WO2024103579A1 - Preparation method for coronavirus pseudovirus particle - Google Patents

Preparation method for coronavirus pseudovirus particle Download PDF

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WO2024103579A1
WO2024103579A1 PCT/CN2023/081026 CN2023081026W WO2024103579A1 WO 2024103579 A1 WO2024103579 A1 WO 2024103579A1 CN 2023081026 W CN2023081026 W CN 2023081026W WO 2024103579 A1 WO2024103579 A1 WO 2024103579A1
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plasmid
preparation
protein
coronavirus
gene
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刘骏
周春艳
金丽
谢伟建
金雨霏
陈雨菲
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复百澳(苏州)生物医药科技有限公司
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    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
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Abstract

Provided is a preparation method for a coronavirus pseudovirus particle. By means of constructing a plasmid (1): a retroviral vector that removes RT and INT genes, in which a gene sequence of an NC protein of the retroviral vector is replaced by a gene sequence of an N protein of a coronavirus; and a plasmid (2): a plasmid expressing a spike protein of the coronavirus and a tag protein, the coronavirus pseudovirus particle is prepared. The pseudovirus particle prepared by the preparation method can efficiently load RNA, thereby enhancing an immune response to SARS-CoV-2-Spike in mice, improving the generation of antibodies, and having very good clinical application prospects.

Description

一种冠状病毒假病毒颗粒的制备方法A method for preparing coronavirus pseudoviral particles 技术领域Technical Field
本发明属于生物技术领域，具体涉及一种冠状病毒假病毒颗粒的制备方法。The invention belongs to the field of biotechnology, and in particular relates to a method for preparing coronavirus pseudovirus particles.
背景技术Background technique
冠状病毒直径约80-120nm，基因组5′端具有甲基化的帽状结构，3′端具有poly(A)尾，基因组全长约27-32kb，是已知RNA病毒中基因组最大的病毒。冠状病毒粒子外有脂肪膜，膜表面一般有三种糖蛋白：刺突蛋白(S，Spike Protein，是受体结合位点、溶细胞作用和主要抗原位点)；包膜蛋白(E，Envelope Protein，较小，与包膜结合的蛋白)；膜蛋白(M，Membrane Protein，负责营养物质的跨膜运输、新生病毒出芽释放与病毒外包膜的形成)。少数种类还有血凝素糖蛋白(HE蛋白，Haemaglutinin-esterase)。冠状病毒的核酸为非节段单链(+)RNA，长27-31kb，是RNA病毒中最长的RNA核酸链，具有正链RNA特有的重要结构特征：即RNA链5’端有甲基化“帽子”，3’端有PolyA“尾巴”结构。这一结构与真核mRNA非常相似，也是其基因组RNA自身可以发挥翻译模板作用的重要结构基础。The diameter of coronavirus is about 80-120nm, the 5′ end of the genome has a methylated cap structure, the 3′ end has a poly(A) tail, and the total length of the genome is about 27-32kb, which is the largest genome among known RNA viruses. The coronavirus particles are covered with a fat membrane, and the membrane surface generally has three types of glycoproteins: spike protein (S, Spike Protein, which is the receptor binding site, cytolytic effect and main antigenic site); envelope protein (E, Envelope Protein, smaller, protein bound to the envelope); membrane protein (M, Membrane Protein, responsible for the transmembrane transport of nutrients, the budding release of newborn viruses and the formation of the outer envelope of the virus). A few species also have hemagglutinin glycoprotein (HE protein, Haemaglutinin-esterase). The nucleic acid of coronavirus is non-segmented single-stranded (+) RNA, 27-31kb long, which is the longest RNA nucleic acid chain among RNA viruses, and has important structural characteristics unique to positive-strand RNA: that is, there is a methylated "cap" at the 5' end of the RNA chain and a PolyA "tail" structure at the 3' end. This structure is very similar to eukaryotic mRNA and is also an important structural basis for its genomic RNA to serve as a translation template.
新冠病毒基因组全长约29.9kb，该病毒含有四种主要的结构蛋白，除膜表面的三种糖蛋白外，还包括核衣壳蛋白(Nucleocapsid，N蛋白)。S蛋白是冠状病毒非常重要的表面蛋白，与病毒的传染能力密切相关，S蛋白包含S1、S2和受体结合域(RBD)，N蛋白在冠状病毒中含量丰富，是一种高度免疫原性蛋白且相对保守，突变率较低，N蛋白从受感染的细胞中释放出来后会自由漂浮。自由漂浮的蛋白引起强烈的免疫反应，导致保护性抗体的产生，N蛋白也是冠状病毒的抗原检测的靶蛋白之一。The total length of the novel coronavirus genome is about 29.9kb. The virus contains four major structural proteins, including nucleocapsid protein (N protein) in addition to the three glycoproteins on the membrane surface. S protein is a very important surface protein of coronavirus and is closely related to the infectious ability of the virus. S protein contains S1, S2 and receptor binding domain (RBD). N protein is abundant in coronavirus and is a highly immunogenic protein that is relatively conservative and has a low mutation rate. N protein floats freely after being released from infected cells. Free-floating proteins cause strong immune responses, leading to the production of protective antibodies. N protein is also one of the target proteins for antigen detection of coronavirus.
目前，针对冠状病毒开发的疫苗主要包括灭活疫苗、腺病毒载体疫苗、减毒流感疫苗、重组蛋白疫苗以及核酸疫苗等。除了灭活疫苗外，其余都是针对S蛋白进行免疫的。由于该病毒从开始至今一直处于不断突变的过程中，单一毒株疫苗对其保护效率较低。已有报道，基于慢病毒载体***通过替换其ENV蛋白构建病毒样颗粒(Virus Like particles，VLP)用于疫苗制备和基因递送等方向的应用(Virus-like particles preparation,immunogenicity and their roles as nanovaccines and drug nanocarriers)。At present, the vaccines developed for coronavirus mainly include inactivated vaccines, adenovirus vector vaccines, attenuated influenza vaccines, recombinant protein vaccines and nucleic acid vaccines. Except for inactivated vaccines, the rest are immunized against S protein. Since the virus has been in a process of continuous mutation since its inception, the protection efficiency of a single strain vaccine is low. It has been reported that virus-like particles (VLPs) constructed by replacing the ENV protein based on the lentiviral vector system are used for vaccine preparation and gene delivery (Virus-like particles preparation, immunogenicity and their roles as nanovaccines and drug nanocarriers).
申请号为CN202210120315的专利中公开了用于预防SARS-CoV-2奥密克戎株的腺病毒载体疫苗。采用密码子偏好性进行优化得到新的S基因序列，其能高效在人源细胞内高效表 达，免疫机体后可高效表达S抗原，产生针对奥密克戎株SARS-CoV-2的中和抗体，可以有效保护机体免受奥密克戎株的侵染。但其针对性较强，因此实际应用受限。Patent application number CN202210120315 discloses an adenovirus vector vaccine for preventing the SARS-CoV-2 Omicron strain. The new S gene sequence was optimized by codon preference, which can be efficiently expressed in human cells. After the immunization, the S antigen can be efficiently expressed, and neutralizing antibodies against the Omicron strain of SARS-CoV-2 can be produced, which can effectively protect the body from infection by the Omicron strain. However, its specificity is relatively strong, so its practical application is limited.
申请号为CN201080020498的专利中公开了非整合型、非复制型逆转录病毒载体，当向动物宿主施用时其在宿主中引起免疫反应。载体转导宿主中的细胞，在那里它们产生病毒样颗粒(VLP)，所述病毒样颗粒在从细胞释放时在宿主中刺激另外的免疫反应。载体是非整合型、非复制型逆转录病毒载体，包括长末端重复序列、包装序列和与共同编码病毒的结构蛋白的一种或多种多核苷酸序列可操作地连接的异源启动子。还公开了制造和使用所述载体的方法。但其主要针对的是HIV疫苗，该方法针对冠状病毒特别是SARS-CoV-2的应用仍需要进一步改进。Patent application number CN201080020498 discloses a non-integrating, non-replicating retroviral vector that causes an immune response in an animal host when administered to the host. The vector transduces cells in the host, where they produce virus-like particles (VLPs), which stimulate additional immune responses in the host when released from the cells. The vector is a non-integrating, non-replicating retroviral vector comprising a long terminal repeat sequence, a packaging sequence, and a heterologous promoter operably linked to one or more polynucleotide sequences encoding structural proteins of the virus. A method for making and using the vector is also disclosed. However, it is mainly aimed at HIV vaccines, and the application of this method to coronaviruses, especially SARS-CoV-2, still needs further improvement.
发明内容Summary of the invention
为了解决上述问题，本发明基于二代慢病毒载体***，分别对psPAX2、pMGD2.0***质粒进行改造，并使用真核表达载体替代病毒表达载体，制备了一种新型病毒样颗粒。In order to solve the above problems, the present invention is based on the second-generation lentiviral vector system, respectively transforms the psPAX2 and pMGD2.0 system plasmids, and uses eukaryotic expression vectors to replace viral expression vectors to prepare a new type of virus-like particle.
本发明中，“HA标签”是一段来源于人流感病毒血凝素(HA)蛋白第98-106位氨基酸的短序列，分子量为1.1KDa，是目前广泛应用的表位标签之一。In the present invention, the "HA tag" is a short sequence derived from amino acids 98-106 of the human influenza virus hemagglutinin (HA) protein, with a molecular weight of 1.1 KDa, and is one of the epitope tags currently widely used.
一方面，本发明提供了一种冠状病毒假病毒颗粒的制备方法。In one aspect, the present invention provides a method for preparing coronavirus pseudoviral particles.
所述的制备方法中包括质粒构建，所述的质粒包括：The preparation method includes plasmid construction, and the plasmid includes:
质粒(1)、去除RT基因和INT基因的逆转录病毒载体，并使用冠状病毒的N蛋白基因序列替换逆转录病毒载体的NC蛋白的基因序列；Plasmid (1), a retroviral vector with the RT gene and the INT gene removed, and the gene sequence of the NC protein of the retroviral vector is replaced with the N protein gene sequence of a coronavirus;
和质粒(2)、表达冠状病毒刺突蛋白和标签蛋白的质粒。and plasmid (2), a plasmid expressing coronavirus spike protein and tag protein.
所述的标签蛋白为HA。The tag protein is HA.
所述的质粒(2)上还包括冠状病毒的5’UTR和3’UTR序列。The plasmid (2) also includes the 5'UTR and 3'UTR sequences of coronavirus.
优选地，所述的逆转录病毒载体为慢病毒载体。Preferably, the retroviral vector is a lentiviral vector.
进一步优选地，所述的慢病毒载体为慢病毒包装***质粒psPAX2。Further preferably, the lentiviral vector is a lentiviral packaging system plasmid psPAX2.
优选地，所述的冠状病毒为SARS-COV-2。Preferably, the coronavirus is SARS-COV-2.
进一步优选地，所述的N蛋白基因序列为SEQ ID NO.1。Further preferably, the N protein gene sequence is SEQ ID NO.1.
所述的的N蛋白基因序列与检测标签6×His融合。The N protein gene sequence is fused with the detection tag 6×His.
在一些具体的实施例中，所述的质粒(1)的构建方法为：将通用的慢病毒包装***质粒psPAX2，通过EcoR 1酶切，获得去除了RT基因和INT基因的空载体，通过基因合成的方法获得SEQ IDNO.1并克隆至该载体上，获得psPAX2-SARS-COV-2-N质粒，即质粒(1)。In some specific embodiments, the construction method of the plasmid (1) is as follows: the universal lentiviral packaging system plasmid psPAX2 is cut with EcoR 1 to obtain an empty vector with the RT gene and INT gene removed, SEQ IDNO.1 is obtained by gene synthesis and cloned into the vector to obtain the psPAX2-SARS-COV-2-N plasmid, i.e., plasmid (1).
所述的EcoR 1酶切位置为382bp和4752bp处。 The EcoR 1 enzyme cutting positions are 382 bp and 4752 bp.
优选地，所述的质粒(2)表达冠状病毒刺突蛋白的基因序列为：SEQ ID NO.2。Preferably, the gene sequence of the coronavirus spike protein expressed by the plasmid (2) is: SEQ ID NO.2.
优选地，所述的质粒(2)标签蛋白的基因序列为：SEQ ID NO.3。Preferably, the gene sequence of the plasmid (2) tag protein is: SEQ ID NO.3.
优选地，所述的质粒(2)的载体骨架为pCDNA3.1载体。Preferably, the vector backbone of the plasmid (2) is a pCDNA3.1 vector.
优选地，所述的质粒(2)的5’UTR序列为SEQ ID NO.4，3’UTR序列为SEQ ID NO.5。Preferably, the 5’UTR sequence of the plasmid (2) is SEQ ID NO.4, and the 3’UTR sequence is SEQ ID NO.5.
在一些具体的实施例中，所述的质粒(2)的制备方法为：合成基因SEQ IDNO.5-SEQ IDNO.2-SEQ IDNO.6-SEQ ID NO.3并克隆构建至pCDNA3.1载体，获得pCDNA3.1-SARS-COV-2-Spike，即质粒(2)。In some specific embodiments, the preparation method of the plasmid (2) is: synthesize the gene SEQ ID NO.5-SEQ ID NO.2-SEQ ID NO.6-SEQ ID NO.3 and clone it into the pCDNA3.1 vector to obtain pCDNA3.1-SARS-COV-2-Spike, that is, plasmid (2).
具体地，所述的制备方法目的在于制备冠状病毒假病毒颗粒，因此还包括转染细胞。Specifically, the preparation method aims to prepare coronavirus pseudoviral particles, and therefore also includes transfecting cells.
优选地，所述的细胞为293T细胞。Preferably, the cells are 293T cells.
优选的，转染细胞的质粒为混合转染质粒，所述的混合转染质粒中包括质粒(1)、质粒(2)和pMD2.G。Preferably, the plasmid used to transfect cells is a mixed transfection plasmid, which includes plasmid (1), plasmid (2) and pMD2.G.
本发明基于二代慢病毒载体***，使用SARS-COV-2-N替换psPAX2质粒的NC蛋白的基因序列，同时去除了整合酶和逆转录酶的部分序列，构建了VLP的骨架结构蛋白表达载体psPAX2-SARS-COV-2-N，结构示意图如图2，其中psPAX2***质粒包括NC蛋白，SARS-COV-2-N为新冠病毒核蛋白基因序列。The present invention is based on the second-generation lentiviral vector system, uses SARS-COV-2-N to replace the gene sequence of the NC protein of the psPAX2 plasmid, and removes part of the sequences of integrase and reverse transcriptase to construct a VLP backbone protein expression vector psPAX2-SARS-COV-2-N. The structural schematic diagram is shown in Figure 2, wherein the psPAX2 system plasmid includes the NC protein, and SARS-COV-2-N is the new coronavirus nucleoprotein gene sequence.
本发明基于pCDNA3.1载体构建了pCDNA3.1-SARS-COV-2-Spike-HA的表达质粒，用于转录出可以被荷载的mRNA及翻译出被递呈与VLP表面新冠病毒刺突蛋白(SARS-COV-2-Spike)同时C端融合标签蛋白HA。设计结构如图3。The present invention constructs an expression plasmid of pCDNA3.1-SARS-COV-2-Spike-HA based on the pCDNA3.1 vector, which is used to transcribe the mRNA that can be loaded and translate the HA protein that is presented to the surface of the VLP and fused to the C-terminal tag protein SARS-COV-2-Spike. The design structure is shown in Figure 3.
本发明假病毒颗粒具体的制备流程如图4。The specific preparation process of the pseudovirus particles of the present invention is shown in FIG4 .
本发明的假病毒颗粒上含有SARS-COV-2-N氨基酸序列SEQ ID NO.6和SARS-COV-2-Spike氨基酸序列SEQ ID NO.7。The pseudovirus particles of the present invention contain the SARS-COV-2-N amino acid sequence SEQ ID NO.6 and the SARS-COV-2-Spike amino acid sequence SEQ ID NO.7.
另一方面，本发明提供了前述的制备方法制备得到的假病毒颗粒。On the other hand, the present invention provides pseudovirus particles prepared by the aforementioned preparation method.
本发明提供的新型病毒样颗粒具有以下结构特点：The novel virus-like particles provided by the present invention have the following structural characteristics:
(1)病毒囊膜上同时含有新冠病毒Spike蛋白和水泡型口炎病毒糖黏蛋白VSV-G；(2)病毒囊膜包裹有新冠病毒结构蛋白N，并通过其荷载SARS-COV-2-Spike的mRNA；(3)SARS-COV-2-Spike基因前后含有5’UTR和3’UTR。(1) The viral envelope contains both the SARS-CoV-2 Spike protein and the vesicular stomatitis virus (VSV-G) glycoprotein; (2) The viral envelope wraps around the SARS-CoV-2 structural protein N, through which it carries the SARS-COV-2-Spike mRNA; (3) The SARS-COV-2-Spike gene contains a 5’UTR and a 3’UTR.
本发明构建的假病毒颗粒结构示意如图1。The structure of the pseudovirus particle constructed by the present invention is schematically shown in FIG1 .
再一方面，本发明提供了前述的假病毒颗粒在基因递送中的应用。In another aspect, the present invention provides use of the aforementioned pseudovirus particle in gene delivery.
优选地，所述的基因递送为RNA递送。Preferably, the gene delivery is RNA delivery.
所述的RNA可以为核酸药物。 The RNA can be a nucleic acid drug.
又一方面，本发明提供了前述的假病毒颗粒在制备治疗和/或预防冠状病毒的药物中的应用。In yet another aspect, the present invention provides use of the aforementioned pseudovirus particles in the preparation of drugs for treating and/or preventing coronavirus.
优选地，所述的药物为疫苗。Preferably, the drug is a vaccine.
进一步优选地，所述的疫苗为SARS-COV-2疫苗。Further preferably, the vaccine is a SARS-COV-2 vaccine.
又一方面，本发明提供了前述的假病毒颗粒在制备抗原检测质控品中的应用。In yet another aspect, the present invention provides the use of the aforementioned pseudovirus particles in the preparation of quality control products for antigen detection.
优选地，所述的抗原检测质控品为冠状病毒的抗原检测质控品。Preferably, the antigen detection quality control product is a coronavirus antigen detection quality control product.
进一步优选地，所述的冠状病毒为SARS-COV-2。Further preferably, the coronavirus is SARS-COV-2.
又一方面，本发明提供了包括前述的假病毒颗粒的药物。In yet another aspect, the present invention provides a medicament comprising the aforementioned pseudoviral particle.
优选地，所述的药物为疫苗。Preferably, the drug is a vaccine.
进一步优选地，所述的疫苗为SARS-COV-2疫苗。Further preferably, the vaccine is a SARS-COV-2 vaccine.
又一方面，本发明提供了包括前述的假病毒颗粒的抗原检测质控品。In yet another aspect, the present invention provides an antigen detection quality control product comprising the aforementioned pseudovirus particles.
优选地，所述的抗原检测质控品为冠状病毒的抗原检测质控品。Preferably, the antigen detection quality control product is a coronavirus antigen detection quality control product.
进一步优选地，所述的冠状病毒为SARS-COV-2。Further preferably, the coronavirus is SARS-COV-2.
本发明的有益效果：Beneficial effects of the present invention:
(1)和新冠病毒灭活疫苗和蛋白疫苗相比，通过在VLP囊膜上表达spike蛋白，具有更好的结构完整性和构象一致性，能更好的模拟病毒进入机体后引起的免疫应答。(1) Compared with the inactivated COVID-19 vaccine and protein vaccine, by expressing the spike protein on the VLP envelope, it has better structural integrity and conformational consistency, and can better simulate the immune response caused by the virus entering the body.
(2)所获得的VLP通过VSV-G和SARS-COV-2-Spike分别和LDL及ACE2受体结合，介导病毒进入细胞内，实现SARS-COV-2-Spike mRNA的细胞内递送，再翻译成SARS-COV-2-Spike蛋白展示于细胞表面，刺激机体进一步产生免疫应答，实现mRNA疫苗的作用。(2) The obtained VLPs bind to LDL and ACE2 receptors through VSV-G and SARS-COV-2-Spike, respectively, mediating the virus entry into cells and achieving intracellular delivery of SARS-COV-2-Spike mRNA, which is then translated into SARS-COV-2-Spike protein and displayed on the cell surface, stimulating the body to further produce an immune response and achieve the effect of mRNA vaccine.
(3)所获得的VLP同时含有刺突蛋白(Spike protein，S蛋白)和核衣壳蛋白(Nucleocapsid，N蛋白)获得双重免疫作用。(3) The obtained VLPs contain both spike protein (S protein) and nucleocapsid protein (N protein) to achieve dual immune effects.
(4)所获得的VLP颗粒同时具备蛋白免疫、mRNA免疫等多重功能。(4) The obtained VLP particles have multiple functions such as protein immunity and mRNA immunity.
(5)所获得的VLP可以通过ENV蛋白实现靶向递送mRNA。(5) The obtained VLPs can achieve targeted delivery of mRNA through ENV protein.
(6)所获得的VLP实行递送功能时，不具有基因组整合等安全风险。(6) The obtained VLPs do not pose safety risks such as genome integration when performing delivery functions.
附图说明BRIEF DESCRIPTION OF THE DRAWINGS
图1为本发明构建的假病毒颗粒结构示意。FIG1 is a schematic diagram of the structure of the pseudovirion particle constructed in the present invention.
图2为psPAX2-SARS-COV-2-N设计结构。 Figure 2 shows the designed structure of psPAX2-SARS-COV-2-N.
图3为pCDNA3.1-SARS-COV-2-Spike相关质粒设计结构。Figure 3 shows the design structure of pCDNA3.1-SARS-COV-2-Spike related plasmids.
图4为本发明假病毒颗粒具体的制备流程。FIG4 is a specific preparation process of pseudoviral particles of the present invention.
图5为psPAX2-SARS-COV-2-N、pCDNA3.1-SARS-COV-2-Spike相关质粒电泳结果。Figure 5 shows the electrophoresis results of psPAX2-SARS-COV-2-N and pCDNA3.1-SARS-COV-2-Spike related plasmids.
图6为QPCR方法对病毒所荷载的RNA进行相对定量结果。FIG. 6 shows the relative quantitative results of RNA carried by the virus using the QPCR method.
图7为WB检测VLP相关结构蛋白的表达结果。FIG. 7 is the expression result of VLP-related structural proteins detected by WB.
图8为VLP细胞内RNA的递送效率。FIG. 8 shows the delivery efficiency of VLP RNA into cells.
图9为免疫后小鼠血清效价ELISA测定结果。FIG. 9 shows the results of ELISA determination of serum titer in mice after immunization.
具体实施方式Detailed ways
下面结合具体实施例，对本发明作进一步详细的阐述，下述实施例不用于限制本发明，仅用于说明本发明。以下实施例中所使用的实验方法如无特殊说明，实施例中未注明具体条件的实验方法，通常按照常规条件，下述实施例中所使用的材料、试剂等，如无特殊说明，均可从商业途径得到。The present invention will be further described in detail below in conjunction with specific examples. The following examples are not intended to limit the present invention, but are only intended to illustrate the present invention. The experimental methods used in the following examples are generally conventional, unless otherwise specified, and the materials, reagents, etc. used in the following examples are commercially available, unless otherwise specified.
逆转录病毒载体主要特征之一是在两端具有长的同向末端重复序列(Long terminal repeat，LTR)，LTR反转录转座子的典型结构包括GAG(病毒颗粒结构蛋白类似物)和POL区段，其中POL区段包括逆转录酶基因(RT)和DDE整合酶基因(INT)。One of the main characteristics of retroviral vectors is that they have long terminal repeats (LTR) at both ends. The typical structure of LTR retrotransposon includes GAG (analog of viral particle structural protein) and POL segment, among which the POL segment includes reverse transcriptase gene (RT) and DDE integrase gene (INT).
所述的“FBS”指胎牛血清，是一种浅黄色澄清、无溶血、无异物稍粘稠液体，血清中所含的抗体、补体等对细胞有害的成分较少，含有丰富的细胞生长必须的营养成份，常用于动物细胞的体外培养。The "FBS" mentioned above refers to fetal bovine serum, which is a light yellow, clear, non-hemolytic, foreign-matter-free, slightly viscous liquid. The serum contains fewer components harmful to cells, such as antibodies and complement, and is rich in nutrients necessary for cell growth. It is often used for in vitro culture of animal cells.
“DMEM培养基”是一种含各种氨基酸和葡萄糖的培养基，是在MEM培养基的基础上研制的，为本领域常用的培养基。"DMEM medium" is a culture medium containing various amino acids and glucose. It is developed on the basis of MEM medium and is a commonly used culture medium in this field.
“PEI”指聚乙烯亚胺(Polyethyleneimine，PEI)，又称聚氮杂环丙烷，是一种水溶性高分子聚合物，多用于生物学实验中的转染。"PEI" refers to polyethyleneimine (PEI), also known as polyaziridine, which is a water-soluble high molecular polymer and is mostly used for transfection in biological experiments.
“OMEM”指Opti MEM培养基，该培养基中无血清，不含大量蛋白组分，不干扰DNA与转染试剂形成复合物。OMEM有利于细胞恢复健康状态，为本领域常用的转染用培养基。"OMEM" refers to Opti MEM medium, which is serum-free, does not contain a large amount of protein components, and does not interfere with the formation of complexes between DNA and transfection reagents. OMEM is conducive to the recovery of cells to a healthy state and is a commonly used transfection medium in this field.
“BCA定量”是一种蛋白定量方法，BCA(bicinchonininc acid)与二价铜离子的硫酸铜等其他试剂组成的试剂，混合一起即成为苹果绿，即BCA工作试剂。在碱性条件下，BCA与蛋白质结合时，蛋白质将Cu2+还原为Cu+，一个Cu+螯合二个BCA分子，工作试剂由原来的苹果绿形成紫色复合物，562nm处有最高的吸收值，可在540-595nm测定其吸收值，颜色的深浅与蛋白质浓度成正比，与标准曲线对比，即可计算待测蛋白的浓度。 "BCA quantification" is a protein quantification method. BCA (bicinchonininc acid) and other reagents such as copper sulfate of divalent copper ions are mixed together to form apple green, which is the BCA working reagent. Under alkaline conditions, when BCA binds to protein, the protein reduces Cu 2+ to Cu + . One Cu + chelates two BCA molecules. The working reagent changes from the original apple green to a purple complex with the highest absorption value at 562nm. Its absorption value can be measured at 540-595nm. The depth of the color is proportional to the protein concentration. By comparing with the standard curve, the concentration of the protein to be tested can be calculated.
实施例1病毒制备相关质粒构建Example 1 Virus preparation related plasmid construction
(1)全基因合成的方法获得以下基因序列。
(1) The following gene sequence was obtained by whole gene synthesis.
(2)将通用的慢病毒包装***质粒psPAX2(Addgene#12259)，通过EcoR 1酶切(载体上位置为382bp和4752bp处)，获得去除了RT基因和INT基因的空载体，通过基因合成的方法获得SEQ IDNO.1并克隆至该载体上，获得psPAX2-SARS-COV-2-N质粒。(2) The universal lentiviral packaging system plasmid psPAX2 (Addgene #12259) was digested with EcoR 1 (at positions 382 bp and 4752 bp on the vector) to obtain an empty vector with the RT gene and INT gene removed. SEQ ID NO.1 was obtained by gene synthesis and cloned into the vector to obtain the psPAX2-SARS-COV-2-N plasmid.
(3)通过基因合成的方式获得SARS-COV-2-Spike相关序列SEQ IDNO.2并克隆构建至pCDNA3.1载体，命名为：pCDNA3.1-SARS-COV-2-Spike。(3) The SARS-COV-2-Spike related sequence SEQ ID NO.2 was obtained by gene synthesis and cloned into the pCDNA3.1 vector and named: pCDNA3.1-SARS-COV-2-Spike.
并通过琼脂糖凝胶电泳和质粒一代测序进行序列验证。The sequences were verified by agarose gel electrophoresis and plasmid sequencing.
psPAX2-SARS-COV-2-N和pCDNA3.1-SARS-COV-2-Spike的电泳结果如图5。The electrophoresis results of psPAX2-SARS-COV-2-N and pCDNA3.1-SARS-COV-2-Spike are shown in Figure 5.
实施例2病毒样颗粒包装实验Example 2 Virus-like particle packaging experiment
(1)转染前，显微镜下观察293T细胞的状态，确定细胞状态良好、密度90％左右，无污染；(1) Before transfection, observe the state of 293T cells under a microscope to ensure that the cells are in good condition, have a density of about 90%, and are free of contamination;
(2)每盘细胞更换10mL含2％FBS的DMEM培养基；(2) Replace each cell dish with 10 mL of DMEM medium containing 2% FBS;
(3)实验分组及转染体如下：

(3) Experimental groups and transfectants are as follows:

(4)各组分别操作：加入转染试剂1×PEI 30μL/盘，混合转染质粒加入到含500μL OMEM的EP管中，室温静置15min；(4) Each group was operated separately: add transfection reagent 1×PEI 30μL/plate, add the mixed transfection plasmid into the EP tube containing 500μL OMEM, and let it stand at room temperature for 15min;
(5)将转染体系分别缓慢滴加至细胞培养皿中，保持培养皿水平，晃动培养皿，使得转染复合物均匀分布于细胞表面；(5) Slowly add the transfection system to the cell culture dish, keep the dish level, and shake the dish to make the transfection complex evenly distributed on the cell surface;
(6)转染后8h，每盘细胞更换10mL含2％FBS的DMEM培养基；(6) 8 h after transfection, replace each cell plate with 10 mL of DMEM medium containing 2% FBS;
(7)转染后48h收集细胞上清液，该上清液中已经含对应的病毒样颗粒；(7) 48 h after transfection, the cell supernatant was collected, and the supernatant already contained the corresponding virus-like particles;
(8)上清液按照500μL/组留样进行病毒样颗粒RNA荷载量检测；(8) 500 μL/group of supernatant was retained for virus-like particle RNA loading detection;
(9)按照50,000g/min×3H，进行超速离心；(9) ultracentrifugation at 50,000 g/min × 3 h;
(10)用PBS重悬离心后沉淀，过滤除菌，冻存-80℃冰箱。(10) Resuspend the pellet after centrifugation with PBS, filter and sterilize, and store in a -80°C freezer.
实施例3病毒样颗粒RNA荷载量检测Example 3 Detection of RNA loading of virus-like particles
(1)使用病毒RNA提取试剂盒(Beaverbio，Cat.NO.70406)进行病毒上清液的中RNA的提取，提取实验按照实验说明书执行；(1) Use a viral RNA extraction kit (Beaverbio, Cat. NO. 70406) to extract RNA from the viral supernatant. The extraction experiment is performed according to the experimental instructions;
(2)病毒RNA提取后使用一步法QPCR检测试剂盒(诺唯赞，HiScript II One Step qRT-PCR SYBR Green Kit，货号：Q221-01)进行QPCR检测，该试剂盒可以同时完成RT-PCR将RNA反转成cDNA和QPCR实时定量检测。(2) After viral RNA was extracted, QPCR detection was performed using a one-step QPCR detection kit (Novozymes, HiScript II One Step qRT-PCR SYBR Green Kit, Catalog No.: Q221-01). This kit can simultaneously complete RT-PCR to convert RNA into cDNA and QPCR real-time quantitative detection.
(3)QPCR反应体系配制如下：
(3) The QPCR reaction system was prepared as follows:
QPCR的引物序列信息
Primer sequence information for QPCR
通过QPCR方法对病毒所荷载的RNA进行相对定量，结果显示，Group 2即psPAX2-SARS-COV-2-N、pMD2.G和pCDNA3.1-SARS-COV-2-Spike所制备的VLP含量较其他组别，可以高效荷载RNA。结果见图6。The RNA carried by the virus was relatively quantified by the QPCR method. The results showed that the VLPs prepared by Group 2, i.e., psPAX2-SARS-COV-2-N, pMD2.G, and pCDNA3.1-SARS-COV-2-Spike, could load RNA more efficiently than those prepared by other groups. The results are shown in Figure 6.
实施例4病毒样颗粒相关蛋白检测Example 4 Detection of virus-like particle-related proteins
(1)按照浓度为沉积胶5％，分离胶10％进行配制SDS-PAGE用胶；(1) Prepare SDS-PAGE gel at a concentration of 5% for deposition gel and 10% for separation gel;
(2)将不同来源的VLP经过BCA定量，按照10μg/孔取样与6×SDS上样缓冲液混合；(2) VLPs from different sources were quantified by BCA, and 10 μg/well was sampled and mixed with 6× SDS loading buffer;
(3)金属浴，100℃热变性处理5min；(3) Metal bath, thermal denaturation treatment at 100°C for 5 min;
(4)上样电泳，浓缩胶电泳电压为120V，分离胶电泳电压调200V，当指示剂溴酚蓝跑至距凝胶下缘约0.5cm处时，结束电泳；(4) Load the sample for electrophoresis. The voltage of the concentrated gel electrophoresis is 120 V, and the voltage of the separation gel electrophoresis is adjusted to 200 V. When the indicator bromophenol blue runs to about 0.5 cm from the bottom edge of the gel, the electrophoresis is terminated.
(5)常规转膜、封闭、一抗孵育，洗脱，二抗孵育，洗脱和显影。(5) Routine transfer, blocking, primary antibody incubation, elution, secondary antibody incubation, elution and development.
WB(Wstern Blot)检测VLP相关结构蛋白的表达结果见图7。The results of WB (Western Blot) detection of the expression of VLP-related structural proteins are shown in Figure 7.
实施例5VLP感染细胞Example 5 VLP infection of cells
(1)实验前24H，接种293T-ACE2细胞于24孔板中，约1×105个/孔，500μL/孔，于37℃，5％CO2条件下培养；(1) 24 hours before the experiment, 293T-ACE2 cells were inoculated in a 24-well plate at approximately 1×10 5 cells/well, 500 μL/well, and cultured at 37°C and 5% CO 2 ;
(2)实验前在显微镜下，对实验用细胞进行观察，确定细胞饱满、分部均匀、无污染后再进行后续实验；(2) Before the experiment, observe the experimental cells under a microscope to ensure that the cells are full, evenly distributed, and free of contamination before conducting subsequent experiments;
(3)根据病毒核酸检测的拷贝数，按照MOI＝10将所获得的病毒样颗粒感染24孔板中293T细胞，三个复孔，同时设置一组无病毒样颗粒侵染的空细胞作为对照；(3) According to the copy number of viral nucleic acid detected, the obtained virus-like particles were infected with 293T cells in a 24-well plate at an MOI of 10, in triplicate, and a group of empty cells without virus-like particle infection was set as a control;
(4)24H后收取细胞总RNA，通过QPCR检测外源基因的含量。(4) After 24 hours, the total cell RNA was collected and the content of the exogenous gene was detected by QPCR.
收集细胞Total RNA通过QPCR的方法检测VLP细胞内RNA的递送效率，结果(见图8)显示，通过Group 2即psPAX2-SARS-COV-2-N、pMD2.G和pCDNA3.1-SARS-COV-2-Spike所获得VLP可以实现细胞内的RNA递送。 Total cell RNA was collected and the VLP intracellular RNA delivery efficiency was detected by QPCR. The results (see Figure 8) showed that the VLPs obtained by Group 2, i.e., psPAX2-SARS-COV-2-N, pMD2.G and pCDNA3.1-SARS-COV-2-Spike, could achieve intracellular RNA delivery.
实施例6小鼠免疫和抗体检测Example 6 Mouse immunization and antibody detection
(1)将不同来源(实施例2的不同组别)的VLP经过BCA定量作为抗原，用生理盐水将抗原稀释到2倍最终浓度(按每针次10μg/50μL用量配制)；(1) VLPs from different sources (different groups in Example 2) were used as antigens after BCA quantification, and the antigens were diluted to twice the final concentration with physiological saline (prepared at 10 μg/50 μL per injection);
(2)无菌条件下取出所需用量混匀佐剂(按每针次50μL，复百澳生物，货号：FBC2591)与抗原按体积比1:1迅速混匀；(2) Under sterile conditions, take out the required amount of adjuvant (50 μL per injection, Fubaiao Biological, catalog number: FBC2591) and mix it with the antigen in a volume ratio of 1:1;
(3)通过后腿小腿肌肉注射免疫小鼠，每只小鼠注射100μL；(3) Immunize mice by intramuscular injection into the hind leg calf, injecting 100 μL per mouse;
(4)第14天按同样方式加强免疫一针；(4) A booster shot on the 14th day in the same manner;
(5)第21天眼眶取血进行ELISA测定。(5) On the 21st day, blood was collected from the eye sockets for ELISA assay.
经免疫后小鼠血清效价ELISA测定结果(图9)显示，通过Group 2即psPAX2-SARS-COV-2-N、pMD2.G和pCDNA3.1-SARS-COV-2-Spike所获得VLP可以增强小鼠对SARS-CoV-2-Spike免疫反应，提高抗体产生。 The results of ELISA determination of mouse serum titer after immunization ( Figure 9 ) showed that the VLPs obtained by Group 2, i.e., psPAX2-SARS-COV-2-N, pMD2.G, and pCDNA3.1-SARS-COV-2-Spike, could enhance the immune response of mice to SARS-CoV-2-Spike and increase antibody production.

Claims (23)

  1. 一种冠状病毒假病毒颗粒的制备方法，其特征在于，包括质粒构建，所述的质粒包括：A method for preparing coronavirus pseudoviral particles, characterized in that it includes plasmid construction, wherein the plasmid includes:
    质粒(1)、去除RT基因和INT基因的逆转录病毒载体，并使用冠状病毒的N蛋白基因序列替换逆转录病毒载体的NC蛋白的基因序列；Plasmid (1), a retroviral vector with the RT gene and the INT gene removed, and the gene sequence of the NC protein of the retroviral vector is replaced with the N protein gene sequence of a coronavirus;
    和质粒(2)、表达冠状病毒刺突蛋白和标签蛋白的质粒。and plasmid (2), a plasmid expressing coronavirus spike protein and tag protein.
  2. 根据权利要求1所述的制备方法，其特征在于，所述的标签蛋白为HA。The preparation method according to claim 1, characterized in that the tag protein is HA.
  3. 根据权利要求2所述的制备方法，其特征在于，所述的质粒(2)上还包括冠状病毒的5’UTR和3’UTR序列。The preparation method according to claim 2, characterized in that the plasmid (2) also includes 5'UTR and 3'UTR sequences of coronavirus.
  4. 根据权利要求3所述的制备方法，其特征在于，所述的逆转录病毒载体为慢病毒载体。The preparation method according to claim 3, characterized in that the retroviral vector is a lentiviral vector.
  5. 根据权利要求4所述的制备方法，其特征在于，所述的慢病毒载体为慢病毒包装***质粒psPAX2。The preparation method according to claim 4, characterized in that the lentiviral vector is a lentiviral packaging system plasmid psPAX2.
  6. 根据权利要求5所述的制备方法，其特征在于，所述的冠状病毒为SARS-COV-2。The preparation method according to claim 5, characterized in that the coronavirus is SARS-COV-2.
  7. 根据权利要求6所述的制备方法，其特征在于，所述的N蛋白基因序列为SEQ ID NO.1。The preparation method according to claim 6 is characterized in that the N protein gene sequence is SEQ ID NO.1.
  8. 根据权利要求7所述的制备方法，其特征在于，所述的的N蛋白基因序列与检测标签6×His融合。The preparation method according to claim 7 is characterized in that the N protein gene sequence is fused with the detection tag 6×His.
  9. 根据权利要求8所述的制备方法，其特征在于，所述的质粒(1)的构建方法为：将通用的慢病毒包装***质粒psPAX2，通过EcoR 1酶切，获得去除了RT基因和INT基因的空载体，通过基因合成的方法获得SEQ ID NO.1并克隆至该载体上，获得psPAX2-SARS-COV-2-N质粒，即质粒(1)。The preparation method according to claim 8 is characterized in that the construction method of the plasmid (1) is as follows: the universal lentiviral packaging system plasmid psPAX2 is cut by EcoR 1 to obtain an empty vector with the RT gene and INT gene removed, SEQ ID NO.1 is obtained by gene synthesis and cloned into the vector to obtain the psPAX2-SARS-COV-2-N plasmid, i.e., plasmid (1).
  10. 根据权利要求9所述的制备方法，其特征在于，EcoR 1酶切位置为382bp和4752bp处。The preparation method according to claim 9 is characterized in that the EcoR 1 enzyme cutting positions are 382bp and 4752bp.
  11. 根据权利要求6所述的制备方法，其特征在于，所述的质粒(2)表达冠状病毒刺突蛋白的基因序列为：SEQ ID NO.2。The preparation method according to claim 6 is characterized in that the gene sequence of the coronavirus spike protein expressed by the plasmid (2) is: SEQ ID NO.2.
  12. 根据权利要求11所述的制备方法，其特征在于，所述的质粒(2)HA标签蛋白的基因序列为：SEQ ID NO.3。The preparation method according to claim 11 is characterized in that the gene sequence of the plasmid (2) HA tag protein is: SEQ ID NO.3.
  13. 根据权利要求12所述的制备方法，其特征在于，所述的质粒(2)的5’UTR序列为SEQ ID NO.4，3’UTR序列为SEQ ID NO.5。The preparation method according to claim 12 is characterized in that the 5’UTR sequence of the plasmid (2) is SEQ ID NO.4, and the 3’UTR sequence is SEQ ID NO.5.
  14. 根据权利要求根据权利要求13所述的制备方法，其特征在于，所述的质粒(2)的制备方法为：合成基因SEQ IDNO.5-SEQ IDNO.2-SEQ IDNO.6-SEQ ID NO.3并克隆构建至 pCDNA3.1载体，获得pCDNA3.1-SARS-COV-2-Spike，即质粒(2)。According to the preparation method according to claim 13, it is characterized in that the preparation method of the plasmid (2) is: synthesizing the gene SEQ ID NO.5-SEQ ID NO.2-SEQ ID NO.6-SEQ ID NO.3 and cloning and constructing it pCDNA3.1 vector, to obtain pCDNA3.1-SARS-COV-2-Spike, i.e. plasmid (2).
  15. 根据权利要求6所述的制备方法，其特征在于，还包括转染细胞，所述的细胞为293T细胞。The preparation method according to claim 6 is characterized in that it also includes transfecting cells, and the cells are 293T cells.
  16. 根据权利要求15所述的制备方法，其特征在于，转染细胞的质粒为混合转染质粒，所述的混合转染质粒中包括质粒(1)、质粒(2)和pMD2.G。The preparation method according to claim 15 is characterized in that the plasmid used to transfect cells is a mixed transfection plasmid, and the mixed transfection plasmid includes plasmid (1), plasmid (2) and pMD2.G.
  17. 权利要求1-16任一项所述的制备方法制备得到的假病毒颗粒。Pseudovirus particles prepared by the preparation method according to any one of claims 1 to 16.
  18. 权利要求17所述的假病毒颗粒在基因递送或基因编辑中的应用。Use of the pseudovirus particles described in claim 17 in gene delivery or gene editing.
  19. 权利要求17所述的假病毒颗粒在制备治疗和/或预防冠状病毒的药物中的应用。Use of the pseudovirus particles described in claim 17 in the preparation of drugs for treating and/or preventing coronavirus.
  20. 根据权利要求19所述的应用，其特征在于，所述的药物为疫苗。The use according to claim 19, characterized in that the drug is a vaccine.
  21. 权利要求17所述的假病毒颗粒在制备冠状病毒的抗原检测质控品中的应用。Use of the pseudovirus particles described in claim 17 in the preparation of coronavirus antigen detection quality control products.
  22. 包括权利要求17所述的假病毒颗粒的药物。A medicament comprising the pseudovirion of claim 17.
  23. 包括权利要求17所述的假病毒颗粒的抗原检测质控品。 An antigen detection quality control product comprising the pseudovirus particles described in claim 17.
PCT/CN2023/081026 2022-11-18 2023-03-13 Preparation method for coronavirus pseudovirus particle WO2024103579A1 (en)

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