其包含與序列編號3、12、14、16或18所表示之鹼基序列具有約90%以上之同一性之鹼基序列;
[5]如[4]所記載之DNA,其具有序列編號3、12、14、16或18所表示之鹼基序列;
[6]一種核酸構建物,其含有於人類中發揮功能之啟動子、及與上述啟動子功能性連結之如[1]至[5]中任一項所記載之DNA;
[7]如[6]所記載之核酸構建物,其進而含有與如[1]至[5]中任一項所記載之DNA功能性連結之轉錄終止序列;
[8]如[6]或[7]所記載之核酸構建物,其係組入至載體而成;
[9]如[8]所記載之核酸構建物,其係組入至選自質體載體、噬菌體載體及病毒載體中之載體而成;
[10]一種醫藥組合物,其含有如[6]至[9]中任一項所記載之核酸構建物;
[11]如[10]所記載之醫藥組合物,其進而包含佐劑;
[12]如[10]或[11]所記載之醫藥組合物,其係冠狀病毒感染症之疫苗;
[13]一種冠狀病毒感染症之預防、症狀之減輕、或治療方法,其包括向對象投予如[12]所記載之醫藥組合物;
[14]如[1]所記載之DNA,其編碼刺突蛋白之C末端缺失片段;
[15]如[1]所記載之DNA,其編碼包含K986P及/或V987P突變之刺突蛋白或其片段;
等。
[發明之效果]
藉由本發明,提供一種能夠於人類細胞內以高效率表現SARS-CoV-2之刺突蛋白之DNA、保持該DNA之核酸構建物、包含該核酸構建物之醫藥組合物。本發明之醫藥組合物可用於SARS-CoV-2病毒感染症之預防、症狀之減輕、或治療。
1.本發明之DNA
本發明之DNA能夠於人類中表現冠狀病毒(SARS-CoV-2)之刺突蛋白或其片段,而用作誘導針對上述病毒之免疫之DNA疫苗。DNA疫苗不同於向生物體投予病原體本身或特定之來自病原體之成分的先前疫苗,係以編碼病原體之抗原性蛋白質之DNA作為必須構成成分之疫苗。DNA疫苗無需處理活病原體即可製造,基於安全性以及經濟性之觀點考慮,係一種被寄予厚望之技術。
本發明之DNA之特徵在於:其係編碼SARS-CoV-2之刺突蛋白或其片段者,且上述DNA中所含之一部分或全部密碼子就人類之表現得到最佳化。藉由上述特徵,本發明之DNA能夠於人類體內以高效率表現刺突蛋白,適於製造高效價之DNA疫苗。
冠狀病毒係具有正鏈RNA基因組之被膜病毒,被認為係諸如鼻炎之相對輕微之疾病之致病病毒,但2003年發現之SARS冠狀病毒(SARS-CoV)被證明會導致嚴重之呼吸道疾病。於冠狀病毒之被膜中存在負責與受體結合及細胞侵入之刺突蛋白。本發明之DNA能夠使SARS-CoV-2所具有之刺突蛋白(以下,有時僅記載為刺突蛋白)或其片段於人類細胞內高效率地表現。
本發明之DNA可為編碼包含約1300個胺基酸之刺突蛋白之全長者,或者亦可為編碼包含其一部分之片段者。作為上述「片段」,只要具備能夠誘導針對SARS-CoV-2之免疫之抗原性,則並無特別限定,可以例示刺突蛋白之約1/2以上、較佳為約2/3以上、更佳為約3/4以上、進而較佳為約90%以上之大小之片段。又,該片段可為全長刺突蛋白之包含N末端之片段、包含C末端之片段、或包含中央區之片段之任一種。已提示,缺失了C末端之19個胺基酸之SARS-CoV之刺突蛋白之片段相較於真核細胞內不具有上述缺失之刺突蛋白,表現量有所提昇(Journal of General Biology, 2005, Vol. 86, p2269 - 2274),從而編碼導入有同樣缺失之SARS-CoV-2刺突蛋白之DNA亦可用於本發明。作為本發明之較佳形態,可例舉編碼刺突蛋白之全長之DNA。
於基因庫(GeneBank)中以NC_045512之登錄號公開了在武漢(Wuhan)被單離出之SARS-CoV-2之資訊。於本說明書中,將該Wuhan株之SARS-CoV-2刺突蛋白之胺基酸序列記載為「野生型之胺基酸序列」。作為本發明之DNA所編碼之SARS-CoV-2之刺突蛋白,可例示具有野生型刺突蛋白或其片段之胺基酸序列者,但並不限定於此,亦可為具有來自各種突變病毒株之刺突蛋白或其片段之胺基酸序列者。本發明亦包含編碼如下修飾刺突蛋白或其片段之DNA,該修飾刺突蛋白或其片段係於上述胺基酸序列中置換、缺失(例如胺基酸序列內部之胺基酸之缺失)、***、及/或附加1個以上、例如1個或數個胺基酸殘基所得。此處,「數個」例如包含2~15個、較佳為2~10個。例如,編碼自SARS-CoV-2感染患者分離出之突變體病毒所具有之具有D614G突變之刺突蛋白(Pathogen, 2020, Vol. 9, 324)之DNA、或者編碼導入有已知有助於冠狀病毒刺突蛋白之高表現之K986P/V987P突變(2P突變;bioRxiv, Preprint. 2020 Jun 11)之刺突蛋白的DNA亦可用於本發明。作為上述修飾刺突蛋白,可例示具有與序列表之序列編號1、11、13、15或17所表示之胺基酸序列或其等之片段具有約90%以上、較佳為約95%以上、更佳為約98%以上、進而較佳為約99%以上之同一性之胺基酸序列的刺突蛋白或其片段。
於本發明中,就人類之表現之密碼子之最佳化例如藉由下述方式來達成:將人類中使用頻度較低之密碼子替換為人類中使用頻度較高之密碼子。密碼子之替換可參照公知之方法(Nucleic Acids Res.,第30卷,e43,2002年;國際公開第2004/059556號說明書;國際公開第2007/102578號說明書等)來實施。又,公開了與人類中密碼子之使用頻度相關之資訊(http://www.kazusa.or.jp/codon/cgi-bin/showcodon.cgi?species=9606)。參照此,將編碼SARS-CoV-2之刺突蛋白之野生型鹼基序列、即天然之病毒基因組RNA之鹼基序列中所存在的人類中使用頻度較低之密碼子替換為人類中出現頻度較高之密碼子,藉此能夠獲得本發明之DNA之鹼基序列。於本發明之DNA中,SARS-CoV-2之基因組RNA上所存在的編碼刺突蛋白或其片段之鹼基序列中所含之密碼子的約50%以上、較佳為約60%以上、更佳為約70%以上被替換為適於在人類中表現之密碼子,從而當導入至人類細胞時能夠以高效率表現刺突蛋白。本發明之DNA之一個較佳形態係編碼刺突蛋白之全長之DNA,例如可例示具有序列編號3所表示之鹼基序列之DNA。又,作為另一形態,提供一種編碼對序列編號1之胺基酸序列進行修飾(胺基酸之置換、缺失、***或附加)所獲得之胺基酸序列的DNA(例如序列編號12、14、16及18之鹼基序列之DNA)。進而,作為本發明之較佳形態,亦例示:包含與序列編號3、12、14、16或18之鹼基序列具有約90%以上之同一性之鹼基序列的DNA、包含與序列編號3、12、14、16或18之鹼基序列具有較佳為約95%以上之同一性之鹼基序列的DNA、包含與序列編號3、12、14、16或18之鹼基序列具有更佳為約98%以上之同一性之鹼基序列的DNA、包含與序列編號3、12、14、16或18之鹼基序列具有進而較佳為約99%以上之同一性之鹼基序列的DNA。
藉由替換密碼子所獲得之於人類中之刺突蛋白表現效率之提昇可藉由下述方式來確認:製作包含本發明之DNA且適於刺突蛋白於人類細胞內表現之核酸構建物後,將該構建物導入至人類細胞中,繼而調查該細胞中之刺突蛋白之表現狀態。上述之刺突蛋白之表現狀態例如可藉由下述方式來調查,即測定導入有上述核酸構建物之細胞中之刺突蛋白mRNA之轉錄量、或測定細胞表面出現之刺突蛋白之量。作為上述mRNA測定方法,例如可例舉RT-PCR(Reverse Transcription-Polymerase Chain Reaction,反轉錄-聚合酶鏈反應)法或微陣列法,又,作為刺突蛋白之測定方法,例如可例舉使用抗刺突蛋白抗體之免疫學方法。進而,於以免疫學方式測定刺突蛋白時,可利用公知之方法、例如ELISA(enzyme linked immunosorbent assay,酶聯免疫吸附測度)法或使用細胞分選儀之方法。
2.本發明之核酸構建物
本發明之核酸構建物係包含上述本發明之DNA,且能夠於細胞內自該DNA轉錄與刺突蛋白或其片段對應之mRNA之構建物。具體而言,係含有於人類中發揮功能之啟動子、及位於其下游之本發明之DNA的核酸構建物。
於本發明之核酸構建物中,本發明之DNA係與在人類中發揮功能之啟動子功能性連結。此處,所謂「功能性連結」,係指控制基因表現之序列存在於能夠對編碼所需蛋白質之序列發揮作用之位置處。例如,所謂「與啟動子功能性連結」,係指在相對於編碼蛋白質之DNA能夠自該DNA開始轉錄之位置、即該DNA之上游處配置有啟動子。
本發明之核酸構建物所使用之啟動子並無特別限定,只要能夠於人類中指示自DNA轉錄RNA即可,例如可使用來自哺乳動物之啟動子(PGK(phosphoglycerate kinase,磷酸甘油酸激酶)啟動子、EF1-α(elongation factor 1-α,延長因子1-α)啟動子、β-血球蛋白啟動子等)、來自病毒之啟動子(CMV(cytomegalovirus,巨細胞病毒)啟動子、SV40(simian vacuolating virus 40,猴空泡病毒)啟動子、MMLV-LTR(Moloney murine leukemia virus-long terminal repeat,莫洛尼鼠白血病病毒-末端長重複序列)啟動子、HIV-LTR(human immunodeficiency virus-long terminal repeat,人類免疫缺乏病毒-末端長重複序列)啟動子等)、人工構建之啟動子(CAG啟動子等)。本發明中可使用構成性啟動子或誘導性啟動子之任一種。於使用後者之情形時,通常併用適當之誘導物質。
於較佳形態中,本發明之核酸構建物於本發明之DNA之下游具備轉錄終止序列。作為轉錄終止序列,例如可例舉於人類中發揮功能之聚(A)附加訊息。本發明之核酸構建物可使用之聚(A)附加訊息並無特別限定,例如可例舉:來自SV40病毒之聚(A)附加訊息序列、來自牛生長激素基因之聚(A)附加訊息序列、或人工合成之聚(A)附加訊息。進而,亦可於本發明之核酸構建物搭載除上述者以外之表現控制因子、例如強化子序列等。該等表現控制因子亦與本發明之DNA或所併用之其他因子功能性連結,只要與本發明之DNA或所併用之其他因子功能性連結,則本發明之核酸構建物中之該表現控制因子之位置並無特別限定。
進而,本發明之核酸構建物亦可包含複數個分子之本發明之DNA。該情形時,上述複數個DNA可分別與不同之啟動子功能性連結,或者亦可藉由一個啟動子而多順反子性地達成來自複數個DNA之刺突蛋白或其片段之表現。為了多順反子性之蛋白質之表現,可利用公知之要素、例如內部核糖體進入位點(internal ribosome entry site;IRES)或轉譯後自動被切斷之肽(P2A肽、T2A肽等)。上述複數個DNA可為相同分子,亦可為不同分子(例如,編碼刺突蛋白全長之DNA與編碼刺突蛋白片段之DNA之組合、編碼野生型胺基酸序列之刺突蛋白之DNA與編碼具有突變或經修飾之刺突蛋白之DNA的組合等)。
本發明之核酸構建物可以DNA片段之形式使用,例如可將該DNA片段投予至人類而使刺突蛋白表現,但亦可搭載於各種載體而使用。作為能夠搭載本發明之核酸構建物之載體,並無限定,可例舉:質體載體、噬菌體載體、病毒載體、人工染色體載體等。上述載體根據本發明之核酸構建物之使用形態來選擇適當者即可,可使用能夠獲取之公知載體或新設計之載體。再者,本發明之核酸構建物中之啟動子或其他表現控制因子可利用由載體所保持者。例如,於保持啟動子之表現載體之對該啟動子而言適當之位置處連接本發明之DNA,藉此能夠製作本發明之核酸構建物。當然,亦可將載體原本不具有之啟動子或表現控制因子與本發明之DNA一起組入至載體。
於需要於人類中暫時性表現刺突蛋白之情形時,例如於質體載體或適於暫時性表現之病毒載體(腺病毒載體、腺相關病毒載體等)搭載本發明之核酸構建物即可。於期望長期表現刺突蛋白之情形時,可利用能夠於人類細胞內複製之游離型載體、或具有將所搭載之基因組入至染色體之能力之病毒載體(反轉錄病毒載體、慢病毒載體等)。
搭載有本發明之核酸構建物之載體中,除了該核酸構建物以外,還可包含其他要素。作為上述其他要素,例如可例舉維持作為載體之功能所需者(例如複製起始點或病毒載體之包裝序列等),但並不限定於該等。亦可將顯示於靶細胞中導入有載體之報導基因(例如螢光蛋白基因、酶基因、細胞表面蛋白基因等)、或可用於排除於載體製造時未保持載體之宿主細胞的抗藥性基因(抗安比西林性基因、抗康黴素性基因等)搭載於載體。
本發明之核酸構建物根據其形式可利用業者所周知之方法進行製造。為核酸片段之形式之核酸構建物可利用公知之核酸擴增法、例如PCR(Polymerase Chain Reaction,聚合酶鏈反應)法或各種等溫核酸擴增法,不使用活細胞而大量地製造。於核酸構建物搭載於載體之情形時,可於該載體所適合之宿主內使載體複製及擴增而製造。於本發明之核酸構建物搭載於質體載體之情形時,於該載體能夠複製之宿主(例如大腸桿菌)內促進質體之複製,繼而利用公知之方法自宿主細胞中提取及純化質體,藉此能夠獲得單離出之質體。例如,自大腸桿菌中純化質體可自藉由SDS(sodium dodecylsulfate,十二烷基硫酸鈉)鹼裂解法所獲得之粗質體製備物,組合核糖核酸酶處理、管柱層析、超過濾等操作來實施。又,基因工程領域中所利用之大多數病毒載體之製造方法亦為業者所周知,適於宿主之細胞亦大多在市場上有售。製作保持有產生病毒載體所需之組分之宿主細胞(病毒產生細胞),並將其進行培養,藉此能夠使病毒載體於細胞內或培養上清液中產生。因此,亦可容易地製造搭載於病毒載體之本發明之核酸構建物。
3.本發明之醫藥組合物
本發明之醫藥組合物係含有上述本發明之核酸構建物且能夠向人類投予之組合物。該組合物之特徵在於含有本發明之核酸構建物、以及藥學上所容許之載體。本發明之醫藥組合物只要為與其投予路徑對應之劑型即可,通常製成注射劑、點滴劑、或其他劑型之非經口製劑。作為該非經口製劑可含有之載體,例如可例舉:如包含生理鹽水、葡萄糖或其他輔助藥(D-山梨醇、D-甘露醇、氯化鈉等)之等張液之注射用水性液。本發明之醫藥組合物亦可進而含有例如緩衝劑(例如,磷酸鹽緩衝液、乙酸鈉緩衝液)、鎮痛劑(例如,氯化苄烷銨、鹽酸普魯卡因等)、穩定劑(例如,人類血清白蛋白、聚乙二醇等)、保存劑、抗氧化劑等。進而,本發明之醫藥組合物亦可利用冷凍乾燥法等製成固體製劑,該固體製劑可於使用時溶解於適當之水性溶劑中來使用。
本發明之醫藥組合物之代表性用途係針對冠狀病毒感染症之DNA疫苗製劑。就使作為疫苗之性能提昇、即在投予時使免疫應答活化之觀點而言,本發明之醫藥組合物中,除了本發明之核酸構建物以外,還可包含例如佐劑作為有效成分。作為傳統之佐劑,已知有氫氧化鋁、完全弗氏佐劑、不完全弗氏佐劑、百日咳菌佐劑等,還已知脂質體、作為雙鏈RNA之聚(I:C)、CpG寡核苷酸等亦具有佐劑效果。
於本發明之醫藥組合物中還可添加促進本發明之核酸構建物向細胞侵入之成分。作為上述成分,例如可使用作為公知之核酸導入促進劑之陽離子性聚合物、陽離子性脂質、陽離子性脂質體等。又,亦可將本發明之核酸構建物封入至脂質體(包含修飾脂質體)、病毒樣粒子(virus like particle;VLP)、或中空AAV(adeno-associated virus,腺相關病毒)衣殼粒子(例如國際公開第2012/144446號說明書)等。藉由與此種物質組合,亦能夠對本發明之醫藥組合物賦予對特定細胞或組織之指向性。例如,組入至具有感染人類細胞之能力之病毒載體中的本發明之核酸構建物能夠被該病毒載體所能感染之細胞主動地吸收。
若向人類投予本發明之醫藥組合物,則於人類體內由本發明之核酸構建物表現之刺突蛋白或其片段被識別為抗原。其結果,於人類中誘導針對SARS-CoV-2之免疫,從而達成該病毒之感染之預防及/或該病毒感染症之症狀之減輕。本發明之醫藥組合物之投予量及投予次數並無特別限定,根據所發揮之效果適宜調整即可。投予路徑亦無特別限定,但就發揮其效果之觀點而言,較佳為非經口投予。通常藉由注射點滴或其他方法向組織內(例如肌肉內)、靜脈內、皮內、皮下、腹腔內等投予。
以下例舉實施例進一步具體地說明本發明,但本發明並不僅限定於以下之實施例。
[實施例]
實施例1 編碼SARS-CoV-2刺突蛋白之DNA之製作
與Wuhan株之SARS-CoV-2之基因有關之資訊於基因庫中以NC_045512之登錄號公開。與SARS-CoV-2刺突蛋白之胺基酸序列(序列編號1)、以及編碼上述胺基酸序列之核酸序列(序列編號2)有關之資訊可自上述基因庫中取得。以化學方式合成與編碼刺突蛋白之病毒基因組RNA之鹼基序列對應之序列(序列編號2)之DNA。此時,於序列編號2之起始密碼子之5'端隔著5'-CACC-3'之4個鹼基附加有限制酶NheI之識別序列,於3'端與終止密碼子(TAA)相鄰地附加有限制酶XbaI之識別序列。
其次,設計出如下DNA,其係將序列編號2中之SARS-CoV-2刺突蛋白之開放閱讀框架之密碼子替換為適於在人類中表現的密碼子而成。藉由將除終止密碼子以外之全部1273個密碼子中之967個在不置換胺基酸之情況下替換為其他密碼子,而獲得序列編號3所表示之鹼基序列。繼而,以化學方式合成上述鹼基序列之DNA。於該DNA之起始密碼子之5'端隔著5'-CACC-3'之4個鹼基附加有限制酶NheI之識別序列,於3'端與終止密碼子(TAA)相鄰地附加有限制酶XbaI之識別序列。再者,上述5'-CACC-3'之序列與緊接在前面之NheI識別序列中之2個鹼基(GC)一起形成Kozak序列。
實施例2 刺突蛋白表現質體之製作
利用限制酶NheI及XbaI(均為TAKARA BIO公司製造)分別將實施例1中所製作之包含序列編號2之鹼基序列之DNA、以及包含序列編號3之鹼基序列之DNA進行雙酶切。將所獲得之DNA片段***至質體載體pVAX1(Thermo Fisher Scientific公司製造)之NheI及XbaI位點間,而製作重組質體。選擇連接有SARS-CoV-2刺突蛋白之開放閱讀框架之質體以適合藉由pVAX1之巨細胞病毒(CMV)啟動子進行轉錄、轉譯,將包含序列編號2之鹼基序列之DNA者命名為質體WT,將包含序列編號3之鹼基序列之DNA者命名為質體CO。再者,於該等質體中,於開放閱讀框架之5'端(CMV啟動子之下游)具有作為搭載於pVAX1之要素之T7啟動子,於開放閱讀框架之3'端具有來自牛生長激素因子基因之多聚腺苷酸化序列。將質體CO之全鹼基序列表示為序列編號4。
將分別經質體WT或質體CO轉形之大腸桿菌HST08株(TAKARA BIO公司製造)於含有康黴素之液體培養基中進行培養。使用NucleoBond XtraMidi(MACHEREY-NAGEL公司製造),自所獲得之大腸桿菌菌體中純化質體,並用於以下之實驗。
實施例3 mRNA定量用引子之製作
製作用於定量於分別導入有質體WT或質體CO之細胞內轉錄之SARS-CoV-2刺突蛋白之mRNA的引子對。關於由質體WT轉錄之mRNA,合成CoV-S1-WT-Q1_F(序列編號5)及CoV-S1-WT-Q1_R(序列編號6)之對(以下,記載為引子對WT-1)。又,關於由質體CO轉錄之mRNA,合成CoV-S1-CO-Q3_F(序列編號7)及CoV-S1-CO-Q3_R(序列編號8)之對(以下,記載為引子對CO-3)。進而,合成針對作為內部標準進行測定之人類GAPDH基因mRNA之引子GAPDH_F(序列編號9)及GAPDH_R(序列編號10)之對(以下,記載為引子對GAPDH)。
實施例4 自重組質體之mRNA轉錄之確認
將1.6×10
5個HEK293T細胞接種至加入有含有10%胎牛血清(FBS)及1%青黴素-鏈黴素之DMEM(Dulbecco's Modified Eagle Medium,杜爾貝科改良伊格爾培養基)之細胞培養用板之孔中。繼而,使用TransIt-293(Mirus公司製造),利用300 ng之質體WT、質體CO、或pVAX1將上述細胞進行轉形。亦準備未經質體轉形之細胞(mock)。將該等細胞於37℃、5%CO
2之條件下進行培養。
使用NucleoSpin RNA plus(MACHEREY-NAGEL公司製造),自培養第2天之細胞中純化RNA後,藉由使用實施例3中所製作之引子對之RT-PCR,定量於細胞內轉錄之SARS-CoV-2刺突蛋白之mRNA。RT-PCR中,將PrimeScript RT Master Mix(TAKARA BIO公司製造)及TB Green Premix Ex Taq II(Tli RNaseH Plus)(TAKARA BIO公司製造)按照其等之使用說明書加以使用。表1中示出以來自各細胞之RNA及各引子組之組合所測得之Ct值。
如表1所示,僅在質體WT與引子對WT-1之組合、質體CO與引子對CO-3之組合時檢測出顯著量之刺突蛋白mRNA。又,利用ΔΔCt法對經質體WT轉形之細胞與經質體CO轉形之細胞的mRNA轉錄量進行比較,結果為後者高出46倍。
[表1]
質體 | 引子對 | ||
WT-1 | CO-3 | GAPDH | |
pVAX1 | 34.285 | 未檢測出 | 17.215 |
WT | 19.165 | 37.41 | 17.015 |
CO | 32.72 | 13.84 | 17.22 |
mock | 34.5 | 未檢測出 | 16.82 |
實施例5 刺突蛋白表現之確認(1)
藉由與實施例4同樣之操作,製備經質體WT、質體CO、或pVAX1轉形之HEK293T細胞。再者,經質體WT轉形之細胞係製備2組。將培養第2天之細胞供於利用流式細胞儀(BD FACS CantoII;BD生物科學公司製造)之分析,算出SARS-CoV-2刺突蛋白陽性率。刺突蛋白係將小鼠抗刺突單株抗體(抗-SARS-CoV-2刺突,小鼠-單株抗體(1A9);GeneTex公司製造)與PE標記抗小鼠IgG抗體(小鼠免疫球蛋白/RPE;Agilent公司製造)組合而檢測出。
將各細胞中之刺突蛋白陽性細胞之比率示於表2。於經質體WT轉形之細胞中,刺突蛋白陽性細胞之比率與背景(導入有pVAX1之細胞中之比率)為相同程度,但關於質體CO,於超過40%之細胞中確認到刺突蛋白之表現。
[表2]
質體 | 陽性率 |
pVAX1 | 0.2% |
WT(1) | 0.1% |
WT(2) | 0.2% |
CO | 40.6% |
實施例6 刺突蛋白表現之確認(2)
針對實施例2中所製作之質體CO以及新製備之3批次之質體CO(分別為<1>、<2>、<3>),使用HEK293T細胞來驗證表現刺突蛋白之能力。
除於條件A(於1.6×10
5個HEK293T細胞中添加200 ng之質體)、條件B(於0.8×10
5個HEK293T細胞中添加500 ng之質體)這2個條件下進行轉形以外,利用與實施例5同樣之操作實施細胞之製備以及刺突蛋白之表現測定。將各細胞中之刺突蛋白陽性細胞之比率及PE之平均螢光強度示於表3。又,將於條件B下經pVAX1、質體CO<1>、<2>、<3>轉形所獲得之細胞中之刺突蛋白表現量之柱狀圖分別示於圖1~4。由此可以明確,質體CO不受其製造批次影響,於所導入之細胞中均成功穩定地誘導刺突蛋白之表現。又,刺突蛋白表現之陽性率及PE之平均螢光強度均與所使用之質體量之間存在關聯。
[表3]
質體 | 條件A | 條件B | |||
陽性率 | 平均螢光強度 | 陽性率 | 平均螢光強度 | ||
pVAX1 | 0.1% | - | 0.1% | ||
CO(實施例2) | 44.8% | 8051 | 未測試 | 未測試 | |
CO<1> | 37.7% | 7670 | 77.5% | 10979 | |
CO<2> | 36.5% | 7411 | 72.8% | 10792 | |
CO<3> | 39.7% | 7426 | 73.8% | 11157 |
實施例7 利用重組質體之免疫誘導之確認(1)
針對實施例2中所製作之質體CO,評價有佐劑或無佐劑時之免疫誘導能力。
準備2組SD大鼠(自CLEA Japan股份有限公司購入),每組6隻,以每隻投予666.6 μg/400 μL之質體CO、或每隻投予666.6 μg/200 μL之質體CO及200 μL之ALUM佐劑(InVivoGen公司製造)的方式分別投予至各組之大鼠之前脛骨肌肉(左右)。大鼠係於能夠自由地攝取餌料及水之環境下飼養。又,作為對照組準備3隻大鼠,不進行處置而直接於相同條件下飼養。於投予2週後,自大鼠之尾靜脈採集血液,製備血清。利用以下所示之操作測定血清中之抗刺突蛋白抗體。
將塗敷有重組2019-nCoV刺突S1+S2蛋白(ECD,His tag;Beta Lifescience公司製造)之96孔板利用阻斷溶液[含有5%脫脂乳之PBS-T(PBS-T係包含0.05% Tween20之磷酸緩衝生理鹽水)]阻斷後,添加利用阻斷溶液稀釋8倍後之血清並於4℃下靜置一晩。第二天,將孔洗淨,添加HRP(horse radish peroxidase,辣根過氧化酶)標記抗大鼠抗體(GH Healthcare公司製造)並於室溫下靜置3小時。利用PBS-T將孔洗淨後,向孔中添加3,3'-5,5'-四甲基聯苯胺(Sigma-Aldrich公司製造)並於室溫下靜置30分鐘後,向孔中添加0.5 N之硫酸而使顯色反應終止。針對各孔,測定450 nm之吸光度,評價血清中之抗刺突蛋白抗體。
將結果示於圖5。於質體CO投予組中,無論有無ALUM佐劑均產生與刺突蛋白結合之抗體,但於併用佐劑之組中抗體效價呈變高趨勢。
實施例8 利用重組質體之免疫誘導之確認(2)
對於6隻SD大鼠,每隻投予666.6 μg之質體CO及66.7 μl之ALUM佐劑。實施3次投予,每次間隔2週,在投予開始後第4週、第6週、第8週採集血液,實施血清中之抗刺突蛋白抗體量之測定。關於抗體之測定,除增加了使用重組2019-nCoV刺突蛋白(RBD;Beta Lifescience公司製造)作為抗原之試驗以外,利用與實施例7同樣之操作進行。其結果,可明確與S1+S2結合之抗體、及與RBD(受體結合區)結合之抗體均於大鼠內產生,兩抗體於投予開始第4週之血清中已檢測出,且於第4週至第8週可見抗體效價之上升。
進而,利用塗敷有重組2019-nCoV刺突(S1-D614G;Sino Biological公司製造)之板實施同樣之測定,藉此亦確認到藉由質體CO所產生之抗體識別具有D614G突變之刺突蛋白。
實施例9 細胞性免疫誘導之確認
使用T細胞ELISPOT套組(INF-γ及IL-4;U-CyTech生物科學公司製造),依據該套組之說明書所記載之操作來調查質體CO對細胞性免疫之效果。
於底部具備PVDF(Polyvinylidene fluoride,聚偏二氟乙烯)膜之96孔板(Millipore公司製造)上塗敷有套組所附帶之抗IFNγ捕捉抗體或抗IL4捕捉抗體。利用阻斷溶液將板洗淨。將藉由慣例自經質體CO免疫之大鼠製備之脾臟細胞3×10
5個與重組2019-nCoV刺突S1+S2蛋白或重組2019-nCoV刺突蛋白(RBD)一起接種至上述板之孔,於37℃下保溫48小時。於保溫結束後,利用PBS-T將孔洗淨,添加生物素化抗大鼠IFNγ抗體或生物素化抗大鼠IL4抗體並於4℃下放置2小時。其次,添加HRP標記卵白素並靜置1小時,進而添加HRP受質溶液而檢測HRP之活性。其結果,表明因利用S1+S2之刺激或利用RBD之刺激各者,而使得自已免疫之大鼠之脾臟細胞之干擾素-γ(INFγ)之產生顯著增加,又,介白素-4(IL4)之產生亦少許增加。根據以上之結果,可明確質體CO誘導Th1型之細胞性免疫。
實施例10 毒性之評價
自經質體CO免疫後第7週之大鼠採集肺、肝臟、腎臟及心臟之組織,製作經HE(hematoxylin-eosin,蘇木精-伊紅)染色之組織切片標本。於顯微鏡下進行觀察,結果未確認到顯示毒性之跡象。又,根據該大鼠之血清中之各種生物化學標記測定,亦未獲得脫離正常範圍之值。
實施例11 搭載有編碼經修飾之刺突蛋白之DNA之質體之製作
針對由世界衛生組織(WHO)報告之各種SARS-CoV-2突變株(variant),驗證其刺突蛋白之胺基酸序列。自其中選擇B.1.1.7系統、B.1.351系統、P.1系統、B.1.617.2系統(分別被賦予Alpha、Beta、Gamma、Delta之WHO標籤),設計出將各者中所確認到之幾種突變導入至野生型刺突蛋白之胺基酸序列(序列編號1)中而成之4種胺基酸序列。各胺基酸序列(分別命名為4GP、SA、BR、IN)中,除了來自各系統之突變以外,亦包含已知有助於刺突蛋白之高表現之K986P/V987P突變以及C末端之19個胺基酸之缺失。繼而,設計出編碼該等胺基酸序列且適於在人類中表現之DNA鹼基序列。
表4中示出新設計出之胺基酸序列之資訊、各胺基酸序列及編碼各胺基酸序列之DNA鹼基序列之序列編號。再者,表4中之胺基酸之位置係基於序列編號1所表示之胺基酸序列。於編碼該等DNA中之上述修飾部分以外之鹼基序列與編碼野生型SARS-CoV-2之刺突蛋白之基因組RNA之序列相比之情形時,密碼子之70%以上被替換為適於在人類中表現者。
[表4]
名稱 | 導入至序列編號1之修飾 | 序列編號 | |
胺基酸序列 | DNA鹼基序列 | ||
4GP | 胺基酸置換:D614G、K986P及V987P 缺失:C末端19個胺基酸 | 11 | 12 |
SA | 胺基酸置換:L18F、D80A、D215G、R246I、K417N、E484K、N501Y、D614G、A701V、K986P及V987P 缺失:C末端19個胺基酸 | 13 | 14 |
BR | 胺基酸置換:L18F、T20N、P26S、D138Y、R190S、K417T、E484K、N501Y、D614G、H655Y、K986P、V987P、T1027I及V1176F 缺失:C末端19個胺基酸 | 15 | 16 |
IN | 胺基酸置換:T19R、G142D、E156G、L452R、T478K、D614G、P681R、D950N、K986P及V987P 缺失:F157、R158及C末端19個胺基酸 | 17 | 18 |
以化學方式合成序列編號12、14、16、18之鹼基序列之DNA。繼而,用該等DNA對搭載於質體CO之SARS-CoV-2刺突蛋白之開放閱讀框架區進行置換,藉此製作能夠表現修飾刺突蛋白之質體。將所獲得之質體分別命名為質體4GP、質體SA、質體BR、質體IN。利用各質體將大腸桿菌HST08株進行轉形,並於含有康黴素之液體培養基中進行培養。使用NucleoBond XtraMidi(MACHEREY-NAGEL公司製造),自所獲得之大腸桿菌菌體中純化質體,並用於以下之實驗。
實施例12 修飾刺突蛋白表現之確認
藉由與實施例4同樣之操作,分別利用質體CO以及質體4GP、SA、BR、及IN將HEK293T細胞進行轉形,並進行培養。將培養第2天之細胞供於利用流式細胞儀(BD FACS CantoII)之分析,調查SARS-CoV-2刺突蛋白之表現(分別4組)。於刺突蛋白之檢測中,將抗S1抗體:SARS-CoV-2(2019-nCoV)刺突S1抗體(兔單抗;SinoBiological公司製造)、及抗S2抗體:抗-SARS-CoV-2刺突、小鼠-單株抗體(1A9)分別與抗兔二次抗體、及抗小鼠二次抗體(均為經螢光標記者)組合而使用。
使用抗S1抗體及抗S2抗體來分析經各質體轉形之細胞中之刺突蛋白陽性細胞,將所得之結果分別示於圖6及圖7。圖中,橫軸表示刺突蛋白陽性細胞相對於全部細胞之比率,縱軸表示試驗中所測得之來自標記之螢光之平均螢光強度,CO、4GP、SA、BR及IN分別表示質體CO、4GP、SA、BR及IN投予組。如圖6及7所示,於以抗S1抗體實施檢測之情形與以抗S2抗體實施檢測之情形時,結果存在偏差,但綜合來看,認為經質體4GP、SA、BR、IN轉形之細胞中之刺突蛋白表現量不會低於經質體CO轉形者。
實施例13 利用修飾刺突蛋白之免疫誘導之確認
針對質體4GP,藉由實施例7中所記載之方法來評價免疫誘導。表明於質體4GP投予組之大鼠中,投予2週後之抗刺突蛋白抗體之產生與質體CO投予組相比較高。進而,繼續飼養並調查抗體產生之狀況,結果於兩質體間未確認到抗體產生量之差。可認為其原因在於產生量飽和。
實施例14 藉由修飾刺突蛋白所產生之抗體之分析
使用質體CO及質體SA來評價免疫誘導能力。準備3組SD大鼠(自CLEA Japan股份有限公司購入),每組3~8隻。製備將666.6 μg/333.3 μL之pVAX(對照組)、質體CO、或質體SA、與ALUM佐劑(InVivogen公司製造)66.7 μL混合而成者,對各組之大鼠每隻投予400 μL(對大鼠之左右之前脛骨肌肉分別投予各200 μL)。大鼠係於能夠自由地攝取餌料及水之環境下飼養。又,作為未處置組準備3隻大鼠,不投予質體而直接於相同條件下飼養。於投予2週後自大鼠之尾靜脈採集血液,製備血清。利用以下所示之操作測定血清中之抗刺突蛋白抗體。
測定上述4種血清中所含之抗體對於各種SARS-CoV-2刺突蛋白之抗體效價。作為刺突蛋白,使用具有來自Wuhan株、Alpha株、Beta株、及Gamma株之胺基酸序列之刺突蛋白(均為ACRO Biosystems公司製造;附有His-Tag)。將塗敷有上述刺突蛋白之96孔板利用阻斷溶液[含有5%脫脂乳之PBS-T]阻斷後,添加利用阻斷溶液自50倍開始連續稀釋後所得之血清並於4℃下靜置一晩。第二天,將孔洗淨,添加HRP標記抗大鼠抗體(GH Healthcare公司製造)並於室溫下靜置3小時。利用PBS-T將孔洗淨後,向孔中添加3,3'-5,5'-四甲基聯苯胺(Sigma-Aldrich公司製造)並於室溫下靜置30分鐘後,向孔中添加0.5 N之硫酸而使顯色反應終止。針對各孔,測定450 nm之吸光度,評價血清中之抗刺突蛋白抗體。
將結果示於圖8。圖中,橫軸表示投予至製備血清之大鼠之質體之種類(AG0302意指質體CO,AG0304意指質體SA,Normal意指質體未投予組)。又,圖中表示刺突蛋白之種類之Wuhan、UK、SA、BR分別意指Wuhan株、Alpha株、Beta株、Gamma株之刺突蛋白。如圖8所示,於投予有質體CO及質體SA之大鼠之血清中,產生了可與所使用之全部刺突蛋白反應之抗體。又,觀察到投予有質體SA之大鼠中所產生之抗體與藉由質體CO所產生之抗體相比,呈如下傾向,即對於Wuhan株及Alpha株之刺突蛋白顯示同等或略高之抗體效價,並且對於Beta株及Gamma株之刺突蛋白顯示更高之反應性。
[序列表非關鍵文字]
SEQ ID NO: 1:SARS-CoV-2刺突蛋白之胺基酸序列
SEQ ID NO: 2:對應於編碼SARS-CoV-2刺突蛋白之野生型RNA之核苷酸序列
SEQ ID NO: 3:編碼SARS-CoV-2刺突蛋白之密碼子修飾DNA序列
SEQ ID NO: 4:質體CO之全長核苷酸序列
SEQ ID NO: 5:引子CoV-S1-WT-Q1_F
SEQ ID NO: 6:引子CoV-S1-WT-Q1_R
SEQ ID NO: 7:引子CoV-S1-CO-Q3_F
SEQ ID NO: 8:引子CoV-S1-CO-Q3_R
SEQ ID NO: 9:引子GAPDH_F
SEQ ID NO: 10:引子GAPDH_R
SEQ ID NO: 11:修飾SARS-CoV-2刺突蛋白之胺基酸序列(4GP)
SEQ ID NO: 12:編碼修飾SARS-CoV-2刺突蛋白之核苷酸序列(4GP)
SEQ ID NO: 13:修飾SARS-CoV-2刺突蛋白之胺基酸序列(SA)
SEQ ID NO: 14:編碼修飾SARS-CoV-2刺突蛋白之核苷酸序列(SA)
SEQ ID NO: 15:修飾SARS-CoV-2刺突蛋白之胺基酸序列(BR)
SEQ ID NO: 16:編碼修飾SARS-CoV-2刺突蛋白之核苷酸序列(BR)
SEQ ID NO: 17:修飾SARS-CoV-2刺突蛋白之胺基酸序列(IN)
SEQ ID NO: 18:編碼修飾SARS-CoV-2刺突蛋白之核苷酸序列(IN)
圖1係表示對照之細胞中的刺突蛋白之表現之圖。
圖2係表示導入有本發明之核酸構建物之細胞中的刺突蛋白之表現之圖。
圖3係表示導入有本發明之核酸構建物之細胞中的刺突蛋白之表現之圖。
圖4係表示導入有本發明之核酸構建物之細胞中的刺突蛋白之表現之圖。
圖5係表示投予有本發明之核酸構建物之大鼠中的抗刺突蛋白抗體之產生之圖。
圖6係表示導入有本發明之核酸構建物之細胞中的刺突蛋白陽性率及螢光強度之圖。
圖7係表示導入有本發明之核酸構建物之細胞中的刺突蛋白陽性率及螢光強度之圖。
圖8係表示投予有本發明之核酸構建物之大鼠中所產生的抗體與刺突蛋白之反應之圖。
<![CDATA[<110> 國立大學法人大阪大學(OSAKA UNIVERSITY)]]> 日商寶生物股份有限公司(TAKARA BIO INC.) 日商安增生股份有限公司(AnGes, Inc.) <![CDATA[<120> 針對SARS-CoV-2之改良型DNA疫苗]]> <![CDATA[<130> 676423]]> <![CDATA[<150> JP2020-167854]]> <![CDATA[<151> 2020-10-02]]> <![CDATA[<160> 18 ]]> <![CDATA[<170> PatentIn第3.5版]]> <![CDATA[<210> 1]]> <![CDATA[<211> 1273]]> <![CDATA[<212> PRT]]> <![CDATA[<213> SARS-CoV-2]]> <![CDATA[<400> 1]]> Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln Cys Val 1 5 10 15 Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr Asn Ser Phe 20 25 30 Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser Ser Val Leu 35 40 45 His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn Val Thr Trp 50 55 60 Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys Arg Phe Asp 65 70 75 80 Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala Ser Thr Glu 85 90 95 Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr Leu Asp Ser 100 105 110 Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn Val Val Ile 115 120 125 Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu Gly Val Tyr 130 135 140 Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe Arg Val Tyr 145 150 155 160 Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln Pro Phe Leu 165 170 175 Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu Arg Glu Phe 180 185 190 Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser Lys His Thr 195 200 205 Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe Ser Ala Leu Glu 210 215 220 Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg Phe Gln Thr 225 230 235 240 Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp Ser Ser Ser 245 250 255 Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr Leu Gln Pro 260 265 270 Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp Ala 275 280 285 Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu Lys 290 295 300 Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val 305 310 315 320 Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys 325 330 335 Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala 340 345 350 Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu 355 360 365 Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro 370 375 380 Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe 385 390 395 400 Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly 405 410 415 Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys 420 425 430 Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn 435 440 445 Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe 450 455 460 Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys 465 470 475 480 Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly 485 490 495 Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val 500 505 510 Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys 515 520 525 Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn Phe Asn 530 535 540 Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys Phe Leu 545 550 555 560 Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp Ala Val 565 570 575 Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys Ser Phe 580 585 590 Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr Ser Asn Gln Val 595 600 605 Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Glu Val Pro Val Ala Ile 610 615 620 His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr Ser Thr Gly Ser 625 630 635 640 Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly Ala Glu His Val 645 650 655 Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly Ile Cys Ala 660 665 670 Ser Tyr Gln Thr Gln Thr Asn Ser Pro Arg Arg Ala Arg Ser Val Ala 675 680 685 Ser Gln Ser Ile Ile Ala Tyr Thr Met Ser Leu Gly Ala Glu Asn Ser 690 695 700 Val Ala Tyr Ser Asn Asn Ser Ile Ala Ile Pro Thr Asn Phe Thr Ile 705 710 715 720 Ser Val Thr Thr Glu Ile Leu Pro Val Ser Met Thr Lys Thr Ser Val 725 730 735 Asp Cys Thr Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys Ser Asn Leu 740 745 750 Leu Leu Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg Ala Leu Thr 755 760 765 Gly Ile Ala Val Glu Gln Asp Lys Asn Thr Gln Glu Val Phe Ala Gln 770 775 780 Val Lys Gln Ile Tyr Lys Thr Pro Pro Ile Lys Asp Phe Gly Gly Phe 785 790 795 800 Asn Phe Ser Gln Ile Leu Pro Asp Pro Ser Lys Pro Ser Lys Arg Ser 805 810 815 Phe Ile Glu Asp Leu Leu Phe Asn Lys Val Thr Leu Ala Asp Ala Gly 820 825 830 Phe Ile Lys Gln Tyr Gly Asp Cys Leu Gly Asp Ile Ala Ala Arg Asp 835 840 845 Leu Ile Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu Pro Pro Leu 850 855 860 Leu Thr Asp Glu Met Ile Ala Gln Tyr Thr Ser Ala Leu Leu Ala Gly 865 870 875 880 Thr Ile Thr Ser Gly Trp Thr Phe Gly Ala Gly Ala Ala Leu Gln Ile 885 890 895 Pro Phe Ala Met Gln Met Ala Tyr Arg Phe Asn Gly Ile Gly Val Thr 900 905 910 Gln Asn Val Leu Tyr Glu Asn Gln Lys Leu Ile Ala Asn Gln Phe Asn 915 920 925 Ser Ala Ile Gly Lys Ile Gln Asp Ser Leu Ser Ser Thr Ala Ser Ala 930 935 940 Leu Gly Lys Leu Gln Asp Val Val Asn Gln Asn Ala Gln Ala Leu Asn 945 950 955 960 Thr Leu Val Lys Gln Leu Ser Ser Asn Phe Gly Ala Ile Ser Ser Val 965 970 975 Leu Asn Asp Ile Leu Ser Arg Leu Asp Lys Val Glu Ala Glu Val Gln 980 985 990 Ile Asp Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln Thr Tyr Val 995 1000 1005 Thr Gln Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala Ser Ala Asn 1010 1015 1020 Leu Ala Ala Thr Lys Met Ser Glu Cys Val Leu Gly Gln Ser Lys 1025 1030 1035 Arg Val Asp Phe Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro 1040 1045 1050 Gln Ser Ala Pro His Gly Val Val Phe Leu His Val Thr Tyr Val 1055 1060 1065 Pro Ala Gln Glu Lys Asn Phe Thr Thr Ala Pro Ala Ile Cys His 1070 1075 1080 Asp Gly Lys Ala His Phe Pro Arg Glu Gly Val Phe Val Ser Asn 1085 1090 1095 Gly Thr His Trp Phe Val Thr Gln Arg Asn Phe Tyr Glu Pro Gln 1100 1105 1110 Ile Ile Thr Thr Asp Asn Thr Phe Val Ser Gly Asn Cys Asp Val 1115 1120 1125 Val Ile Gly Ile Val Asn Asn Thr Val Tyr Asp Pro Leu Gln Pro 1130 1135 1140 Glu Leu Asp Ser Phe Lys Glu Glu Leu Asp Lys Tyr Phe Lys Asn 1145 1150 1155 His Thr Ser Pro Asp Val Asp Leu Gly Asp Ile Ser Gly Ile Asn 1160 1165 1170 Ala Ser Val Val Asn Ile Gln Lys Glu Ile Asp Arg Leu Asn Glu 1175 1180 1185 Val Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu Gln Glu Leu 1190 1195 1200 Gly Lys Tyr Glu Gln Tyr Ile Lys Trp Pro Trp Tyr Ile Trp Leu 1205 1210 1215 Gly Phe Ile Ala Gly Leu Ile Ala Ile Val Met Val Thr Ile Met 1220 1225 1230 Leu Cys Cys Met Thr Ser Cys Cys Ser Cys Leu Lys Gly Cys Cys 1235 1240 1245 Ser Cys Gly Ser Cys Cys Lys Phe Asp Glu Asp Asp Ser Glu Pro 1250 1255 1260 Val Leu Lys Gly Val Lys Leu His Tyr Thr 1265 1270 <![CDATA[<210> 2]]> <![CDATA[<211> 3822]]> <![CDATA[<212> DNA]]> <![CDATA[<213> SARS-CoV-2]]> <![CDATA[<400> 2]]> atgtttgttt ttcttgtttt attgccacta gtctctagtc agtgtgttaa tcttacaacc 60 agaactcaat taccccctgc atacactaat tctttcacac gtggtgttta ttaccctgac 120 aaagttttca gatcctcagt tttacattca actcaggact tgttcttacc tttcttttcc 180 aatgttactt ggttccatgc tatacatgtc tctgggacca atggtactaa gaggtttgat 240 aaccctgtcc taccatttaa tgatggtgtt tattttgctt ccactgagaa gtctaacata 300 ataagaggct ggatttttgg tactacttta gattcgaaga cccagtccct acttattgtt 360 aataacgcta ctaatgttgt tattaaagtc tgtgaatttc aattttgtaa tgatccattt 420 ttgggtgttt attaccacaa aaacaacaaa agttggatgg aaagtgagtt cagagtttat 480 tctagtgcga ataattgcac ttttgaatat gtctctcagc cttttcttat ggaccttgaa 540 ggaaaacagg gtaatttcaa aaatcttagg gaatttgtgt ttaagaatat tgatggttat 600 tttaaaatat attctaagca cacgcctatt aatttagtgc gtgatctccc tcagggtttt 660 tcggctttag aaccattggt agatttgcca ataggtatta acatcactag gtttcaaact 720 ttacttgctt tacatagaag ttatttgact cctggtgatt cttcttcagg ttggacagct 780 ggtgctgcag cttattatgt gggttatctt caacctagga cttttctatt aaaatataat 840 gaaaatggaa ccattacaga tgctgtagac tgtgcacttg accctctctc agaaacaaag 900 tgtacgttga aatccttcac tgtagaaaaa ggaatctatc aaacttctaa ctttagagtc 960 caaccaacag aatctattgt tagatttcct aatattacaa acttgtgccc ttttggtgaa 1020 gtttttaacg ccaccagatt tgcatctgtt tatgcttgga acaggaagag aatcagcaac 1080 tgtgttgctg attattctgt cctatataat tccgcatcat tttccacttt taagtgttat 1140 ggagtgtctc ctactaaatt aaatgatctc tgctttacta atgtctatgc agattcattt 1200 gtaattagag gtgatgaagt cagacaaatc gctccagggc aaactggaaa gattgctgat 1260 tataattata aattaccaga tgattttaca ggctgcgtta tagcttggaa ttctaacaat 1320 cttgattcta aggttggtgg taattataat tacctgtata gattgtttag gaagtctaat 1380 ctcaaacctt ttgagagaga tatttcaact gaaatctatc aggccggtag cacaccttgt 1440 aatggtgttg aaggttttaa ttgttacttt cctttacaat catatggttt ccaacccact 1500 aatggtgttg gttaccaacc atacagagta gtagtacttt cttttgaact tctacatgca 1560 ccagcaactg tttgtggacc taaaaagtct actaatttgg ttaaaaacaa atgtgtcaat 1620 ttcaacttca atggtttaac aggcacaggt gttcttactg agtctaacaa aaagtttctg 1680 cctttccaac aatttggcag agacattgct gacactactg atgctgtccg tgatccacag 1740 acacttgaga ttcttgacat tacaccatgt tcttttggtg gtgtcagtgt tataacacca 1800 ggaacaaata cttctaacca ggttgctgtt ctttatcagg atgttaactg cacagaagtc 1860 cctgttgcta ttcatgcaga tcaacttact cctacttggc gtgtttattc tacaggttct 1920 aatgtttttc aaacacgtgc aggctgttta ataggggctg aacatgtcaa caactcatat 1980 gagtgtgaca tacccattgg tgcaggtata tgcgctagtt atcagactca gactaattct 2040 cctcggcggg cacgtagtgt agctagtcaa tccatcattg cctacactat gtcacttggt 2100 gcagaaaatt cagttgctta ctctaataac tctattgcca tacccacaaa ttttactatt 2160 agtgttacca cagaaattct accagtgtct atgaccaaga catcagtaga ttgtacaatg 2220 tacatttgtg gtgattcaac tgaatgcagc aatcttttgt tgcaatatgg cagtttttgt 2280 acacaattaa accgtgcttt aactggaata gctgttgaac aagacaaaaa cacccaagaa 2340 gtttttgcac aagtcaaaca aatttacaaa acaccaccaa ttaaagattt tggtggtttt 2400 aatttttcac aaatattacc agatccatca aaaccaagca agaggtcatt tattgaagat 2460 ctacttttca acaaagtgac acttgcagat gctggcttca tcaaacaata tggtgattgc 2520 cttggtgata ttgctgctag agacctcatt tgtgcacaaa agtttaacgg ccttactgtt 2580 ttgccacctt tgctcacaga tgaaatgatt gctcaataca cttctgcact gttagcgggt 2640 acaatcactt ctggttggac ctttggtgca ggtgctgcat tacaaatacc atttgctatg 2700 caaatggctt ataggtttaa tggtattgga gttacacaga atgttctcta tgagaaccaa 2760 aaattgattg ccaaccaatt taatagtgct attggcaaaa ttcaagactc actttcttcc 2820 acagcaagtg cacttggaaa acttcaagat gtggtcaacc aaaatgcaca agctttaaac 2880 acgcttgtta aacaacttag ctccaatttt ggtgcaattt caagtgtttt aaatgatatc 2940 ctttcacgtc ttgacaaagt tgaggctgaa gtgcaaattg ataggttgat cacaggcaga 3000 cttcaaagtt tgcagacata tgtgactcaa caattaatta gagctgcaga aatcagagct 3060 tctgctaatc ttgctgctac taaaatgtca gagtgtgtac ttggacaatc aaaaagagtt 3120 gatttttgtg gaaagggcta tcatcttatg tccttccctc agtcagcacc tcatggtgta 3180 gtcttcttgc atgtgactta tgtccctgca caagaaaaga acttcacaac tgctcctgcc 3240 atttgtcatg atggaaaagc acactttcct cgtgaaggtg tctttgtttc aaatggcaca 3300 cactggtttg taacacaaag gaatttttat gaaccacaaa tcattactac agacaacaca 3360 tttgtgtctg gtaactgtga tgttgtaata ggaattgtca acaacacagt ttatgatcct 3420 ttgcaacctg aattagactc attcaaggag gagttagata aatattttaa gaatcataca 3480 tcaccagatg ttgatttagg tgacatctct ggcattaatg cttcagttgt aaacattcaa 3540 aaagaaattg accgcctcaa tgaggttgcc aagaatttaa atgaatctct catcgatctc 3600 caagaacttg gaaagtatga gcagtatata aaatggccat ggtacatttg gctaggtttt 3660 atagctggct tgattgccat agtaatggtg acaattatgc tttgctgtat gaccagttgc 3720 tgtagttgtc tcaagggctg ttgttcttgt ggatcctgct gcaaatttga tgaagacgac 3780 tctgagccag tgctcaaagg agtcaaatta cattacacat aa 3822 <![CDATA[<210> 3]]> <![CDATA[<211> 3822]]> <![CDATA[<212> DNA]]> <![CDATA[<213> 人工]]> <![CDATA[<220>]]> <![CDATA[<223> 編碼SARS-CoV-2刺突蛋白之密碼子修飾DNA序列]]> <![CDATA[<400> 3]]> atgttcgtgt tcctggtgct gctgcccctg gtgagcagcc agtgcgtgaa cctgaccacc 60 agaacccagc tgccccccgc ctacaccaac agcttcacca gaggcgtgta ctaccccgac 120 aaggtgttca gaagcagcgt gctgcacagc acccaggacc tgttcctgcc cttcttcagc 180 aacgtgacct ggttccacgc catccacgtg agcggcacca acggcaccaa gagattcgac 240 aaccccgtgc tgcccttcaa cgacggcgtg tacttcgcca gcaccgagaa gagcaacatc 300 atcagaggct ggatcttcgg caccaccctg gacagcaaga cccagagcct gctgatcgtg 360 aacaacgcca ccaacgtggt gatcaaggtg tgcgagttcc agttctgcaa cgaccccttc 420 ctgggcgtgt actaccacaa gaacaacaag agctggatgg agagcgagtt cagagtgtac 480 agcagcgcca acaactgcac cttcgagtac gtgagccagc ccttcctgat ggacctggag 540 ggcaagcagg gcaacttcaa gaacctgaga gagttcgtgt tcaagaacat cgacggctac 600 ttcaagatct acagcaagca cacccccatc aacctggtga gagacctgcc tcagggcttt 660 agcgccctgg agccactggt ggacctgcca atcggcatca acatcaccag attccagacc 720 ctgctggccc tgcacagaag ctacctgaca ccaggcgatt ctagctctgg atggacagcc 780 ggcgccgctg cctattacgt gggctacctg cagcctagaa ccttcctgct gaagtacaac 840 gagaacggca ccatcaccga tgccgtggac tgcgccctgg atcccctgag cgagaccaag 900 tgtaccctga agagcttcac cgtggagaag ggcatctacc agaccagcaa cttcagagtg 960 cagcccaccg agagcatcgt gagattcccc aacatcacca acctgtgccc cttcggcgag 1020 gtgttcaacg ccaccagatt cgccagcgtg tacgcctgga acagaaagag aatcagcaac 1080 tgcgtggccg actacagcgt gctgtacaac agcgccagct tcagcacctt caagtgctac 1140 ggcgtgagcc ccaccaagct gaacgacctg tgcttcacca acgtgtacgc cgacagcttc 1200 gtgatcagag gcgacgaggt gagacagatt gcccctggcc agaccggcaa gatcgccgac 1260 tacaactaca agctgcccga cgacttcacc ggctgcgtga tcgcctggaa cagcaacaac 1320 ctggacagca aggtgggcgg caactacaac tacctgtaca gactgttcag aaagagcaac 1380 ctgaagccct tcgagagaga catcagcacc gagatctacc aggccggctc taccccatgc 1440 aatggcgtgg agggcttcaa ttgctacttc cccctgcaga gctacggctt ccagcccacc 1500 aacggcgtgg gctaccagcc ctacagagtg gtggtgctga gctttgaact gctgcacgcc 1560 cctgccaccg tgtgcggccc aaagaagagc accaatctgg tgaagaacaa gtgcgtgaac 1620 ttcaacttca acggcctgac cggcaccggc gtgctgaccg agagcaacaa gaagttcctg 1680 cccttccagc agttcggcag agacatcgcc gacaccaccg atgccgtgag agatccccag 1740 accctggaga tcctggacat caccccctgt agctttggcg gcgtgagcgt gattaccccc 1800 ggcaccaata ccagcaacca ggtggccgtg ctgtaccagg acgtgaactg caccgaggtg 1860 ccagtggcca tccatgccga ccagctgacc ccaacctgga gagtgtacag caccggcagc 1920 aacgtgttcc agacaagagc cggctgtctg attggcgccg agcacgtgaa taacagctac 1980 gagtgcgata tcccaatcgg cgccggcatc tgtgccagct atcagaccca gaccaatagc 2040 cccagaagag ccagaagcgt ggccagccag agcatcatcg cctacaccat gagcctgggc 2100 gccgagaaca gcgtggccta cagcaacaac agcatcgcca tccccaccaa cttcaccatc 2160 agcgtgacca ccgagatcct gcccgtgagc atgaccaaga ccagcgtgga ctgcaccatg 2220 tacatctgcg gcgacagcac cgagtgcagc aacctgctgc tgcagtacgg cagcttctgc 2280 acccagctga acagagccct gaccggcatc gccgtggagc aggacaagaa cacccaggag 2340 gtgttcgccc aggtgaagca gatctacaag acccccccca tcaaggactt cggcggcttc 2400 aacttcagcc agatcctgcc cgaccccagc aagcccagca agagaagctt catcgaggac 2460 ctgctgttca acaaggtgac cctggccgac gccggcttca tcaagcagta cggcgactgc 2520 ctgggcgaca tcgccgccag agacctgatc tgcgcccaga agtttaatgg actgacagtg 2580 ctgccacccc tgctgaccga tgagatgatc gcccagtaca ccagcgctct gctggccggc 2640 acaatcacca gcggctggac atttggagcc ggagccgctc tgcagatccc atttgccatg 2700 cagatggcct acagattcaa cggcatcggc gtgacccaga acgtgctgta cgagaaccag 2760 aagctgatcg ccaaccagtt caacagcgcc atcggcaaga tccaggacag cctgtctagc 2820 acagcctctg ccctgggcaa gctgcaggat gtggtgaacc agaacgccca ggccctgaac 2880 accctggtga agcagctgag cagcaacttc ggcgccatca gcagcgtgct gaacgacatc 2940 ctgagcagac tggacaaggt ggaggccgag gtgcagatcg acagactgat caccggcaga 3000 ctgcagagcc tgcagaccta cgtgacccag cagctgatca gagccgccga aatcagagcc 3060 agcgccaatc tggccgccac caagatgagc gagtgcgtgc tgggccagag caagagagtg 3120 gacttctgcg gcaagggcta ccacctgatg agctttcccc agagcgcccc tcacggcgtg 3180 gtgtttctgc acgtgaccta cgtgcctgcc caggagaaga acttcaccac cgcccctgcc 3240 atctgccacg atggcaaggc ccacttccct agagagggcg tgttcgtgag caacggcacc 3300 cactggttcg tgacccagag aaacttctac gagccccaga tcatcaccac cgacaacacc 3360 ttcgtgagcg gcaactgcga cgtggtgatc ggcatcgtga acaacaccgt gtacgacccc 3420 ctgcagcccg agctggacag cttcaaggag gagctggaca agtacttcaa gaaccacacc 3480 agccccgatg tggacctggg cgatatcagc ggcatcaatg ccagcgtggt gaacatccag 3540 aaggagatcg accggctcaa tgaggtggcc aagaacctga acgagagcct gatcgacctg 3600 caggaactgg gcaaatatga gcagtacatc aagtggccct ggtacatctg gctgggcttc 3660 atcgccggcc tgatcgccat cgtgatggtg accatcatgc tgtgctgcat gaccagctgc 3720 tgcagctgcc tgaagggctg ctgcagctgc gggtcttgtt gcaagttcga cgaggacgac 3780 agcgagcccg tgctgaaggg cgtgaagctg cactacacct aa 3822 <![CDATA[<210> 4]]> <![CDATA[<211> 6734]]> <![CDATA[<212> DNA]]> <![CDATA[<213> 人工]]> <![CDATA[<220>]]> <![CDATA[<223> 質體CO]]> <![CDATA[<400> 4]]> gctgcttcgc gatgtacggg ccagatatac gcgttgacat tgattattga ctagttatta 60 atagtaatca attacggggt cattagttca tagcccatat atggagttcc gcgttacata 120 acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat 180 aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga 240 gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc 300 ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt 360 atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtgat 420 gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg gatttccaag 480 tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac gggactttcc 540 aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg tacggtggga 600 ggtctatata agcagagctc tctggctaac tagagaaccc actgcttact ggcttatcga 660 aattaatacg actcactata gggagaccca agctggctag ccaccatgtt cgtgttcctg 720 gtgctgctgc ccctggtgag cagccagtgc gtgaacctga ccaccagaac ccagctgccc 780 cccgcctaca ccaacagctt caccagaggc gtgtactacc ccgacaaggt gttcagaagc 840 agcgtgctgc acagcaccca ggacctgttc ctgcccttct tcagcaacgt gacctggttc 900 cacgccatcc acgtgagcgg caccaacggc accaagagat tcgacaaccc cgtgctgccc 960 ttcaacgacg gcgtgtactt cgccagcacc gagaagagca acatcatcag aggctggatc 1020 ttcggcacca ccctggacag caagacccag agcctgctga tcgtgaacaa cgccaccaac 1080 gtggtgatca aggtgtgcga gttccagttc tgcaacgacc ccttcctggg cgtgtactac 1140 cacaagaaca acaagagctg gatggagagc gagttcagag tgtacagcag cgccaacaac 1200 tgcaccttcg agtacgtgag ccagcccttc ctgatggacc tggagggcaa gcagggcaac 1260 ttcaagaacc tgagagagtt cgtgttcaag aacatcgacg gctacttcaa gatctacagc 1320 aagcacaccc ccatcaacct ggtgagagac ctgcctcagg gctttagcgc cctggagcca 1380 ctggtggacc tgccaatcgg catcaacatc accagattcc agaccctgct ggccctgcac 1440 agaagctacc tgacaccagg cgattctagc tctggatgga cagccggcgc cgctgcctat 1500 tacgtgggct acctgcagcc tagaaccttc ctgctgaagt acaacgagaa cggcaccatc 1560 accgatgccg tggactgcgc cctggatccc ctgagcgaga ccaagtgtac cctgaagagc 1620 ttcaccgtgg agaagggcat ctaccagacc agcaacttca gagtgcagcc caccgagagc 1680 atcgtgagat tccccaacat caccaacctg tgccccttcg gcgaggtgtt caacgccacc 1740 agattcgcca gcgtgtacgc ctggaacaga aagagaatca gcaactgcgt ggccgactac 1800 agcgtgctgt acaacagcgc cagcttcagc accttcaagt gctacggcgt gagccccacc 1860 aagctgaacg acctgtgctt caccaacgtg tacgccgaca gcttcgtgat cagaggcgac 1920 gaggtgagac agattgcccc tggccagacc ggcaagatcg ccgactacaa ctacaagctg 1980 cccgacgact tcaccggctg cgtgatcgcc tggaacagca acaacctgga cagcaaggtg 2040 ggcggcaact acaactacct gtacagactg ttcagaaaga gcaacctgaa gcccttcgag 2100 agagacatca gcaccgagat ctaccaggcc ggctctaccc catgcaatgg cgtggagggc 2160 ttcaattgct acttccccct gcagagctac ggcttccagc ccaccaacgg cgtgggctac 2220 cagccctaca gagtggtggt gctgagcttt gaactgctgc acgcccctgc caccgtgtgc 2280 ggcccaaaga agagcaccaa tctggtgaag aacaagtgcg tgaacttcaa cttcaacggc 2340 ctgaccggca ccggcgtgct gaccgagagc aacaagaagt tcctgccctt ccagcagttc 2400 ggcagagaca tcgccgacac caccgatgcc gtgagagatc cccagaccct ggagatcctg 2460 gacatcaccc cctgtagctt tggcggcgtg agcgtgatta cccccggcac caataccagc 2520 aaccaggtgg ccgtgctgta ccaggacgtg aactgcaccg aggtgccagt ggccatccat 2580 gccgaccagc tgaccccaac ctggagagtg tacagcaccg gcagcaacgt gttccagaca 2640 agagccggct gtctgattgg cgccgagcac gtgaataaca gctacgagtg cgatatccca 2700 atcggcgccg gcatctgtgc cagctatcag acccagacca atagccccag aagagccaga 2760 agcgtggcca gccagagcat catcgcctac accatgagcc tgggcgccga gaacagcgtg 2820 gcctacagca acaacagcat cgccatcccc accaacttca ccatcagcgt gaccaccgag 2880 atcctgcccg tgagcatgac caagaccagc gtggactgca ccatgtacat ctgcggcgac 2940 agcaccgagt gcagcaacct gctgctgcag tacggcagct tctgcaccca gctgaacaga 3000 gccctgaccg gcatcgccgt ggagcaggac aagaacaccc aggaggtgtt cgcccaggtg 3060 aagcagatct acaagacccc ccccatcaag gacttcggcg gcttcaactt cagccagatc 3120 ctgcccgacc ccagcaagcc cagcaagaga agcttcatcg aggacctgct gttcaacaag 3180 gtgaccctgg ccgacgccgg cttcatcaag cagtacggcg actgcctggg cgacatcgcc 3240 gccagagacc tgatctgcgc ccagaagttt aatggactga cagtgctgcc acccctgctg 3300 accgatgaga tgatcgccca gtacaccagc gctctgctgg ccggcacaat caccagcggc 3360 tggacatttg gagccggagc cgctctgcag atcccatttg ccatgcagat ggcctacaga 3420 ttcaacggca tcggcgtgac ccagaacgtg ctgtacgaga accagaagct gatcgccaac 3480 cagttcaaca gcgccatcgg caagatccag gacagcctgt ctagcacagc ctctgccctg 3540 ggcaagctgc aggatgtggt gaaccagaac gcccaggccc tgaacaccct ggtgaagcag 3600 ctgagcagca acttcggcgc catcagcagc gtgctgaacg acatcctgag cagactggac 3660 aaggtggagg ccgaggtgca gatcgacaga ctgatcaccg gcagactgca gagcctgcag 3720 acctacgtga cccagcagct gatcagagcc gccgaaatca gagccagcgc caatctggcc 3780 gccaccaaga tgagcgagtg cgtgctgggc cagagcaaga gagtggactt ctgcggcaag 3840 ggctaccacc tgatgagctt tccccagagc gcccctcacg gcgtggtgtt tctgcacgtg 3900 acctacgtgc ctgcccagga gaagaacttc accaccgccc ctgccatctg ccacgatggc 3960 aaggcccact tccctagaga gggcgtgttc gtgagcaacg gcacccactg gttcgtgacc 4020 cagagaaact tctacgagcc ccagatcatc accaccgaca acaccttcgt gagcggcaac 4080 tgcgacgtgg tgatcggcat cgtgaacaac accgtgtacg accccctgca gcccgagctg 4140 gacagcttca aggaggagct ggacaagtac ttcaagaacc acaccagccc cgatgtggac 4200 ctgggcgata tcagcggcat caatgccagc gtggtgaaca tccagaagga gatcgaccgg 4260 ctcaatgagg tggccaagaa cctgaacgag agcctgatcg acctgcagga actgggcaaa 4320 tatgagcagt acatcaagtg gccctggtac atctggctgg gcttcatcgc cggcctgatc 4380 gccatcgtga tggtgaccat catgctgtgc tgcatgacca gctgctgcag ctgcctgaag 4440 ggctgctgca gctgcgggtc ttgttgcaag ttcgacgagg acgacagcga gcccgtgctg 4500 aagggcgtga agctgcacta cacctaatct agagggcccg tttaaacccg ctgatcagcc 4560 tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg 4620 accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat 4680 tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag 4740 gattgggaag acaatagcag gcatgctggg gatgcggtgg gctctatggc ttctactggg 4800 cggttttatg gacagcaagc gaaccggaat tgccagctgg ggcgccctct ggtaaggttg 4860 ggaagccctg caaagtaaac tggatggctt tcttgccgcc aaggatctga tggcgcaggg 4920 gatcaagctc tgatcaagag acaggatgag gatcgtttcg catgattgaa caagatggat 4980 tgcacgcagg ttctccggcc gcttgggtgg agaggctatt cggctatgac tgggcacaac 5040 agacaatcgg ctgctctgat gccgccgtgt tccggctgtc agcgcagggg cgcccggttc 5100 tttttgtcaa gaccgacctg tccggtgccc tgaatgaact gcaagacgag gcagcgcggc 5160 tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag 5220 cgggaaggga ctggctgcta ttgggcgaag tgccggggca ggatctcctg tcatctcacc 5280 ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat gcggcggctg catacgcttg 5340 atccggctac ctgcccattc gaccaccaag cgaaacatcg catcgagcga gcacgtactc 5400 ggatggaagc cggtcttgtc gatcaggatg atctggacga agagcatcag gggctcgcgc 5460 cagccgaact gttcgccagg ctcaaggcga gcatgcccga cggcgaggat ctcgtcgtga 5520 cccatggcga tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt tctggattca 5580 tcgactgtgg ccggctgggt gtggcggacc gctatcagga catagcgttg gctacccgtg 5640 atattgctga agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg 5700 ccgctcccga ttcgcagcgc atcgccttct atcgccttct tgacgagttc ttctgaatta 5760 ttaacgctta caatttcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac 5820 accgcatcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc 5880 taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa 5940 tagcacgtgc taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat 6000 aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta 6060 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa 6120 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt 6180 tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag 6240 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta 6300 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca 6360 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag 6420 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa 6480 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga 6540 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc 6600 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc 6660 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt 6720 gctcacatgt tctt 6734 <![CDATA[<210> 5]]> <![CDATA[<211> 20]]> <![CDATA[<212> DNA]]> <![CDATA[<213> 人工]]> <![CDATA[<220>]]> <![CDATA[<223> 引子]]> <![CDATA[<400> 5]]> ctacatgcac cagcaactgt 20 <![CDATA[<210> 6]]> <![CDATA[<211> 20]]> <![CDATA[<212> DNA]]> <![CDATA[<213> 人工]]> <![CDATA[<220>]]> <![CDATA[<223> 引子]]> <![CDATA[<400> 6]]> cacctgtgcc tgttaaacca 20 <![CDATA[<210> 7]]> <![CDATA[<211> 20]]> <![CDATA[<212> DNA]]> <![CDATA[<213> 人工]]> <![CDATA[<220>]]> <![CDATA[<223> 引子]]> <![CDATA[<400> 7]]> agccagagca tcatcgccta 20 <![CDATA[<210> 8]]> <![CDATA[<211> 20]]> <![CDATA[<212> DNA]]> <![CDATA[<213> 人工]]> <![CDATA[<220>]]> <![CDATA[<223> 引子]]> <![CDATA[<400> 8]]> tggtcacgct gatggtgaag 20 <![CDATA[<210> 9]]> <![CDATA[<211> 20]]> <![CDATA[<212> DNA]]> <![CDATA[<213> 人工]]> <![CDATA[<220>]]> <![CDATA[<223> 引子]]> <![CDATA[<400> 9]]> gcaccgtcaa ggctgagaac 20 <![CDATA[<210> 10]]> <![CDATA[<211> 19]]> <![CDATA[<212> DNA]]> <![CDATA[<213> 人工]]> <![CDATA[<220>]]> <![CDATA[<223> 引子]]> <![CDATA[<400> 10]]> tggtgaagac gccagtgga 19 <![CDATA[<210> 11]]> <![CDATA[<211> 1254]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工]]> <![CDATA[<220>]]> <![CDATA[<223> 修飾SARS-CoV-2刺突蛋白之胺基酸序列(4GP)]]> <![CDATA[<400> 11]]> Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln Cys Val 1 5 10 15 Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr Asn Ser Phe 20 25 30 Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser Ser Val Leu 35 40 45 His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn Val Thr Trp 50 55 60 Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys Arg Phe Asp 65 70 75 80 Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala Ser Thr Glu 85 90 95 Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr Leu Asp Ser 100 105 110 Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn Val Val Ile 115 120 125 Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu Gly Val Tyr 130 135 140 Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe Arg Val Tyr 145 150 155 160 Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln Pro Phe Leu 165 170 175 Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu Arg Glu Phe 180 185 190 Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser Lys His Thr 195 200 205 Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe Ser Ala Leu Glu 210 215 220 Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg Phe Gln Thr 225 230 235 240 Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp Ser Ser Ser 245 250 255 Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr Leu Gln Pro 260 265 270 Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp Ala 275 280 285 Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu Lys 290 295 300 Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val 305 310 315 320 Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys 325 330 335 Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala 340 345 350 Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu 355 360 365 Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro 370 375 380 Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe 385 390 395 400 Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly 405 410 415 Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys 420 425 430 Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn 435 440 445 Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe 450 455 460 Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys 465 470 475 480 Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly 485 490 495 Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val 500 505 510 Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys 515 520 525 Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn Phe Asn 530 535 540 Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys Phe Leu 545 550 555 560 Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp Ala Val 565 570 575 Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys Ser Phe 580 585 590 Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr Ser Asn Gln Val 595 600 605 Ala Val Leu Tyr Gln Gly Val Asn Cys Thr Glu Val Pro Val Ala Ile 610 615 620 His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr Ser Thr Gly Ser 625 630 635 640 Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly Ala Glu His Val 645 650 655 Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly Ile Cys Ala 660 665 670 Ser Tyr Gln Thr Gln Thr Asn Ser Pro Arg Arg Ala Arg Ser Val Ala 675 680 685 Ser Gln Ser Ile Ile Ala Tyr Thr Met Ser Leu Gly Ala Glu Asn Ser 690 695 700 Val Ala Tyr Ser Asn Asn Ser Ile Ala Ile Pro Thr Asn Phe Thr Ile 705 710 715 720 Ser Val Thr Thr Glu Ile Leu Pro Val Ser Met Thr Lys Thr Ser Val 725 730 735 Asp Cys Thr Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys Ser Asn Leu 740 745 750 Leu Leu Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg Ala Leu Thr 755 760 765 Gly Ile Ala Val Glu Gln Asp Lys Asn Thr Gln Glu Val Phe Ala Gln 770 775 780 Val Lys Gln Ile Tyr Lys Thr Pro Pro Ile Lys Asp Phe Gly Gly Phe 785 790 795 800 Asn Phe Ser Gln Ile Leu Pro Asp Pro Ser Lys Pro Ser Lys Arg Ser 805 810 815 Phe Ile Glu Asp Leu Leu Phe Asn Lys Val Thr Leu Ala Asp Ala Gly 820 825 830 Phe Ile Lys Gln Tyr Gly Asp Cys Leu Gly Asp Ile Ala Ala Arg Asp 835 840 845 Leu Ile Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu Pro Pro Leu 850 855 860 Leu Thr Asp Glu Met Ile Ala Gln Tyr Thr Ser Ala Leu Leu Ala Gly 865 870 875 880 Thr Ile Thr Ser Gly Trp Thr Phe Gly Ala Gly Ala Ala Leu Gln Ile 885 890 895 Pro Phe Ala Met Gln Met Ala Tyr Arg Phe Asn Gly Ile Gly Val Thr 900 905 910 Gln Asn Val Leu Tyr Glu Asn Gln Lys Leu Ile Ala Asn Gln Phe Asn 915 920 925 Ser Ala Ile Gly Lys Ile Gln Asp Ser Leu Ser Ser Thr Ala Ser Ala 930 935 940 Leu Gly Lys Leu Gln Asp Val Val Asn Gln Asn Ala Gln Ala Leu Asn 945 950 955 960 Thr Leu Val Lys Gln Leu Ser Ser Asn Phe Gly Ala Ile Ser Ser Val 965 970 975 Leu Asn Asp Ile Leu Ser Arg Leu Asp Pro Pro Glu Ala Glu Val Gln 980 985 990 Ile Asp Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln Thr Tyr Val 995 1000 1005 Thr Gln Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala Ser Ala Asn 1010 1015 1020 Leu Ala Ala Thr Lys Met Ser Glu Cys Val Leu Gly Gln Ser Lys 1025 1030 1035 Arg Val Asp Phe Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro 1040 1045 1050 Gln Ser Ala Pro His Gly Val Val Phe Leu His Val Thr Tyr Val 1055 1060 1065 Pro Ala Gln Glu Lys Asn Phe Thr Thr Ala Pro Ala Ile Cys His 1070 1075 1080 Asp Gly Lys Ala His Phe Pro Arg Glu Gly Val Phe Val Ser Asn 1085 1090 1095 Gly Thr His Trp Phe Val Thr Gln Arg Asn Phe Tyr Glu Pro Gln 1100 1105 1110 Ile Ile Thr Thr Asp Asn Thr Phe Val Ser Gly Asn Cys Asp Val 1115 1120 1125 Val Ile Gly Ile Val Asn Asn Thr Val Tyr Asp Pro Leu Gln Pro 1130 1135 1140 Glu Leu Asp Ser Phe Lys Glu Glu Leu Asp Lys Tyr Phe Lys Asn 1145 1150 1155 His Thr Ser Pro Asp Val Asp Leu Gly Asp Ile Ser Gly Ile Asn 1160 1165 1170 Ala Ser Val Val Asn Ile Gln Lys Glu Ile Asp Arg Leu Asn Glu 1175 1180 1185 Val Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu Gln Glu Leu 1190 1195 1200 Gly Lys Tyr Glu Gln Tyr Ile Lys Trp Pro Trp Tyr Ile Trp Leu 1205 1210 1215 Gly Phe Ile Ala Gly Leu Ile Ala Ile Val Met Val Thr Ile Met 1220 1225 1230 Leu Cys Cys Met Thr Ser Cys Cys Ser Cys Leu Lys Gly Cys Cys 1235 1240 1245 Ser Cys Gly Ser Cys Cys 1250 <![CDATA[<210> 12]]> <![CDATA[<211> 3765]]> <![CDATA[<212> DNA]]> <![CDATA[<213> 人工]]> <![CDATA[<220>]]> <![CDATA[<223> 編碼修飾SARS-CoV-2刺突蛋白之核苷酸序列(4GP) ]]> <![CDATA[<400> 12]]> atgttcgtgt tcctggtgct gctgcccctg gtgagcagcc agtgcgtgaa cctgaccacc 60 agaacccagc tgccccccgc ctacaccaac agcttcacca gaggcgtgta ctaccccgac 120 aaggtgttca gaagcagcgt gctgcacagc acccaggacc tgttcctgcc cttcttcagc 180 aacgtgacct ggttccacgc catccacgtg agcggcacca acggcaccaa gagattcgac 240 aaccccgtgc tgcccttcaa cgacggcgtg tacttcgcca gcaccgagaa gagcaacatc 300 atcagaggct ggatcttcgg caccaccctg gacagcaaga cccagagcct gctgatcgtg 360 aacaacgcca ccaacgtggt gatcaaggtg tgcgagttcc agttctgcaa cgaccccttc 420 ctgggcgtgt actaccacaa gaacaacaag agctggatgg agagcgagtt cagagtgtac 480 agcagcgcca acaactgcac cttcgagtac gtgagccagc ccttcctgat ggacctggag 540 ggcaagcagg gcaacttcaa gaacctgaga gagttcgtgt tcaagaacat cgacggctac 600 ttcaagatct acagcaagca cacccccatc aacctggtga gagacctgcc tcagggcttt 660 agcgccctgg agccactggt ggacctgcca atcggcatca acatcaccag attccagacc 720 ctgctggccc tgcacagaag ctacctgaca ccaggcgatt ctagctctgg atggacagcc 780 ggcgccgctg cctattacgt gggctacctg cagcctagaa ccttcctgct gaagtacaac 840 gagaacggca ccatcaccga tgccgtggac tgcgccctgg atcccctgag cgagaccaag 900 tgtaccctga agagcttcac cgtggagaag ggcatctacc agaccagcaa cttcagagtg 960 cagcccaccg agagcatcgt gagattcccc aacatcacca acctgtgccc cttcggcgag 1020 gtgttcaacg ccaccagatt cgccagcgtg tacgcctgga acagaaagag aatcagcaac 1080 tgcgtggccg actacagcgt gctgtacaac agcgccagct tcagcacctt caagtgctac 1140 ggcgtgagcc ccaccaagct gaacgacctg tgcttcacca acgtgtacgc cgacagcttc 1200 gtgatcagag gcgacgaggt gagacagatt gcccctggcc agaccggcaa gatcgccgac 1260 tacaactaca agctgcccga cgacttcacc ggctgcgtga tcgcctggaa cagcaacaac 1320 ctggacagca aggtgggcgg caactacaac tacctgtaca gactgttcag aaagagcaac 1380 ctgaagccct tcgagagaga catcagcacc gagatctacc aggccggctc taccccatgc 1440 aatggcgtgg agggcttcaa ttgctacttc cccctgcaga gctacggctt ccagcccacc 1500 aacggcgtgg gctaccagcc ctacagagtg gtggtgctga gctttgaact gctgcacgcc 1560 cctgccaccg tgtgcggccc aaagaagagc accaatctgg tgaagaacaa gtgcgtgaac 1620 ttcaacttca acggcctgac cggcaccggc gtgctgaccg agagcaacaa gaagttcctg 1680 cccttccagc agttcggcag agacatcgcc gacaccaccg atgccgtgag agatccccag 1740 accctggaga tcctggacat caccccctgt agctttggcg gcgtgagcgt gattaccccc 1800 ggcaccaata ccagcaacca ggtggccgtg ctgtaccagg gcgtgaactg caccgaggtg 1860 ccagtggcca tccatgccga ccagctgacc ccaacctgga gagtgtacag caccggcagc 1920 aacgtgttcc agacaagagc cggctgtctg attggcgccg agcacgtgaa taacagctac 1980 gagtgcgata tcccaatcgg cgccggcatc tgtgccagct atcagaccca gaccaatagc 2040 cccagaagag ccagaagcgt ggccagccag agcatcatcg cctacaccat gagcctgggc 2100 gccgagaaca gcgtggccta cagcaacaac agcatcgcca tccccaccaa cttcaccatc 2160 agcgtgacca ccgagatcct gcccgtgagc atgaccaaga ccagcgtgga ctgcaccatg 2220 tacatctgcg gcgacagcac cgagtgcagc aacctgctgc tgcagtacgg cagcttctgc 2280 acccagctga acagagccct gaccggcatc gccgtggagc aggacaagaa cacccaggag 2340 gtgttcgccc aggtgaagca gatctacaag acccccccca tcaaggactt cggcggcttc 2400 aacttcagcc agatcctgcc cgaccccagc aagcccagca agagaagctt catcgaggac 2460 ctgctgttca acaaggtgac cctggccgac gccggcttca tcaagcagta cggcgactgc 2520 ctgggcgaca tcgccgccag agacctgatc tgcgcccaga agtttaatgg actgacagtg 2580 ctgccacccc tgctgaccga tgagatgatc gcccagtaca ccagcgctct gctggccggc 2640 acaatcacca gcggctggac atttggagcc ggagccgctc tgcagatccc atttgccatg 2700 cagatggcct acagattcaa cggcatcggc gtgacccaga acgtgctgta cgagaaccag 2760 aagctgatcg ccaaccagtt caacagcgcc atcggcaaga tccaggacag cctgtctagc 2820 acagcctctg ccctgggcaa gctgcaggat gtggtgaacc agaacgccca ggccctgaac 2880 accctggtga agcagctgag cagcaacttc ggcgccatca gcagcgtgct gaacgacatc 2940 ctgagcagac tggacccccc tgaggccgag gtgcagatcg acagactgat caccggcaga 3000 ctgcagagcc tgcagaccta cgtgacccag cagctgatca gagccgccga aatcagagcc 3060 agcgccaatc tggccgccac caagatgagc gagtgcgtgc tgggccagag caagagagtg 3120 gacttctgcg gcaagggcta ccacctgatg agctttcccc agagcgcccc tcacggcgtg 3180 gtgtttctgc acgtgaccta cgtgcctgcc caggagaaga acttcaccac cgcccctgcc 3240 atctgccacg atggcaaggc ccacttccct agagagggcg tgttcgtgag caacggcacc 3300 cactggttcg tgacccagag aaacttctac gagccccaga tcatcaccac cgacaacacc 3360 ttcgtgagcg gcaactgcga cgtggtgatc ggcatcgtga acaacaccgt gtacgacccc 3420 ctgcagcccg agctggacag cttcaaggag gagctggaca agtacttcaa gaaccacacc 3480 agccccgatg tggacctggg cgatatcagc ggcatcaatg ccagcgtggt gaacatccag 3540 aaggagatcg accggctcaa tgaggtggcc aagaacctga acgagagcct gatcgacctg 3600 caggaactgg gcaaatatga gcagtacatc aagtggccct ggtacatctg gctgggcttc 3660 atcgccggcc tgatcgccat cgtgatggtg accatcatgc tgtgctgcat gaccagctgc 3720 tgcagctgcc tgaagggctg ctgcagctgc gggtcttgtt gctaa 3765 <![CDATA[<210> 13]]> <![CDATA[<211> 1254]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工]]> <![CDATA[<220>]]> <![CDATA[<223> 修飾SARS-CoV-2刺突蛋白之胺基酸序列(SA)]]> <![CDATA[<400> 13]]> Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln Cys Val 1 5 10 15 Asn Phe Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr Asn Ser Phe 20 25 30 Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser Ser Val Leu 35 40 45 His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn Val Thr Trp 50 55 60 Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys Arg Phe Ala 65 70 75 80 Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala Ser Thr Glu 85 90 95 Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr Leu Asp Ser 100 105 110 Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn Val Val Ile 115 120 125 Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu Gly Val Tyr 130 135 140 Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe Arg Val Tyr 145 150 155 160 Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln Pro Phe Leu 165 170 175 Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu Arg Glu Phe 180 185 190 Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser Lys His Thr 195 200 205 Pro Ile Asn Leu Val Arg Gly Leu Pro Gln Gly Phe Ser Ala Leu Glu 210 215 220 Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg Phe Gln Thr 225 230 235 240 Leu Leu Ala Leu His Ile Ser Tyr Leu Thr Pro Gly Asp Ser Ser Ser 245 250 255 Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr Leu Gln Pro 260 265 270 Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp Ala 275 280 285 Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu Lys 290 295 300 Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val 305 310 315 320 Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys 325 330 335 Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala 340 345 350 Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu 355 360 365 Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro 370 375 380 Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe 385 390 395 400 Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly 405 410 415 Asn Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys 420 425 430 Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn 435 440 445 Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe 450 455 460 Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys 465 470 475 480 Asn Gly Val Lys Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly 485 490 495 Phe Gln Pro Thr Tyr Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val 500 505 510 Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys 515 520 525 Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn Phe Asn 530 535 540 Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys Phe Leu 545 550 555 560 Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp Ala Val 565 570 575 Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys Ser Phe 580 585 590 Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr Ser Asn Gln Val 595 600 605 Ala Val Leu Tyr Gln Gly Val Asn Cys Thr Glu Val Pro Val Ala Ile 610 615 620 His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr Ser Thr Gly Ser 625 630 635 640 Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly Ala Glu His Val 645 650 655 Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly Ile Cys Ala 660 665 670 Ser Tyr Gln Thr Gln Thr Asn Ser Pro Arg Arg Ala Arg Ser Val Ala 675 680 685 Ser Gln Ser Ile Ile Ala Tyr Thr Met Ser Leu Gly Val Glu Asn Ser 690 695 700 Val Ala Tyr Ser Asn Asn Ser Ile Ala Ile Pro Thr Asn Phe Thr Ile 705 710 715 720 Ser Val Thr Thr Glu Ile Leu Pro Val Ser Met Thr Lys Thr Ser Val 725 730 735 Asp Cys Thr Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys Ser Asn Leu 740 745 750 Leu Leu Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg Ala Leu Thr 755 760 765 Gly Ile Ala Val Glu Gln Asp Lys Asn Thr Gln Glu Val Phe Ala Gln 770 775 780 Val Lys Gln Ile Tyr Lys Thr Pro Pro Ile Lys Asp Phe Gly Gly Phe 785 790 795 800 Asn Phe Ser Gln Ile Leu Pro Asp Pro Ser Lys Pro Ser Lys Arg Ser 805 810 815 Phe Ile Glu Asp Leu Leu Phe Asn Lys Val Thr Leu Ala Asp Ala Gly 820 825 830 Phe Ile Lys Gln Tyr Gly Asp Cys Leu Gly Asp Ile Ala Ala Arg Asp 835 840 845 Leu Ile Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu Pro Pro Leu 850 855 860 Leu Thr Asp Glu Met Ile Ala Gln Tyr Thr Ser Ala Leu Leu Ala Gly 865 870 875 880 Thr Ile Thr Ser Gly Trp Thr Phe Gly Ala Gly Ala Ala Leu Gln Ile 885 890 895 Pro Phe Ala Met Gln Met Ala Tyr Arg Phe Asn Gly Ile Gly Val Thr 900 905 910 Gln Asn Val Leu Tyr Glu Asn Gln Lys Leu Ile Ala Asn Gln Phe Asn 915 920 925 Ser Ala Ile Gly Lys Ile Gln Asp Ser Leu Ser Ser Thr Ala Ser Ala 930 935 940 Leu Gly Lys Leu Gln Asp Val Val Asn Gln Asn Ala Gln Ala Leu Asn 945 950 955 960 Thr Leu Val Lys Gln Leu Ser Ser Asn Phe Gly Ala Ile Ser Ser Val 965 970 975 Leu Asn Asp Ile Leu Ser Arg Leu Asp Pro Pro Glu Ala Glu Val Gln 980 985 990 Ile Asp Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln Thr Tyr Val 995 1000 1005 Thr Gln Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala Ser Ala Asn 1010 1015 1020 Leu Ala Ala Thr Lys Met Ser Glu Cys Val Leu Gly Gln Ser Lys 1025 1030 1035 Arg Val Asp Phe Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro 1040 1045 1050 Gln Ser Ala Pro His Gly Val Val Phe Leu His Val Thr Tyr Val 1055 1060 1065 Pro Ala Gln Glu Lys Asn Phe Thr Thr Ala Pro Ala Ile Cys His 1070 1075 1080 Asp Gly Lys Ala His Phe Pro Arg Glu Gly Val Phe Val Ser Asn 1085 1090 1095 Gly Thr His Trp Phe Val Thr Gln Arg Asn Phe Tyr Glu Pro Gln 1100 1105 1110 Ile Ile Thr Thr Asp Asn Thr Phe Val Ser Gly Asn Cys Asp Val 1115 1120 1125 Val Ile Gly Ile Val Asn Asn Thr Val Tyr Asp Pro Leu Gln Pro 1130 1135 1140 Glu Leu Asp Ser Phe Lys Glu Glu Leu Asp Lys Tyr Phe Lys Asn 1145 1150 1155 His Thr Ser Pro Asp Val Asp Leu Gly Asp Ile Ser Gly Ile Asn 1160 1165 1170 Ala Ser Val Val Asn Ile Gln Lys Glu Ile Asp Arg Leu Asn Glu 1175 1180 1185 Val Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu Gln Glu Leu 1190 1195 1200 Gly Lys Tyr Glu Gln Tyr Ile Lys Trp Pro Trp Tyr Ile Trp Leu 1205 1210 1215 Gly Phe Ile Ala Gly Leu Ile Ala Ile Val Met Val Thr Ile Met 1220 1225 1230 Leu Cys Cys Met Thr Ser Cys Cys Ser Cys Leu Lys Gly Cys Cys 1235 1240 1245 Ser Cys Gly Ser Cys Cys 1250 <![CDATA[<210> 14]]> <![CDATA[<211> 3765]]> <![CDATA[<212> DNA]]> <![CDATA[<213> 人工]]> <![CDATA[<220>]]> <![CDATA[<223> 編碼修飾SARS-CoV-2刺突蛋白之核苷酸序列(SA) ]]> <![CDATA[<400> 14]]> atgttcgtgt tcctggtgct gctgcccctg gtgagcagcc agtgcgtgaa cttcaccacc 60 agaacccagc tgccccccgc ctacaccaac agcttcacca gaggcgtgta ctaccccgac 120 aaggtgttca gaagcagcgt gctgcacagc acccaggacc tgttcctgcc cttcttcagc 180 aacgtgacct ggttccacgc catccacgtg agcggcacca acggcaccaa gagattcgcc 240 aaccccgtgc tgcccttcaa cgacggcgtg tacttcgcca gcaccgagaa gagcaacatc 300 atcagaggct ggatcttcgg caccaccctg gacagcaaga cccagagcct gctgatcgtg 360 aacaacgcca ccaacgtggt gatcaaggtg tgcgagttcc agttctgcaa cgaccccttc 420 ctgggcgtgt actaccacaa gaacaacaag agctggatgg agagcgagtt cagagtgtac 480 agcagcgcca acaactgcac cttcgagtac gtgagccagc ccttcctgat ggacctggag 540 ggcaagcagg gcaacttcaa gaacctgaga gagttcgtgt tcaagaacat cgacggctac 600 ttcaagatct acagcaagca cacccccatc aacctggtga gaggcctgcc tcagggcttt 660 agcgccctgg agccactggt ggacctgcca atcggcatca acatcaccag attccagacc 720 ctgctggccc tgcacatcag ctacctgaca ccaggcgatt ctagctctgg atggacagcc 780 ggcgccgctg cctattacgt gggctacctg cagcctagaa ccttcctgct gaagtacaac 840 gagaacggca ccatcaccga tgccgtggac tgcgccctgg atcccctgag cgagaccaag 900 tgtaccctga agagcttcac cgtggagaag ggcatctacc agaccagcaa cttcagagtg 960 cagcccaccg agagcatcgt gagattcccc aacatcacca acctgtgccc cttcggcgag 1020 gtgttcaacg ccaccagatt cgccagcgtg tacgcctgga acagaaagag aatcagcaac 1080 tgcgtggccg actacagcgt gctgtacaac agcgccagct tcagcacctt caagtgctac 1140 ggcgtgagcc ccaccaagct gaacgacctg tgcttcacca acgtgtacgc cgacagcttc 1200 gtgatcagag gcgacgaggt gagacagatt gcccctggcc agaccggcaa catcgccgac 1260 tacaactaca agctgcccga cgacttcacc ggctgcgtga tcgcctggaa cagcaacaac 1320 ctggacagca aggtgggcgg caactacaac tacctgtaca gactgttcag aaagagcaac 1380 ctgaagccct tcgagagaga catcagcacc gagatctacc aggccggctc taccccatgc 1440 aatggcgtga agggcttcaa ttgctacttc cccctgcaga gctacggctt ccagcccacc 1500 tacggcgtgg gctaccagcc ctacagagtg gtggtgctga gctttgaact gctgcacgcc 1560 cctgccaccg tgtgcggccc aaagaagagc accaatctgg tgaagaacaa gtgcgtgaac 1620 ttcaacttca acggcctgac cggcaccggc gtgctgaccg agagcaacaa gaagttcctg 1680 cccttccagc agttcggcag agacatcgcc gacaccaccg atgccgtgag agatccccag 1740 accctggaga tcctggacat caccccctgt agctttggcg gcgtgagcgt gattaccccc 1800 ggcaccaata ccagcaacca ggtggccgtg ctgtaccagg gcgtgaactg caccgaggtg 1860 ccagtggcca tccatgccga ccagctgacc ccaacctgga gagtgtacag caccggcagc 1920 aacgtgttcc agacaagagc cggctgtctg attggcgccg agcacgtgaa taacagctac 1980 gagtgcgata tcccaatcgg cgccggcatc tgtgccagct atcagaccca gaccaatagc 2040 cccagaagag ccagaagcgt ggccagccag agcatcatcg cctacaccat gagcctgggc 2100 gtggagaaca gcgtggccta cagcaacaac agcatcgcca tccccaccaa cttcaccatc 2160 agcgtgacca ccgagatcct gcccgtgagc atgaccaaga ccagcgtgga ctgcaccatg 2220 tacatctgcg gcgacagcac cgagtgcagc aacctgctgc tgcagtacgg cagcttctgc 2280 acccagctga acagagccct gaccggcatc gccgtggagc aggacaagaa cacccaggag 2340 gtgttcgccc aggtgaagca gatctacaag acccccccca tcaaggactt cggcggcttc 2400 aacttcagcc agatcctgcc cgaccccagc aagcccagca agagaagctt catcgaggac 2460 ctgctgttca acaaggtgac cctggccgac gccggcttca tcaagcagta cggcgactgc 2520 ctgggcgaca tcgccgccag agacctgatc tgcgcccaga agtttaatgg actgacagtg 2580 ctgccacccc tgctgaccga tgagatgatc gcccagtaca ccagcgctct gctggccggc 2640 acaatcacca gcggctggac atttggagcc ggagccgctc tgcagatccc atttgccatg 2700 cagatggcct acagattcaa cggcatcggc gtgacccaga acgtgctgta cgagaaccag 2760 aagctgatcg ccaaccagtt caacagcgcc atcggcaaga tccaggacag cctgtctagc 2820 acagcctctg ccctgggcaa gctgcaggat gtggtgaacc agaacgccca ggccctgaac 2880 accctggtga agcagctgag cagcaacttc ggcgccatca gcagcgtgct gaacgacatc 2940 ctgagcagac tggacccccc tgaggccgag gtgcagatcg acagactgat caccggcaga 3000 ctgcagagcc tgcagaccta cgtgacccag cagctgatca gagccgccga aatcagagcc 3060 agcgccaatc tggccgccac caagatgagc gagtgcgtgc tgggccagag caagagagtg 3120 gacttctgcg gcaagggcta ccacctgatg agctttcccc agagcgcccc tcacggcgtg 3180 gtgtttctgc acgtgaccta cgtgcctgcc caggagaaga acttcaccac cgcccctgcc 3240 atctgccacg atggcaaggc ccacttccct agagagggcg tgttcgtgag caacggcacc 3300 cactggttcg tgacccagag aaacttctac gagccccaga tcatcaccac cgacaacacc 3360 ttcgtgagcg gcaactgcga cgtggtgatc ggcatcgtga acaacaccgt gtacgacccc 3420 ctgcagcccg agctggacag cttcaaggag gagctggaca agtacttcaa gaaccacacc 3480 agccccgatg tggacctggg cgatatcagc ggcatcaatg ccagcgtggt gaacatccag 3540 aaggagatcg accggctcaa tgaggtggcc aagaacctga acgagagcct gatcgacctg 3600 caggaactgg gcaaatatga gcagtacatc aagtggccct ggtacatctg gctgggcttc 3660 atcgccggcc tgatcgccat cgtgatggtg accatcatgc tgtgctgcat gaccagctgc 3720 tgcagctgcc tgaagggctg ctgcagctgc gggtcttgtt gctaa 3765 <![CDATA[<210> 15]]> <![CDATA[<211> 1254]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工]]> <![CDATA[<220>]]> <![CDATA[<223> 修飾SARS-CoV-2刺突蛋白之胺基酸序列(BR)]]> <![CDATA[<400> 15]]> Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln Cys Val 1 5 10 15 Asn Phe Thr Asn Arg Thr Gln Leu Pro Ser Ala Tyr Thr Asn Ser Phe 20 25 30 Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser Ser Val Leu 35 40 45 His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn Val Thr Trp 50 55 60 Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys Arg Phe Asp 65 70 75 80 Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala Ser Thr Glu 85 90 95 Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr Leu Asp Ser 100 105 110 Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn Val Val Ile 115 120 125 Lys Val Cys Glu Phe Gln Phe Cys Asn Tyr Pro Phe Leu Gly Val Tyr 130 135 140 Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe Arg Val Tyr 145 150 155 160 Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln Pro Phe Leu 165 170 175 Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu Ser Glu Phe 180 185 190 Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser Lys His Thr 195 200 205 Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe Ser Ala Leu Glu 210 215 220 Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg Phe Gln Thr 225 230 235 240 Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp Ser Ser Ser 245 250 255 Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr Leu Gln Pro 260 265 270 Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp Ala 275 280 285 Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu Lys 290 295 300 Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val 305 310 315 320 Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys 325 330 335 Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala 340 345 350 Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu 355 360 365 Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro 370 375 380 Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe 385 390 395 400 Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly 405 410 415 Thr Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys 420 425 430 Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn 435 440 445 Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe 450 455 460 Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys 465 470 475 480 Asn Gly Val Lys Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly 485 490 495 Phe Gln Pro Thr Tyr Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val 500 505 510 Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys 515 520 525 Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn Phe Asn 530 535 540 Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys Phe Leu 545 550 555 560 Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp Ala Val 565 570 575 Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys Ser Phe 580 585 590 Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr Ser Asn Gln Val 595 600 605 Ala Val Leu Tyr Gln Gly Val Asn Cys Thr Glu Val Pro Val Ala Ile 610 615 620 His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr Ser Thr Gly Ser 625 630 635 640 Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly Ala Glu Tyr Val 645 650 655 Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly Ile Cys Ala 660 665 670 Ser Tyr Gln Thr Gln Thr Asn Ser Pro Arg Arg Ala Arg Ser Val Ala 675 680 685 Ser Gln Ser Ile Ile Ala Tyr Thr Met Ser Leu Gly Ala Glu Asn Ser 690 695 700 Val Ala Tyr Ser Asn Asn Ser Ile Ala Ile Pro Thr Asn Phe Thr Ile 705 710 715 720 Ser Val Thr Thr Glu Ile Leu Pro Val Ser Met Thr Lys Thr Ser Val 725 730 735 Asp Cys Thr Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys Ser Asn Leu 740 745 750 Leu Leu Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg Ala Leu Thr 755 760 765 Gly Ile Ala Val Glu Gln Asp Lys Asn Thr Gln Glu Val Phe Ala Gln 770 775 780 Val Lys Gln Ile Tyr Lys Thr Pro Pro Ile Lys Asp Phe Gly Gly Phe 785 790 795 800 Asn Phe Ser Gln Ile Leu Pro Asp Pro Ser Lys Pro Ser Lys Arg Ser 805 810 815 Phe Ile Glu Asp Leu Leu Phe Asn Lys Val Thr Leu Ala Asp Ala Gly 820 825 830 Phe Ile Lys Gln Tyr Gly Asp Cys Leu Gly Asp Ile Ala Ala Arg Asp 835 840 845 Leu Ile Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu Pro Pro Leu 850 855 860 Leu Thr Asp Glu Met Ile Ala Gln Tyr Thr Ser Ala Leu Leu Ala Gly 865 870 875 880 Thr Ile Thr Ser Gly Trp Thr Phe Gly Ala Gly Ala Ala Leu Gln Ile 885 890 895 Pro Phe Ala Met Gln Met Ala Tyr Arg Phe Asn Gly Ile Gly Val Thr 900 905 910 Gln Asn Val Leu Tyr Glu Asn Gln Lys Leu Ile Ala Asn Gln Phe Asn 915 920 925 Ser Ala Ile Gly Lys Ile Gln Asp Ser Leu Ser Ser Thr Ala Ser Ala 930 935 940 Leu Gly Lys Leu Gln Asp Val Val Asn Gln Asn Ala Gln Ala Leu Asn 945 950 955 960 Thr Leu Val Lys Gln Leu Ser Ser Asn Phe Gly Ala Ile Ser Ser Val 965 970 975 Leu Asn Asp Ile Leu Ser Arg Leu Asp Pro Pro Glu Ala Glu Val Gln 980 985 990 Ile Asp Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln Thr Tyr Val 995 1000 1005 Thr Gln Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala Ser Ala Asn 1010 1015 1020 Leu Ala Ala Tyr Lys Met Ser Glu Cys Val Leu Gly Gln Ser Lys 1025 1030 1035 Arg Val Asp Phe Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro 1040 1045 1050 Gln Ser Ala Pro His Gly Val Val Phe Leu His Val Thr Tyr Val 1055 1060 1065 Pro Ala Gln Glu Lys Asn Phe Thr Thr Ala Pro Ala Ile Cys His 1070 1075 1080 Asp Gly Lys Ala His Phe Pro Arg Glu Gly Val Phe Val Ser Asn 1085 1090 1095 Gly Thr His Trp Phe Val Thr Gln Arg Asn Phe Tyr Glu Pro Gln 1100 1105 1110 Ile Ile Thr Thr Asp Asn Thr Phe Val Ser Gly Asn Cys Asp Val 1115 1120 1125 Val Ile Gly Ile Val Asn Asn Thr Val Tyr Asp Pro Leu Gln Pro 1130 1135 1140 Glu Leu Asp Ser Phe Lys Glu Glu Leu Asp Lys Tyr Phe Lys Asn 1145 1150 1155 His Thr Ser Pro Asp Val Asp Leu Gly Asp Ile Ser Gly Ile Asn 1160 1165 1170 Ala Ser Phe Val Asn Ile Gln Lys Glu Ile Asp Arg Leu Asn Glu 1175 1180 1185 Val Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu Gln Glu Leu 1190 1195 1200 Gly Lys Tyr Glu Gln Tyr Ile Lys Trp Pro Trp Tyr Ile Trp Leu 1205 1210 1215 Gly Phe Ile Ala Gly Leu Ile Ala Ile Val Met Val Thr Ile Met 1220 1225 1230 Leu Cys Cys Met Thr Ser Cys Cys Ser Cys Leu Lys Gly Cys Cys 1235 1240 1245 Ser Cys Gly Ser Cys Cys 1250 <![CDATA[<210> 16]]> <![CDATA[<211> 3765]]> <![CDATA[<212> DNA]]> <![CDATA[<213> 人工]]> <![CDATA[<220>]]> <![CDATA[<223> 編碼修飾SARS-CoV-2刺突蛋白之核苷酸序列(BR) ]]> <![CDATA[<400> 16]]> atgttcgtgt tcctggtgct gctgcccctg gtgagcagcc agtgcgtgaa cttcaccaac 60 agaacccagc tgcccagcgc ctacaccaac agcttcacca gaggcgtgta ctaccccgac 120 aaggtgttca gaagcagcgt gctgcacagc acccaggacc tgttcctgcc cttcttcagc 180 aacgtgacct ggttccacgc catccacgtg agcggcacca acggcaccaa gagattcgac 240 aaccccgtgc tgcccttcaa cgacggcgtg tacttcgcca gcaccgagaa gagcaacatc 300 atcagaggct ggatcttcgg caccaccctg gacagcaaga cccagagcct gctgatcgtg 360 aacaacgcca ccaacgtggt gatcaaggtg tgcgagttcc agttctgcaa ctaccccttc 420 ctgggcgtgt actaccacaa gaacaacaag agctggatgg agagcgagtt cagagtgtac 480 agcagcgcca acaactgcac cttcgagtac gtgagccagc ccttcctgat ggacctggag 540 ggcaagcagg gcaacttcaa gaacctgagc gagttcgtgt tcaagaacat cgacggctac 600 ttcaagatct acagcaagca cacccccatc aacctggtga gagacctgcc tcagggcttt 660 agcgccctgg agccactggt ggacctgcca atcggcatca acatcaccag attccagacc 720 ctgctggccc tgcacagaag ctacctgaca ccaggcgatt ctagctctgg atggacagcc 780 ggcgccgctg cctattacgt gggctacctg cagcctagaa ccttcctgct gaagtacaac 840 gagaacggca ccatcaccga tgccgtggac tgcgccctgg atcccctgag cgagaccaag 900 tgtaccctga agagcttcac cgtggagaag ggcatctacc agaccagcaa cttcagagtg 960 cagcccaccg agagcatcgt gagattcccc aacatcacca acctgtgccc cttcggcgag 1020 gtgttcaacg ccaccagatt cgccagcgtg tacgcctgga acagaaagag aatcagcaac 1080 tgcgtggccg actacagcgt gctgtacaac agcgccagct tcagcacctt caagtgctac 1140 ggcgtgagcc ccaccaagct gaacgacctg tgcttcacca acgtgtacgc cgacagcttc 1200 gtgatcagag gcgacgaggt gagacagatt gcccctggcc agaccggcac catcgccgac 1260 tacaactaca agctgcccga cgacttcacc ggctgcgtga tcgcctggaa cagcaacaac 1320 ctggacagca aggtgggcgg caactacaac tacctgtaca gactgttcag aaagagcaac 1380 ctgaagccct tcgagagaga catcagcacc gagatctacc aggccggctc taccccatgc 1440 aatggcgtga agggcttcaa ttgctacttc cccctgcaga gctacggctt ccagcccacc 1500 tacggcgtgg gctaccagcc ctacagagtg gtggtgctga gctttgaact gctgcacgcc 1560 cctgccaccg tgtgcggccc aaagaagagc accaatctgg tgaagaacaa gtgcgtgaac 1620 ttcaacttca acggcctgac cggcaccggc gtgctgaccg agagcaacaa gaagttcctg 1680 cccttccagc agttcggcag agacatcgcc gacaccaccg atgccgtgag agatccccag 1740 accctggaga tcctggacat caccccctgt agctttggcg gcgtgagcgt gattaccccc 1800 ggcaccaata ccagcaacca ggtggccgtg ctgtaccagg gcgtgaactg caccgaggtg 1860 ccagtggcca tccatgccga ccagctgacc ccaacctgga gagtgtacag caccggcagc 1920 aacgtgttcc agacaagagc cggctgtctg attggcgccg agtacgtgaa taacagctac 1980 gagtgcgata tcccaatcgg cgccggcatc tgtgccagct atcagaccca gaccaatagc 2040 cccagaagag ccagaagcgt ggccagccag agcatcatcg cctacaccat gagcctgggc 2100 gccgagaaca gcgtggccta cagcaacaac agcatcgcca tccccaccaa cttcaccatc 2160 agcgtgacca ccgagatcct gcccgtgagc atgaccaaga ccagcgtgga ctgcaccatg 2220 tacatctgcg gcgacagcac cgagtgcagc aacctgctgc tgcagtacgg cagcttctgc 2280 acccagctga acagagccct gaccggcatc gccgtggagc aggacaagaa cacccaggag 2340 gtgttcgccc aggtgaagca gatctacaag acccccccca tcaaggactt cggcggcttc 2400 aacttcagcc agatcctgcc cgaccccagc aagcccagca agagaagctt catcgaggac 2460 ctgctgttca acaaggtgac cctggccgac gccggcttca tcaagcagta cggcgactgc 2520 ctgggcgaca tcgccgccag agacctgatc tgcgcccaga agtttaatgg actgacagtg 2580 ctgccacccc tgctgaccga tgagatgatc gcccagtaca ccagcgctct gctggccggc 2640 acaatcacca gcggctggac atttggagcc ggagccgctc tgcagatccc atttgccatg 2700 cagatggcct acagattcaa cggcatcggc gtgacccaga acgtgctgta cgagaaccag 2760 aagctgatcg ccaaccagtt caacagcgcc atcggcaaga tccaggacag cctgtctagc 2820 acagcctctg ccctgggcaa gctgcaggat gtggtgaacc agaacgccca ggccctgaac 2880 accctggtga agcagctgag cagcaacttc ggcgccatca gcagcgtgct gaacgacatc 2940 ctgagcagac tggacccccc tgaggccgag gtgcagatcg acagactgat caccggcaga 3000 ctgcagagcc tgcagaccta cgtgacccag cagctgatca gagccgccga aatcagagcc 3060 agcgccaatc tggccgccat caagatgagc gagtgcgtgc tgggccagag caagagagtg 3120 gacttctgcg gcaagggcta ccacctgatg agctttcccc agagcgcccc tcacggcgtg 3180 gtgtttctgc acgtgaccta cgtgcctgcc caggagaaga acttcaccac cgcccctgcc 3240 atctgccacg atggcaaggc ccacttccct agagagggcg tgttcgtgag caacggcacc 3300 cactggttcg tgacccagag aaacttctac gagccccaga tcatcaccac cgacaacacc 3360 ttcgtgagcg gcaactgcga cgtggtgatc ggcatcgtga acaacaccgt gtacgacccc 3420 ctgcagcccg agctggacag cttcaaggag gagctggaca agtacttcaa gaaccacacc 3480 agccccgatg tggacctggg cgatatcagc ggcatcaatg ccagcttcgt gaacatccag 3540 aaggagatcg accggctcaa tgaggtggcc aagaacctga acgagagcct gatcgacctg 3600 caggaactgg gcaaatatga gcagtacatc aagtggccct ggtacatctg gctgggcttc 3660 atcgccggcc tgatcgccat cgtgatggtg accatcatgc tgtgctgcat gaccagctgc 3720 tgcagctgcc tgaagggctg ctgcagctgc gggtcttgtt gctaa 3765 <![CDATA[<210> 17]]> <![CDATA[<211> 1252]]> <![CDATA[<212> PRT]]> <![CDATA[<213> 人工]]> <![CDATA[<220>]]> <![CDATA[<223> 修飾SARS-CoV-2刺突蛋白之胺基酸序列(IN)]]> <![CDATA[<400> 17]]> Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln Cys Val 1 5 10 15 Asn Leu Arg Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr Asn Ser Phe 20 25 30 Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser Ser Val Leu 35 40 45 His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn Val Thr Trp 50 55 60 Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys Arg Phe Asp 65 70 75 80 Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala Ser Thr Glu 85 90 95 Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr Leu Asp Ser 100 105 110 Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn Val Val Ile 115 120 125 Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu Asp Val Tyr 130 135 140 Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Gly Val Tyr Ser Ser 145 150 155 160 Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln Pro Phe Leu Met Asp 165 170 175 Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu Arg Glu Phe Val Phe 180 185 190 Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser Lys His Thr Pro Ile 195 200 205 Asn Leu Val Arg Asp Leu Pro Gln Gly Phe Ser Ala Leu Glu Pro Leu 210 215 220 Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg Phe Gln Thr Leu Leu 225 230 235 240 Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp Ser Ser Ser Gly Trp 245 250 255 Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr Leu Gln Pro Arg Thr 260 265 270 Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp Ala Val Asp 275 280 285 Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu Lys Ser Phe 290 295 300 Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val Gln Pro 305 310 315 320 Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe 325 330 335 Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn 340 345 350 Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn 355 360 365 Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys 370 375 380 Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile 385 390 395 400 Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly Lys Ile 405 410 415 Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile 420 425 430 Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn 435 440 445 Tyr Arg Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg 450 455 460 Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Lys Pro Cys Asn Gly 465 470 475 480 Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln 485 490 495 Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser 500 505 510 Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys Lys Ser 515 520 525 Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn Phe Asn Gly Leu 530 535 540 Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys Phe Leu Pro Phe 545 550 555 560 Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp Ala Val Arg Asp 565 570 575 Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys Ser Phe Gly Gly 580 585 590 Val Ser Val Ile Thr Pro Gly Thr Asn Thr Ser Asn Gln Val Ala Val 595 600 605 Leu Tyr Gln Gly Val Asn Cys Thr Glu Val Pro Val Ala Ile His Ala 610 615 620 Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr Ser Thr Gly Ser Asn Val 625 630 635 640 Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly Ala Glu His Val Asn Asn 645 650 655 Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly Ile Cys Ala Ser Tyr 660 665 670 Gln Thr Gln Thr Asn Ser Arg Arg Arg Ala Arg Ser Val Ala Ser Gln 675 680 685 Ser Ile Ile Ala Tyr Thr Met Ser Leu Gly Ala Glu Asn Ser Val Ala 690 695 700 Tyr Ser Asn Asn Ser Ile Ala Ile Pro Thr Asn Phe Thr Ile Ser Val 705 710 715 720 Thr Thr Glu Ile Leu Pro Val Ser Met Thr Lys Thr Ser Val Asp Cys 725 730 735 Thr Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys Ser Asn Leu Leu Leu 740 745 750 Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg Ala Leu Thr Gly Ile 755 760 765 Ala Val Glu Gln Asp Lys Asn Thr Gln Glu Val Phe Ala Gln Val Lys 770 775 780 Gln Ile Tyr Lys Thr Pro Pro Ile Lys Asp Phe Gly Gly Phe Asn Phe 785 790 795 800 Ser Gln Ile Leu Pro Asp Pro Ser Lys Pro Ser Lys Arg Ser Phe Ile 805 810 815 Glu Asp Leu Leu Phe Asn Lys Val Thr Leu Ala Asp Ala Gly Phe Ile 820 825 830 Lys Gln Tyr Gly Asp Cys Leu Gly Asp Ile Ala Ala Arg Asp Leu Ile 835 840 845 Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu Pro Pro Leu Leu Thr 850 855 860 Asp Glu Met Ile Ala Gln Tyr Thr Ser Ala Leu Leu Ala Gly Thr Ile 865 870 875 880 Thr Ser Gly Trp Thr Phe Gly Ala Gly Ala Ala Leu Gln Ile Pro Phe 885 890 895 Ala Met Gln Met Ala Tyr Arg Phe Asn Gly Ile Gly Val Thr Gln Asn 900 905 910 Val Leu Tyr Glu Asn Gln Lys Leu Ile Ala Asn Gln Phe Asn Ser Ala 915 920 925 Ile Gly Lys Ile Gln Asp Ser Leu Ser Ser Thr Ala Ser Ala Leu Gly 930 935 940 Lys Leu Gln Asn Val Val Asn Gln Asn Ala Gln Ala Leu Asn Thr Leu 945 950 955 960 Val Lys Gln Leu Ser Ser Asn Phe Gly Ala Ile Ser Ser Val Leu Asn 965 970 975 Asp Ile Leu Ser Arg Leu Asp Pro Pro Glu Ala Glu Val Gln Ile Asp 980 985 990 Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln Thr Tyr Val Thr Gln 995 1000 1005 Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala Ser Ala Asn Leu Ala 1010 1015 1020 Ala Thr Lys Met Ser Glu Cys Val Leu Gly Gln Ser Lys Arg Val 1025 1030 1035 Asp Phe Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro Gln Ser 1040 1045 1050 Ala Pro His Gly Val Val Phe Leu His Val Thr Tyr Val Pro Ala 1055 1060 1065 Gln Glu Lys Asn Phe Thr Thr Ala Pro Ala Ile Cys His Asp Gly 1070 1075 1080 Lys Ala His Phe Pro Arg Glu Gly Val Phe Val Ser Asn Gly Thr 1085 1090 1095 His Trp Phe Val Thr Gln Arg Asn Phe Tyr Glu Pro Gln Ile Ile 1100 1105 1110 Thr Thr Asp Asn Thr Phe Val Ser Gly Asn Cys Asp Val Val Ile 1115 1120 1125 Gly Ile Val Asn Asn Thr Val Tyr Asp Pro Leu Gln Pro Glu Leu 1130 1135 1140 Asp Ser Phe Lys Glu Glu Leu Asp Lys Tyr Phe Lys Asn His Thr 1145 1150 1155 Ser Pro Asp Val Asp Leu Gly Asp Ile Ser Gly Ile Asn Ala Ser 1160 1165 1170 Val Val Asn Ile Gln Lys Glu Ile Asp Arg Leu Asn Glu Val Ala 1175 1180 1185 Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu Gln Glu Leu Gly Lys 1190 1195 1200 Tyr Glu Gln Tyr Ile Lys Trp Pro Trp Tyr Ile Trp Leu Gly Phe 1205 1210 1215 Ile Ala Gly Leu Ile Ala Ile Val Met Val Thr Ile Met Leu Cys 1220 1225 1230 Cys Met Thr Ser Cys Cys Ser Cys Leu Lys Gly Cys Cys Ser Cys 1235 1240 1245 Gly Ser Cys Cys 1250 <![CDATA[<210> 18]]> <![CDATA[<211> 3759]]> <![CDATA[<212> DNA]]> <![CDATA[<213> 人工]]> <![CDATA[<220>]]> <![CDATA[<223> 編碼修飾SARS-CoV-2刺突蛋白之核苷酸序列(IN) ]]> <![CDATA[<400> 18]]> atgttcgtgt tcctggtgct gctgcccctg gtgagcagcc agtgcgtgaa cctgagaacc 60 agaacccagc tgccccccgc ctacaccaac agcttcacca gaggcgtgta ctaccccgac 120 aaggtgttca gaagcagcgt gctgcacagc acccaggacc tgttcctgcc cttcttcagc 180 aacgtgacct ggttccacgc catccacgtg agcggcacca acggcaccaa gagattcgac 240 aaccccgtgc tgcccttcaa cgacggcgtg tacttcgcca gcaccgagaa gagcaacatc 300 atcagaggct ggatcttcgg caccaccctg gacagcaaga cccagagcct gctgatcgtg 360 aacaacgcca ccaacgtggt gatcaaggtg tgcgagttcc agttctgcaa cgaccccttc 420 ctggacgtgt actaccacaa gaacaacaag agctggatgg agagcggcgt gtacagcagc 480 gccaacaact gcaccttcga gtacgtgagc cagcccttcc tgatggacct ggagggcaag 540 cagggcaact tcaagaacct gagagagttc gtgttcaaga acatcgacgg ctacttcaag 600 atctacagca agcacacccc catcaacctg gtgagagacc tgcctcaggg ctttagcgcc 660 ctggagccac tggtggacct gccaatcggc atcaacatca ccagattcca gaccctgctg 720 gccctgcaca gaagctacct gacaccaggc gattctagct ctggatggac agccggcgcc 780 gctgcctatt acgtgggcta cctgcagcct agaaccttcc tgctgaagta caacgagaac 840 ggcaccatca ccgatgccgt ggactgcgcc ctggatcccc tgagcgagac caagtgtacc 900 ctgaagagct tcaccgtgga gaagggcatc taccagacca gcaacttcag agtgcagccc 960 accgagagca tcgtgagatt ccccaacatc accaacctgt gccccttcgg cgaggtgttc 1020 aacgccacca gattcgccag cgtgtacgcc tggaacagaa agagaatcag caactgcgtg 1080 gccgactaca gcgtgctgta caacagcgcc agcttcagca ccttcaagtg ctacggcgtg 1140 agccccacca agctgaacga cctgtgcttc accaacgtgt acgccgacag cttcgtgatc 1200 agaggcgacg aggtgagaca gattgcccct ggccagaccg gcaagatcgc cgactacaac 1260 tacaagctgc ccgacgactt caccggctgc gtgatcgcct ggaacagcaa caacctggac 1320 agcaaggtgg gcggcaacta caactacaga tacagactgt tcagaaagag caacctgaag 1380 cccttcgaga gagacatcag caccgagatc taccaggccg gctctaagcc atgcaatggc 1440 gtggagggct tcaattgcta cttccccctg cagagctacg gcttccagcc caccaacggc 1500 gtgggctacc agccctacag agtggtggtg ctgagctttg aactgctgca cgcccctgcc 1560 accgtgtgcg gcccaaagaa gagcaccaat ctggtgaaga acaagtgcgt gaacttcaac 1620 ttcaacggcc tgaccggcac cggcgtgctg accgagagca acaagaagtt cctgcccttc 1680 cagcagttcg gcagagacat cgccgacacc accgatgccg tgagagatcc ccagaccctg 1740 gagatcctgg acatcacccc ctgtagcttt ggcggcgtga gcgtgattac ccccggcacc 1800 aataccagca accaggtggc cgtgctgtac cagggcgtga actgcaccga ggtgccagtg 1860 gccatccatg ccgaccagct gaccccaacc tggagagtgt acagcaccgg cagcaacgtg 1920 ttccagacaa gagccggctg tctgattggc gccgagcacg tgaataacag ctacgagtgc 1980 gatatcccaa tcggcgccgg catctgtgcc agctatcaga cccagaccaa tagcagaaga 2040 agagccagaa gcgtggccag ccagagcatc atcgcctaca ccatgagcct gggcgccgag 2100 aacagcgtgg cctacagcaa caacagcatc gccatcccca ccaacttcac catcagcgtg 2160 accaccgaga tcctgcccgt gagcatgacc aagaccagcg tggactgcac catgtacatc 2220 tgcggcgaca gcaccgagtg cagcaacctg ctgctgcagt acggcagctt ctgcacccag 2280 ctgaacagag ccctgaccgg catcgccgtg gagcaggaca agaacaccca ggaggtgttc 2340 gcccaggtga agcagatcta caagaccccc cccatcaagg acttcggcgg cttcaacttc 2400 agccagatcc tgcccgaccc cagcaagccc agcaagagaa gcttcatcga ggacctgctg 2460 ttcaacaagg tgaccctggc cgacgccggc ttcatcaagc agtacggcga ctgcctgggc 2520 gacatcgccg ccagagacct gatctgcgcc cagaagttta atggactgac agtgctgcca 2580 cccctgctga ccgatgagat gatcgcccag tacaccagcg ctctgctggc cggcacaatc 2640 accagcggct ggacatttgg agccggagcc gctctgcaga tcccatttgc catgcagatg 2700 gcctacagat tcaacggcat cggcgtgacc cagaacgtgc tgtacgagaa ccagaagctg 2760 atcgccaacc agttcaacag cgccatcggc aagatccagg acagcctgtc tagcacagcc 2820 tctgccctgg gcaagctgca gaacgtggtg aaccagaacg cccaggccct gaacaccctg 2880 gtgaagcagc tgagcagcaa cttcggcgcc atcagcagcg tgctgaacga catcctgagc 2940 agactggacc cccctgaggc cgaggtgcag atcgacagac tgatcaccgg cagactgcag 3000 agcctgcaga cctacgtgac ccagcagctg atcagagccg ccgaaatcag agccagcgcc 3060 aatctggccg ccaccaagat gagcgagtgc gtgctgggcc agagcaagag agtggacttc 3120 tgcggcaagg gctaccacct gatgagcttt ccccagagcg cccctcacgg cgtggtgttt 3180 ctgcacgtga cctacgtgcc tgcccaggag aagaacttca ccaccgcccc tgccatctgc 3240 cacgatggca aggcccactt ccctagagag ggcgtgttcg tgagcaacgg cacccactgg 3300 ttcgtgaccc agagaaactt ctacgagccc cagatcatca ccaccgacaa caccttcgtg 3360 agcggcaact gcgacgtggt gatcggcatc gtgaacaaca ccgtgtacga ccccctgcag 3420 cccgagctgg acagcttcaa ggaggagctg gacaagtact tcaagaacca caccagcccc 3480 gatgtggacc tgggcgatat cagcggcatc aatgccagcg tggtgaacat ccagaaggag 3540 atcgaccggc tcaatgaggt ggccaagaac ctgaacgaga gcctgatcga cctgcaggaa 3600 ctgggcaaat atgagcagta catcaagtgg ccctggtaca tctggctggg cttcatcgcc 3660 ggcctgatcg ccatcgtgat ggtgaccatc atgctgtgct gcatgaccag ctgctgcagc 3720 tgcctgaagg gctgctgcag ctgcgggtct tgttgctaa 3759
Claims (12)
- 一種DNA,其係編碼冠狀病毒(SARS CoV-2)之刺突蛋白或其片段者,且所含之一部分或全部密碼子就人類之表現得到最佳化。
- 如請求項1之DNA,其中SARS CoV-2基因組RNA之編碼刺突蛋白或其片段之鹼基序列中所含之密碼子的約50%以上被替換為其他密碼子。
- 如請求項1之DNA,其編碼刺突蛋白之全長。
- 如請求項1之DNA,其包含與序列編號3、12、14、16或18所表示之鹼基序列具有約90%以上之同一性之鹼基序列。
- 如請求項4之DNA,其具有序列編號3、12、14、16或18所表示之鹼基序列。
- 一種核酸構建物,其含有於人類中發揮功能之啟動子、及與上述啟動子功能性連結之如請求項1至5中任一項之DNA。
- 如請求項6之核酸構建物,其進而含有與如請求項1至5中任一項之DNA功能性連結之轉錄終止序列。
- 如請求項6或7之核酸構建物,其係組入至載體而成。
- 如請求項8之核酸構建物,其係組入至選自質體載體、噬菌體載體及病毒載體中之載體而成。
- 一種醫藥組合物,其含有如請求項6至9中任一項之核酸構建物。
- 如請求項10之醫藥組合物,其進而包含佐劑。
- 如請求項10或11之醫藥組合物,其係冠狀病毒感染症之疫苗。
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WO2023240159A2 (en) * | 2022-06-07 | 2023-12-14 | The Regents Of The University Of California | Sars-cov-2 multi-antigen universal vaccines |
CN115073565B (zh) * | 2022-06-13 | 2023-02-21 | 华素生物科技(北京)有限公司 | 重组新型冠状病毒s蛋白三聚体及其制备方法与应用 |
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DE10260805A1 (de) | 2002-12-23 | 2004-07-22 | Geneart Gmbh | Verfahren und Vorrichtung zum Optimieren einer Nucleotidsequenz zur Expression eines Proteins |
WO2007102578A1 (ja) | 2006-03-09 | 2007-09-13 | Keio University | 塩基配列設計方法 |
JP6007463B2 (ja) | 2011-04-18 | 2016-10-12 | 国立研究開発法人国立精神・神経医療研究センター | 薬剤送達粒子及びその製造方法 |
US10543261B2 (en) | 2012-08-31 | 2020-01-28 | Osaka University | DNA vaccine containing VEGF-specific epitope and/or angiopoietin-2-specific epitope |
CA3041307A1 (en) | 2016-10-21 | 2018-04-26 | Giuseppe Ciaramella | Human cytomegalovirus vaccine |
EP3530674B1 (en) | 2016-10-23 | 2024-04-24 | Denka Company Limited | Polypeptide monomer, associated product formed of said polypeptide monomer having cell penetration function, norovirus component vaccine for subcutaneous, intradermal, percutaneous or intramuscular administration and having said associated product as effective component thereof, and method for producing said associated product |
GB201705765D0 (en) | 2017-04-10 | 2017-05-24 | Univ Oxford Innovation Ltd | HBV vaccine |
RU2731342C9 (ru) * | 2020-08-22 | 2021-10-05 | федеральное государственное бюджетное учреждение "Национальный исследовательский центр эпидемиологии и микробиологии имени почетного академика Н.Ф. Гамалеи" Министерства здравоохранения Российской Федерации | Фармацевтическое средство и способ его использования для индукции специфического иммунитета против вируса тяжелого острого респираторного синдрома SARS-CoV-2 (варианты) |
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