TW202216739A - mRNA and novel coronavirus mRNA vaccine containing the same which is economical and efficient, safer and more efficient, more stable in structure, and more efficient in protein expression - Google Patents

mRNA and novel coronavirus mRNA vaccine containing the same which is economical and efficient, safer and more efficient, more stable in structure, and more efficient in protein expression Download PDF

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TW202216739A
TW202216739A TW109141038A TW109141038A TW202216739A TW 202216739 A TW202216739 A TW 202216739A TW 109141038 A TW109141038 A TW 109141038A TW 109141038 A TW109141038 A TW 109141038A TW 202216739 A TW202216739 A TW 202216739A
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黃英
李波
湯勇
楊錦霞
李濤
李冬麗
張樂
李佑平
熊恒
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大陸商深圳吉諾因生物科技有限公司
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Abstract

The present invention discloses an isolated mRNA, which includes mRNA encoding a novel coronavirus S1 protein, and the amino acid sequence of the S1 protein is represented by SEQ ID NO: 2; it also includes one or more of the following (a) to (d): (a) 5'-cap structure, (b) 3'-polyAdenylic acid, (c) 5'-UTR, (d) 3'-UTR; also disclosed are DNA, compositions comprising the same , liposome nanoparticles, mRNA vaccines, pharmaceutical compositions and kits against the novel coronavirus. The mRNA can be highly expressed in cells; it is economical and efficient, safer and more efficient, more stable in structure, and more efficient in protein expression, and can continuously express the novel coronavirus S1 protein. When it is prepared as liposome nanoparticles/vaccine, a very small dose can be used to achieve sufficient protection effect, the immunogenicity is reduced, and at the same time the body's immune system can be activated to generate humoral immunity and cellular immunity.

Description

一種mRNA及包含其的新型冠狀病毒mRNA疫苗An mRNA and a novel coronavirus mRNA vaccine containing the same

本申請要求申請日為2020/10/27的中國專利申請2020111641442的優先權。本申請引用上述中國專利申請的全文。This application claims the priority of Chinese patent application 2020111641442 with an application date of 2020/10/27. This application cites the full text of the above Chinese patent application.

本發明涉及一種mRNA、可轉錄其的DNA、包含其的組成物、脂質體奈米顆粒、針對新型冠狀病毒的mRNA疫苗、藥物組成物和套組。The present invention relates to an mRNA, a DNA that can transcribe the same, a composition comprising the same, a liposome nanoparticle, an mRNA vaccine against a novel coronavirus, a pharmaceutical composition and a kit.

新冠肺炎是指由新型冠狀病毒(SARS-CoV-2，簡稱新冠病毒)引起的肺炎，是一種急性感染性肺炎，它具有人傳染人的能力，感染初期病人有發熱、乏力、乾咳的症狀,嚴重者可出現呼吸困難、呼吸窘迫症候群或敗血症休克。冠狀病毒(Coronavirus)是一個常見而又古老的病毒大家系，現今已確認的人源冠狀病毒(Human Coronavirus，HCoVs)有6種，其中包括引起急性呼吸道症候群(SARS)的SARS冠狀病毒(SARS-CoV)、引起中東呼吸道症候群(MERS)的MERS冠狀病毒(MERS-CoV)以及引起新冠肺炎(COVID-19)的新型冠狀病毒(SARS-CoV-2)。New coronary pneumonia refers to pneumonia caused by the new coronavirus (SARS-CoV-2, referred to as new coronavirus), which is an acute infectious pneumonia. It has the ability to infect people. In the early stage of infection, patients have symptoms of fever, fatigue, and dry cough. In severe cases, dyspnea, respiratory distress syndrome or septic shock may occur. Coronavirus (Coronavirus) is a common and ancient family of viruses, there are 6 confirmed human coronaviruses (Human Coronavirus, HCoVs), including SARS coronavirus (SARS-CoV) that causes acute respiratory syndrome (SARS). CoV), the MERS coronavirus (MERS-CoV) that causes Middle East Respiratory Syndrome (MERS), and the novel coronavirus (SARS-CoV-2) that causes COVID-19.

新型冠狀病毒SARS-CoV-2的RNA基因組為單股、正鏈RNA，5’端的前約2/3長度區域編碼非結構蛋白，剩餘1/3區域編碼結構蛋白，依次為棘蛋白(Spike蛋白/S蛋白)、套膜蛋白(envelope/E)、膜蛋白(membrane/M)、核殼蛋白(nucleocapsid/N)以及附加蛋白。S蛋白是病毒表面最重要的跨膜糖蛋白，S蛋白又分為S1和S2兩部分，其中N端為S1，S1上含有受體結合區，主要功能是與宿主細胞表面受體結合完成入侵；C端為S2，形成細長突起的柄部分，主要功能媒介細胞間的融合和病毒遺傳物質注入宿主細胞。新型冠狀病毒SARS-CoV-2高度守恆的S蛋白序列含有1273個胺基酸，其中S蛋白中與宿主細胞表面受體ACE2結合區域(RBD，receptor binding domain)為S蛋白的S1次單元，位於1-661位胺基酸。對於病毒而言，中和抗體能夠完全阻止病毒進入人體細胞，病毒蛋白上抗體結合位址的位置和數量是影響人體產生中和抗體能力的重要因素。S1作為受體結合域，除RBD上有抗體結合位址，在RBD附近246-257位胺基酸處也存在一個強抗體結合位址。有研究表明(DNA vaccine encoding Middle East respiratory syndrome coronavirus S1 protein induces protective immune responses in mice, Vaccine, 14 Mar 2017, 35(16):2069-2075)，以S蛋白的S1次單元為標的區域設計的疫苗，同樣能引起身體的體液免疫和/或細胞免疫，避免或減緩冠狀病毒對身體的感染，不僅如此，相比於S蛋白或RBD標的區域疫苗，以S1結構域為標的區域設計的疫苗可能引發身體產生更多的中和抗體，並且在小鼠中，S1疫苗引發的IgG2a/IgG1更加平衡，安全性更高。除了中和抗體，人體還依賴細胞毒性CD8+T細胞和輔助CD4+T細胞來完全清除病毒。有研究預測(Binbin Chen et al., Potential T-cell and B-cell Epitopes of 2019-nCoV, bioRxiv, 2020)，SARS-CoV-2病毒基因組上可能存在405個T細胞抗原決定位，其中S1部分的T細胞抗原決定位覆蓋率相當高，可見除RBD外的S1序列區域仍然含有大量不可小覷的B細胞/T細胞抗原決定位。除此之外，有研究表明(Site-specific analysis of the SARS-CoV-2 glycan shield, DOI: 10.1101/2020.03.26.010322)，S1蛋白序列部分上包含13個的糖基化位址，其中RBD區域2個，糖基化參與調控蛋白質在組織和細胞中的定位、功能和活性，會影響細胞辨識、細胞分化、訊號傳導、免疫反應等多種重要的生命活動。 The RNA genome of the new coronavirus SARS-CoV-2 is single-stranded, positive-strand RNA, the first 2/3 of the 5'-end region encodes non-structural proteins, and the remaining 1/3 region encodes structural proteins, followed by spike proteins (Spike proteins). /S protein), envelope protein (envelope/E), membrane protein (membrane/M), nucleocapsid/N, and additional proteins. The S protein is the most important transmembrane glycoprotein on the surface of the virus. The S protein is divided into two parts, S1 and S2, of which the N-terminus is S1, and the S1 contains a receptor binding region. Its main function is to bind to the host cell surface receptor to complete the invasion. ; The C-terminus is S2, forming the stalk part of the elongated protrusion, and the main function is to mediate the fusion between cells and the injection of viral genetic material into the host cell. The highly conserved S protein sequence of the new coronavirus SARS-CoV-2 contains 1273 amino acids, of which the receptor binding domain (RBD, receptor binding domain) in the S protein and the host cell surface receptor ACE2 is the S1 subunit of the S protein, located in 1-661 amino acid. For viruses, neutralizing antibodies can completely prevent the virus from entering human cells. The location and number of antibody binding sites on viral proteins are important factors that affect the ability of the human body to produce neutralizing antibodies. S1 serves as the receptor binding domain. In addition to the antibody binding site on the RBD, there is also a strong antibody binding site at the 246-257 amino acid near the RBD. Studies have shown that (DNA vaccine encoding Middle East respiratory syndrome coronavirus S1 protein induces protective immune responses in mice, Vaccine, 14 Mar 2017, 35(16):2069-2075), the vaccine designed with the S1 subunit of S protein as the target area , can also cause the body's humoral immunity and/or cellular immunity, avoid or slow down the infection of the coronavirus to the body, not only that, compared with the S protein or RBD-targeted regional vaccines, vaccines designed with the S1 domain as the target region may cause The body produced more neutralizing antibodies, and in mice, the IgG2a/IgG1 elicited by the S1 vaccine was more balanced and safer. In addition to neutralizing antibodies, the body relies on cytotoxic CD8+ T cells and helper CD4+ T cells for complete virus clearance. Some studies predict (Binbin Chen et al ., Potential T-cell and B-cell Epitopes of 2019-nCoV, bioRxiv, 2020) that there may be 405 T cell epitopes on the SARS-CoV-2 virus genome, of which the S1 part The coverage of T cell epitopes is quite high, and it can be seen that the S1 sequence region other than RBD still contains a large number of B cell/T cell epitopes that cannot be underestimated. In addition, studies have shown (Site-specific analysis of the SARS-CoV-2 glycan shield, DOI: 10.1101/2020.03.26.010322) that the S1 protein sequence contains 13 glycosylation sites, of which the RBD region 2. Glycosylation is involved in regulating the localization, function and activity of proteins in tissues and cells, and affects various important life activities such as cell identification, cell differentiation, signal transduction, and immune response.

疫苗是指為了預防、控制傳染病的發生、流行，用於人體預防接種的疫苗類預防性生物製品。自2020年1月11日中國向全球分享了新型冠狀病毒的基因序列開始，各國的疫苗研發工作都在緊鑼密鼓地進行著。根據世界衛生組織的資料，現在全球有70多種針對新冠病毒的疫苗正在積極研發，其中有8個候選疫苗已進入臨床階段，主要包括以病毒載體疫苗、減毒/去活化疫苗、重組蛋白疫苗為代表的傳統疫苗和以mRNA疫苗、DNA疫苗為代表的新型核酸疫苗。其中，去活化疫苗、病毒載體疫苗、DNA疫苗都是透過將病原體本身或部分或其抗原基因注入身體內引起免疫反應。去活化疫苗目前存在的主要缺陷在於：1)一般免疫效果較弱，只能誘導產生體液免疫；2)誘導產生免疫反應持續時間較短，需要多次接種；3)去活化劑對病毒抗原有著不同程度的影響；4)各個抗原成分之間的疫苗反應不平衡，可能誘發其他疾病。腺病毒載體疫苗目前存在的主要缺陷在於：1)誘導產生免疫反應持續時間較短；2)缺乏靶向性，可能會感染正常細胞引起不良反應；3)若身體本身存在對腺病毒載體的免疫力，身體內的腺病毒抗體會轉而攻擊載體，令疫苗失效。DNA疫苗目前存在的主要缺陷在於：1)表現效率可能不高；2)病毒基因有併入到宿主染色體的風險；3)DNA需要入細胞核，再經轉錄、轉譯等過程，過程長起效慢。Vaccines refer to vaccine-type preventive biological products used for human vaccination in order to prevent and control the occurrence and prevalence of infectious diseases. Since China shared the genetic sequence of the new coronavirus with the world on January 11, 2020, vaccine research and development work in various countries has been in full swing. According to the World Health Organization, more than 70 vaccines against the new coronavirus are being actively developed around the world, of which 8 candidate vaccines have entered the clinical stage, mainly including viral vector vaccines, attenuated/deactivated vaccines, and recombinant protein vaccines as Representative traditional vaccines and new nucleic acid vaccines represented by mRNA vaccines and DNA vaccines. Among them, deactivated vaccines, viral vector vaccines, and DNA vaccines all induce immune responses by injecting pathogens themselves or parts or their antigenic genes into the body. The main defects of deactivated vaccines are: 1) the general immune effect is weak, and can only induce humoral immunity; 2) the duration of the induced immune response is short, requiring multiple vaccinations; 3) the deactivator has a strong effect on viral antigens. Different degrees of influence; 4) Unbalanced vaccine responses among various antigenic components may induce other diseases. The main drawbacks of adenovirus vector vaccines are: 1) The duration of the immune response induced is short; 2) The lack of targeting may infect normal cells and cause adverse reactions; 3) If the body itself has immunity to adenovirus vectors force, the adenovirus antibodies in the body will turn to attack the vector, rendering the vaccine ineffective. The main defects of DNA vaccines are: 1) The performance efficiency may not be high; 2) The virus gene has the risk of being incorporated into the host chromosome; 3) The DNA needs to enter the nucleus, and then undergo transcription, translation and other processes, and the process is long and slow. .

mRNA疫苗是將抗原蛋白的基因序列結合修飾序列，透過體外轉錄、純化等程序製備得到的mRNA，透過特定的遞送系統(或載體)包裹mRNA，將其導入身體細胞(例如注射至人體)，隨後mRNA在細胞內釋放並表現目標蛋白、活化身體的免疫系統，從而誘導特異性的體液免疫和細胞免疫反應，從而使身體獲得免疫保護的一種核酸製劑。具體為：免疫系統中最強大的抗原呈現細胞一樹突狀細胞(DC細胞)，一方面將完整的抗原呈現給B細胞，啟動體液免疫反應，產生抗體，阻止病毒侵染宿主細胞，起預防作用；另一方面透過蛋白酶水解抗原，呈現給主要組織相容性複合物(MHC)上的CD8+和CD4+T細胞，啟動細胞免疫反應，消滅已被病毒侵染的宿主細胞，起治療作用。mRNA疫苗作為新興疫苗，有著相比於其他疫苗的優勢，但目前mRNA疫苗的研發仍處於臨床研究階段，還沒有獲批上市的mRNA疫苗，由此可見mRNA疫苗的研發仍存在不少困難點。mRNA vaccines are mRNAs prepared by combining the gene sequences of antigenic proteins with modified sequences, through in vitro transcription, purification and other procedures, wrapping the mRNA through a specific delivery system (or carrier), and introducing it into body cells (such as injection into the human body), followed by mRNA is a nucleic acid preparation that is released in cells and expresses target proteins, activates the body's immune system, and induces specific humoral and cellular immune responses, thereby enabling the body to obtain immune protection. Specifically: Dendritic cells (DC cells) are the most powerful antigen-presenting cells in the immune system. On the one hand, they present complete antigens to B cells, initiate humoral immune responses, produce antibodies, and prevent viruses from infecting host cells. On the other hand, the protease hydrolyzes the antigen and presents it to the CD8+ and CD4+ T cells on the major histocompatibility complex (MHC) to initiate a cellular immune response to eliminate the host cells that have been infected by the virus, and play a therapeutic role. As an emerging vaccine, mRNA vaccine has advantages over other vaccines. However, the research and development of mRNA vaccine is still in the clinical research stage, and there is no mRNA vaccine approved for marketing. This shows that there are still many difficulties in the development of mRNA vaccine.

mRNA疫苗的研製可分為mRNA疫苗的結構設計、mRNA的生產、mRNA的純化和mRNA的遞送幾大步驟，其中mRNA疫苗的結構設計關係mRNA的穩定性和目標蛋白的表現能力，mRNA的遞送技術關係到mRNA的轉染效率和免疫反應的能力及類型，這兩點是目前mRNA疫苗領域的開發困難點與核心技術。The development of mRNA vaccine can be divided into several major steps: structural design of mRNA vaccine, production of mRNA, purification of mRNA and delivery of mRNA. The structural design of mRNA vaccine is related to the stability of mRNA and the expression ability of target protein, and the delivery technology of mRNA. It is related to the transfection efficiency of mRNA and the ability and type of immune response, which are the current difficulties and core technologies in the development of mRNA vaccines.

首先，mRNA疫苗結構多樣化，mRNA中的帽子結構(Cap)的類型和加帽方式、5’UTR區的長度和來源、編碼抗原蛋白的開放閱讀框(open reading frame，ORF)、3’UTR區的長度和來源和Poly(A)尾的長度和加尾方式等都會影響到mRNA的穩定性和蛋白表現的效率。新型冠狀病毒SARS-CoV-2的RNA基因組編碼多種抗原蛋白，如棘蛋白(Spike蛋白/S蛋白)、套膜蛋白(envelope /E蛋白)、膜蛋白(membrane /M蛋白)、核殼蛋白(nucleocapsid/N蛋白)等，如何選擇能達到更好免疫效果的抗原蛋白是mRNA疫苗的首要困難點。mRNA帽子結構(Cap)有三種：CAP 0型、CAP I型和CAP II型，UTR可來自任何自然界存在基因的UTR，即使是人源蛋白基因的UTR也有十幾萬種，因此疫苗設計需要從上億種UTR組合中選擇合適的組合來穩定mRNA、提高蛋白表現。除此之外，mRNA在體外轉錄過程中，轉錄質體、加帽、加尾、幾十種轉錄基質的選擇都會影響mRNA的穩定性、轉錄效率和蛋白表現能力。其次，如何實現mRNA的高效轉運也是阻礙mRNA疫苗研發的技術困難點。由於mRNA自身穩定性差，易被體內外的核酸酶降解，所以在保證其安全性的基礎上，有效和耐受性好的遞送技術對於充分發揮mRNA疫苗的潛力是不可或缺的。目前mRNA疫苗遞送技術有十幾種，常用的安全的遞送方式為載體遞送，遞送載體主要包括病毒載體，如慢病毒、腺病毒和仙台病毒等；非病毒載體，如奈米脂質體顆粒(LNP)、無機奈米粒子、樹狀大分子等。即使是主流的LNP奈米脂質體粒子包裹RNA法，也需要從眾多種脂質體材料中選擇的最合適的材料及其配比。First, the structure of mRNA vaccine is diversified, the type and capping method of cap structure (Cap) in mRNA, the length and source of 5'UTR region, the open reading frame (ORF) encoding antigenic protein, 3'UTR The length and source of the region and the length and tailing method of the Poly(A) tail will affect the stability of mRNA and the efficiency of protein expression. The RNA genome of the new coronavirus SARS-CoV-2 encodes a variety of antigenic proteins, such as spike protein (Spike protein/S protein), envelope protein (envelope/E protein), membrane protein (membrane/M protein), nucleocapsid protein ( nucleocapsid/N protein), etc., how to select antigenic proteins that can achieve better immune effect is the primary difficulty of mRNA vaccines. There are three types of mRNA cap structures (Caps): CAP type 0, CAP type I and CAP type II. UTRs can be derived from UTRs of any naturally occurring gene, and there are hundreds of thousands of UTRs even for human protein genes. Therefore, vaccine design needs to start from Choose the right combination from hundreds of millions of UTR combinations to stabilize mRNA and improve protein expression. In addition, in the process of mRNA transcription in vitro, the choice of transcription plastids, capping, tailing, and dozens of transcription substrates will affect the stability, transcription efficiency and protein expression ability of mRNA. Secondly, how to achieve efficient mRNA transport is also a technical difficulty that hinders the development of mRNA vaccines. Since mRNA has poor stability and is easily degraded by nucleases in vivo and in vitro, effective and well-tolerated delivery technology is indispensable for fully exploiting the potential of mRNA vaccines on the basis of ensuring its safety. At present, there are more than a dozen mRNA vaccine delivery technologies. The commonly used safe delivery method is vector delivery, which mainly includes viral vectors, such as lentivirus, adenovirus and Sendai virus; ), inorganic nanoparticles, dendrimers, etc. Even the mainstream LNP nanoliposome particle-encapsulated RNA method requires the most suitable materials and their ratios selected from a variety of liposome materials.

考慮到上述傳統疫苗安全性低、療效不佳、生產週期長和產生免疫所需時間長等原因，而且疫苗研發所需時間長，各種疫苗存在著各自的優缺點和技術壁壘，進入臨床的疫苗的安全性和有效性還未經證明，仍有失敗的風險，目前仍然沒有新冠病毒肺炎疫苗獲批上市，也沒有確定哪種疫苗的效果最好，因此開發新的冠狀病毒的治療藥物及疫苗、積極防治新型冠狀病毒感染(SARS-CoV-2)迫在眉睫。Considering the above-mentioned reasons such as low safety, poor efficacy, long production cycle and long time required to generate immunity, and the long time required for vaccine development, various vaccines have their own advantages, disadvantages and technical barriers. The safety and efficacy of SARS-CoV-2 have not been proven, and there is still a risk of failure. At present, there is still no new coronavirus pneumonia vaccine approved for marketing, and it has not been determined which vaccine has the best effect. Therefore, the development of new coronavirus therapeutic drugs and vaccines , Active prevention and control of novel coronavirus infection (SARS-CoV-2) is imminent.

本發明所要解決的技術問題是為了克服現有技術中商品化的新冠病毒疫苗不足等缺陷，提供了一種mRNA、可轉錄其的DNA、包含其的組成物、脂質體奈米顆粒、針對新冠病毒的mRNA疫苗、藥物組成物和套組。本發明的經過密碼子最適化後或者進一步經過修飾所得的mRNA在細胞中可以高度表現；從而可以獲得分子層級精准設計的類似於完全加工成熟的mRNA分子產品；不僅經濟高效，而且所述mRNA更加安全、高效，結構更加穩定，蛋白表現效率更高，能夠持續地表現新冠病毒S1蛋白。將本發明的mRNA製備成脂質體奈米顆粒/疫苗時，可以實現使用極小劑量就能達到足夠的保護效果，且免疫原性有所降低，同時能夠活化身體免疫系統產生體液免疫和細胞免疫。將本發明的mRNA製備成疫苗時，成本較低、生產週期短，有利於大規模工業生產。The technical problem to be solved by the present invention is to overcome the deficiencies of the commercialized new coronavirus vaccine in the prior art, and provide a mRNA, a DNA that can transcribe the same, a composition containing the same, a liposome nanoparticle, and a vaccine against the new coronavirus. mRNA vaccines, pharmaceutical compositions and kits. The mRNA obtained after codon optimization or further modification of the present invention can be highly expressed in cells; thus, a molecular product similar to a fully processed mature mRNA molecular product can be obtained with precise design at the molecular level; not only is economical and efficient, but also the mRNA is more Safe, efficient, more stable in structure, more efficient in protein expression, and able to continuously express the new coronavirus S1 protein. When the mRNA of the present invention is prepared into liposome nanoparticles/vaccine, a very small dose can be used to achieve sufficient protection effect, and the immunogenicity is reduced, and at the same time, the body's immune system can be activated to generate humoral immunity and cellular immunity. When the mRNA of the present invention is prepared into a vaccine, the cost is low and the production period is short, which is favorable for large-scale industrial production.

本領域技術人員公知，mRNA不具有感染性和併入性，在體內是透過正常的途徑降解的，不存在潛在的感染或***突變的風險。完整結構的mRNA包含以下幾個必要的元件，依次為帽子結構(Cap)、5’UTR區、編碼抗原蛋白的開放閱讀框(open reading frame，ORF)、3’UTR區和Poly(A)尾結構，其中帽子結構(Cap)、Poly(A)的長度和加尾方式以及UTR的長度和來源都會影響到mRNA的穩定性和蛋白表現的效率。mRNA疫苗結構設計需要從大量的元件資料庫中篩選到提高mRNA的安全性、穩定性、有效性和轉譯效率的結構。而本發明人透過大量實驗和摸索，從上億種mRNA疫苗可能的結構中，分析得到了一種能夠安全、高效、穩定表現新冠病毒S1蛋白的mRNA序列，將其製備成疫苗等時能夠同時活化身體免疫系統產生體液免疫和細胞免疫。It is well known to those skilled in the art that mRNA is non-infectious and non-incorporating, and is degraded in vivo through normal pathways, without the risk of potential infection or insertional mutation. The complete structure of mRNA contains the following necessary elements, which are cap structure (Cap), 5'UTR region, open reading frame (ORF) encoding antigenic protein, 3'UTR region and Poly (A) tail. The structure, including the cap structure (Cap), the length and tailing of Poly (A), and the length and source of UTR, all affect the stability of mRNA and the efficiency of protein expression. The structural design of mRNA vaccines needs to be screened from a large number of component databases to improve the safety, stability, efficacy and translation efficiency of mRNA structures. Through a lot of experiments and explorations, the inventors have analyzed and obtained an mRNA sequence that can safely, efficiently and stably express the S1 protein of the new coronavirus from hundreds of millions of possible structures of mRNA vaccines, which can be simultaneously activated when preparing vaccines, etc. The body's immune system produces humoral and cellular immunity.

為了解決上述技術問題，本發明第一方面提供了一種分離的mRNA，其包括編碼來源於SARS-CoV-2病毒的S1蛋白或其免疫原性片段的mRNA，其中，所述S1蛋白的胺基酸序列如SEQ ID NO: 2所示；In order to solve the above-mentioned technical problems, the first aspect of the present invention provides an isolated mRNA comprising mRNA encoding S1 protein or an immunogenic fragment thereof derived from SARS-CoV-2 virus, wherein the amine group of the S1 protein The acid sequence is shown in SEQ ID NO: 2;

所述分離的mRNA還包括以下(a)~(d)中的一種或多種(在某一較佳實施例中可同時含有5’-帽結構、3’端聚腺苷酸序列、5’UTR和3’UTR)： (a)5’-帽結構，優選為CAP 0型、CAP I型或CAP II型的帽結構或其類似物；所述CAP 0型的帽結構優選為7-甲基鳥苷帽結構(又可寫為7Me Gppp Pu(Pu為嘌呤核苷)或m7G5'ppp5'Np)，所述CAP I型的帽結構優選為N7mGpppAm(又可寫為7Me Gppp XMe或m7G5'ppp5'NmpNp)；本發明中，所述的CAP I型的帽結構可以是透過選擇目前常用的、穩定的重組痘苗病毒衍生酶和2’-O甲基轉移酶兩步酶法進行mRNA的體外加帽反應。從而使得加帽穩定，加帽效率高，不會出現部分mRNA加帽失敗的結果，也不會在體外轉錄過程中與GTP發生競爭減少mRNA產量。 (b)3’-聚腺苷酸，其序列優選為包含16-150個腺苷核苷酸的序列，更優選包含50-120個腺苷核苷酸的序列； (c)5’-UTR，所述5’-UTR優選為核糖體蛋白L32的5’-UTR，例如為人核糖體蛋白L32的5’-UTR，其序列優選如SEQ ID NO: 3(5’-GGGGCGCTGCCTACGGAGGTGGCAGCCATCTCCTTCTCGGCATCAAGCTTACC-3’)所示； (d)3’-UTR，所述3’-UTR的序列優選為β球蛋白的3’-UTR和α球蛋白的3’-UTR，例如為人β球蛋白(HBB)的3’-UTR和人α球蛋白(HBA)的3’-UTR，其序列優選如SEQ ID NO: 4(5’-GCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGC-3’)或SEQ ID NO: 7(5’-TGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC-3’)所示。本發明中，所述的UTR(非編碼區，Untranslated Region)可來源於脊椎動物、哺乳動物等，考慮到疫苗的安全性，優選人源蛋白基因UTR。 The isolated mRNA also includes one or more of the following (a) to (d) (in a preferred embodiment, it may simultaneously contain a 5'-cap structure, a 3'-terminal polyadenylation sequence, a 5'UTR and 3'UTR): (a) a 5'-cap structure, preferably a cap structure of CAP 0 type, CAP I type or CAP II type or an analog thereof; the cap structure of said CAP 0 type is preferably a 7-methylguanosine cap structure (also Can be written as 7Me Gppp Pu (Pu is a purine nucleoside) or m7G5'ppp5'Np), the cap structure of the CAP I type is preferably N7mGpppAm (can be written as 7Me Gppp XMe or m7G5'ppp5'NmpNp); the present invention , the cap structure of the CAP I type can be the capping reaction of mRNA in vitro by selecting the commonly used, stable recombinant vaccinia virus-derived enzyme and 2'-O methyltransferase two-step enzymatic method. As a result, capping is stable, the capping efficiency is high, and there will be no failure of partial mRNA capping, and it will not compete with GTP during in vitro transcription to reduce mRNA production. (b) 3'-polyadenylic acid, the sequence of which is preferably a sequence comprising 16-150 adenosine nucleotides, more preferably a sequence comprising 50-120 adenosine nucleotides; (c) 5'-UTR, the 5'-UTR is preferably the 5'-UTR of ribosomal protein L32, such as the 5'-UTR of human ribosomal protein L32, the sequence of which is preferably as shown in SEQ ID NO: 3(5 '-GGGGCGCTGCCTACGGAGGTGGCAGCCATCTCCTTCTCGGCATCAAGCTTACC-3'); (d) 3'-UTR, the sequence of the 3'-UTR is preferably the 3'-UTR of β-globulin and the 3'-UTR of α-globulin, such as the 3'-UTR of human β-globulin (HBB)和人α球蛋白(HBA)的3'-UTR，其序列優選如SEQ ID NO: 4(5'-GCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGC-3')或SEQ ID NO: 7(5'-TGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC-3')所示。 In the present invention, the UTR (non-coding region, Untranslated Region) can be derived from vertebrates, mammals, etc. In consideration of the safety of the vaccine, the UTR of the human protein gene is preferred.

較佳地，所述免疫原性片段為所述S1蛋白的RBD結構域(receptor binding domain，RBD，受體結合結構域，負責辨識細胞的受體)。Preferably, the immunogenic fragment is the RBD domain (receptor binding domain, RBD, receptor binding domain, the receptor responsible for recognizing cells) of the S1 protein.

較佳地，所述的3’-聚腺苷酸透過體外酵素催化反應添加到所述編碼來源於SARS-CoV-2病毒的S1蛋白的mRNA上。Preferably, the 3'-polyadenylic acid is added to the mRNA encoding the S1 protein derived from the SARS-CoV-2 virus through an in vitro enzyme-catalyzed reaction.

較佳地，所述的3’-聚腺苷酸透過將其連接到包含所述編碼來源於SARS-CoV-2病毒的S1蛋白的mRNA的轉錄模板DNA之後進行轉錄(通常是將所述的3’-聚腺苷酸提前設計在所述mRNA的轉錄模板DNA質體上，然後進行轉錄)。Preferably, the 3'-polyadenylic acid is transcribed by connecting it to the transcription template DNA comprising the mRNA encoding the S1 protein derived from the SARS-CoV-2 virus (usually the 3'-polyadenylation is designed in advance on the transcription template DNA plastid of the mRNA, followed by transcription).

較佳地，所述分離的mRNA進一步包含以下(e)~(f)中的一種或兩種： (e)訊號胜肽，所述訊號胜肽優選為人IgE訊號胜肽或人IgG訊號胜肽或小鼠IgK訊號胜肽；所述人IgE訊號胜肽的序列如SEQ ID NO: 5(MDWTWILFLVAAATRVHS)所示；所述人IgG訊號胜肽的序列如SEQ ID NO: 8(MGWSCIILFLVATATGVHS)所示；所述小鼠IgK訊號胜肽的序列如SEQ ID NO: 9(METDTLLLWVLLLWVPGSTGD)所示；其通常位於起始密碼子之後，為一段編碼疏水性胺基酸序列的RNA區域，負責把蛋白質引導到細胞含不同膜結構的次細胞胞器內。 (f)多核苷酸修飾，所述多核苷酸優選N1-甲基假尿苷(m1ΨTP)、假尿苷(Ψ)和5-甲基尿苷(m5U)中的一種或多種。 Preferably, the isolated mRNA further comprises one or both of the following (e) to (f): (e) a signal peptide, the signal peptide is preferably a human IgE signal peptide or a human IgG signal peptide or a mouse IgK signal peptide; the sequence of the human IgE signal peptide is as shown in SEQ ID NO: 5 (MDWTWILFLVAAATRVHS ); the sequence of the human IgG signal peptide is shown in SEQ ID NO: 8 (MGWSCIILFLVATATGVHS); the sequence of the mouse IgK signal peptide is shown in SEQ ID NO: 9 (METDTLLLWVLLLWVPGSTGD); it is usually located in After the start codon, there is an RNA region encoding a hydrophobic amino acid sequence responsible for directing proteins to subcellular organelles containing different membrane structures. (f) modification of a polynucleotide, the polynucleotide is preferably one or more of N1-methylpseudouridine (m1ΨTP), pseudouridine (Ψ) and 5-methyluridine (m5U).

在某一較佳實施例中，所述的分離的mRNA，任選地從5’末端到3’末端，包括CAP I型的帽結構、人核糖體蛋白L32的5’-UTR、所述的編碼來源於SARS-CoV-2病毒的S1蛋白或其免疫原性片段的mRNA或其轉錄模板DNA或其變體、人α珠蛋白的3’-UTR和包含120個腺苷核苷酸的3’-聚腺苷酸。例如所述的分離的mRNA從5’末端到3’末端依次由CAP I型的帽結構、人核糖體蛋白L32的5’-UTR、所述的編碼來源於SARS-CoV-2病毒的S1蛋白或其免疫原性片段的mRNA或其轉錄模板DNA或其變體、人α珠蛋白的3’-UTR和包含120個腺苷核苷酸的3’-聚腺苷酸組成(例如本發明實施例部分所述的SARS-CoV-2-A)。In a preferred embodiment, the isolated mRNA, optionally from the 5' end to the 3' end, includes the cap structure of type CAP I, the 5'-UTR of human ribosomal protein L32, the mRNA encoding the S1 protein derived from SARS-CoV-2 virus or its immunogenic fragment or its transcription template DNA or its variant, the 3'-UTR of human alpha globin and the 3'-UTR comprising 120 adenosine nucleotides '-polyAdenylate. For example, the isolated mRNA is composed of the cap structure of CAP I, the 5'-UTR of human ribosomal protein L32, and the S1 protein encoding the SARS-CoV-2 virus from the 5' end to the 3' end. The mRNA of its immunogenic fragment or its transcription template DNA or its variant, the 3'-UTR of human alpha globin and the 3'-polyadenylic acid comprising 120 adenosine nucleotides (for example, the implementation of the present invention) SARS-CoV-2-A described in the Examples section).

在某一較佳實施例中，所述的分離的mRNA，任選地從5’末端到3’末端，包括CAP I型的帽結構、人核糖體蛋白L32的5’-UTR、所述的編碼來源於SARS-CoV-2病毒的S1蛋白或其免疫原性片段的mRNA或其轉錄模板DNA或其變體、人β珠蛋白的3’-UTR和包含120個腺苷核苷酸的3’-聚腺苷酸。例如所述的分離的mRNA從5’末端到3’末端依次由CAP I型的帽結構、人核糖體蛋白L32的5’-UTR、所述的編碼來源於SARS-CoV-2病毒的S1蛋白或其免疫原性片段的mRNA或其轉錄模板DNA或其變體、人β珠蛋白的3’-UTR和包含120個腺苷核苷酸的3’-聚腺苷酸組成(例如本發明實施例部分所述的SARS-CoV-2-B、SARS-CoV-2-C或SARS-CoV-2-D)。In a preferred embodiment, the isolated mRNA, optionally from the 5' end to the 3' end, includes the cap structure of type CAP I, the 5'-UTR of human ribosomal protein L32, the mRNA encoding the S1 protein derived from SARS-CoV-2 virus or its immunogenic fragment or its transcription template DNA or its variant, the 3'-UTR of human β-globin and the 3'-UTR comprising 120 adenosine nucleotides '-polyAdenylate. For example, the isolated mRNA is composed of the cap structure of CAP I, the 5'-UTR of human ribosomal protein L32, and the S1 protein encoding the SARS-CoV-2 virus from the 5' end to the 3' end. The mRNA of its immunogenic fragment or its transcription template DNA or its variant, the 3'-UTR of human beta globin and the 3'-polyadenylic acid comprising 120 adenosine nucleotides (such as the implementation of the present invention) SARS-CoV-2-B, SARS-CoV-2-C or SARS-CoV-2-D as described in the Examples section).

在某一較佳實施例中，所述的分離的mRNA，任選地從5’末端到3’末端，包括CAP I型的帽結構、人核糖體蛋白L32的5’-UTR、所述的編碼來源於SARS-CoV-2病毒的S1蛋白或其免疫原性片段的mRNA或其轉錄模板DNA或其變體、人β珠蛋白的3’-UTR和包含50個腺苷核苷酸的3’-聚腺苷酸。例如所述的分離的mRNA從5’末端到3’末端依次由CAP I型的帽結構、人核糖體蛋白L32的5’-UTR、所述的編碼來源於SARS-CoV-2病毒的S1蛋白或其免疫原性片段的mRNA或其轉錄模板DNA或其變體、人β珠蛋白的3’-UTR和包含50個腺苷核苷酸的3’-聚腺苷酸組成(例如本發明實施例部分所述的SARS-CoV-2-E)。In a preferred embodiment, the isolated mRNA, optionally from the 5' end to the 3' end, includes the cap structure of type CAP I, the 5'-UTR of human ribosomal protein L32, the mRNA encoding the S1 protein derived from SARS-CoV-2 virus or its immunogenic fragment or its transcription template DNA or its variant, the 3'-UTR of human beta globin and the 3'-UTR comprising 50 adenosine nucleotides '-polyAdenylate. For example, the isolated mRNA is composed of the cap structure of CAP I, the 5'-UTR of human ribosomal protein L32, and the S1 protein encoding the SARS-CoV-2 virus from the 5' end to the 3' end. The mRNA of its immunogenic fragment or its transcription template DNA or its variant, the 3'-UTR of human beta globin and the 3'-polyadenylic acid comprising 50 adenosine nucleotides (such as the implementation of the present invention) SARS-CoV-2-E as described in the Examples section).

在某一較佳實施例中，所述的分離的mRNA，任選地從5’末端到3’末端，包括CAP I型的帽結構、人核糖體蛋白L32的5’-UTR、所述的編碼來源於SARS-CoV-2病毒的S1蛋白或其免疫原性片段的mRNA或其轉錄模板DNA或其變體、人β珠蛋白的3’-UTR和包含16個腺苷核苷酸的3’-聚腺苷酸。例如所述的分離的mRNA從5’末端到3’末端依次由CAP I型的帽結構、人核糖體蛋白L32的5’-UTR、所述的編碼來源於SARS-CoV-2病毒的S1蛋白或其免疫原性片段的mRNA或其轉錄模板DNA或其變體、人β珠蛋白的3’-UTR和包含16個腺苷核苷酸的3’-聚腺苷酸組成(例如本發明實施例部分所述的SARS-CoV-2-F)。In a preferred embodiment, the isolated mRNA, optionally from the 5' end to the 3' end, includes the cap structure of type CAP I, the 5'-UTR of human ribosomal protein L32, the mRNA encoding the S1 protein derived from SARS-CoV-2 virus or its immunogenic fragment or its transcription template DNA or its variant, the 3'-UTR of human β-globin, and the 3'-UTR comprising 16 adenosine nucleotides '-polyAdenylate. For example, the isolated mRNA is composed of the cap structure of CAP I, the 5'-UTR of human ribosomal protein L32, and the S1 protein encoding the SARS-CoV-2 virus from the 5' end to the 3' end. The mRNA of its immunogenic fragment or its transcription template DNA or its variant, the 3'-UTR of human beta globin and the 3'-polyadenylic acid comprising 16 adenosine nucleotides (for example, the implementation of the present invention) SARS-CoV-2-F described in the Examples section).

在某一較佳實施例中，所述的分離的mRNA，任選地從5’末端到3’末端，包括CAP I型的帽結構、人核糖體蛋白L32的5’-UTR、人IgE訊號胜肽、所述的編碼來源於SARS-CoV-2病毒的S1蛋白或其免疫原性片段的mRNA或其轉錄模板DNA或其變體、人β珠蛋白的3’-UTR和包含120個腺苷核苷酸的3’-聚腺苷酸。例如所述的分離的mRNA從5’末端到3’末端依次由CAP I型的帽結構、人核糖體蛋白L32的5’-UTR、人IgE訊號胜肽、所述的編碼來源於SARS-CoV-2病毒的S1蛋白或其免疫原性片段的mRNA或其轉錄模板DNA或其變體、人β珠蛋白的3’-UTR和包含120個腺苷核苷酸的3’-聚腺苷酸組成(例如本發明實施例部分所述的SARS-CoV-2-G)。In a preferred embodiment, the isolated mRNA, optionally from the 5' end to the 3' end, includes the cap structure of CAP I, the 5'-UTR of human ribosomal protein L32, the human IgE signal Peptide, said mRNA encoding S1 protein derived from SARS-CoV-2 virus or its immunogenic fragment or its transcription template DNA or its variant, 3'-UTR of human beta globin and 120 glands 3'-polyadenylation of glycoside nucleotides. For example, the isolated mRNA is composed of the cap structure of CAP I type, the 5'-UTR of human ribosomal protein L32, the human IgE signal peptide from the 5' end to the 3' end, and the coding is derived from SARS-CoV. -2 mRNA of S1 protein of virus or its immunogenic fragment or its transcription template DNA or its variant, 3'-UTR of human β-globin and 3'-polyadenylate comprising 120 adenosine nucleotides composition (such as SARS-CoV-2-G as described in the Examples section of the present invention).

在某一較佳實施例中，所述的分離的mRNA，任選地從5’末端到3’末端，包括CAP I型的帽結構、人核糖體蛋白L32的5’-UTR、人IgG訊號胜肽、所述的編碼來源於SARS-CoV-2病毒的S1蛋白或其免疫原性片段的mRNA或其轉錄模板DNA或其變體、人β珠蛋白的3’-UTR和包含120個腺苷核苷酸的3’-聚腺苷酸。例如所述的分離的mRNA從5’末端到3’末端依次由CAP I型的帽結構、人核糖體蛋白L32的5’-UTR、人IgG訊號胜肽、所述的編碼來源於SARS-CoV-2病毒的S1蛋白或其免疫原性片段的mRNA或其轉錄模板DNA或其變體、人β珠蛋白的3’-UTR和包含120個腺苷核苷酸的3’-聚腺苷酸組成(例如本發明實施例部分所述的SARS-CoV-2-H)。In a preferred embodiment, the isolated mRNA, optionally from the 5' end to the 3' end, includes the cap structure of CAP type I, the 5'-UTR of human ribosomal protein L32, the human IgG signal Peptide, said mRNA encoding S1 protein derived from SARS-CoV-2 virus or its immunogenic fragment or its transcription template DNA or its variant, 3'-UTR of human beta globin and 120 glands 3'-polyadenylation of glycoside nucleotides. For example, the isolated mRNA is composed of the cap structure of CAP I type, the 5'-UTR of human ribosomal protein L32, the human IgG signal peptide from the 5' end to the 3' end, and the coding is derived from SARS-CoV. -2 mRNA of S1 protein of virus or its immunogenic fragment or its transcription template DNA or its variant, 3'-UTR of human β-globin and 3'-polyadenylate comprising 120 adenosine nucleotides composition (such as SARS-CoV-2-H as described in the Examples section of the present invention).

在某一較佳實施例中，所述的分離的mRNA，任選地從5’末端到3’末端，包括CAP I型的帽結構、人核糖體蛋白L32的5’-UTR、鼠IgK訊號胜肽、所述的編碼來源於SARS-CoV-2病毒的S1蛋白或其免疫原性片段的mRNA或其轉錄模板DNA或其變體、人β珠蛋白的3’-UTR和包含120個腺苷核苷酸的3’-聚腺苷酸。例如所述的分離的mRNA從5’末端到3’末端依次由CAP I型的帽結構、人核糖體蛋白L32的5’-UTR、鼠IgK訊號胜肽、所述的編碼來源於SARS-CoV-2病毒的S1蛋白或其免疫原性片段的mRNA或其轉錄模板DNA或其變體、人β珠蛋白的3’-UTR和包含120個腺苷核苷酸的3’-聚腺苷酸組成(例如本發明實施例部分所述的SARS-CoV-2-I)。In a preferred embodiment, the isolated mRNA, optionally from the 5' end to the 3' end, includes the cap structure of CAP I, the 5'-UTR of human ribosomal protein L32, the murine IgK signal Peptide, said mRNA encoding S1 protein derived from SARS-CoV-2 virus or its immunogenic fragment or its transcription template DNA or its variant, 3'-UTR of human beta globin and 120 glands 3'-polyadenylation of glycoside nucleotides. For example, the isolated mRNA is composed of the cap structure of CAP I type, the 5'-UTR of human ribosomal protein L32, the murine IgK signal peptide, and the coding is derived from SARS-CoV from the 5' end to the 3' end. -2 mRNA of S1 protein of virus or its immunogenic fragment or its transcription template DNA or its variant, 3'-UTR of human β-globin and 3'-polyadenylate comprising 120 adenosine nucleotides composition (such as SARS-CoV-2-I as described in the Examples section of the present invention).

在某一較佳實施例中，所述的分離的mRNA，任選地從5’末端到3’末端，包括CAP I型的帽結構、人核糖體蛋白L32的5’-UTR、所述的編碼來源於SARS-CoV-2病毒的S1蛋白或其免疫原性片段的mRNA或其轉錄模板DNA或其變體、人β珠蛋白的3’-UTR和包含120個腺苷核苷酸的3’-聚腺苷酸，且經N1-甲基假尿苷的修飾。例如所述的分離的mRNA從5’末端到3’末端依次由CAP I型的帽結構、人核糖體蛋白L32的5’-UTR、所述的編碼來源於SARS-CoV-2病毒的S1蛋白或其免疫原性片段的mRNA或其轉錄模板DNA或其變體、人β珠蛋白的3’-UTR和包含120個腺苷核苷酸的3’-聚腺苷酸組成，且經N1-甲基假尿苷的修飾(例如本發明實施例部分所述的SARS-CoV-2-G1)。In a preferred embodiment, the isolated mRNA, optionally from the 5' end to the 3' end, includes the cap structure of type CAP I, the 5'-UTR of human ribosomal protein L32, the mRNA encoding the S1 protein derived from SARS-CoV-2 virus or its immunogenic fragment or its transcription template DNA or its variant, the 3'-UTR of human β-globin and the 3'-UTR comprising 120 adenosine nucleotides '-polyA, and modified with N1-methylpseudouridine. For example, the isolated mRNA is composed of the cap structure of CAP I, the 5'-UTR of human ribosomal protein L32, and the S1 protein encoding the SARS-CoV-2 virus from the 5' end to the 3' end. The mRNA of its immunogenic fragment or its transcription template DNA or its variant, the 3'-UTR of human β-globin and the 3'-polyadenylic acid comprising 120 adenosine nucleotides are composed of N1- Modification of methyl pseudouridine (eg SARS-CoV-2-G1 as described in the Examples section of the present invention).

在某一較佳實施例中，所述的分離的mRNA，任選地從5’末端到3’末端，包括CAP I型的帽結構、人核糖體蛋白L32的5’-UTR、所述的編碼來源於SARS-CoV-2病毒的S1蛋白或其免疫原性片段的mRNA或其轉錄模板DNA或其變體、人β珠蛋白的3’-UTR和包含120個腺苷核苷酸的3’-聚腺苷酸，且經假尿苷的修飾。例如所述的分離的mRNA從5’末端到3’末端依次由CAP I型的帽結構、人核糖體蛋白L32的5’-UTR、所述的編碼來源於SARS-CoV-2病毒的S1蛋白或其免疫原性片段的mRNA或其轉錄模板DNA或其變體、人β珠蛋白的3’-UTR和包含120個腺苷核苷酸的3’-聚腺苷酸組成，且經假尿苷的修飾(例如本發明實施例部分所述的SARS-CoV-2-G2)。In a preferred embodiment, the isolated mRNA, optionally from the 5' end to the 3' end, includes the cap structure of type CAP I, the 5'-UTR of human ribosomal protein L32, the mRNA encoding the S1 protein derived from SARS-CoV-2 virus or its immunogenic fragment or its transcription template DNA or its variant, the 3'-UTR of human β-globin and the 3'-UTR comprising 120 adenosine nucleotides '-polyA, and modified with pseudouridine. For example, the isolated mRNA is composed of the cap structure of CAP I, the 5'-UTR of human ribosomal protein L32, and the S1 protein encoding the SARS-CoV-2 virus from the 5' end to the 3' end. The mRNA of its immunogenic fragment or its transcription template DNA or its variant, the 3'-UTR of human β-globin and the 3'-polyadenylic acid comprising 120 adenosine nucleotides, and the Modification of glycosides (eg SARS-CoV-2-G2 as described in the Examples section of the present invention).

在某一較佳實施例中，所述的分離的mRNA，任選地從5’末端到3’末端，包括CAP I型的帽結構、人核糖體蛋白L32的5’-UTR、所述的編碼來源於SARS-CoV-2病毒的S1蛋白或其免疫原性片段的mRNA或其轉錄模板DNA或其變體、人β珠蛋白的3’-UTR和包含120個腺苷核苷酸的3’-聚腺苷酸，且經5-甲基尿苷的修飾。例如所述的分離的mRNA從5’末端到3’末端依次由CAP I型的帽結構、人核糖體蛋白L32的5’-UTR、所述的編碼來源於SARS-CoV-2病毒的S1蛋白或其免疫原性片段的mRNA或其轉錄模板DNA或其變體、人β珠蛋白的3’-UTR和包含120個腺苷核苷酸的3’-聚腺苷酸組成，且經5-甲基尿苷的修飾(例如本發明實施例部分所述的SARS-CoV-2-G3)。In a preferred embodiment, the isolated mRNA, optionally from the 5' end to the 3' end, includes the cap structure of type CAP I, the 5'-UTR of human ribosomal protein L32, the mRNA encoding the S1 protein derived from SARS-CoV-2 virus or its immunogenic fragment or its transcription template DNA or its variant, the 3'-UTR of human β-globin and the 3'-UTR comprising 120 adenosine nucleotides '-polyA, and modified with 5-methyluridine. For example, the isolated mRNA is composed of the cap structure of CAP I, the 5'-UTR of human ribosomal protein L32, and the S1 protein encoding the SARS-CoV-2 virus from the 5' end to the 3' end. The mRNA of its immunogenic fragment or its transcription template DNA or its variant, the 3'-UTR of human β-globin, and the 3'-polyadenylic acid comprising 120 adenosine nucleotides are composed of 5- Modifications of methyluridine (eg SARS-CoV-2-G3 as described in the Examples section of the present invention).

本發明中，所述的分離的mRNA通常可以適合作為疫苗進行使用。In the present invention, the isolated mRNA is generally suitable for use as a vaccine.

本發明中，所述的分離的mRNA通常可以為人工合成所得。In the present invention, the isolated mRNA can usually be obtained by artificial synthesis.

本發明中，所述的分離的mRNA通常還可以進一步經過其他修飾。In the present invention, the isolated mRNA can usually be further modified.

本發明中，為了保證蛋白完整的免疫原性和其穩定性，選擇了新冠病毒的S1蛋白基因序列為本發明核酸的ORF(開放閱讀框，open reading frame)。In the present invention, in order to ensure the complete immunogenicity and stability of the protein, the S1 protein gene sequence of the new coronavirus is selected as the ORF (open reading frame) of the nucleic acid of the present invention.

在本發明某一較佳實施例中，所述分離的mRNA的序列如SEQ ID NO: 10所示。In a preferred embodiment of the present invention, the sequence of the isolated mRNA is shown in SEQ ID NO: 10.

為了解決上述技術問題，本發明第二方面提供了一種分離的DNA，所述分離的DNA可轉錄如本發明第一方面所述的分離的mRNA。In order to solve the above technical problem, the second aspect of the present invention provides an isolated DNA, which can transcribe the isolated mRNA described in the first aspect of the present invention.

較佳地，所述分離的DNA的核苷酸序列如SEQ ID NO: 6所示，或與其具有85%、90%、95%、96%、97%、98%、99%或以上同源性的序列所示。Preferably, the nucleotide sequence of the isolated DNA is shown in SEQ ID NO: 6, or has 85%, 90%, 95%, 96%, 97%, 98%, 99% or more homology with it shown in the sequence of sex.

為了解決上述技術問題，本發明第三方面提供了一種重組表現載體，所述重組表現載體中含有如本發明第一方面所述的分離的mRNA、如本發明第二方面所述的DNA。In order to solve the above technical problems, the third aspect of the present invention provides a recombinant expression vector, which contains the isolated mRNA described in the first aspect of the present invention and the DNA described in the second aspect of the present invention.

較佳地，所述重組表現載體的骨架載體為pAAV-MCS、pcDNA 3.1(+)、pCMV-MCS、pEGFP-CTSB或pLVX-PAX1。在某一較佳實施例中，所述重組表現載體的骨架載體為pcDNA 3.1(+)。Preferably, the backbone vector of the recombinant expression vector is pAAV-MCS, pcDNA 3.1(+), pCMV-MCS, pEGFP-CTSB or pLVX-PAX1. In a preferred embodiment, the backbone vector of the recombinant expression vector is pcDNA 3.1(+).

較佳地，所述重組表現載體的啟動子為Lac乳糖操縱子、TAC啟動子、TRC啟動子或T7啟動子。Preferably, the promoter of the recombinant expression vector is Lac lactose operon, TAC promoter, TRC promoter or T7 promoter.

為了解決上述技術問題，本發明第四方面提供了一種轉形體，在宿主中導入如本發明第一方面所述的分離的mRNA、或如本發明第二方面所述的DNA、或如本發明第三方面所述的重組表現載體。In order to solve the above-mentioned technical problems, the fourth aspect of the present invention provides a transformant, in which the isolated mRNA described in the first aspect of the present invention, or the DNA described in the second aspect of the present invention, or the The recombinant expression vector described in the third aspect.

所述轉形體的製備方法可為本領域常規的製備方法，例如為：將上述重組表現載體轉形至宿主細胞中製得。所述轉形體的宿主細胞為本領域常規的各種宿主細胞，只要能滿足使上述重組表現載體穩定地自行複製，且所攜帶所述的核酸可被有效表現即可。優選地，所述宿主細胞為原核細胞和/或真核細胞，優選為分離的哺乳動物細胞，更優選為哺乳動物受試者的細胞，進一步優選為哺乳動物受試者的分離的細胞，最優選為人受試者的分離的細胞。將前述重組表現質體轉形至宿主細胞中，即可得本發明優選的轉形體。其中所述轉形方法為本領域常規轉形方法，較佳地為化學轉形法，熱刺激法或電轉形法。The preparation method of the transformant can be a conventional preparation method in the field, for example, by transforming the above-mentioned recombinant expression vector into a host cell. The host cells of the transformants are various conventional host cells in the art, as long as the above-mentioned recombinant expression vector can stably replicate itself and the nucleic acid carried by it can be effectively expressed. Preferably, the host cell is a prokaryotic cell and/or a eukaryotic cell, preferably an isolated mammalian cell, more preferably a mammalian subject's cell, further preferably a mammalian subject's isolated cell, most preferably Isolated cells of a human subject are preferred. The preferred transformants of the present invention can be obtained by transforming the aforementioned recombinant expression plasmids into host cells. The transformation method is a conventional transformation method in the field, preferably a chemical transformation method, a thermal stimulation method or an electrical transformation method.

為了解決上述技術問題，本發明第五方面提供了組成物，其包含如本發明第一方面所述的分離的mRNA、或如本發明第二方面所述的DNA。In order to solve the above technical problems, the fifth aspect of the present invention provides a composition comprising the isolated mRNA as described in the first aspect of the present invention, or the DNA as described in the second aspect of the present invention.

為了解決上述技術問題，本發明第六方面提供了一種脂質體奈米顆粒(或稱為脂質體、脂質複合物/體、LNP)，其包含如本發明第一方面所述的分離的mRNA、如本發明第二方面所述的DNA和/或如本發明第五方面所述的組成物。In order to solve the above-mentioned technical problems, the sixth aspect of the present invention provides a liposome nanoparticle (or referred to as liposome, lipid complex/body, LNP), which comprises the isolated mRNA as described in the first aspect of the present invention, The DNA according to the second aspect of the invention and/or the composition according to the fifth aspect of the invention.

較佳地，所述脂質體奈米顆粒為長循環陽離子脂質體奈米顆粒，優選為經PEG或其衍生物修飾的長循環陽離子脂質體奈米顆粒；所述PEG的相對分子質量優選為2000~5000，例如為2000、3000、4000或5000。Preferably, the liposome nanoparticles are long-circulating cationic liposome nanoparticles, preferably long-circulating cationic liposome nanoparticles modified by PEG or its derivatives; the relative molecular mass of the PEG is preferably 2000. ~5000, for example 2000, 3000, 4000 or 5000.

更佳地，所述脂質體奈米顆粒還包括脂質體遞送系統，其優選包括陽離子脂質、結構脂質、輔助脂質和表面活性劑，更優選包括20%~70%的陽離子脂質、10%～65%的結構脂質、5%~25%的輔助脂質和1%~10%的表面活性劑。More preferably, the liposome nanoparticle also includes a liposome delivery system, which preferably includes cationic lipids, structured lipids, helper lipids and surfactants, more preferably includes 20% to 70% of cationic lipids, 10% to 65% of cationic lipids. % structural lipids, 5%~25% auxiliary lipids and 1%~10% surfactants.

其中，所述陽離子脂質可以是優選自N,N-二烯基-N,N-二甲基氯化銨(DODAC)、N,N-二硬脂基-N,N-二甲基溴化銨(DDAB)、N,N-二甲基-2,3-二甲氧基丙胺(DODMA)、1,2-二甲基氧基-3-(二甲胺基)乙氧基丙烷(Dlin-DAC)、2,2-二甲基苯胺-4-(2-二甲基氨基甲基)-[1,3]-二氧戊環(Dlin-KC2-DMA)和二亞油基甲基-4-二甲基氨基丁酸酯(Dlin-MC3-DMA)中的一種或多種，更優選自Dlin-KC2-DMA和Dlin-MC3-DMA中的一種或兩種，最優選為Dlin-MC3-DMA；所述陽離子脂質的莫耳百分比優選為30%~60%，更優選為45%~55%，例如50%。其中，所述結構脂質可以是優選自膽固醇、膽固醇酯、固醇類激素、固醇類維生素和植物甾醇中的一種或多種，更優選自膽固醇、膽固醇酯和植物甾醇中的一種或多種，最優選為膽固醇；所述結構脂質的莫耳百分比優選為15%~50%，例如23%、28%、30%、33%、35%、38.5%、40%或48%。其中，所述輔助脂質可以是優選自二硬脂醯基磷脂醯膽鹼(DSPC)、二油醯基卵磷脂(DOPC)、二棕櫚醯磷脂醯甘油(DPPG)、二油醯基磷脂醯絲胺酸(DOPS)和二油醯磷脂醯乙醇胺(DOPE)中的一種或多種，更優選為DSPC和/或DOPS，最優選為DSPC；所述輔助脂質的莫耳百分比優選為10%~20%，例如13%或15%。其中，所述表面活性劑可以是優選自DAG-PEG、DAA-PEG、DMG-PEG、Cer-PEG和DSPE-PEG中的一種或多種，更優選為PEG-DMG(1,2-二肉豆蔻醯-rac-甘油-3-甲氧基聚乙二醇)；所述表面活性劑的莫耳百分比優選為1%~5%；更優選為1%~3%，例如為1.5%或2%。Wherein, the cationic lipid can be preferably selected from N,N-dienyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethyl bromide Ammonium (DDAB), N,N-dimethyl-2,3-dimethoxypropylamine (DODMA), 1,2-dimethyloxy-3-(dimethylamino)ethoxypropane (Dlin -DAC), 2,2-dimethylaniline-4-(2-dimethylaminomethyl)-[1,3]-dioxolane (Dlin-KC2-DMA) and dilinoleylmethyl One or more of -4-dimethylaminobutyrate (Dlin-MC3-DMA), more preferably one or both of Dlin-KC2-DMA and Dlin-MC3-DMA, most preferably Dlin-MC3 -DMA; the molar percentage of the cationic lipid is preferably 30% to 60%, more preferably 45% to 55%, such as 50%. Wherein, the structural lipid can be preferably one or more selected from cholesterol, cholesterol ester, sterol hormone, sterol vitamin and phytosterol, more preferably one or more selected from cholesterol, cholesterol ester and phytosterol, most preferably Preferably cholesterol; the molar percentage of the structural lipid is preferably 15% to 50%, such as 23%, 28%, 30%, 33%, 35%, 38.5%, 40% or 48%. Wherein, the auxiliary lipid may be preferably selected from the group consisting of distearyl phospholipid choline (DSPC), dioleyl phosphatidylcholine (DOPC), dipalmitoyl phospholipid glycerol (DPPG), dioleyl phospholipid filament One or more of amino acid (DOPS) and dioleophosphatidylethanolamine (DOPE), more preferably DSPC and/or DOPS, most preferably DSPC; the molar percentage of the auxiliary lipid is preferably 10%～20% , such as 13% or 15%. Wherein, the surfactant can be preferably one or more selected from DAG-PEG, DAA-PEG, DMG-PEG, Cer-PEG and DSPE-PEG, more preferably PEG-DMG (1,2-Dimyr acyl-rac-glycerol-3-methoxypolyethylene glycol); the molar percentage of the surfactant is preferably 1% to 5%; more preferably 1% to 3%, such as 1.5% or 2% .

在某一較佳實施例中，所述脂質體奈米顆粒中的脂質體遞送系統包括45%～55%的Dlin-MC3-DMA、30%～40%的膽固醇、10%～15%的DSPC和1%～3%的DMG-PEG 2000；所述百分比為莫耳百分比。在此配比範圍內的脂質體的粒徑分佈更集中，包封率和入胞率更高，效果更佳。 In a preferred embodiment, the liposome delivery system in the liposome nanoparticle comprises 45%-55% Dlin-MC3-DMA, 30%-40% cholesterol, 10%-15% DSPC and 1% to 3% of DMG-PEG 2000 ; the percentages are molar percentages. The particle size distribution of liposomes within this ratio range is more concentrated, the encapsulation efficiency and cell entry rate are higher, and the effect is better.

在某一較佳實施例中，所述脂質體奈米顆粒中的脂質體遞送系統由20%的Dlin-MC3-DMA、65%的膽固醇、13%的DSPC和2%的DMG-PEG 2000組成。 In a certain preferred embodiment, the liposome delivery system in the liposome nanoparticle is composed of 20% Dlin-MC3-DMA, 65% cholesterol, 13% DSPC and 2% DMG-PEG 2000 .

在某一較佳實施例中，所述脂質體奈米顆粒中的脂質體遞送系統由70%的Dlin-MC3-DMA、15%的膽固醇、13%的DSPC和2%的DMG-PEG 2000組成。 In a certain preferred embodiment, the liposome delivery system in the liposome nanoparticle is composed of 70% Dlin-MC3-DMA, 15% cholesterol, 13% DSPC and 2% DMG-PEG 2000 .

在某一較佳實施例中，所述脂質體奈米顆粒中的脂質體遞送系統由50%的Dlin-MC3-DMA、35%的膽固醇、13%的DSPC和2%的DMG-PEG 2000組成。 In a certain preferred embodiment, the liposome delivery system in the liposome nanoparticle is composed of 50% Dlin-MC3-DMA, 35% cholesterol, 13% DSPC and 2% DMG-PEG 2000 .

在某一較佳實施例中，所述脂質體奈米顆粒中的脂質體遞送系統由50%的Dlin-MC3-DMA、35%的膽固醇、10%的DSPC和5%的DMG-PEG 2000組成。 In a certain preferred embodiment, the liposome delivery system in the liposome nanoparticle is composed of 50% Dlin-MC3-DMA, 35% cholesterol, 10% DSPC and 5% DMG-PEG 2000 .

在某一較佳實施例中，所述脂質體奈米顆粒中的脂質體遞送系統由50%的Dlin-MC3-DMA、35%的膽固醇、13%的DOPC和2%的DMG-PEG 2000組成。 In a certain preferred embodiment, the liposome delivery system in the liposome nanoparticle is composed of 50% Dlin-MC3-DMA, 35% cholesterol, 13% DOPC and 2% DMG-PEG 2000 .

在某一較佳實施例中，所述脂質體奈米顆粒中的脂質體遞送系統由50%的Dlin-MC3-DMA、28%的膽固醇、20%的DSPC和2%的DMG-PEG 2000組成。 In a certain preferred embodiment, the liposome delivery system in the liposome nanoparticle is composed of 50% Dlin-MC3-DMA, 28% cholesterol, 20% DSPC and 2% DMG-PEG 2000 .

在某一較佳實施例中，所述脂質體奈米顆粒中的脂質體遞送系統由50%的Dlin-MC3-DMA、23%的膽固醇、25%的DSPC和2%的DMG-PEG 2000組成。 In a certain preferred embodiment, the liposome delivery system in the liposome nanoparticle is composed of 50% Dlin-MC3-DMA, 23% cholesterol, 25% DSPC and 2% DMG-PEG 2000 .

在某一較佳實施例中，所述脂質體奈米顆粒中的脂質體遞送系統由50%的Dlin-MC3-DMA、38.5%的膽固醇、10%的DSPC和1.5%的DMG-PEG 2000組成。 In a certain preferred embodiment, the liposome delivery system in the liposome nanoparticle is composed of 50% Dlin-MC3-DMA, 38.5% cholesterol, 10% DSPC and 1.5% DMG-PEG 2000 .

較佳地，所述脂質體遞送系統與所述mRNA、所述DNA和/或所述組成物的質量比為(5-30):1，優選為(10-20):1；例如15:1。Preferably, the mass ratio of the liposome delivery system to the mRNA, the DNA and/or the composition is (5-30): 1, preferably (10-20): 1; for example 15: 1.

針對上述的mRNA疫苗的遞送系統通常可以經過程序的改良，比如調整使用的輔料脂質體的組成，莫耳比，與mRNA混合的條件等。本領域人員應當理解的是，這些均應當在本申請的保護範圍之內。The delivery system for the above-mentioned mRNA vaccine can usually be improved by procedures, such as adjusting the composition of the used excipient liposome, the molar ratio, and the conditions of mixing with mRNA, etc. It should be understood by those skilled in the art that these should all fall within the protection scope of the present application.

在某一較佳實施例中，所得脂質體奈米顆粒(以mRNA-LNP的形式)的平均粒徑可以為50-100nm，包封率可以≥80%，可以將其作為新型冠狀病毒SARS-CoV-2 mRNA的核酸疫苗進行注射。In a certain preferred embodiment, the average particle size of the obtained liposome nanoparticles (in the form of mRNA-LNP) can be 50-100 nm, and the encapsulation efficiency can be ≥ 80%, which can be used as the new coronavirus SARS- CoV-2 mRNA nucleic acid vaccine for injection.

在某一較佳實施例中，所述的脂質體奈米顆粒可透過以下方法製得：將上述的mRNA溶解於pH6.8-7.4的PBS緩衝液，此為水相。將陽離子脂質奈米顆粒Dlin-MC3-DMA、膽固醇、DSPC和DMG-PEG 2000，按莫耳百分比50:13:35:2溶於無水乙醇，此為有機相。採用微流體混合儀將有機相與水相按1:5的體積比混合，混合後除去溶液中的乙醇並濃縮mRNA濃度至0.5mg/mL。 In a preferred embodiment, the liposome nanoparticles can be prepared by the following method: dissolving the above-mentioned mRNA in a PBS buffer of pH 6.8-7.4, which is an aqueous phase. The cationic lipid nanoparticles Dlin-MC3-DMA, cholesterol, DSPC and DMG-PEG 2000 were dissolved in absolute ethanol according to the molar percentage of 50:13:35:2, which was the organic phase. The organic phase and the aqueous phase were mixed in a volume ratio of 1:5 using a microfluidic mixer. After mixing, the ethanol in the solution was removed and the mRNA concentration was concentrated to 0.5 mg/mL.

本發明中，將mRNA以安全高效的LNP脂質體奈米顆粒的形式進行遞送，不僅可以保護核酸不被降解，延長釋放時間持續增強免疫反應，同時，透過改變脂質體的粒徑和電荷，結合肌肉注射給藥方式，實現更加精准的靶向給藥，有助於提高mRNA疫苗的穩定性，延長身體免疫反時間、增加免疫反應類型。不僅可以產生強大的免疫反應，而且改善了mRNA疫苗耐受性，使其更好的發揮作用。In the present invention, the mRNA is delivered in the form of safe and efficient LNP liposome nanoparticles, which can not only protect the nucleic acid from being degraded, prolong the release time and continuously enhance the immune response, but at the same time, by changing the particle size and charge of the liposome, the combination of The intramuscular injection method enables more precise targeted drug delivery, which helps to improve the stability of mRNA vaccines, prolong the immune response time of the body, and increase the types of immune responses. Not only can it generate a strong immune response, but also improve the tolerance of mRNA vaccines, making them work better.

為了解決上述技術問題，本發明第七方面提供了一種mRNA疫苗，其包含如本發明第一方面所述的分離的mRNA、如本發明第二方面所述的DNA、如本發明第五方面所述的組成物和/或如本發明第六方面所述的脂質體奈米顆粒(從而引起適應性免疫反應)。該疫苗通常可以是針對SARS-CoV-2病毒的疫苗。In order to solve the above-mentioned technical problems, the seventh aspect of the present invention provides an mRNA vaccine, which comprises the isolated mRNA described in the first aspect of the present invention, the DNA described in the second aspect of the present invention, and the DNA described in the fifth aspect of the present invention. The composition described above and/or the liposomal nanoparticle according to the sixth aspect of the present invention (thereby eliciting an adaptive immune response). The vaccine can generally be a vaccine against the SARS-CoV-2 virus.

較佳地，所述mRNA疫苗還包括佐劑。Preferably, the mRNA vaccine further includes an adjuvant.

為了解決上述技術問題，本發明第八方面提供了一種藥物組成物，其包含如本發明第一方面所述的分離的mRNA、如本發明第二方面所述的DNA、如本發明第五方面所述的組成物、如本發明第六方面所述的脂質體奈米顆粒和/或如本發明第七方面所述的mRNA疫苗，和任選地藥學上可接受的載體。In order to solve the above technical problems, the eighth aspect of the present invention provides a pharmaceutical composition, which comprises the isolated mRNA described in the first aspect of the present invention, the DNA described in the second aspect of the present invention, and the fifth aspect of the present invention. The composition, the liposome nanoparticle according to the sixth aspect of the present invention and/or the mRNA vaccine according to the seventh aspect of the present invention, and optionally a pharmaceutically acceptable carrier.

為了解決上述技術問題，本發明第九方面提供了一種套組，其包含如本發明第一方面所述的分離的mRNA、如本發明第二方面所述的DNA、如本發明第五方面所述的組成物、如本發明第六方面所述的脂質體奈米顆粒、如本發明第七方面所述的mRNA疫苗和/或如本發明第八方面所述的藥物組成物。In order to solve the above-mentioned technical problems, the ninth aspect of the present invention provides a kit, which comprises the isolated mRNA described in the first aspect of the present invention, the DNA described in the second aspect of the present invention, and the DNA described in the fifth aspect of the present invention. The composition, the liposome nanoparticle according to the sixth aspect of the present invention, the mRNA vaccine according to the seventh aspect of the present invention, and/or the pharmaceutical composition according to the eighth aspect of the present invention.

較佳地，所述套組還包含：使用說明、用於轉染的細胞、輔劑、施用所述藥物組成物的工具、藥學可接受的載體和/或用於溶解或稀釋所述mRNA、所述DNA、所述組成物、所述脂質體奈米顆粒、所述疫苗或所述藥物組成物的藥學可接受的溶液。Preferably, the set also comprises: instructions for use, cells for transfection, adjuvants, tools for administering the pharmaceutical composition, pharmaceutically acceptable carriers and/or for dissolving or diluting the mRNA, A pharmaceutically acceptable solution of the DNA, the composition, the liposomal nanoparticle, the vaccine, or the pharmaceutical composition.

為了解決上述技術問題，本發明第十方面提供了如本發明第一方面所述的分離的mRNA、如本發明第二方面所述的DNA、如本發明第五方面所述的組成物、如本發明第六方面所述的脂質體奈米顆粒、如本發明第七方面所述的mRNA疫苗和/或如本發明第八方面所述的藥物組成物在製備用於預防和/或治療SARS-CoV-2病毒感染或SARS-CoV-2病毒感染所致疾病的藥物中的應用。In order to solve the above technical problems, the tenth aspect of the present invention provides the isolated mRNA according to the first aspect of the present invention, the DNA according to the second aspect of the present invention, the composition according to the fifth aspect of the present invention, the The liposome nanoparticle described in the sixth aspect of the present invention, the mRNA vaccine described in the seventh aspect of the present invention, and/or the pharmaceutical composition described in the eighth aspect of the present invention are prepared for preventing and/or treating SARS. - CoV-2 virus infection or the use of medicines for diseases caused by SARS-CoV-2 virus infection.

此外，本發明還提供了一種如本發明第一方面所述的分離的mRNA、如本發明第二方面所述的DNA、如本發明第五方面所述的組成物、如本發明第六方面所述的脂質體奈米顆粒、如本發明第七方面所述的mRNA疫苗和/或如本發明第八方面所述的藥物組成物在製備套組中的應用。In addition, the present invention also provides the isolated mRNA according to the first aspect of the present invention, the DNA according to the second aspect of the present invention, the composition according to the fifth aspect of the present invention, and the sixth aspect of the present invention. Application of the liposome nanoparticle, the mRNA vaccine according to the seventh aspect of the present invention and/or the pharmaceutical composition according to the eighth aspect of the present invention in preparing a kit.

此外，本發明還提供了一種生物材料，所述生物材料包括編碼所述mRNA的DNA分子，所述DNA分子能夠轉錄得到所述mRNA；所述生物材料還可以是含有上述DNA分子的表現盒或載體，例如含有編碼所述mRNA的質體，以用來貯存所述mRNA的序列資訊，或用於選殖所述DNA分子或表現DNA分子編碼的蛋白；所述生物材料還可以為含有上述mRNA和/或DNA分子的微生物和/或細胞，以表現所述mRNA或DNA編碼的蛋白，或實現DNA分子的選殖。所述DNA的核苷酸序列可以是優選如SEQ ID NO: 6所示。In addition, the present invention also provides a biological material, the biological material includes a DNA molecule encoding the mRNA, and the DNA molecule can be transcribed to obtain the mRNA; the biological material can also be an expression cassette containing the above DNA molecule or A vector, for example, contains a plastid encoding the mRNA, for storing the sequence information of the mRNA, or for colonizing the DNA molecule or expressing the protein encoded by the DNA molecule; the biological material can also contain the above mRNA and/or DNA molecules to express the mRNA or DNA encoded proteins, or to effect the colonization of DNA molecules. The nucleotide sequence of the DNA may preferably be as shown in SEQ ID NO:6.

此外，本發明還提供了一種治療和/或預防SARS-CoV-2病毒感染或SARS-CoV-2病毒感染所致疾病的方法，其包括(例如向個體受試者)施用如本發明第一方面所述的分離的mRNA、如本發明第二方面所述的DNA、如本發明第五方面所述的組成物、如本發明第六方面所述的脂質體奈米顆粒、如本發明第七方面所述的mRNA疫苗、如本發明第八方面所述的藥物組成物和/或如本發明第九方面所述的套組。In addition, the present invention also provides a method of treating and/or preventing SARS-CoV-2 virus infection or a disease caused by SARS-CoV-2 virus infection, comprising (eg, to an individual subject) administering the first method of the present invention The isolated mRNA according to the aspect, the DNA according to the second aspect of the present invention, the composition according to the fifth aspect of the present invention, the liposome nanoparticle according to the sixth aspect of the present invention, and the liposome nanoparticle according to the sixth aspect of the present invention. The mRNA vaccine according to the seventh aspect, the pharmaceutical composition according to the eighth aspect of the present invention and/or the kit according to the ninth aspect of the present invention.

此外，本發明還提供了如本發明第一方面所述的分離的mRNA、如本發明第二方面所述的DNA、如本發明第五方面所述的組成物、如本發明第六方面所述的脂質體奈米顆粒、如本發明第七方面所述的mRNA疫苗、如本發明第八方面所述的藥物組成物和/或如本發明第九方面所述的套組在治療和/或預防SARS-CoV-2病毒感染或SARS-CoV-2病毒感染所致疾病中的應用。In addition, the present invention also provides the isolated mRNA according to the first aspect of the present invention, the DNA according to the second aspect of the present invention, the composition according to the fifth aspect of the present invention, and the sixth aspect of the present invention. The described liposome nanoparticle, the mRNA vaccine as described in the seventh aspect of the present invention, the pharmaceutical composition as described in the eighth aspect of the present invention and/or the set as described in the ninth aspect of the present invention are used in treatment and/or Or the application in the prevention of SARS-CoV-2 virus infection or diseases caused by SARS-CoV-2 virus infection.

此外，本發明還提供了一種在受試者中誘導免疫反應的方法，其包括對所述受試者施用如本發明第一方面所述的分離的mRNA、如本發明第二方面所述的DNA、如本發明第五方面所述的組成物、如本發明第六方面所述的脂質體奈米顆粒、如本發明第七方面所述的mRNA疫苗、如本發明第八方面所述的藥物組成物和/或如本發明第九方面所述的套組的步驟。較佳地，其中所述施用步驟可為本領域常規，例如可以為肌內施用、皮下施用、皮內施用、鼻內、***內或直腸內施用、局部施用等。Furthermore, the present invention also provides a method of inducing an immune response in a subject, comprising administering to the subject an isolated mRNA as described in the first aspect of the present invention, an isolated mRNA as described in the second aspect of the present invention DNA, the composition according to the fifth aspect of the present invention, the liposome nanoparticle according to the sixth aspect of the present invention, the mRNA vaccine according to the seventh aspect of the present invention, and the eighth aspect of the present invention The steps of the pharmaceutical composition and/or kit according to the ninth aspect of the present invention. Preferably, the administration step may be conventional in the art, such as intramuscular administration, subcutaneous administration, intradermal administration, intranasal, intravaginal or rectal administration, topical administration and the like.

本發明中，所述“免疫原性片段”是指當施用於受試者時具有免疫原性並引起保護性免疫反應的蛋白質的一部分。在一個實施例中，所述的“免疫原性”或“免疫原性的”是指當蛋白質、胜肽、核酸、抗原或生物體施用於動物時，蛋白質、胜肽、核酸、抗原或生物體在動物體內引起免疫反應的先天能力。因此，在一個實施例中，“免疫原性”有所增強，通常是指當蛋白質、胜肽、核酸、抗原或生物體施用於動物時，增加蛋白質、胜肽、核酸、抗原或生物體在動物體內引起免疫反應的能力。在一個實施例中，蛋白質、胜肽、核酸、抗原或生物體引起免疫反應的能力的增加可以透過針對蛋白質、胜肽、核酸、抗原或生物體的更大數量的抗體，針對抗原或生物體的多樣性更大的抗體，對蛋白質、胜肽、核酸、抗原或生物體具有特異性的更大數量的T細胞，對蛋白質、胜肽、核酸、抗原或生物體具有更強的細胞毒性或輔助T細胞反應等來測量。在一個實施例中，免疫原性片段也是抗原性的。在另一個實施例中，“抗原性”是指能夠與免疫系統的抗原辨識分子例如免疫球蛋白(抗體)或T細胞抗原受體特異性相互作用的片段。在另一個實施例中，抗原性片段包含至少約8個胺基酸(AA)的抗原決定位。具有抗原性的分子本身不必具有免疫原性，即能夠在沒有載體的情況下引發免疫反應。In the present invention, the "immunogenic fragment" refers to a part of a protein that is immunogenic and elicits a protective immune response when administered to a subject. In one embodiment, "immunogenic" or "immunogenic" refers to a protein, peptide, nucleic acid, antigen or organism when the protein, peptide, nucleic acid, antigen or organism is administered to an animal The body's innate ability to elicit an immune response in an animal. Thus, in one embodiment, "immunogenicity" is enhanced, and generally refers to an increase in the protein, peptide, nucleic acid, antigen, or organism's presence in an animal when administered to an animal. The ability of an animal to elicit an immune response. In one embodiment, the increased ability of a protein, peptide, nucleic acid, antigen or organism to elicit an immune response may be through a greater number of antibodies directed against the protein, peptide, nucleic acid, antigen or organism, against the antigen or organism greater diversity of antibodies, greater numbers of T cells specific for proteins, peptides, nucleic acids, antigens or organisms, greater cytotoxicity for proteins, peptides, nucleic acids, antigens or organisms or Helper T cell responses, etc. In one embodiment, the immunogenic fragment is also antigenic. In another embodiment, "antigenic" refers to a fragment capable of specifically interacting with an antigen-recognition molecule of the immune system, such as an immunoglobulin (antibody) or T cell antigen receptor. In another embodiment, the antigenic fragment comprises an epitope of at least about 8 amino acids (AA). Antigenic molecules do not have to be immunogenic themselves, that is, to be able to elicit an immune response in the absence of a carrier.

本發明中，所述“變體”是指與群體的大部分不同但仍與常見模式足夠相似以被視為其中一種例如剪接變體的胺基酸或核酸序列(或在其他實施例中為生物體或組織)。在一個實施例中，變體可以是序列守恆變體，而在另一實施例中，變體可以是功能性守恆變體。在一個實施例中，變體可以包含一個或多個胺基酸的添加、缺失或取代。In the present invention, the "variant" refers to an amino acid or nucleic acid sequence (or in other embodiments, an amino acid or nucleic acid sequence that differs from the majority of the population but is sufficiently similar to a common pattern to be considered as one of, eg, a splice variant). organism or tissue). In one embodiment, the variant may be a sequence-conserving variant, while in another embodiment, the variant may be a functionally-conserving variant. In one embodiment, a variant may comprise one or more additions, deletions or substitutions of amino acids.

本發明中，所述“包括或包含”可以是指除了包括後面所列舉的成分，還存在其他成分；也可以是指“由……組成”，即只包括後面所列舉的成分而不存在其他成分。In the present invention, the "comprising or comprising" may mean that in addition to the components listed below, there are other components; it may also mean "consisting of", that is, only the components listed below are included and no other components are present. Element.

本發明中，所述的“一種或多種”通常可以是指一種、兩種、三種或更多種。In the present invention, the "one or more" can generally refer to one, two, three or more.

在符合本領域常識的基礎上，上述各優選條件，可任意組合，即得本發明各較佳實例。On the basis of conforming to common knowledge in the art, the above preferred conditions can be combined arbitrarily to obtain preferred examples of the present invention.

本發明所用試劑和原料均市售可得。The reagents and raw materials used in the present invention are all commercially available.

本發明的積極進步效果在於： 本發明的經過密碼子最適化後或者進一步經過修飾所得的mRNA在細胞中可以高度表現；從而可以獲得分子層級精准設計的類似於完全加工成熟的mRNA分子產品；不僅經濟高效，而且所述mRNA更加安全、高效，結構更加穩定，蛋白表現效率更高，能夠持續地表現新冠病毒S1蛋白。將本發明的mRNA製備成脂質體奈米顆粒/疫苗時，可以實現使用極小劑量就能達到足夠的保護效果，且免疫原性有所降低，同時能夠活化身體免疫系統產生體液免疫和細胞免疫。將本發明的mRNA製備成疫苗時，成本較低、生產週期短，有利於大規模工業生產。 The positive progressive effect of the present invention is: The mRNA obtained after codon optimization or further modification of the present invention can be highly expressed in cells; thus, a molecular product similar to a fully processed mature mRNA molecular product can be obtained with precise design at the molecular level; not only is economical and efficient, but also the mRNA is more Safe, efficient, more stable in structure, more efficient in protein expression, and able to continuously express the new coronavirus S1 protein. When the mRNA of the present invention is prepared into liposome nanoparticles/vaccine, a very small dose can be used to achieve sufficient protection effect, and the immunogenicity is reduced, and at the same time, the body's immune system can be activated to generate humoral immunity and cellular immunity. When the mRNA of the present invention is prepared into a vaccine, the cost is low and the production period is short, which is favorable for large-scale industrial production.

具體實施方式Detailed ways

下面透過實施例的方式進一步說明本發明，但並不因此將本發明限制在所述的實施例範圍之中。下列實施例中未註明具體條件的實驗方法，按照常規方法和條件，或按照商品說明書選擇。 實施例 1 新型冠 狀病毒 SARS-CoV-2 mRNA 的 ORF 序列最適化 The present invention is further described below by way of examples, but the present invention is not limited to the scope of the described examples. The experimental methods that do not specify specific conditions in the following examples are selected according to conventional methods and conditions, or according to the product description. Example 1 Optimization of ORF sequence of novel coronavirus SARS-CoV-2 mRNA

本實施例從四個公開的資料來源(CNGBdb/GenBank/Genome Warehouse/GISAID)收集到69株全基因組資料，其中有10株來源GenBank的資料可同時獲取到其蛋白序列，其餘59株只有全基因組資料，使用病毒基因組ORF閱讀器VIGOR對基因組序列進行注釋，得到其餘59株的S蛋白序列。VIGOR(病毒基因組ORF閱讀器)是一種用於在流感病毒、輪狀病毒、鼻病毒和冠狀病毒亞型中進行基因預測的網絡應用工具。VIGOR基於序列相似性搜索來檢測蛋白質編碼區，並且可以準確地檢測基因組特定特徵，例如移碼、重疊基因、嵌入基因，並可以在單個多胜肽開放閱讀框內預測成熟的胜肽段。該程序內置了針對流感和輪狀病毒的基因分型功能。對於測試的RNA病毒基因組，VIGOR的特異性和敏感性大於99%。In this example, the whole genome data of 69 strains were collected from four public data sources (CNGBdb/GenBank/Genome Warehouse/GISAID), of which 10 strains were obtained from GenBank data and their protein sequences could be obtained at the same time, and the remaining 59 strains only had whole genomes Using the viral genome ORF reader VIGOR to annotate the genome sequence, the S protein sequences of the remaining 59 strains were obtained. VIGOR (Viral Genome ORF Reader) is a web application tool for gene prediction in influenza virus, rotavirus, rhinovirus and coronavirus subtypes. VIGOR detects protein-coding regions based on sequence similarity searches, and can accurately detect genome-specific features such as frameshifts, overlapping genes, embedded genes, and predict mature peptide segments within a single polypeptide open reading frame. The program has built-in genotyping capabilities for influenza and rotavirus. The specificity and sensitivity of VIGOR was greater than 99% for the tested RNA virus genomes.

Clustal Omega是一款新的多序列比對程序，它使用種子引導樹和HMM輪廓圖技術來生成三個或更多序列之間的比對。採用Clustal Omerga線上工具對69株S蛋白序列進行多序列比對分析，發現69株中的6株(8.70%)S蛋白S1序列共存在3個多型性位址，且多型性頻率最高不超過4.35%，表明S蛋白S1序列的守恆性高。因此，後續分析使用S蛋白(序列長度1273個胺基酸)守恆性最高的其中一株病毒株(GenBank:MN908947)為基礎進行抗原決定位預測與疫苗的設計。Clustal Omega is a new multiple sequence alignment program that uses seed-guided tree and HMM contour map techniques to generate alignments between three or more sequences. The Clustal Omerga online tool was used to conduct multiple sequence alignment analysis of the S protein sequences of 69 strains. It was found that 6 of the 69 strains (8.70%) had 3 polymorphic sites in the S protein S1 sequence, and the polymorphism frequency was the highest. More than 4.35%, indicating that the conservation of the S1 sequence of the S protein is high. Therefore, the subsequent analysis used one of the most conserved virus strains (GenBank: MN908947) of the S protein (sequence length of 1273 amino acids) as the basis for epitope prediction and vaccine design.

隨著疫情全球爆發，公共資料庫積累了越來越多的SARS-CoV-2全基因組序列，持續從公共資料庫收集了411株新型冠狀病毒SARS-CoV-2的全基因組序列，病毒株採集地點包括中國、東亞、美國、澳大利亞和歐洲，採集日期從2019年12月23日到2020年03月24日。411株新型冠狀病毒SARS-CoV-2的全基因組序列，其中來源GenBank的資料可同時獲取到其蛋白序列，其餘全基因組序列經VIGOR工具進行基因組注釋，Clustal Omega進行多序列比對，發現119株(28.95%)新型冠狀病毒2019-nCOV的S蛋白S1序列共存在27個多型性位址，且只有1個位址的多型性頻率為20.19%，其餘位址的多型性頻率均不超過1.22%，進一步說明S蛋白S1序列的守恆性高。截止2020年04月15日，國家生物資訊中心2019新型冠狀病毒資訊庫基於5554條高品質人源新冠病毒全基因組序列變異分析，所有統計基於與2019-nCoV (MN908947)序列的比較分析鑒定，S蛋白S1序列的胺基酸變化位址為145個，且只有1個位址的變異頻率為58.12%，其餘位址的變異頻率均不超過0.59%，說明選擇MN908947的S蛋白S1序列作為該核酸疫苗的ORF，守恆性高，可以覆蓋38.84%的病毒株。With the global outbreak of the epidemic, more and more whole genome sequences of SARS-CoV-2 have been accumulated in public databases, and the whole genome sequences of 411 new coronavirus SARS-CoV-2 strains have been continuously collected from public databases. Locations include China, East Asia, the United States, Australia, and Europe, and the collection dates are from December 23, 2019 to March 24, 2020. The whole genome sequences of 411 new coronavirus SARS-CoV-2 strains, of which the data from GenBank can be obtained at the same time, and the protein sequences can be obtained at the same time. (28.95%) There are 27 polymorphic addresses in the S protein S1 sequence of the new coronavirus 2019-nCOV, and only one address has a polymorphic frequency of 20.19%, and the polymorphic frequencies of the other addresses are not More than 1.22%, further indicating that the S1 sequence of the S protein is highly conserved. As of April 15, 2020, the 2019 new coronavirus information database of the National Bioinformatics Center is based on the analysis of 5,554 high-quality human new coronavirus whole genome sequences. There are 145 amino acid change addresses in the protein S1 sequence, and only one address has a mutation frequency of 58.12%, and the mutation frequency of the other addresses does not exceed 0.59%, indicating that the S protein S1 sequence of MN908947 was selected as the nucleic acid. The ORF of the vaccine is highly conserved and can cover 38.84% of the virus strains.

因此，確定SARS-CoV-2的S1蛋白所對應的基因序列為本發明核酸疫苗mRNA的ORF，序列如SEQ ID NO: 1所示，其胺基酸序列如SEQ ID NO: 2所示。 實施例 2 新型冠 狀病毒 SARS-CoV-2 mRNA 的 3’UTR 序列最適化 Therefore, it is determined that the gene sequence corresponding to the S1 protein of SARS-CoV-2 is the ORF of the nucleic acid vaccine mRNA of the present invention, the sequence is shown in SEQ ID NO: 1, and the amino acid sequence thereof is shown in SEQ ID NO: 2. Example 2 Optimization of the 3'UTR sequence of the novel coronavirus SARS-CoV-2 mRNA

本實施例中的SARS-CoV-2 mRNA的3’UTR序列最佳化方案如表1所示，具體如下： SARS-CoV-2 mRNA序列關鍵元件是採用本領域常規的雙酶法Cap1(利用牛痘病毒加帽酶及其他組份，將7-甲基鳥苷帽結構(Cap 0)加到RNA的5’末端，然後使用2’-O-甲基轉移酶和SAM上的甲基轉移至Cap0上形成Cap1)、人核糖體蛋白L32(PRL32)的部分5’UTR序列(序列如SEQ ID NO: 3所示：5’-GGGGCGCTGCCTACGGAGGTGGCAGCCATCTCCTTCTCGGCATCAAGCTTACC-3’)、ORF為上述GenBank為MN908947的毒株基因組序列中編碼S1蛋白的基因序列(如SEQ ID NO: 1所示)和polyA序列(120個A，在質體上提前加尾)。區別在於SARS-CoV-2-A mRNA採用人β珠蛋白(HBB)的3’UTR序列(序列如SEQ ID NO: 4所示：5’-GCTCGCTTTCTTG CTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGC-3’)，SARS-CoV-2-B mRNA採用人α珠蛋白(HBA)的3’UTR序列(序列如SEQ ID NO: 7所示：TGATAATAGGCTGGAGCCTCGG TGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC)。 表1  SARS-CoV-2 mRNA疫苗的3’UTR的最適化設計 mRNA 5’帽子結構 5’UTR ORF 3’UTR polyA SARS-CoV-2-A Cap1 PRL32部分5’UTR序列(SEQ ID NO: 3) ORF 人α珠蛋白3’UTR序列(SEQ ID NO:  7) 120A SARS-CoV-2-B Cap1 PRL32部分5’UTR序列(SEQ ID NO: 3) ORF 人β珠蛋白3’UTR序列(SEQ ID NO:  4) 120A The optimization scheme of the 3' UTR sequence of SARS-CoV-2 mRNA in this example is shown in Table 1, and the details are as follows: Vaccinia virus capping enzyme and other components, add a 7-methylguanosine cap structure (Cap 0) to the 5' end of RNA, then use 2'-O-methyltransferase and the methyl group on the SAM to transfer to Cap1 is formed on Cap0), the partial 5'UTR sequence of human ribosomal protein L32 (PRL32) (sequence is shown in SEQ ID NO: 3: 5'-GGGGCGCTGCCTACGGAGGTGGCAGCCATCTCCTTCTCGGCATCAAGCTTACC-3'), ORF is the genome of the above-mentioned GenBank strain of MN908947 The gene sequence encoding S1 protein in the sequence (as shown in SEQ ID NO: 1) and the polyA sequence (120 A, tailed in advance on the plastid). The difference is that SARS-CoV-2-A mRNA adopts the 3'UTR sequence of human beta globin (HBB) (the sequence is shown in SEQ ID NO: 4: 5'-GCTCGCTTTCTTG CTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGC-3'), SARS-CoV-2- B mRNA adopts the 3'UTR sequence of human alpha globin (HBA) (the sequence is shown in SEQ ID NO: 7: TGATAATAGGCTGGAGCCTCGG TGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC). Table 1 Optimal design of 3'UTR of SARS-CoV-2 mRNA vaccine mRNA 5' hat construction 5'UTR ORF 3'UTR polyA SARS-CoV-2-A Cap1 PRL32 partial 5'UTR sequence (SEQ ID NO: 3) ORF Human alpha globin 3'UTR sequence (SEQ ID NO: 7) 120A SARS-CoV-2-B Cap1 PRL32 partial 5'UTR sequence (SEQ ID NO: 3) ORF Human beta globin 3' UTR sequence (SEQ ID NO: 4) 120A

體外轉錄工藝製備出來的上述SARS-CoV-2-A mRNA和SARS-CoV-2-B mRNA分別瞬時轉染HEK 293T細胞，並用合適的抗S1蛋白抗體(義翹神州)染色，用FITC偶聯的二級抗體(Alpha Diagnostic International)進行複染。轉染後24小時，HEK 293T細胞用合適的抗S1蛋白抗體和用FITC偶聯的二級抗體(1:500)染色，然後用流式細胞術(FACS)在BeckMan-CytoFLEX上使用進行分析。結果如圖1所示(其中wfi組為空白對照，注射的注射用水)。The above-mentioned SARS-CoV-2-A mRNA and SARS-CoV-2-B mRNA prepared by in vitro transcription process were transiently transfected into HEK 293T cells, respectively, and stained with an appropriate anti-S1 protein antibody (Yiqiao Shenzhou), coupled with FITC The secondary antibody (Alpha Diagnostic International) was counterstained. Twenty-four hours after transfection, HEK 293T cells were stained with the appropriate anti-S1 protein antibody and with a FITC-conjugated secondary antibody (1:500) and analyzed by flow cytometry (FACS) on a BeckMan-CytoFLEX. The results are shown in Figure 1 (the wfi group is the blank control, and the injected water is used for injection).

從圖中結果可以看出，兩組的S1蛋白表現量均較高，其中，SARS-CoV-2-B mRNA轉染組轉染細胞中的S1蛋白表現量高於SARS-CoV-2-A mRNA轉染組(SARS-CoV-2-B mRNA轉染組的表現量約為SARS-CoV-2-A mRNA轉染組的1.5倍)，可見人β珠蛋白3’UTR序列比該人α珠蛋白3’UTR序列更有助於提高蛋白表現量。因此，確定人β珠蛋白3’UTR序列為本發明後續實驗中優選的3’UTR。 實施例 3 新型冠 狀病毒 SARS-CoV-2 mRNA 的 Poly(A) 的最適化 It can be seen from the results in the figure that the expression of S1 protein in both groups is higher, and the expression of S1 protein in the transfected cells of the SARS-CoV-2-B mRNA transfection group is higher than that of SARS-CoV-2-A mRNA transfection group (the expression level of SARS-CoV-2-B mRNA transfection group is about 1.5 times that of SARS-CoV-2-A mRNA transfection group), it can be seen that the 3'UTR sequence of human β-globin is higher than that of human α-globin. The globin 3'UTR sequence is more helpful to improve protein expression. Therefore, the 3'UTR sequence of human β-globin was determined to be the preferred 3'UTR in the subsequent experiments of the present invention. Example 3 Optimization of Poly(A) of Novel Coronavirus SARS-CoV-2 mRNA

本實施例中的SARS-CoV-2 mRNA的序列最適化方案如表2所示，具體如下： SARS-CoV-2 mRNA序列關鍵元件是採用雙酶法Cap1、人核糖體蛋白L32(PRL32)的部分5’UTR序列、ORF為上述GenBank為MN908947的毒株基因組序列中編碼S1蛋白的基因序列(如SEQ ID NO: 1所示)、人β珠蛋白(HBB)3’UTR序列。 The sequence optimization scheme of SARS-CoV-2 mRNA in this example is shown in Table 2, and the details are as follows: The key elements of the SARS-CoV-2 mRNA sequence are the double-enzyme method Cap1, the partial 5'UTR sequence of human ribosomal protein L32 (PRL32), and the ORF is the gene sequence encoding the S1 protein in the genome sequence of the above-mentioned GenBank strain of MN908947 ( As shown in SEQ ID NO: 1), human beta globin (HBB) 3'UTR sequence.

SARS-CoV-2-C和SARS-CoV-2-D的序列部分是一樣的，區別在於SARS-CoV-2-C mRNA採用在pcDNA 3.1(+)質體上提前加尾120A的方式，SARS-CoV-2-D mRNA採用體外酶素催化加尾120A的方式。SARS-CoV-2-C與SARS-CoV-2-E、SARS-CoV-2-F的除polyA外其他序列部分一樣，也同樣採用質體上提前加尾的，區別在於polyA的數量分別為120A、50A和16A。 表2 SARS-CoV-2 mRNA疫苗Poly(A)的最適化設計 mRNA 5’帽子結構 5’UTR ORF 3’UTR polyA SARS-CoV-2-C Cap1 PRL32部分5’UTR序列(SEQ ID NO: 3) ORF 人β珠蛋白3’UTR序列(SEQ ID NO: 4) 120A SARS-CoV-2-D Cap1 PRL32部分5’UTR序列(SEQ ID NO: 3) ORF 人β珠蛋白3’UTR序列(SEQ ID NO: 4) 120A SARS-CoV-2-E Cap1 PRL32部分5’UTR序列(SEQ ID NO: 3) ORF 人β珠蛋白3’UTR序列(SEQ ID NO: 4) 50A SARS-CoV-2-F Cap1 PRL32部分5’UTR序列(SEQ ID NO: 3) ORF 人β珠蛋白3’UTR序列(SEQ ID NO: 4) 16A The sequence parts of SARS-CoV-2-C and SARS-CoV-2-D are the same, the difference is that SARS-CoV-2-C mRNA adopts the method of adding tail 120A to pcDNA 3.1(+) plastid in advance. -CoV-2-D mRNA was tailed by 120A catalyzed by in vitro enzyme. SARS-CoV-2-C is the same as SARS-CoV-2-E and SARS-CoV-2-F except for the other sequence parts except polyA, and also adopts the tailing on the plastid in advance. The difference is that the number of polyA is 120A, 50A and 16A. Table 2 Optimal design of SARS-CoV-2 mRNA vaccine Poly(A) mRNA 5' hat construction 5'UTR ORF 3'UTR polyA SARS-CoV-2-C Cap1 PRL32 partial 5'UTR sequence (SEQ ID NO: 3) ORF Human beta globin 3' UTR sequence (SEQ ID NO: 4) 120A SARS-CoV-2-D Cap1 PRL32 partial 5'UTR sequence (SEQ ID NO: 3) ORF Human beta globin 3' UTR sequence (SEQ ID NO: 4) 120A SARS-CoV-2-E Cap1 PRL32 partial 5'UTR sequence (SEQ ID NO: 3) ORF Human beta globin 3' UTR sequence (SEQ ID NO: 4) 50A SARS-CoV-2-F Cap1 PRL32 partial 5'UTR sequence (SEQ ID NO: 3) ORF Human beta globin 3' UTR sequence (SEQ ID NO: 4) 16A

體外轉錄工藝製備出來的上述SARS-CoV-2-C mRNA、SARS-CoV-2-D mRNA、SARS-CoV-2-E mRNA和SARS-CoV-2-F mRNA分別瞬時轉染HEK 293T細胞，並用合適的抗S1蛋白抗體(義翹神州)染色，用FITC偶聯的二級抗體(Alpha Diagnostic International)進行複染。轉染後24小時，HEK 293T細胞用合適的抗S1蛋白抗體和用FITC偶聯的二級抗體(1:500)染色，然後用流式細胞術(FACS)在BeckMan-CytoFLEX上使用進行分析。結果如圖2所示。The above-mentioned SARS-CoV-2-C mRNA, SARS-CoV-2-D mRNA, SARS-CoV-2-E mRNA and SARS-CoV-2-F mRNA prepared by in vitro transcription process were respectively transiently transfected into HEK 293T cells, And stained with appropriate anti-S1 protein antibody (Yiqiao Shenzhou) and counterstained with FITC-conjugated secondary antibody (Alpha Diagnostic International). Twenty-four hours after transfection, HEK 293T cells were stained with the appropriate anti-S1 protein antibody and with a FITC-conjugated secondary antibody (1:500) and analyzed by flow cytometry (FACS) on a BeckMan-CytoFLEX. The results are shown in Figure 2.

從圖中結果可以看出，上述幾組的S1蛋白表現量都較對照組顯著提高，其中，SARS-CoV-2-C mRNA轉染組轉染細胞中的S1蛋白表現量明顯高於SARS-CoV-2-E mRNA轉染組(約為其1.5倍)和SARS-CoV-2-F mRNA轉染組(約為其3倍)，可見Poly(A)尾的長度對mRNA的影響更為顯著。除此之外，SARS-CoV-2-C mRNA轉染組轉染細胞中的S1蛋白表現量高於SARS-CoV-2-D mRNA轉染組(約為其2倍)，說明加尾的方式同樣對mRNA有影響。綜上考慮，本發明後續實驗中優選質體上提前加尾120A的方式。 實施例 4 新型冠 狀病毒 SARS-CoV-2 mRNA 的訊號胜肽的最適化 It can be seen from the results in the figure that the expression of S1 protein in the above groups was significantly higher than that in the control group. In the CoV-2-E mRNA transfection group (about 1.5 times its value) and the SARS-CoV-2-F mRNA transfection group (about 3 times its value), it can be seen that the length of the Poly(A) tail has a greater effect on mRNA. Remarkably. In addition, the expression level of S1 protein in the transfected cells of the SARS-CoV-2-C mRNA transfection group was higher than that of the SARS-CoV-2-D mRNA transfection group (about 2 times), indicating that the tailed The way also affects mRNA. To sum up, the method of adding tail 120A to the plastid in advance is preferred in the subsequent experiments of the present invention. Example 4 Optimization of the Signaling Peptide of Novel Coronavirus SARS-CoV-2 mRNA

本實施例中的SARS-CoV-2 mRNA的序列最適化方案如表3所示，具體如下： SARS-CoV-2 mRNA序列關鍵元件是採用雙酶法Cap1、人核糖體蛋白L32(PRL32)的部分5’UTR序列、人β珠蛋白(HBB)3’UTR序列和質體加尾120A。 The sequence optimization scheme of SARS-CoV-2 mRNA in this example is shown in Table 3, and the details are as follows: The key elements of the SARS-CoV-2 mRNA sequence are the double-enzyme method Cap1, the partial 5'UTR sequence of human ribosomal protein L32 (PRL32), the 3'UTR sequence of human beta globin (HBB), and the plastid tail 120A.

SARS-CoV-2-G、SARS-CoV-2-H和SARS-CoV-2-I的其他序列是一樣的，區別在於SARS-CoV-2-G mRNA、SARS-CoV-2-H和SARS-CoV-2-I分別在上述GenBank為MN908947的毒株基因組序列中編碼S1蛋白基因前端分別加上了人IgE訊號胜肽(序列如SEQ ID NO: 5所示：MDWTWILFLVAAATRVHS)、人IgG訊號胜肽(序列如SEQ ID NO: 8所示：MGWSCIILFLVATATGVHS)和鼠IgK(序列如SEQ ID NO: 9所示：METDTLLLWVLLLWVPGSTGD)訊號胜肽，得到三個不同的ORF，分別對應表3中的“ORF-1”、“ORF-2”和“ORF-3”。 表3 SARS-CoV-2 mRNA疫苗訊號胜肽的最適化設計 mRNA 5’帽子結構 5’UTR ORF 3’UTR polyA SARS-CoV-2-G Cap1 PRL32部分5’UTR序列 ORF-1 人β珠蛋白3’UTR序列 120A SARS-CoV-2-H Cap1 PRL32部分5’UTR序列 ORF-2 人β珠蛋白3’UTR序列 120A SARS-CoV-2-I Cap1 PRL32部分5’UTR序列 ORF-3 人β珠蛋白3’UTR序列 120A The other sequences of SARS-CoV-2-G, SARS-CoV-2-H and SARS-CoV-2-I are the same, the difference is SARS-CoV-2-G mRNA, SARS-CoV-2-H and SARS -CoV-2-1 is respectively added with human IgE signal peptide (sequence shown in SEQ ID NO: 5: MDWTWILFLVAAATRVHS), human IgG signal peptide in the front end of the gene encoding S1 protein in the genome sequence of the above-mentioned GenBank strain as MN908947. Peptide (sequence as shown in SEQ ID NO: 8: MGWSCIILFLVATATGVHS) and murine IgK (sequence as shown in SEQ ID NO: 9: METDTLLLWVLLLWVPGSTGD) signal peptides to obtain three different ORFs, corresponding to "ORF- 1", "ORF-2" and "ORF-3". Table 3 Optimal design of signal peptides for SARS-CoV-2 mRNA vaccines mRNA 5' hat construction 5'UTR ORF 3'UTR polyA SARS-CoV-2-G Cap1 PRL32 partial 5'UTR sequence ORF-1 Human β-globin 3'UTR sequence 120A SARS-CoV-2-H Cap1 PRL32 partial 5'UTR sequence ORF-2 Human β-globin 3'UTR sequence 120A SARS-CoV-2-I Cap1 PRL32 partial 5'UTR sequence ORF-3 Human β-globin 3'UTR sequence 120A

體外轉錄工藝製備出來的上述SARS-CoV-2-G mRNA、SARS-CoV-2-H mRNA和SARS-CoV-2-I mRNA分別瞬時轉染HEK 293T細胞，並用合適的抗S1蛋白抗體(義翹神州)染色，用FITC偶聯的二級抗體(Alpha Diagnostic International)進行複染。轉染後24小時，HEK 293T細胞用合適的抗S1蛋白抗體和用FITC偶聯的二級抗體(1:500)染色，然後用流式細胞術(FACS)在BeckMan-CytoFLEX上使用進行分析。結果如圖3所示。The above-mentioned SARS-CoV-2-G mRNA, SARS-CoV-2-H mRNA and SARS-CoV-2-I mRNA prepared by the in vitro transcription process were transiently transfected into HEK 293T cells, respectively, and a suitable anti-S1 protein antibody (sense) was used. (Qiao Shenzhou) staining and counterstaining with FITC-conjugated secondary antibody (Alpha Diagnostic International). Twenty-four hours after transfection, HEK 293T cells were stained with the appropriate anti-S1 protein antibody and with a FITC-conjugated secondary antibody (1:500) and analyzed by flow cytometry (FACS) on a BeckMan-CytoFLEX. The results are shown in Figure 3.

從圖中結果可以看出，上述幾組的S1蛋白表現量都較對照組顯著提高，其中，SARS-CoV-2-G mRNA轉染組轉染細胞中的S1蛋白表現量要高於SARS-CoV-2-H mRNA轉染組(約為其2倍)和SARS-CoV-2-I mRNA轉染組(約為其1.5倍)，可見使用人IgE訊號胜肽比人IgG訊號胜肽和鼠IgK訊號胜肽更能提高蛋白表現量，因此本發明選擇SARS-CoV-2-G作為後續實驗mRNA疫苗的mRNA序列的來源，SARS-CoV-2-G的序列為如SEQ ID NO: 6所示的DNA序列。 實施例 5 新型冠 狀病毒 SARS-CoV-2 mRNA 的體外轉錄基質的最適化 It can be seen from the results in the figure that the expression of S1 protein in the above groups was significantly higher than that in the control group. In the CoV-2-H mRNA transfection group (about 2 times that) and the SARS-CoV-2-I mRNA transfection group (about 1.5 times), it can be seen that the use of human IgE signal peptides is higher than that of human IgG signal peptides and The murine IgK signal peptide can improve the protein expression. Therefore, the present invention selects SARS-CoV-2-G as the source of the mRNA sequence of the mRNA vaccine in the subsequent experiments. The sequence of SARS-CoV-2-G is as SEQ ID NO: 6 DNA sequence shown. Example 5 Optimization of the in vitro transcription substrate of the novel coronavirus SARS-CoV-2 mRNA

本實施例中的SARS-CoV-2 mRNA來自於實施例3中最適化得到的序列SARS-CoV-2-G，序列為SEQ ID NO: 6，將其(序列SEQ ID NO: 6後面還包括限制酶切割位址AGAAGAGC)從載體上的T7啟動子轉錄起始位址轉錄下來的mRNA的序列如SEQ ID NO: 10所示。The SARS-CoV-2 mRNA in this example comes from the optimized sequence SARS-CoV-2-G obtained in Example 3, and the sequence is SEQ ID NO: 6. Restriction enzyme cleavage address AGAAGAGC) The sequence of the mRNA transcribed from the T7 promoter transcription start address on the vector is shown in SEQ ID NO: 10.

選擇三種人工修飾轉錄基質核苷酸替換傳統的基質NTP，即N1-甲基假尿苷(m1ΨTP)、假尿苷(Ψ)、5-甲基尿苷(m5U)(兆維科)分別對應SARS-CoV-2-G1、SARS-CoV-2-G2和SARS-CoV-2-G3，對照採用傳統基質NTP，為SARS-CoV-2-G4。Three artificially modified transcription substrate nucleotides were selected to replace the traditional substrate NTP, namely N1-methylpseudouridine (m1ΨTP), pseudouridine (Ψ), and 5-methyluridine (m5U) (Zhaoweike), respectively. SARS-CoV-2-G1, SARS-CoV-2-G2 and SARS-CoV-2-G3, the control used traditional matrix NTP, SARS-CoV-2-G4.

體外轉錄工藝製備出來的上述SARS-CoV-2-G1 mRNA、SARS-CoV-2-G2 mRNA、SARS-CoV-2-G3和SARS-CoV-2-G4的mRNA分別瞬時轉染HEK 293T細胞，並用合適的抗S1蛋白抗體(義翹神州)染色，用FITC偶聯的二級抗體(Alpha Diagnostic International)進行複染。轉染後24小時，HEK 293T細胞用合適的抗S1蛋白抗體和用FITC偶聯的二級抗體(1:500)染色，然後用流式細胞術(FACS)在BeckMan-CytoFLEX上使用進行分析。結果如圖4所示。The above-mentioned SARS-CoV-2-G1 mRNA, SARS-CoV-2-G2 mRNA, SARS-CoV-2-G3 and SARS-CoV-2-G4 mRNA prepared by in vitro transcription process were transiently transfected into HEK 293T cells, respectively, And stained with appropriate anti-S1 protein antibody (Yiqiao Shenzhou) and counterstained with FITC-conjugated secondary antibody (Alpha Diagnostic International). Twenty-four hours after transfection, HEK 293T cells were stained with the appropriate anti-S1 protein antibody and with a FITC-conjugated secondary antibody (1:500) and analyzed by flow cytometry (FACS) on a BeckMan-CytoFLEX. The results are shown in Figure 4.

從圖中結果可以看出，SARS-CoV-2-G1 mRNA、SARS-CoV-2-G2 mRNA和SARS-CoV-2-G3 mRNA轉染組轉染細胞中的S1蛋白表現量均高於SARS-CoV-2-G4轉染組，可見使用人工修飾轉錄基質核苷酸替換傳統的基質NTP可以提高蛋白表現量。進一步地，SARS-CoV-2-G1 mRNA轉染組的S1蛋白表現量最高，說明N1-甲基假尿苷(m1ΨTP)的效果最好，因此後續實驗中選擇N1-甲基假尿苷(m1ΨTP)替換傳統轉錄基質核苷酸NTP。 實施例 6 新型冠 狀病毒 SARS-CoV-2 核酸疫苗的製備方法 It can be seen from the results in the figure that the expression of S1 protein in the transfected cells of the SARS-CoV-2-G1 mRNA, SARS-CoV-2-G2 mRNA and SARS-CoV-2-G3 mRNA transfection groups was higher than that of SARS-CoV-2 -CoV-2-G4 transfection group, it can be seen that the use of artificially modified transcription matrix nucleotides to replace traditional matrix NTPs can improve protein expression. Further, the expression of S1 protein in the SARS-CoV-2-G1 mRNA transfection group was the highest, indicating that N1-methylpseudouridine (m1ΨTP) had the best effect. Therefore, N1-methylpseudouridine (m1ΨTP) was selected in subsequent experiments. m1ΨTP) replaces the traditional transcription substrate nucleotide NTP. Embodiment 6 The preparation method of novel coronavirus SARS-CoV-2 nucleic acid vaccine

將實施例5中的SARS-CoV-2-G1體外轉錄純化得到的mRNA溶解於pH6.8-7.4的PBS緩衝液，此為水相。 表4 SARS-CoV-2 mRNA疫苗LNP脂質體遞送系統的最適化設計 組別 陽離子脂質種類及其莫耳百分比 輔助脂質種類及其莫耳百分比 結構脂質種類及其莫耳百分比 表面活性劑種類及其莫耳百分比 A Dlin-MC3-DMA 20% DSPC 13% 膽固醇 65% DMG-PEG 20002% B Dlin-MC3-DMA 70% DSPC 13% 膽固醇 15% DMG-PEG 20002% C Dlin-MC3-DMA 50% DSPC 13% 膽固醇 35% DMG-PEG 20002% D Dlin-MC3-DMA 50% DSPC 10% 膽固醇 35% DMG-PEG 20005% E Dlin-MC3-DMA 50% DOPS 13% 膽固醇 35% DMG-PEG 20002% F Dlin-MC3-DMA 50% DSPC 20% 膽固醇 28% DMG-PEG 20002% G Dlin-MC3-DMA 50% DSPC 25% 膽固醇 23% DMG-PEG 20002% H Dlin-MC3-DMA 50% DSPC 10% 膽固醇 38.5% DMG-PEG 20001.5% The mRNA obtained by in vitro transcription and purification of SARS-CoV-2-G1 in Example 5 was dissolved in a PBS buffer of pH 6.8-7.4, which was an aqueous phase. Table 4 Optimal design of SARS-CoV-2 mRNA vaccine LNP liposome delivery system group Cationic lipid species and their molar percentages Helper lipid species and their molar percentages Structural lipid species and their molar percentages Types of surfactants and their molar percentages A Dlin-MC3-DMA 20% DSPC 13% Cholesterol 65% DMG-PEG 2000 2% B Dlin-MC3-DMA 70% DSPC 13% Cholesterol 15% DMG-PEG 2000 2% C Dlin-MC3-DMA 50% DSPC 13% Cholesterol 35% DMG-PEG 2000 2% D Dlin-MC3-DMA 50% DSPC 10% Cholesterol 35% DMG-PEG 2000 5% E Dlin-MC3-DMA 50% DOPS 13% Cholesterol 35% DMG-PEG 2000 2% F Dlin-MC3-DMA 50% DSPC 20% Cholesterol 28% DMG-PEG 2000 2% G Dlin-MC3-DMA 50% DSPC 25% Cholesterol 23% DMG-PEG 2000 2% H Dlin-MC3-DMA 50% DSPC 10% Cholesterol 38.5% DMG-PEG 2000 1.5%

將LNP脂質體遞送系統的四種組分按照上述表4中的莫耳百分比溶於無水乙醇，此為有機相。The four components of the LNP liposome delivery system were dissolved in absolute ethanol according to the molar percentages in Table 4 above, which was the organic phase.

採用微流體混合儀，以10 mL/min的流速將有機相與水相按1:5的體積比混合，混合後除去溶液中的乙醇並濃縮mRNA濃度至0.5 mg/mL，最終得到包裹SARS-CoV-2-G1 mRNA的脂質體奈米顆粒，其中LNP：mRNA(w/w)＝15:1，即為新型冠狀病毒SARS-CoV-2 mRNA核酸疫苗。檢測粒徑、PDI、包封率和入胞率，實驗組A~G和組H(參考Corbett, Kizzmekia S., et al. "SARS-CoV-2 mRNA Vaccine Development Enabled by Prototype Pathogen Preparedness." bioRxiv (2020))的檢測結果如表5所示。 表5 不同組分LNP的粒徑、PDI、包封率和入胞率檢測結果 組別 粒徑(nm) PDI 包封率(%) 入胞率(%) A 105.47±4.06 0.071±0.06 39.9±5.58 82.17±3.66 B 63.30±5.42 0.068±0.02 20.8±5.47 77.56±4.30 C 86.11±1.28 0.067±0.03 90.3±3.01 92.51±2.52 D 74.81±1.99 0.065±0.05 92.4±3.73 87.39±4.58 E 90.34±9.89 0.109±0.05 83.4±4.14 88.93±3.38 F 81.39±7.03 0.191±0.04 87.6±4.86 90.93±3.03 G 85.83±1.90 0.296±0.07 32.9±5.74 77.28±2.26 H 80.20±5.52 0.209±0.08 91.4±3.21 87.66±3.64 Using a microfluidic mixer, the organic phase and the aqueous phase were mixed at a volume ratio of 1:5 at a flow rate of 10 mL/min. After mixing, the ethanol in the solution was removed and the mRNA concentration was concentrated to 0.5 mg/mL. Finally, a packaged SARS- The liposome nanoparticle of CoV-2-G1 mRNA, wherein LNP:mRNA(w/w)=15:1, is the new coronavirus SARS-CoV-2 mRNA nucleic acid vaccine. To detect particle size, PDI, encapsulation efficiency and cell entrapment rate, experimental group A~G and group H (refer to Corbett, Kizzmekia S., et al. "SARS-CoV-2 mRNA Vaccine Development Enabled by Prototype Pathogen Preparedness." bioRxiv (2020)) detection results are shown in Table 5. Table 5 Detection results of particle size, PDI, encapsulation efficiency and cell entry rate of LNPs with different components group Particle size (nm) PDI Encapsulation Efficiency (%) Cell rate (%) A 105.47±4.06 0.071±0.06 39.9±5.58 82.17±3.66 B 63.30±5.42 0.068±0.02 20.8±5.47 77.56±4.30 C 86.11±1.28 0.067±0.03 90.3±3.01 92.51±2.52 D 74.81±1.99 0.065±0.05 92.4±3.73 87.39±4.58 E 90.34±9.89 0.109±0.05 83.4±4.14 88.93±3.38 F 81.39±7.03 0.191±0.04 87.6±4.86 90.93±3.03 G 85.83±1.90 0.296±0.07 32.9±5.74 77.28±2.26 H 80.20±5.52 0.209±0.08 91.4±3.21 87.66±3.64

從表5可以看出，陽離子脂質的含量對LNP的粒徑大小有一些影響，適量DSPC輔助脂質的加入可以提高奈米脂質體顆粒的包封率和入胞率，綜合考慮奈米脂質體顆粒的粒徑、PDI、包封率和入胞率發現，上述所有組的表現均較好，其中C組的綜合表現相對最好，因此選擇C組的組分配比作為本mRNA疫苗的LNP奈米脂質體遞送系統。 實施例 7 中和抗體效價測定 It can be seen from Table 5 that the content of cationic lipid has some influence on the particle size of LNP. The addition of an appropriate amount of DSPC auxiliary lipid can improve the encapsulation efficiency and cell entry rate of nanoliposome particles. Considering nanoliposome particles comprehensively The particle size, PDI, encapsulation rate and cell entrapment rate of the mRNA vaccine showed that all the above groups performed well, and the comprehensive performance of group C was relatively the best. Therefore, the group distribution ratio of group C was selected as the LNP nanoparticle of this mRNA vaccine. Liposome delivery system. Example 7 Determination of Neutralizing Antibody Titer

mRNA選用實例5中的SARS-CoV-2-G1 mRNA進行小鼠免疫實驗，疫苗載體按照實施例6中的配方製備。24隻(雌雄各半)健康的BALB/c小鼠(南京集萃藥康)隨機分成4組，每組3雌3雄。同6隻人源化ACE2小鼠(南京集萃藥康)組成的一組作為試驗系統。對照組注射市售PBS，BALB/c實驗組分別免疫mRNA-3 10 μg/隻、mRNA-3 30 μg/隻、mRNA-3 100 μg/隻，如表6所示。 表6 小鼠免疫原性實驗分組及給藥設計表 組 對照品/供試品 劑量(μg/隻) 動物數 隻(雌雄各半) 1 輔料對照組 0 6 2 mRNA-3低劑量組 10 6 3 mRNA-3中劑量組 30 6 4 mRNA-3高劑量組 100 6 The mRNA was selected from the SARS-CoV-2-G1 mRNA in Example 5 to carry out the mouse immunization experiment, and the vaccine vector was prepared according to the formula in Example 6. Twenty-four (half male and half) healthy BALB/c mice (Nanjing Jicuiyaokang) were randomly divided into 4 groups, each with 3 females and 3 males. A group consisting of 6 humanized ACE2 mice (Nanjing Jicui Yaokang) was used as the experimental system. The control group was injected with commercially available PBS, and the BALB/c experimental group was immunized with mRNA-3 10 μg/a, mRNA-3 30 μg/a, and mRNA-3 100 μg/a, respectively, as shown in Table 6. Table 6 Mouse immunogenicity experimental grouping and dosing design table Group reference/test Dosage (μg/only) number of animals only (half male and female) 1 Excipient control group 0 6 2 mRNA-3 low dose group 10 6 3 mRNA-3 medium dose group 30 6 4 mRNA-3 high dose group 100 6

免疫實驗共3次，均為小鼠後肢肌肉注射，每間隔3周免疫一次。第一次免疫為D1，在D18和D36異氟烷麻醉後從眼眶靜脈取血，D57異氟烷麻醉後腹主動脈取血，分離血清。為監測每次免疫後疫苗組小鼠血清抗體的消長情況，採用間接ELISA法測定D18，D36，D57血清抗SARS-CoV-2 IgG 抗體效價，結果如圖5所示： 結果表明，在D18可以檢測到一定程度的小鼠免疫血清抗SARS-CoV-2 IgG抗體，並且抗體效價與劑量高低有正相關關係。在D36、D57可以看到抗體位準隨免疫次數的增加以及時間的延長逐漸升高。第三次免疫後，在100μg/隻的高劑量組小鼠免疫血清抗SARS-CoV-2 IgG抗體效價最高可達1:1479。 A total of 3 immunization experiments were performed, all of which were intramuscularly injected into the hind limbs of mice, and immunized once every 3 weeks. The first immunization was D1. Blood was collected from the orbital vein after isoflurane anesthesia at D18 and D36, and blood was collected from the abdominal aorta after isoflurane anesthesia at D57, and serum was separated. In order to monitor the fluctuation of serum antibodies of the mice in the vaccine group after each immunization, indirect ELISA method was used to determine the anti-SARS-CoV-2 IgG antibody titers of D18, D36 and D57 serum. The results are shown in Figure 5: The results showed that a certain degree of anti-SARS-CoV-2 IgG antibodies in mouse immune serum could be detected at D18, and the antibody titer was positively correlated with the dose. At D36 and D57, it can be seen that the antibody level gradually increases with the increase of immunization times and the prolongation of time. After the third immunization, the titer of anti-SARS-CoV-2 IgG antibody in the immune serum of mice in the high-dose group of 100 μg/mouse was up to 1:1479.

於D57實驗終點，疫苗免疫組和對照組小鼠各3隻，分離小鼠脾臟細胞，以SARS-CoV-2的S1蛋白作為特異性刺激物進行IFN-γ ELISpot檢測，以刺激物存在時的SFU數與無刺激物時的SFU數的差值作圖，結果如圖6所示： 結果表明，經SARS-CoV-2的S1蛋白特異性抗原刺激後，疫苗免疫組小鼠脾細胞分泌IFN-γ斑點形成細胞的數量均高於對照組，並且隨著劑量增加表現出升高。進一步地，結果表明本mRNA疫苗可以有效活化小鼠免疫系統產生體液免疫和細胞免疫，產生大量中和抗體，顯示出良好的安全性、有效性和長效性。 At the end of the D57 experiment, there were 3 mice in each of the vaccine immunization group and the control group, the spleen cells of the mice were isolated, and the S1 protein of SARS-CoV-2 was used as a specific stimulator for IFN-γ ELISpot detection. The difference between the number of SFU and the number of SFU without the stimulus is plotted, and the results are shown in Figure 6: The results showed that after stimulation with the S1 protein-specific antigen of SARS-CoV-2, the number of IFN-γ speck-forming cells secreting IFN-γ in the spleen cells of the mice in the vaccine immunization group was higher than that in the control group, and increased with the increase of the dose. Further, the results show that the mRNA vaccine can effectively activate the immune system of mice to generate humoral immunity and cellular immunity, and generate a large number of neutralizing antibodies, showing good safety, efficacy and long-term effect.

雖然以上描述了本發明的具體實施方式，但是本領域的技術人員應當理解，這些僅是舉例說明，在不背離本發明的原理和實質的前提下，可以對這些實施方式做出多種變更或修改。因此，本發明的保護範圍由所附申請專利範圍限定。Although the specific embodiments of the present invention are described above, those skilled in the art should understand that these are only examples, and various changes or modifications can be made to these embodiments without departing from the principle and essence of the present invention . Therefore, the protection scope of the present invention is defined by the appended claims.

圖1為實施例2中SARS-CoV-2 mRNA轉染細胞的S1蛋白表現位準。Figure 1 shows the expression level of S1 protein in SARS-CoV-2 mRNA-transfected cells in Example 2.

圖2為實施例3中SARS-CoV-2 mRNA轉染細胞的S1蛋白表現位準。Figure 2 shows the expression level of S1 protein in SARS-CoV-2 mRNA-transfected cells in Example 3.

圖3為實施例4中SARS-CoV-2 mRNA轉染細胞的S1蛋白表現位準。FIG. 3 is the expression level of S1 protein in SARS-CoV-2 mRNA-transfected cells in Example 4.

圖4為實施例5中SARS-CoV-2 mRNA轉染細胞的S1蛋白表現位準。FIG. 4 is the expression level of S1 protein in SARS-CoV-2 mRNA-transfected cells in Example 5.

圖5為實施例7中mRNA-3疫苗免疫組小鼠血清抗SARS-COV-2 IgG抗體效價(以上數值均為雙重複孔的平均值)。Fig. 5 is the anti-SARS-COV-2 IgG antibody titer of the serum of mice in the mRNA-3 vaccine immunization group in Example 7 (the above values are the average values of double-replicated wells).

圖6為實施例7中mRNA-3疫苗免疫組小鼠脾細胞IFN-γ酶聯免疫斑點分析(以上數值均為雙重複孔的平均值)。Figure 6 is the IFN-γ enzyme-linked immunospot analysis of spleen cells of mice in the mRNA-3 vaccine immunization group in Example 7 (the above values are the average values of double-replicated wells).

Figure 12_A0101_SEQ_0001
Figure 12_A0101_SEQ_0001

Figure 12_A0101_SEQ_0002
Figure 12_A0101_SEQ_0002

Figure 12_A0101_SEQ_0003
Figure 12_A0101_SEQ_0003

Figure 12_A0101_SEQ_0004
Figure 12_A0101_SEQ_0004

Figure 12_A0101_SEQ_0005
Figure 12_A0101_SEQ_0005

Figure 12_A0101_SEQ_0006
Figure 12_A0101_SEQ_0006

Figure 12_A0101_SEQ_0007
Figure 12_A0101_SEQ_0007

Figure 12_A0101_SEQ_0008
Figure 12_A0101_SEQ_0008

Figure 12_A0101_SEQ_0009
Figure 12_A0101_SEQ_0009

Figure 12_A0101_SEQ_0010
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Figure 12_A0101_SEQ_0011
Figure 12_A0101_SEQ_0011

Figure 12_A0101_SEQ_0012
Figure 12_A0101_SEQ_0012

Figure 12_A0101_SEQ_0013
Figure 12_A0101_SEQ_0013

Claims (11)

一種分離的mRNA，其包括編碼來源於SARS-CoV-2病毒的S1蛋白的mRNA，其中，所述S1蛋白的胺基酸序列如SEQ ID NO: 2所示； 所述分離的mRNA還包括以下(a)~(d)中的一種或多種： (a) 5’-帽結構，優選為CAP 0型、CAP I型或CAP II型的帽結構；所述CAP 0型的帽結構優選為7-甲基鳥苷帽結構，所述CAP I型的帽結構優選為N7mGpppAm； (b) 3’-聚腺苷酸，其序列優選為包含16-150個腺苷核苷酸的序列，更優選包含50-120個腺苷核苷酸的序列； (c) 5’-UTR，所述5’-UTR優選為核糖體蛋白L32的5’-UTR，例如為人核糖體蛋白L32的5’-UTR，其序列優選如SEQ ID NO: 3所示； (d) 3’-UTR，所述3’-UTR的序列優選為β珠蛋白的3’-UTR和α珠蛋白的3’-UTR，例如為人β珠蛋白的3’-UTR和人α珠蛋白的3’-UTR，其序列優選如SEQ ID NO: 4或SEQ ID NO: 7所示； 較佳地： 所述的3’-聚腺苷酸透過體外酶素催化反應添加到所述編碼來源於SARS-CoV-2病毒的S1蛋白的mRNA上，或者透過將其連接到包含所述編碼來源於SARS-CoV-2病毒的S1蛋白的mRNA的轉錄模板DNA之後進行轉錄。 An isolated mRNA comprising the mRNA encoding the S1 protein of the SARS-CoV-2 virus, wherein the amino acid sequence of the S1 protein is shown in SEQ ID NO: 2; The isolated mRNA also includes one or more of the following (a) to (d): (a) a 5'-cap structure, preferably a CAP 0 type, CAP I type or CAP II type cap structure; the CAP 0 type cap structure is preferably a 7-methylguanosine cap structure, the CAP I type The cap structure is preferably N7mGpppAm; (b) 3'-polyadenylic acid, the sequence of which is preferably a sequence comprising 16-150 adenosine nucleotides, more preferably a sequence comprising 50-120 adenosine nucleotides; (c) 5'-UTR, the 5'-UTR is preferably the 5'-UTR of ribosomal protein L32, such as the 5'-UTR of human ribosomal protein L32, the sequence of which is preferably as shown in SEQ ID NO: 3 ; (d) 3'-UTR, the sequence of the 3'-UTR is preferably the 3'-UTR of β-globin and the 3'-UTR of α-globin, such as the 3'-UTR of human β-globin and human α The 3'-UTR of globin, the sequence of which is preferably shown in SEQ ID NO: 4 or SEQ ID NO: 7; Preferably: The 3'-polyadenylic acid is added to the mRNA encoding the S1 protein derived from the SARS-CoV-2 virus through an in vitro enzyme-catalyzed reaction, or by connecting it to the mRNA encoding the S1 protein derived from the SARS-CoV-2 virus. The transcription of the mRNA of the S1 protein of the CoV-2 virus is followed by the transcription of the template DNA. 如請求項1所述的分離的mRNA，其特徵在於，其進一步包含以下(e)~(f)中的一種或兩種： (e) 訊號胜肽，所述訊號胜肽優選為人IgE訊號胜肽、人IgG訊號胜肽或小鼠IgK訊號胜肽；所述人IgE訊號胜肽的序列優選如SEQ ID NO: 5所示；所述人IgG訊號胜肽的序列優選如SEQ ID NO: 8所示；所述小鼠IgK訊號胜肽的序列優選如SEQ ID NO: 9所示； (f) 多核苷酸修飾，所述多核苷酸優選自N1-甲基假尿苷、假尿苷和5-甲基尿苷中的一種或多種； 較佳地： 所述的分離的mRNA，任選地從5’末端到3’末端，選自以下組： (1) 包括CAP I型的帽結構、人核糖體蛋白L32的5’-UTR、所述編碼來源於SARS-CoV-2病毒的S1蛋白的mRNA、人α珠蛋白的3’-UTR和包含120個腺苷核苷酸的3’-聚腺苷酸； (2) 包括CAP I型的帽結構、人核糖體蛋白L32的5’-UTR、所述編碼來源於SARS-CoV-2病毒的S1蛋白的mRNA、人β珠蛋白的3’-UTR和包含120個腺苷核苷酸的3’-聚腺苷酸； (3) 包括CAP I型的帽結構、人核糖體蛋白L32的5’-UTR、所述編碼來源於SARS-CoV-2病毒的S1蛋白的mRNA、人β珠蛋白的3’-UTR和包含50個腺苷核苷酸的3’-聚腺苷酸； (4) 包括CAP I型的帽結構、人核糖體蛋白L32的5’-UTR、所述編碼來源於SARS-CoV-2病毒的S1蛋白的mRNA、人β珠蛋白的3’-UTR和包含16個腺苷核苷酸的3’-聚腺苷酸； (5) 包括CAP I型的帽結構、人核糖體蛋白L32的5’-UTR、人IgE訊號胜肽、所述編碼來源於SARS-CoV-2病毒的S1蛋白的mRNA、人β珠蛋白的3’-UTR和包含120個腺苷核苷酸的3’-聚腺苷酸； (6) 包括CAP I型的帽結構、人核糖體蛋白L32的5’-UTR、人IgG訊號胜肽、所述編碼來源於SARS-CoV-2病毒的S1蛋白的mRNA、人β珠蛋白的3’-UTR和包含120個腺苷核苷酸的3’-聚腺苷酸； (7) 包括CAP I型的帽結構、人核糖體蛋白L32的5’-UTR、鼠IgK訊號胜肽、所述編碼來源於SARS-CoV-2病毒的S1蛋白的mRNA、人β珠蛋白的3’-UTR和包含120個腺苷核苷酸的3’-聚腺苷酸； (8) 包括CAP I型的帽結構、人核糖體蛋白L32的5’-UTR、所述編碼來源於SARS-CoV-2病毒的S1蛋白的mRNA、人β珠蛋白的3’-UTR和包含120個腺苷核苷酸的3’-聚腺苷酸，且經N1-甲基假尿苷的修飾； (9) 包括CAP I型的帽結構、人核糖體蛋白L32的5’-UTR、所述編碼來源於SARS-CoV-2病毒的S1蛋白的mRNA、人β珠蛋白的3’-UTR和包含120個腺苷核苷酸的3’-聚腺苷酸，且經假尿苷的修飾； (10) 包括CAP I型的帽結構、人核糖體蛋白L32的5’-UTR、所述編碼來源於SARS-CoV-2病毒的S1蛋白的mRNA、人β珠蛋白的3’-UTR和包含120個腺苷核苷酸的3’-聚腺苷酸，且經5-甲基尿苷的修飾； 更佳地，所述分離的mRNA的序列如SEQ ID NO: 10所示。 The isolated mRNA of claim 1, further comprising one or both of the following (e) to (f): (e) a signal peptide, the signal peptide is preferably a human IgE signal peptide, a human IgG signal peptide or a mouse IgK signal peptide; the sequence of the human IgE signal peptide is preferably as shown in SEQ ID NO: 5 shown; the sequence of the human IgG signal peptide is preferably shown in SEQ ID NO: 8; the sequence of the mouse IgK signal peptide is preferably shown in SEQ ID NO: 9; (f) modification of polynucleotides, preferably one or more of N1-methylpseudouridine, pseudouridine and 5-methyluridine; Preferably: The isolated mRNA, optionally from the 5' end to the 3' end, is selected from the group consisting of: (1) Including the cap structure of CAP I type, the 5'-UTR of human ribosomal protein L32, the mRNA encoding the S1 protein derived from the SARS-CoV-2 virus, the 3'-UTR of human alpha globin and the 3'-polyA of 120 adenosine nucleotides; (2) Including the cap structure of CAP I type, the 5'-UTR of human ribosomal protein L32, the mRNA encoding the S1 protein derived from the SARS-CoV-2 virus, the 3'-UTR of human beta globin and the 3'-polyA of 120 adenosine nucleotides; (3) Including the cap structure of CAP I type, the 5'-UTR of human ribosomal protein L32, the mRNA encoding the S1 protein derived from the SARS-CoV-2 virus, the 3'-UTR of human beta globin and the 3'-polyadenylation of 50 adenosine nucleotides; (4) Including the cap structure of CAP I type, the 5'-UTR of human ribosomal protein L32, the mRNA encoding the S1 protein derived from the SARS-CoV-2 virus, the 3'-UTR of human beta globin and the 3'-polyadenylation of 16 adenosine nucleotides; (5) Including the cap structure of CAP I type, the 5'-UTR of human ribosomal protein L32, the human IgE signal peptide, the mRNA encoding the S1 protein derived from the SARS-CoV-2 virus, the human β-globin 3'-UTR and 3'-polyA containing 120 adenosine nucleotides; (6) Including the cap structure of CAP I type, the 5'-UTR of human ribosomal protein L32, the human IgG signal peptide, the mRNA encoding the S1 protein derived from the SARS-CoV-2 virus, and the human β-globin 3'-UTR and 3'-polyA containing 120 adenosine nucleotides; (7) Including the cap structure of CAP I type, the 5'-UTR of human ribosomal protein L32, the murine IgK signal peptide, the mRNA encoding the S1 protein derived from the SARS-CoV-2 virus, the human β-globin 3'-UTR and 3'-polyA containing 120 adenosine nucleotides; (8) Including the cap structure of CAP type I, the 5'-UTR of human ribosomal protein L32, the mRNA encoding the S1 protein derived from the SARS-CoV-2 virus, the 3'-UTR of human beta globin and the 3'-polyadenylate of 120 adenosine nucleotides, and modified with N1-methylpseudouridine; (9) including the cap structure of CAP I type, the 5'-UTR of human ribosomal protein L32, the mRNA encoding the S1 protein derived from the SARS-CoV-2 virus, the 3'-UTR of human beta globin, and the 3'-polyA of 120 adenosine nucleotides, modified with pseudouridine; (10) Including the cap structure of CAP I type, the 5'-UTR of human ribosomal protein L32, the mRNA encoding the S1 protein derived from the SARS-CoV-2 virus, the 3'-UTR of human beta globin and the 3'-polyadenylate of 120 adenosine nucleotides, modified with 5-methyluridine; More preferably, the sequence of the isolated mRNA is shown in SEQ ID NO: 10. 一種分離的DNA，其核苷酸序列如SEQ ID NO: 6所示。An isolated DNA whose nucleotide sequence is shown in SEQ ID NO:6. 一種重組表現載體，其特徵在於，所述重組表現載體中含有如請求項1或2所述的分離的mRNA、或、如請求項3所述的DNA； 較佳地： 所述重組表現載體的骨架載體為pAAV-MCS、pcDNA 3.1(+)、pCMV-MCS、pEGFP-CTSB或pLVX-PAX1；和/或，所述重組表現載體的啟動子為Lac乳糖操縱子、TAC啟動子、TRC啟動子或T7啟動子。 A recombinant expression vector, characterized in that the recombinant expression vector contains the isolated mRNA as described in claim 1 or 2, or the DNA as described in claim 3; Preferably: The backbone vector of the recombinant expression vector is pAAV-MCS, pcDNA 3.1(+), pCMV-MCS, pEGFP-CTSB or pLVX-PAX1; and/or, the promoter of the recombinant expression vector is Lac lactose operon, TAC promoter, TRC promoter or T7 promoter. 一種轉形體，其特徵在於，在宿主中導入如請求項1或2所述的分離的mRNA、如請求項3所述的DNA、或、如請求項4所述的重組表現載體； 較佳地，所述轉形體的宿主細胞為原核細胞和/或真核細胞，優選為分離的哺乳動物細胞，更優選為哺乳動物受試者的細胞，進一步優選為哺乳動物受試者的分離的細胞，最優選為人受試者的分離的細胞。 A transformant, characterized in that the isolated mRNA as described in claim 1 or 2, the DNA as described in claim 3, or the recombinant expression vector as described in claim 4 are introduced into a host; Preferably, the host cell of the transformant is a prokaryotic cell and/or a eukaryotic cell, preferably an isolated mammalian cell, more preferably a cell of a mammalian subject, further preferably an isolated mammalian subject cells, most preferably isolated cells from a human subject. 一種組成物，其包含如請求項1或2所述的分離的mRNA和/或如請求項3所述的DNA。A composition comprising the isolated mRNA as claimed in claim 1 or 2 and/or the DNA as claimed in claim 3. 一種脂質體奈米顆粒，其包含如請求項1或2所述的分離mRNA、如請求項3所述的DNA、和/或如請求項6所述的組成物； 較佳地： 所述脂質體奈米顆粒為長循環陽離子脂質體奈米顆粒，優選為經PEG或其衍生物修飾的長循環陽離子脂質體奈米顆粒；所述PEG的相對分子質量優選為2000~5000，例如為2000、3000、4000或5000； 更佳地： 所述脂質體奈米顆粒還包括脂質體遞送系統，其優選包括陽離子脂質、結構脂質、輔助脂質和表面活性劑，更優選包括20%~70%的陽離子脂質、10%～65%的結構脂質、5%~25%的輔助脂質和1%~10%的表面活性劑； 進一步更佳地： 所述陽離子脂質優選自DODAC、DDAB、DODMA、Dlin-DAC、Dlin-KC2-DMA和Dlin-MC3-DMA中的一種或多種，更優選自Dlin-KC2-DMA和Dlin-MC3-DMA中的一種或兩種，最優選為Dlin-MC3-DMA；其莫耳百分比優選為30%~60%，更優選為45%~55%，例如50%； 和/或，所述結構脂質優選自膽固醇、膽固醇酯、固醇類激素、固醇類維生素和植物甾醇中的一種或多種，更優選自膽固醇、膽固醇酯和植物甾醇中的一種或多種，最優選為膽固醇；其莫耳百分比優選為15%~50%，例如23%、28%、30%、33%、35%、38.5%、40%或48%； 和/或，所述輔助脂質優選自DSPC、DOPC、DPPG、DOPS和DOPE中的一種或多種，更優選為DSPC和/或DOPS，最優選為DSPC；其莫耳百分比優選為10%~20%，例如13%或15%； 和/或，所述表面活性劑優選自DAG-PEG、DAA-PEG、DMG-PEG、Cer-PEG和DSPE-PEG中的一種或多種，更優選為PEG-DMG；其莫耳百分比優選為1%~5%；更優選為1%~3%，例如為1.5%或2%； 和/或，所述脂質體遞送系統與所述mRNA、所述DNA和/或所述組成物的質量比為(5-30):1，優選為(10-20):1；例如15:1。 A liposomal nanoparticle comprising the isolated mRNA as claimed in claim 1 or 2, the DNA as claimed in claim 3, and/or the composition as claimed in claim 6; Preferably: The liposome nanoparticle is a long-circulating cationic liposome nanoparticle, preferably a long-circulating cationic liposome nanoparticle modified by PEG or a derivative thereof; the relative molecular mass of the PEG is preferably 2000-5000, for example is 2000, 3000, 4000 or 5000; Better yet: The liposome nanoparticle also includes a liposome delivery system, which preferably includes cationic lipids, structured lipids, helper lipids, and surfactants, and more preferably includes 20% to 70% of cationic lipids, 10% to 65% of structured lipids , 5%~25% auxiliary lipid and 1%~10% surfactant; Even better: The cationic lipid is preferably selected from one or more of DODAC, DDAB, DODMA, Dlin-DAC, Dlin-KC2-DMA and Dlin-MC3-DMA, more preferably one or more of Dlin-KC2-DMA and Dlin-MC3-DMA Or two kinds, most preferably Dlin-MC3-DMA; Its molar percentage is preferably 30%～60%, more preferably 45%～55%, for example 50%; And/or, the structural lipid is preferably selected from one or more of cholesterol, cholesterol esters, steroid hormones, sterol vitamins and phytosterols, more preferably from one or more of cholesterol, cholesterol esters and phytosterols, most preferably Preferably it is cholesterol; its molar percentage is preferably 15% to 50%, such as 23%, 28%, 30%, 33%, 35%, 38.5%, 40% or 48%; And/or, the auxiliary lipid is preferably selected from one or more of DSPC, DOPC, DPPG, DOPS and DOPE, more preferably DSPC and/or DOPS, most preferably DSPC; its molar percentage is preferably 10%～20% , such as 13% or 15%; And/or, the surfactant is preferably selected from one or more of DAG-PEG, DAA-PEG, DMG-PEG, Cer-PEG and DSPE-PEG, more preferably PEG-DMG; its molar percentage is preferably 1 %~5%; more preferably 1%~3%, such as 1.5% or 2%; And/or, the mass ratio of the liposome delivery system to the mRNA, the DNA and/or the composition is (5-30): 1, preferably (10-20): 1; for example 15: 1. 一種mRNA疫苗，其特徵在於，其包含如請求項1或2所述的分離的mRNA、如請求項3所述的DNA、如請求項6所述的組成物和/或如請求項7所述的脂質體奈米顆粒； 較佳地，所述mRNA疫苗還包括佐劑。 An mRNA vaccine, characterized in that it comprises the isolated mRNA as described in claim 1 or 2, the DNA as described in claim 3, the composition as described in claim 6 and/or as described in claim 7 liposomal nanoparticles; Preferably, the mRNA vaccine further includes an adjuvant. 一種藥物組成物，其包含如請求項1或2所述的分離的mRNA、如請求項3所述的DNA、如請求項6所述的組成物、如請求項7所述的脂質體奈米顆粒和/或如請求項8所述的mRNA疫苗，和任選地藥學上可接受的載體。A pharmaceutical composition comprising the isolated mRNA as claimed in claim 1 or 2, the DNA as claimed in claim 3, the composition as claimed in claim 6, and the liposome nanoparticle as claimed in claim 7 Particles and/or the mRNA vaccine as claimed in claim 8, and optionally a pharmaceutically acceptable carrier. 一種套組，其包含如請求項1或2所述的分離的mRNA、如請求項3所述的DNA、如請求項6所述的組成物、如請求項7所述的脂質體奈米顆粒、如請求項8所述的mRNA疫苗和/或如請求項9所述的藥物組成物； 較佳地，所述套組還包含：使用說明、用於轉染的細胞、輔劑、施用所述藥物組成物的工具、藥學可接受的載體和/或用於溶解或稀釋所述mRNA、所述DNA、所述組成物、所述脂質體奈米顆粒、所述疫苗或所述藥物組成物的藥學可接受的溶液。 A kit comprising the isolated mRNA as claimed in claim 1 or 2, the DNA as claimed in claim 3, the composition as claimed in claim 6, and the liposome nanoparticle as claimed in claim 7 , the mRNA vaccine as claimed in claim 8 and/or the pharmaceutical composition as claimed in claim 9; Preferably, the set also comprises: instructions for use, cells for transfection, adjuvants, tools for administering the pharmaceutical composition, pharmaceutically acceptable carriers and/or for dissolving or diluting the mRNA, A pharmaceutically acceptable solution of the DNA, the composition, the liposomal nanoparticle, the vaccine, or the pharmaceutical composition. 如請求項1或2所述的分離的mRNA、如請求項3所述的DNA、如請求項6所述的組成物、如請求項7所述的脂質體奈米顆粒、如請求項8所述的mRNA疫苗和/或如請求項9所述的藥物組成物在製備用於預防和/或治療SARS-CoV-2病毒感染或SARS-CoV-2病毒感染所致疾病的藥物中的應用。The isolated mRNA according to claim 1 or 2, the DNA according to claim 3, the composition according to claim 6, the liposomal nanoparticle according to claim 7, and the 8 claim Application of the mRNA vaccine and/or the pharmaceutical composition according to claim 9 in the preparation of a medicine for preventing and/or treating SARS-CoV-2 virus infection or diseases caused by SARS-CoV-2 virus infection.
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CIARAMELLA et al. Patent 3003103 Summary