TWI833281B - Fragments of coronavirus nucleocapsid protein for increasing gene expression and application thereof - Google Patents

Abstract

The present invention relates to fragments of coronavirus nucleocapsid protein, which are used for increasing gene expression. The fragments include at least the dimerization domain and/or the C-terminal domain (CTD) of the coronavirus nucleocapsid protein. The present invention also relates to polynucleotides encoding the protein fragments, plasmids including the polynucleotides, cells including the protein fragments, the polynucleotides, or the plasmids, and methods for increasing expression of a target gene or methods for screening antiviral drugs using the protein fragments, the polynucleotides, the plasmids, or the cells.

Description

Coronavirus nucleocapsid protein fragments used to increase gene expression and their applications

The present invention relates to a coronavirus nucleocapsid protein fragment for increasing gene expression, particularly a coronavirus nucleocapsid protein fragment that at least contains the dimerization domain and/or the carboxyl-terminal domain (C-terminal domain) of the coronavirus nucleocapsid protein. CTD) protein fragment.

Ebola virus (EBOV) belongs to the Filoviridae family and is classified as a biological hazard level 4 virus. The fatality rate of Ebola virus infection in humans is as high as 40% to 90%, so it is regarded as a potential tool for bioterrorism attacks. The Ebola virus RNA genome is about 19 kb long and can produce eight viral proteins, four of which, nucleocapsid protein (NP), polymerase protein (L), VP30 protein, and VP35 protein, will The complex formed plays a role in helping Ebola virus replication and transcription. With the help of this complex, three types of Ebola virus RNA are simultaneously produced in cells, namely viral RNA (viral RNA, vRNA), messenger RNA (mRNA), and complementary RNA (cRNA). It is known that the stability of RNA produced during the replication and transcription process of Ebola virus is related to the above four proteins. The EBOV minigenome system was developed based on such characteristics. As shown in Figure 1, the system includes a minigenome (as shown in Figure 1 EBOV-GFP vRNA) and expression plasmids containing genes encoding NP protein, L protein, VP30 protein, and VP35 protein respectively. The minigenome uses a reporter gene (see Figure 1 as an example, the reporter gene is green fluorescent protein (GFP)) to replace all the genes in the Ebola virus genome (that is, the open reading frame (open reading frame)). reading frame)), and the reporter gene is flanked by the 5'-untranslated region (UTR) and 3'-UTR of the Ebola virus, which contains the packaging, transcription, and replication of the Ebola virus genome, and All regulatory elements required for packaging into progeny viruses. Next, the plasmid containing the minigenome and the expression plasmid containing the genes encoding the four proteins (NP, L, VP30, and VP35 proteins) were simultaneously transfected into cells. In the cell, the reporter gene in the minigenome is expressed driven by the expression of the four proteins. Therefore, when cells express the reporter gene, it means that Ebola virus can replicate and transcribe in this environment. During the operation, the Ebola virus minigenome system does not produce Ebola virus particles, so it can be operated in biosafety level 2 (BSL-2) laboratories and can be used as a screening method to inhibit Ebola virus replication. and tools for converting drugs to green. In short, the Ebola virus minigenome system is a safe and close-up tool for observing replication and transcription processes. In addition, this system can also be used as a verification tool for proteins related to replication and transcriptional regulation.

The inventor of this case unexpectedly discovered that when the expression plasmid carrying the nucleocapsid protein gene encoding the novel coronavirus (Severe acute respiratory syndrome coronavirus 2, SARS-CoV-2) was transfected into host cells together with the Ebola virus minigenome system, , SARS-CoV-2 nucleocapsid protein can increase the expression of reporter genes in the Ebola virus minigenome system.

In one aspect, the present invention provides a protein fragment for increasing gene expression, comprising a dimerization domain of a coronavirus nucleocapsid protein and/or a carboxyl-terminal domain of a coronavirus nucleocapsid protein. (CTD).

In another aspect, the invention provides a polynucleotide encoding a protein fragment of the invention.

In another aspect, the present invention provides a plasmid comprising a polynucleotide sequence encoding the protein fragment of the present invention, and a promoter sequence at the 5' end of the nucleotide sequence.

In another aspect, the invention provides a cell comprising a protein fragment of the invention, a polynucleotide encoding a protein fragment of the invention, or a polynucleotide sequence encoding a protein fragment of the invention and in the nucleus The 5' end of the nucleotide sequence is a promoter sequence of the plasmid.

On the other hand, the present invention provides a method for increasing the expression of a target gene, comprising the following steps: (1) providing a polynucleotide sequence containing a protein fragment encoding the present invention and a polynucleotide at the 5' end of the nucleotide sequence a plasmid of a promoter sequence, (2) providing an expression vector containing the target gene, (3) co-transfecting the plastid of step (1) and the expression vector of step (2) into an expression host cell, and (4) Provide an appropriate condition to express the target gene.

In another aspect, the present invention provides a method for increasing the expression of a target gene, comprising the following steps: (1) providing a cell comprising a protein fragment of the present invention, a polynucleotide encoding a protein fragment of the present invention, or A plasmid containing a polynucleotide sequence encoding the protein fragment of the present invention and a promoter sequence at the 5' end of the nucleotide sequence, (2) providing an expression vector containing the target gene, (3) combining the steps (2) The expression vector is transfected into the cells of step (1), and (4) an appropriate condition is provided to express the target gene.

On the other hand, the present invention provides a method for screening drugs that inhibit viruses, comprising the following steps: (1) providing a polynucleotide sequence containing a protein fragment encoding the present invention and a polynucleotide sequence at the 5' end of the nucleotide sequence A plasmid with a promoter sequence, (2) providing a viral minigenome system containing a reporter gene, (3) co-transfecting the plasmid of step (1) with the viral minigenome system of step (2) Infect a expressing host cell, (4) add a candidate drug to the expressing host cell, and (5) observe the expression of the reporter gene. If the reporter gene does not express, the candidate drug has the effect of inhibiting the virus.

In another aspect, the present invention provides a method for screening drugs that inhibit viruses, comprising the following steps: (1) providing a cell comprising a protein fragment of the present invention, a polynucleotide encoding a protein fragment of the present invention, or A plasmid containing a polynucleotide sequence encoding a protein fragment of the present invention and a promoter sequence at the 5' end of the nucleotide sequence, (2) providing a viral minigenome system containing a reporter gene, (3) Transfect the viral minigenome system of step (2) into the cells of step (1), (4) add a candidate drug to the cells, (5) observe the expression of the reporter gene, if the reporter gene does not express, then This drug candidate has virus-inhibiting effects.

The present invention is illustrated by the following examples and drawings, but the present invention is not limited by the following examples.

The inventor of this case unexpectedly discovered that when the expressoplast carrying the gene encoding the SARS-CoV-2 nucleocapsid protein was transfected into host cells together with the Ebola virus minigenome system, the SARS-CoV-2 nucleocapsid protein could increase the number of Ebola viruses. Expression of reporter genes in the Lavirus minigenome system. Further research by the inventor of this case found that in addition to SARS-CoV-2, severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1), Middle East respiratory syndrome–related coronavirus , MERS-CoV), human coronavirus HKU1 (Human coronavirus HKU1, HCoV HKU1), and the nucleocapsid protein or fragments thereof of human coronavirus OC43 (HCoV OC43) can also promote the expression of a target gene. In addition, the inventor of this case also discovered that the main active region of the coronavirus nucleocapsid protein that increases gene expression includes the dimerization domain, and the secondary active region includes the carboxyl-terminal domain (CTD).

The mechanism of action of the present invention is shown in Figure 2. An expression plasmid (SARS2-NP) containing a gene encoding SARS-CoV-2 nucleocapsid protein was added to the Ebola virus minigenome system, and combined with the Ebola virus minigenome system (including a minigenome with a GFP reporter gene, and expression plasmids containing genes encoding NP protein, L protein, VP30 protein, and VP35 protein respectively) were simultaneously transfected into the cells. In cells, under the influence of the expression of these four proteins and the SARS-CoV-2 nucleocapsid protein, the mRNA and cRNA of the reporter gene in the minigenome increase, and the protein expression of the reporter gene increases.

Therefore, the present invention provides a protein fragment for increasing gene expression, comprising a dimerization domain of a coronavirus nucleocapsid protein and/or a carboxyl-terminal domain (CTD) of the coronavirus nucleocapsid protein.

In certain embodiments, the coronavirus is severe acute respiratory syndrome coronavirus (SARS-CoV-1), novel coronavirus (SARS-CoV-2), Middle East respiratory syndrome coronavirus (MERS-CoV), human coronavirus virus HKU1 (HCoV HKU1), or human coronavirus OC43 (HCoV OC43).

In certain embodiments, in addition to the dimerization domain of the nucleocapsid protein of the coronavirus and/or the carboxyl-terminal domain (CTD) of the nucleocapsid protein of the coronavirus, the protein fragment of the present invention also contains It includes at least one of the following domains: the N-terminal domain (NTD) of the nucleocapsid protein of the coronavirus, the RNA binding domain (RBD) of the nucleocapsid protein of the coronavirus, and The linker of the nucleocapsid protein of the coronavirus. In certain embodiments, the protein fragment of the invention includes the NTD, RBD, linker, and dimerization domain of the nucleocapsid protein. In certain embodiments, the protein fragment of the invention includes the RBD, linker, dimerization domain, and CTD of the nucleocapsid protein. In certain embodiments, the protein fragment of the invention includes the dimerization domain and CTD of the nucleocapsid protein. In certain embodiments, the protein fragment of the invention includes the dimerization domain of the nucleocapsid protein. In certain embodiments, the protein fragment of the invention includes the CTD of the nucleocapsid protein. In certain embodiments, the protein fragment of the invention includes the NTD and dimerization domain of the nucleocapsid protein. In certain embodiments, the protein fragment of the invention includes the NTD, dimerization domain, and CTD of the nucleocapsid protein. In certain embodiments, the protein fragments of the invention comprise the full-length sequence of the nucleocapsid protein. In certain embodiments, the protein fragments of the invention comprise at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or Amino acid sequence with 99% similarity: SEQ ID NO: 1, 12, 13, 15, 17, 18, 19, 20, 21, 22, 23, 28, 29, 30, 31, 32, 33, 34, 35, and 36. In certain embodiments, the protein fragment of the present invention has one of the following amino acid sequences: SEQ ID NO: 1, 12, 13, 15, 17, 18, 19, 20, 21, 22, 23, 28, 29 , 30, 31, 32, 33, 34, 35, and 36.

The present invention also provides a polynucleotide encoding any of the above-mentioned protein fragments.

The polynucleotide encoding any of the above-mentioned protein fragments of the present invention is derived from the amino acid sequence of the protein fragment used to increase gene expression of the present invention. Each amino acid on the amino acid sequence of the protein fragment used to increase gene expression of the present invention is replaced with the nucleotide sequence encoding the amino acid listed in the genetic code table (including various simplified The polynucleotide sequence provided by the present invention can be obtained by degenerate codons (also known as synonymous codons). For example, the proline in the amino acid sequence of the protein fragment used to increase gene expression of the present invention can be encoded by nucleotide sequences such as CCA, CCC, CCG, and CCT.

The present invention also provides a plasmid, comprising a nucleotide sequence encoding any of the above-mentioned protein fragments of the present invention, and a promoter sequence at the 5' end of the nucleotide sequence.

In certain embodiments, the promoter is a strong promoter. Examples of strong promoters include, but are not limited to, retrovirus long terminal repeat (LTR), cytomegalovirus (CMV), murine stem cell virus (MSCV) U3, phosphoglycerate Kinase (Phosphoglycerate kinase, PGK), Actin, ubiquitin protein and simian virus 40 (SV40)/CD43 complex promoter and elongation factor (EF)-1 , and the spleen focus-forming virus (SFFV) promoter. In addition to the strong promoter, the plasmid may further contain regulatory elements that can enhance the expression of genes encoding any of the above-mentioned protein fragments, such as central polypurine track (CPPT), woodchuck hepatitis virus post-transcriptional regulatory elements (Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element, WPRE), CMV enhancer, R-U5' segment in the LTR of HTLV-I, SV40 enhancer, and rabbit -The intronic sequence between exons 2 and 3 of the globin protein.

The invention also provides a cell comprising any of the above-mentioned protein fragments of the invention, a nucleotide sequence encoding any of the above-mentioned protein fragments of the invention, or a nucleotide sequence encoding any of the above-mentioned protein fragments of the invention and in the nucleus The 5' end of the nucleotide sequence is a promoter sequence of the plasmid.

In certain embodiments, any of the above-mentioned protein fragments of the present invention is expressed in a cell line that transiently expresses the protein fragment. Transient transfection is a process in which the nucleic acid introduced into a cell is not integrated into the genome or chromosomal DNA of the cell. They are in fact retained in the cell as extrachromosomal elements (eg, as episomes). The transcription process of the nucleic acid of the cell-free genome is unaffected and the protein encoded by the nucleic acid of the cell-free genome is produced. In certain embodiments, any of the above-mentioned protein fragments of the present invention is expressed in a cell line that stably expresses the protein fragment. Host cells can be appropriately engineered and transformed with vectors containing expression control elements (eg, promoters, enhancers, transcription terminators, polyadenylation sites, etc.) and selectable marker genes. After the introduction of foreign DNA, the cells can be grown in proliferation medium for 1-2 days and then transferred to selective medium. The selectable marker in the recombinant plastid confers resistance to selection and allows cells to stably integrate the plastid into their chromosomes to grow and form variant regions that can be sequentially selected and expanded into cell lines. Methods for constructing and producing stable cell lines are well known in the art, and reagents are generally commercially available.

The present invention also provides a method for increasing the expression of a target gene, which includes the following steps: (1) providing a polynucleotide sequence encoding the protein fragment of the present invention and a promoter sequence at the 5' end of the nucleotide sequence the plasmid, (2) providing an expression vector containing the target gene, (3) co-transfecting the plastid of step (1) and the expression vector of step (2) into an expression host cell, and (4) providing An appropriate condition enables the expression of the target gene.

The present invention also provides a method for increasing the expression of a target gene, which includes the following steps: (1) providing a cell that contains the protein fragment of the present invention, the polynucleotide encoding the protein fragment of the present invention, or the cell containing the protein fragment encoding the present invention. The polynucleotide sequence of the protein fragment and a plasmid of a promoter sequence at the 5' end of the nucleotide sequence, (2) providing an expression vector containing the target gene, (3) converting the step (2) The expression vector is transfected into the cells in step (1), and (4) an appropriate condition is provided to express the target gene.

The present invention also provides a method for screening drugs that inhibit viruses, which includes the following steps: (1) providing a polynucleotide sequence encoding the protein fragment of the present invention and a promoter sequence at the 5' end of the nucleotide sequence the plasmid, (2) providing a viral minigenome system containing a reporter gene, (3) co-transfecting the plastid of step (1) and the viral minigenome system of step (2) into a expressing host cell, ( 4) Add a candidate drug to the expressing host cell, and (5) observe the expression of the reporter gene. If the reporter gene does not express, the candidate drug has the effect of inhibiting the virus.

The present invention also provides a method for screening drugs that inhibit viruses, which includes the following steps: (1) providing a cell that contains the protein fragment of the present invention, the polynucleotide encoding the protein fragment of the present invention, or the cell containing the protein fragment encoding the present invention. a polynucleotide sequence of the protein fragment and a promoter sequence at the 5' end of the nucleotide sequence, (2) providing a viral minigenome system containing a reporter gene, (3) combining step (2) ) is transfected into the cells of step (1), (4) adding a candidate drug to the cells, (5) observing the expression of the reporter gene, if the reporter gene does not express, then the candidate drug has Virus-inhibiting effect.

Examples of reporter genes include, but are not limited to, those encoding glutathione S-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (chloramphenicol) acetyltransferase (CAT), β-galactosidase, β-glucuronidase, luciferase (luciferase), green fluorescent protein (GFP), enhanced green fluorescent protein (enhanced green fluorescent protein) , EGFP), red fluorescent protein (RFP), yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), and autofluorescent proteins (including blue fluorescent protein The gene for blue fluorescent protein (BFP). In some embodiments, the reporter gene is a gene encoding a fluorescent protein, a gene encoding chloramphenicol acetyltransferase (CAT), or a gene encoding secreted alkaline phosphatase (SEAP). The gene, the gene encoding β-galactosidase (β-gal), or the gene encoding luciferase.

In certain embodiments, the viral minigenome system is an Ebola virus minigenome system. The Ebola virus minigenome system includes an expression vector containing an Ebola virus minigenome containing a reporter gene, an expression vector containing a gene encoding Ebola virus nucleocapsid protein (NP), and an expression vector containing an encoding Ebola virus polymer An expression vector containing an enzyme protein (polymerase protein, L) gene, an expression vector containing a gene encoding the Ebola virus VP30 protein, and an expression vector containing a gene encoding the Ebola virus VP35 protein.

Examples of viral minigenome systems include, but are not limited to, Ebola virus minigenome system, Marburg virus minigenome system, Severe fever with thrombocytopenia syndrome virus minigenome system, Rift Valley fever Rift Valley Fever Virus mini-genome system, Nipah Virus mini-genome system, Coronavirus mini-genome system, and Enterovirus mini-genome system, etc.

Unless otherwise defined herein, scientific and technical terms used in connection with this document shall have the meaning commonly understood by one of ordinary skill in the art. Furthermore, unless the context otherwise requires, singular terms shall include the plural and plural terms shall include the singular. The methods and techniques of the present invention can generally be performed according to conventional methods known in the art. In general, the nomenclature described herein is used to link the following techniques, as well as techniques in biochemistry, enzymology, molecular and cellular biology, microbiology, and genetics, all of which are known and commonly used by those in the art. Unless otherwise indicated, the methods and techniques of the present invention may generally be performed according to conventional methods known in the art and are described in the various general and more specific references cited and discussed in this specification.

As used herein, the term "coronavirus" refers to viruses belonging to the family Coronaviridae and the subfamily Orthocoronavirinae . Coronaviruses are enveloped, positive-stranded, single-stranded RNA viruses with a genome size ranging from 26,000 to 32,000 nucleotides, encoding a spike (S), an outer membrane (E), and a membrane (Membrane, M). ), and four structural proteins including nucleocapsid (N) protein. Coronaviruses can infect mammals and birds; in humans and birds, coronaviruses can cause respiratory infections ranging from mild to fatal. There are seven known coronaviruses that infect humans, including Human coronavirus 229E (HCoV 229E), Human coronavirus OC43 (HCoV OC43), Human coronavirus NL63 (HCoV NL63), and Human coronavirus HKU1 (HCoV HKU1) , severe acute respiratory syndrome coronavirus (SARS-CoV-1), novel coronavirus (SARS-CoV-2), Middle East respiratory syndrome coronavirus (MERS-CoV).

As used herein, the term "nucleocapsid protein" refers to the viral protein shell surrounding a viral genome (DNA or RNA). Nucleocapsid protein is the main component of the viral nucleocapsid. It can combine with itself and the viral genome, thereby packaging the viral genome in a closed cavity. In addition to coronaviruses, many viruses also have nucleocapsid proteins, such as Ebola virus and influenza virus. The nucleocapsid protein of coronavirus contains N-terminal domain (NTD), RNA binding domain (RBD), linker, dimerization domain, carboxyl C-terminal domain (CTD).

As used herein, the term "minigenome" refers to an artificial DNA sequence that replaces the open reading frame of a virus with a reporter gene flanked by two 5'-UTR and 3'-UTR. The 5'-UTR and 3'-UTR contain all the regulatory elements required to package, transcribe, replicate, and package the viral genome into progeny viruses. Reporter genes for minigenomes are usually genes encoding fluorescent proteins, chloramphenicol acetyltransferase (CAT), or luciferase. As an example, "Ebola virus minigenome" refers to replacing the open reading frame of Ebola virus with a reporter gene, that is, replacing all the genes in the genome of Ebola virus with a reporter gene, and the reporter gene is flanked by They are the 5'-UTR and 3'-UTR of Ebola virus respectively, which contain all the regulatory elements required to package, transcribe, replicate, and package the Ebola virus genome into progeny viruses. Viruses that can serve as minigenomes include, but are not limited to, Ebola virus, Marburg virus, fever syndrome virus, Rift Valley fever virus, Nipah virus, coronavirus, and enterovirus.

As used herein, the term "minigenome system" refers to a set of plastids that includes an expression plasmid harboring a minigenome and a plastid encoding viral proteins required for the replication and transcription of the viral genome that the minigenome replaces. Expression plastids of genes. When used, these plasmids are co-transfected into cells together. As an example, an "Ebola virus minigenome system" includes an expression plasmid containing an Ebola minigenome with a reporter gene, an expression vector containing a gene encoding Ebola virus nucleocapsid protein (NP), and an expression vector containing a gene encoding Ebola virus nucleocapsid protein (NP). An expression vector for viral polymerase protein (L) gene, an expression vector containing a gene encoding Ebola virus VP30 protein, and an expression vector containing a gene encoding Ebola virus VP35 protein.

As used herein, the term "gene expression" refers to the transformation of information contained in a gene into a gene product. The gene product may be a direct transcription product of a gene (eg, mRNA, tRNA, rRNA, antisense RNA, cRNA, ribozyme, structural RNA, or any other type of RNA) or a protein resulting from translation of the mRNA.

As used herein, the term "nucleotide" is intended to include monomers containing a nitrogen base linked to a sugar phosphate, which includes a sugar, such as ribose or 2'-deoxyribose, linked to one or more Phosphate group. "Polynucleotide" and "nucleic acid" refer to polymers including more than one nucleotide monomer, where the monomers are usually linked by sugar-phosphate bonds in a sugar-phosphate backbone. A polynucleotide need not include only one type of nucleotide monomer. For example, the nucleotides comprising a given polynucleotide may be only ribonucleotides, only 2'-oxyribonucleotides, or both ribonucleotides and 2'-deoxyribonucleotides. combination. Polynucleotides include naturally occurring nucleic acids, such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), as well as nucleic acid analogs containing one or more non-naturally occurring monomers. Polynucleotides can be synthesized, for example, using automated DNA synthesizers. The term "nucleic acid" generally refers to large polynucleotides. It will be understood that when a nucleotide sequence is represented by a DNA sequence (i.e. A, T, G, C), this also includes an RNA sequence (i.e. A, U, G, C) where "U" is substituted for "T ". The term "cDNA" refers to a DNA that is complementary or identical to an mRNA, whether in single-stranded or double-stranded form, but in which "T" is substituted for "U". The term "recombinant nucleic acid" means a polynucleotide or nucleic acid having sequences that are not naturally joined together. The recombinant nucleic acid may be in the form of a vector.

As used herein, the term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that act in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code and those that are later modified, for example, hydroxyproline, gamma carboxyglutamic acid, and O-phosphoserine. For the purposes of this invention, the term "amino acid analogue" refers to a compound that has the same basic chemical structure as a naturally occurring amino acid, i.e., a hydrogen, a carboxyl group, an amino group, and an R group. Carbon, the R group is, for example, homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. These analogs have modified R groups (eg, norleucine) or modified peptide backbones, but retain the same basic chemical structure as the naturally occurring amino acid. For the purposes of the present invention, the term "amino acid mimetic" refers to a compound that has a structure that is different from the general chemical structure of an amino acid, but that acts in a manner similar to naturally occurring amino acids.

The words "about", "approximately" or "approximately" used herein essentially mean that the stated value or range is within 20%, preferably within 10%, and more preferably within 5%. Numerical quantities provided herein are approximations and may be inferred if the terms "about," "approximately," or "approximately" are not used.

As used herein, the articles "a," "an," and "any" refer to grammatical items that refer to one or more than one (ie, at least one) item. For example, "an element" means one element or more than one element.

The invention is further illustrated by the following examples, which are provided for purposes of illustration and not limitation. In light of this disclosure, those skilled in the art will understand that many variations can be made in the specific embodiments disclosed and still obtain the same or similar results without departing from the spirit and scope of the invention.

Example

Example 1 SARS-CoV-2 Effects of nucleocapsid protein on Ebola virus minigenome system

Materials and methods

Preparation of plasmids containing genes encoding SARS-CoV-2 nucleocapsid proteins. After amplifying the gene sequence encoding SARS-CoV-2 nucleocapsid protein (SEQ ID NO: 1) by PCR, the gene sequence was cloned into the pCAGGS vector (Model: V008798, NovoPro Bioscience Company, Shanghai, China) to generate pCAGGS_SARSCoV2_NP plasmid, and transform the pCAGGS_SARSCoV2_NP plasmid into E. coli.

Operation of the Ebola virus minigenome system . The Ebola virus minigenome system contains five plasmids, namely pCAGGS_3E5E_eGFP plasmid (model #103054, Addgene), pCAGGS_L_EBOV plasmid (model #103052, Addgene), pCAGGS_VP30_EBOV plasmid (model #103051, Addgene), pCAGGS_VP35_EBOV plasmid (model #103050, Addgene), and pCAGGS_NP_EBOV plasmid (model #103049, Addgene). The five plasmids were purchased from Addgene platform (Watertown, MA, USA). Prepare the plastid mixture of each group according to the ratio in Table 1 below: Control group: only contains the Ebola virus mini-genome system; Test group 1: Ebola virus mini-genome system + 1000 ng SARS-CoV-2 nucleocapsid protein gene plastid ; and Experimental Group 2: Ebola virus minigenome system + 500 ng SARS-CoV-2 nucleocapsid protein gene plasmid.

Table 1 Proportions of plastid mixtures in each group in Example 1 control group Experimental group 1 Experimental group 2 pCAGGS_3E5E_eGFP plasmid 1500ng 1500ng 1500ng pCAGGS_L_EBOV plasmid 1000ng 1000ng 1000ng pCAGGS_VP30_EBOV plasmid 250ng 250ng 250ng pCAGGS_VP35_EBOV plasmid 750ng 750ng 750ng pCAGGS_NP_EBOV plasmid 250ng 250ng 250ng pCAGGS_SARSCoV2_NP plasmid 0ng 1000ng 500

120 µL serum-reduced medium (GibcoTM Opti-MEM; model: 11058021, Thermo Fisher Scientific, Waltham, MA, USA) and 8 µL T-Pro NTR II were added to the plastid mixture of each group. The reagent set (model: JT97-N002, T-Pro Biotechnology Co., Ltd., New Taipei City, Taiwan) was left to stand for 15 minutes to become the transfection reagent mixture. In addition, 1.6 x 10 ⁵ /well human embryonic kidney cells (293T cells; number: CRL-3216, American Type Culture Collection, ATCC, Manassas, VA , USA) inoculated in Dulbecco's Modified Eagle Medium (Dulbecco's Modified Eagle Medium, USA) containing 10% fetal bovine serum (FBS, model: 89510-186, Avantor Company, Radnor Township, Pennsylvania, USA) DMEM, model: CC103-0500, GeneDireX Company, Taoyuan City, Taiwan) into a 24-well cell culture plate, and add 40 µL transfection reagent mixture for transfection. The transfected cells were placed in a 37 ^° C/5% _CO2 cell culture incubator and cultured for the specified time before analysis.

Fluorescence microscopy analysis . The cells cultured for 24 and 48 hours after transfection (control group and test group 1) were placed on an inverted fluorescence microscope (Olympus IX71, Olympus Corporation, Tokyo, Japan), and excited with excitation light of a wavelength of 475 nm. 509 nm absorbs light for observation.

Flow cytometry analysis . Take out the cells cultured for 24 and 48 hours after transfection (control group, test group 1, test group 2), remove the culture medium in the cell culture plate, and add 150 µL TrypLE Express (Gibco ^TM , model: 12605-028, Thermo Fisher Scientific), add 500 µL Dulbecco's Phosphate Buffered Saline (DPBS, HyClone ^TM , Model: SH30028.03, Cytiva, Marlborough, MA, USA) to neutralize the cells. Mixture. Transfer the cell mixture into a test tube and place on ice until use. Fluorescence signals were analyzed with a flow cytometer (CytoFLEX, Beckman Coulter, Brea, California, USA), and data analysis was performed with flow cytometry analysis tool software (FlowJo v10).

RNA quantification analysis . Take out the cells cultured for 0, 12, 24, 36 and 48 hours after transfection (control group and test group 1), remove the culture medium in the cell culture plate, and add 1 mL TRI Reagent RNA Isolation Reagent (Model: T9424, Sigma -Aldrich Co., St. Louis, MO, USA) reagent to lyse cells and extract RNA. The extracted RNA was passed through a Picodrop ^TM ultramicrovolume spectrophotometer (Picodrop Ltd., Cambridge, UK) to measure RNA concentration. RNA was transcribed into cDNA using Superscript ^™ reverse transcriptase kit (Thermo Fisher Scientific). In addition, the total RNA, mRNA, viral RNA (vRNA), complementary RNA (cRNA), and glyceraldehyde-3-phosphate of the green fluorescent protein (GFP) in the Ebola virus minigenome system were removed. Hydrogenase (Glyceraldehyde 3-phosphate dehydrogenase, GAPDH) gene design primer pair used for quantitative PCR. Use the SYBR Green Master Mix reagent set (Thermo Fisher Scientific) to respectively use the primer pair of GFP total RNA (SEQ ID NOs: 2 and 3), the primer pair of GFP mRNA (SEQ ID NOs: 4 and 5), and the primer pair of GFP vRNA. The primer pair (SEQ ID NOs: 6 and 7), the primer pair of GFP cRNA (SEQ ID NOs: 8 and 9), and the primer pair of GAPDH gene (SEQ ID NOs: 10 and 11) were used to perform quantitative PCR on cDNA, and use The LightCycler 480 II instrument (Roche, Basel, Switzerland) detects individual gene copy numbers. The GAPDH gene was used as the internal control gene (internal control), and the gene expression ratio was calculated.

Statistical analysis . Statistical analysis was performed using Prism 6.01 (GraphPad Software, Inc., San Diego, CA, USA) analysis suite software. The Mann-Whitney U test was used to compare differences between each experimental group and the control group. * p < 0.05, ** p < 0.01, *** p < 0.001.

result

SARS-CoV-2 nucleocapsid protein improves reporter performance in Ebola virus minigenome systems. As shown in Figure 3 and Figure 4, whether 24 or 48 hours after transfection, the expression of the reporter gene (GFP gene) in experimental group 1 (Ebola virus minigenome system + 1000 ng SARS-CoV-2 nucleocapsid protein) The number of cells was significantly higher than that of the control group (which only included the Ebola virus minigenome system), and the fluorescence intensity of the former was higher than that of the latter. The above results show that adding the SARS-CoV-2 nucleocapsid protein gene to the Ebola virus minigenome system increases the number of cells expressing the reporter gene and increases the fluorescence brightness.

In addition, as shown in Figure 5, whether it is experimental group 1 (Ebola virus minigenome system + 1000 ng SARS-CoV-2 nucleocapsid protein) or experimental group 2 (Ebola virus minigenome system + 500 ng SARS-CoV- 2 nucleocapsid protein), 24 and 48 hours after transfection, the number of cells expressing the reporter gene (GFP gene) was statistically significantly greater than that of the control group (only including the Ebola virus minigenome system) ( p ＜ 0.01 or p <0.05). The above results show that adding the SARS-CoV-2 nucleocapsid protein gene to the Ebola virus minigenome system significantly increased the number of cells expressing the reporter gene after 24 hours, and the addition of 500 ng SARS-CoV-2 nucleocapsid protein gene plasmids The effect is equivalent to that of adding 1000 ng SARS-CoV-2 nucleocapsid protein gene plasmid.

SARS-CoV-2 nucleocapsid protein increases the expression of mRNA and cRNA of reporter genes in the Ebola virus minigenome system . As shown in Figure 6A, 24 and 48 hours after transfection, the total RNA expression of the reporter gene (GFP gene) in experimental group 1 (Ebola virus minigenome system + 1000 ng SARS-CoV-2 nucleocapsid protein) was significant increased, and was statistically significantly greater than the total RNA expression of the reporter gene (GFP gene) in the control group (only containing the Ebola virus minigenome system) ( p < 0.05). Further analysis is shown in Figure 6B. After 36 and 48 hours of transfection, the vRNA expression amount of the reporter gene in experimental group 1 was significantly reduced due to cell metabolism; after 24 and 48 hours of transfection, the expression amount of the reporter gene in experimental group 1 The expression amount of mRNA increased significantly, and was statistically significantly greater than the expression amount of mRNA of the reporter gene in the control group ( p <0.05); 24-48 hours after transfection, the expression amount of cRNA of the reporter gene in experimental group 1 increased significantly, And it was statistically significantly higher than the cRNA expression amount of the reporter gene in the control group ( p < 0.05).

The results of this example show that SARS-CoV-2 nucleocapsid protein can promote the transcription (increase of mRNA) and replication (increase of cRNA) of the reporter gene in the Ebola virus minigenome system, thereby increasing the expression of the reporter gene (cells expressing the reporter gene The number increases and the fluorescence brightness increases).

Example 2 SARS-CoV-2 Effect of nucleocapsid protein on antiviral drug screening using Ebola virus minigenome system

Materials and methods

Operation of the Ebola virus minigenome system . Same as described in Example 1. Briefly, 0 ng and 1000 ng pCAGGS_SARSCoV2_NP plasmids were added to the system respectively to form a control group (containing only the Ebola virus minigenome system) and an experimental group (Ebola virus minigenome system + 1000 ng SARS-CoV-2 nucleocapsid protein). Add 120 µL of serum-reduced medium and 8 µL of T-Pro NTR II reagent kit to each group, and let it stand for 15 minutes to form a transfection reagent mixture. Then, the transfection reagent mixture of each group was added to 293T cells for transfection. The transfected cells were placed in a 37 ^° C/5% _CO2 cell culture incubator.

Remdesivir antiviral drug screening . Six hours after the cells were transfected, antiviral drug Remdesivir (Remdesivir) at concentrations of 25 µM, 50 µM, 100 µM, 200 µM, 400 µM, and 800 µM was added to the cell culture plate respectively. The cells treated with remdesivir were placed in a 37 ^° C/5% _CO2 cell culture incubator for 48 hours, and then analyzed by flow cytometry.

Flow cytometry analysis . Same as described in Example 1.

Statistical analysis . Same as described in Example 1.

result

SARS-CoV-2 nucleocapsid protein increases the sensitivity of Ebola virus minigenome system for antiviral drug screening. As shown in Figure 7, whether it is the control group (containing only Ebola virus minigenome system) or the experimental group (Ebola virus minigenome system + 1000 ng SARS-CoV-2 nucleocapsid protein), with the concentration of remdesivir The number of cells expressing the reporter gene (GFP) gradually decreased, indicating that Remdesivir has the ability to inhibit the virus. However, in the control group, although the number of cells expressing the reporter gene in the control group cells treated with remdesivir decreased, it was not comparable to that in the control group cells that were not treated with remdesivir (0 µM). There is a statistically significant difference in the number of cells. In comparison, in the experimental group, the cells in the experimental group only need to be treated with 50 µM remdesivir, and the number of cells expressing the reporter gene can be compared with the number of cells in the experimental group not treated with remdesivir (0 µM). There was a statistically significant difference in the number of cells expressing the reporter gene ( p < 0.05).

The results of this example show that the SARS-CoV-2 nucleocapsid protein can promote the sensitivity of the Ebola virus minigenome system for screening antiviral drugs, making it easier to determine whether candidate drugs have the effect of inhibiting the virus.

Example 3 SARS-CoV-2 Effect of nucleocapsid protein on gene expression

Materials and methods

Cell transfection . Expression plasmids carrying enhanced green fluorescent protein (EGFP) and red fluorescent protein (RFP) reporter genes respectively, pEGFP-N2 plasmid and piRFP682_N1 plasmid, were purchased from Addgene platform (Watertown City , Massachusetts, USA). Prepare the plastid mixture of each group according to the ratio in Table 2 below: Negative control group: cells not transfected with any plastids; Experimental group G1: Cells transfected with EGFP gene only; Experimental group G2: Transfected with EGFP gene + 1000 ng Cells with SARS-CoV-2 nucleocapsid protein gene plasmid; Experimental group R1: cells transfected with RFP gene only; and Experimental group R2: cells transfected with RFP gene + 1000 ng SARS-CoV-2 nucleocapsid protein gene plasmid cells).

Table 2 Proportions of plastid mixtures in each group in Example 3 negative control group Test group G1 Test group G2 Test group R1 Test group R2 pEGFP-N2 plasmid 0ng 500ng 500ng 0ng 0ng piRFP682_N1 plasmid 0ng 0ng 0ng 500ng 500ng pCAGGS_SARSCoV2_NP plasmid (same as Example 1) 0ng 0ng 1000ng 0ng 1000

Add 120 µL serum-reduced medium (Gibco ^TM Opti-MEM; model: 11058021, Thermo Fisher Scientific) and 8 µL T-Pro NTR II reagent kit (model: JT97-N002, T-Pro Biotechnology Co., Ltd.) and let it stand for 15 minutes to become the transfection reagent mixture. In addition, 1.6 x 10 ⁵ cells/well 293T cells (ATCC, No.: CRL-3216) were seeded in a 24-well cell culture plate containing 10% FBS/DMEM, and 40 µL transfection reagent mixture was added for transfection. The transfected cells were placed in a 37 ^° C/5% _CO2 cell culture incubator, and flow cytometry analysis was performed after 24 hours of culture.

Flow cytometry analysis . Same as described in Example 1.

result

SARS-CoV-2 nucleocapsid protein increases reporter expression in expression plasmids . As shown in Figure 8A, compared with the negative control group (cells not transfected with any plastids, the proportion of cells showing green fluorescence was 0.2%) and the experimental group G1 (cells transfected with only the EGFP gene, showing green fluorescence The proportion of cells in the experimental group G2 (cells transfected with EGFP gene + 1000 ng SARS-CoV-2 nucleocapsid protein gene plasmid) was as high as 96.7%, significantly improving the expression of the EGFP gene. . Similarly, as shown in Figure 8B, compared with the negative control group (cells not transfected with any plastid, the proportion of cells showing red fluorescence was 0.04%) and the experimental group R1 (cells transfected with only the RFP gene, the proportion of cells showing red fluorescence was 0.04%) The proportion of cells with red fluorescence was 12.9%), and the proportion of cells with red fluorescence in experimental group R2 (cells transfected with RFP gene + 1000 ng SARS-CoV-2 nucleocapsid protein gene plasmid) was as high as 26.7%, significantly increasing RFP Gene expression.

The results of this example show that SARS-CoV-2 nucleocapsid protein can significantly increase the expression of target genes in the plastid, and is not limited to the green fluorescent protein gene, but is applicable to various target genes.

Example 4 SARS-CoV-2 Active zone analysis of nucleocapsid protein increasing gene expression

Materials and methods

Preparation of plasmids containing gene fragments encoding each segment of SARS-CoV-2 nucleocapsid protein . As shown in Figure 9, the SARS-CoV-2 nucleocapsid protein contains an amine-terminal domain (NTD), an RNA-binding domain (RBD), a linker, a dimerization domain, and a carboxyl-terminal domain (CTD). Use PCR to amplify the gene sequences encoding the following SARS-CoV-2 nucleocapsid protein fragments, and add a DNA sequence (ATG) encoding methionine (Methionine) to the 5' end of the gene sequence of each fragment (except SEQ ID NOs: 12, 14, 16, 19, 20): Amino acids 1-360 (SEQ ID NO: 12); Amino acids 61-419 (SEQ ID NO: 13); Amino acids 1 -180 amino acids (SEQ ID NO: 14); 241-419 amino acids (SEQ ID NO: 15); 1-67 amino acids (SEQ ID NO: 16); 241-363 Amino acids (SEQ ID NO: 17); Amino acids 354-419 (SEQ ID NO: 18); Amino acids 1-67/241-360 (SEQ ID NO: 19); and Amino acids 1-67/241-360 (SEQ ID NO: 19); 1-67/241-419 amino acids (SEQ ID NO: 20). These gene sequences were selected and cloned into pCAGGS vector respectively to produce pCAGGS_SARSCoV2_NP (1-360 aa) plasmid (can express SEQ ID NO: 12 sequence); pCAGGS_SARSCoV2_NP (61-419 aa) plasmid (can express SEQ ID NO: 13 sequence); pCAGGS_SARSCoV2_NP (1-180 aa) plasmid (can express SEQ ID NO: 14 sequence); pCAGGS_SARSCoV2_NP (241-419 aa) plastid (can express SEQ ID NO: 15 sequence); pCAGGS_SARSCoV2_NP (1-67 aa) Plastid (can express SEQ ID NO: 16 sequence); pCAGGS_SARSCoV2_NP (241-361 aa) plastid (can express SEQ ID NO: 17 sequence); pCAGGS_SARSCoV2_NP (354-419 aa) plastid (can express SEQ ID NO: 18 sequence); pCAGGS_SARSCoV2_NP (1-67/241-360 aa) plasmid (can express SEQ ID NO: 19 sequence); and pCAGGS_SARSCoV2_NP (1-67/241-419 aa) plastid (can express SEQ ID NO: 20 sequence) ). And these plasmids were transformed into E. coli respectively.

Operation of the Ebola virus minigenome system . Same as described in Example 1. Briefly, 0 ng and 1000 ng of pCAGGS_SARSCoV2_NP, pCAGGS_SARSCoV2_NP (1-360 aa), pCAGGS_SARSCoV2_NP (61-419 aa), pCAGGS_SARSCoV2_NP (1-180 aa), pCAGGS_SARSCoV2_NP (241-419 aa), pCAGGS_SAR were added to the system respectively. SCoV2_NP (1-67 aa), pCAGGS_SARSCoV2_NP (241-360 aa), pCAGGS_SARSCoV2_NP (354-419 aa), pCAGGS_SARSCoV2_NP (1-67/241-360 aa), and pCAGGS_SARSCoV2_NP (1-67/241-419 aa) plastids, To form control group 1 (containing only Ebola virus minigenome system), control group 2 (Ebola virus minigenome system + 1000 ng SARS-CoV-2 nucleocapsid protein full length), and experimental group AI. Add 120 µL serum-reduced medium and 8 µL T-Pro NTR II reagent kit to each group respectively, and let it stand for 15 minutes to form a transfection reagent mixture. Then, the transfection reagent mixture of each group was added to 293T cells for transfection. The transfected cells were cultured in a 37 ^° C/5% _CO2 cell incubator for 48 hours, and then analyzed by flow cytometry.

Flow cytometry analysis . Same as described in Example 1.

Statistical analysis . Same as described in Example 1.

result

The main active region of SARS-CoV-2 nucleocapsid protein that increases gene expression is the amino acid segment 241-360 , and the secondary active region is the amino acid segment 354-419 . As shown in Figure 9 and Table 3, the groups containing the 241-360th amino acid segment of the SARS-CoV-2 nucleocapsid protein (including control group 2, test group A, test group B, test group D, test The average proportion (n=3) of fluorescent cells (that is, cells expressing the reporter gene (GFP gene)) produced by group F, experimental group H, and experimental group I) was all statistically significantly higher than that of the control group 1 ( The average proportion of fluorescent cells ( p < 0.05) containing only the Ebola virus minigenome system shows that the 241-360 amino acid segments of the SARS-CoV-2 nucleocapsid protein have the activity of increasing gene expression. In addition, the fluorescent cells produced by test group G (Ebola virus minigenome system + 1000 ng SARS-CoV-2 nucleocapsid protein amino acid segment 354-419) were also statistically significantly higher than those in the control group 1 The average proportion of fluorescent cells ( p < 0.05) shows that the 354-419 amino acid segments of the SARS-CoV-2 nucleocapsid protein also have the activity of increasing gene expression, but its activity is less than the 241-360 amino groups acid section. Groups that do not include amino acid segments 241-360 and 354-419 of the SARS-CoV-2 nucleocapsid protein (including experimental group C and experimental group E) cannot be added to the Ebola virus minigenome system. The number of cells expressing the reporter gene (GFP gene).

Table 3 Proportions of fluorescent cells in each group in Example 4 Group Average proportion of fluorescent cells (n=3) standard deviation P value Control group 1 Contains only EBOV minigenome (ST3) 22.2% 0.1 - Control group 2 ST3+ SARSCoV2_NP(1-419aa) 35.6% 1.1 <0.05 Experimental group A ST3+ SARSCoV2_NP(1-361aa) 29.3% 3.6 <0.05 Experimental group B ST3+ SARSCoV2_NP(61-419aa) 27.0% 2.8 <0.05 Experimental group C ST3+ SARSCoV2_NP(1-180aa) 18.5% 1.9 <0.05 Experimental group D ST3+ SARSCoV2_NP(240-419aa) 25.8% 1.4 <0.05 Experimental group E ST3+ SARSCoV2_NP(1-67aa) 24.0% 3.2 >0.05 Test group F ST3+ SARSCoV2_NP(240-361aa) 27.6% 2.7 <0.05 Test group G ST3+ SARSCoV2_NP(352-419aa) 24.7% 0.5 <0.05 Test group H ST3+ SARSCoV2_NP(1-67/240-361aa) 25.0% 0.8 <0.05 Experimental group I ST3+ SARSCoV2_NP(1-67/240-419aa) 28.9% 3.3 <0.05

The results of this example show that the main active region of SARS-CoV-2 nucleocapsid protein that increases gene expression is the 241-360 amino acid segment, which contains the dimerization domain. In addition, the secondary active region of SARS-CoV-2 nucleocapsid protein that increases gene expression is the 354-419th amino acid segment, which contains the carboxyl-terminal domain (CTD).

Example 5 Effects of nucleocapsid proteins of different coronaviruses on the Ebola virus minigenome system

Materials and methods

Preparation of plasmids containing genes encoding coronavirus nucleocapsid proteins . PCR was used to amplify the encoding SARS-CoV-1 nucleocapsid protein (SEQ ID NO: 21), MERS-CoV nucleocapsid protein (SEQ ID NO: 22), HCoV HKU1 nucleocapsid protein (SEQ ID NO: 23), HCoV OC43 nucleocapsid protein (SEQ ID NO: 24), HCoV 229E nucleocapsid protein (SEQ ID NO: 25), HCoV NL63 nucleocapsid protein (SEQ ID NO: 26), influenza A virus (Influenza A) nucleocapsid protein ( SEQ ID NO: 27), these gene sequences were selected and cloned into the pCAGGS vector to generate pCAGGS_SARSCoV1_NP plasmid (which can express the SEQ ID NO: 21 sequence); pCAGGS_MERSCoV_NP plasmid (which can express the SEQ ID NO: 22 sequence) ; Represents SEQ ID NO: 26 sequence); and pCAGGS_InfluenzaA_NP plasmid (can express SEQ ID NO: 27 sequence). And these plasmids were transformed into E. coli respectively.

Operation of the Ebola virus minigenome system . Same as described in Example 1. Briefly, 0 ng and 1000 ng of pCAGGS_SARSCoV1_NP, pCAGGS_SARSCoV2_NP, pCAGGS_MERSCoV_NP, pCAGGS_HCoVHKU1_NP, pCAGGS_HCoVOC43_NP, pCAGGS_HCoV229E_NP, pCAGGS_HCoVNL63_NP, and pCAGGS_InfluenzaA_NP plasmids were added to the system, respectively. , to form a control group (containing only the Ebola virus minigenome system ) and experimental groups 1-9. Add 120 µL of serum-reduced medium and 8 µL of T-Pro NTR II reagent kit to each group, and let it stand for 15 minutes to form a transfection reagent mixture. Then, the transfection reagent mixture of each group was added to 293T cells for transfection. The transfected cells were cultured in a 37 ^° C/5% _CO2 cell incubator for 48 hours, and then analyzed by flow cytometry.

Flow cytometry analysis . Same as described in Example 1.

Statistical analysis . Same as described in Example 1.

result

The nucleocapsid proteins of SARS-CoV-1 , SARS-CoV-2 , MERS-CoV , and HCoV HKU1 can improve the performance of reporters in the Ebola virus minigenome system. As shown in Table 4, it contains SARS-CoV-1 nucleocapsid protein (test group 1), SARS-CoV-2 nucleocapsid protein (test group 2), MERS-CoV nucleocapsid protein (test group 3), HCoV HKU1 core The average proportion of fluorescent cells (i.e., cells expressing the reporter gene (GFP gene)) in the shell protein (test group 4) group (n=3) was statistically significantly higher than that in the control group (only including Ebola viral minigenome system) ( p < 0.05). However, the nucleocapsid proteins of human coronaviruses HCoV OC43, HCoV 229E, HCoV NL63, and influenza A virus were unable to increase the number of cells expressing the reporter gene (GFP gene) in the Ebola virus minigenome system.

Table 4 Proportions of fluorescent cells in each group in Example 5 Group Average proportion of fluorescent cells (n=3) standard deviation P value control group Contains only EBOV minigenome (ST3) 22.2% 0.1 - Experimental group 1 ST3+ SARSCoV1_NP 46.0% 6.4 <0.05 Experimental group 2 ST3+ SARSCoV2_NP 45.4% 3.6 <0.05 Experimental group 3 ST3+ MERSCoV_NP 35.9% 5.9 <0.05 Experimental group 4 ST3+ HCoVHKU1_NP 43.6% 5.2 <0.05 Experimental group 5 ST3+ HCoVOC43_NP 19.1% 1.3 <0.05 Experimental group 6 ST3+ HCoV229E_NP 21.9% 1.0 >0.05 Experimental group 7 ST3+ HCoVNL63_NP 20.4% 0.7 <0.05 Experimental group 8 ST3+ InfluenzaA_NP 14.8% 0.3 <0.05

The results of this example show that adding the nucleocapsid protein genes of SARS-CoV-1, SARS-CoV-2, MERS-CoV, and HCoV HKU1 to the Ebola virus minigenome system will increase the number of cells expressing the reporter gene and also That is, the nucleocapsid proteins of these coronaviruses can improve the performance of reporters in the Ebola virus minigenome system.

Example 6 Effects of active regions of nucleocapsid proteins of different coronaviruses on gene expression

Materials and methods

Preparation of plasmids containing genes encoding the active region of coronavirus nucleocapsid protein . According to the results of Example 4, PCR amplification was performed on the gene sequence encoding the active region (including the dimerization domain and CTD) of each coronavirus nucleocapsid protein that increases gene expression, and was added to the 5' end of each gene sequence. A DNA sequence encoding methionine (ATG). The amplified sequence includes: the gene sequence encoding amino acids 241-422 of SARS-CoV-1 nucleocapsid protein (SEQ ID NO: 28); the gene sequence encoding amino acids 241-419 of SARS-CoV-2 nucleocapsid protein Gene sequence encoding amino acids (SEQ ID NO: 15); Gene sequence encoding MERS-CoV nucleocapsid protein amino acids 232-413 (SEQ ID NO: 29); encoding HCoV HKU1 nucleocapsid protein 245-441 The gene sequence of amino acids (SEQ ID NO: 30); and the gene sequence encoding amino acids 246-448 of HCoV OC43 nucleocapsid protein (SEQ ID NO: 31). Then, these gene sequences were selected and cloned into the pCAGGS vector respectively to generate pCAGGS_SARSCoV1_NP(241-422aa) plasmid (can express SEQ ID NO: 28 sequence); pCAGGS_SARSCoV2_NP(241-419aa) plasmid (can express SEQ ID NO: 15 sequence) (same as the plasmid described in Example 4); pCAGGS_MERSCoV_NP (232-413aa) plasmid (can express SEQ ID NO: 29 sequence); pCAGGS_HCoVHKU1_NP (245-441aa) plasmid (can express SEQ ID NO: 30 sequence) ; pCAGGS_HCoVOC43_NP (246-448aa) plasmid (can express SEQ ID NO: 31 sequence). And these plasmids were transformed into E. coli respectively.

Cell transfection . The following plasmids (1000 ng) were mixed with pEGFP-N2 plasmids (500 ng) carrying the EGFP reporter gene to prepare the plasmid mixture of each group: Control group: only containing pEGFP-N2 plasmids; Experimental group A: pEGFP-N2 plasmid + pCAGGS_SARSCoV1_NP(241-422aa) plasmid; Experimental group B: pEGFP-N2 plasmid + pCAGGS_SARSCoV2_NP(241-419aa) plasmid; Experimental group C: pEGFP-N2 plasmid + pCAGGS_MERSCoV_NP(232- 413aa) plasmid; Experimental group D: pEGFP-N2 plasmid + pCAGGS_MERSCoV_NP (245-441aa) plasmid; and Experimental group E: pEGFP-N2 plasmid + pCAGGS_HCoVOC43_NP (246-448aa) plasmid.

Add 120 µL of serum-reduced medium and 8 µL of T-Pro NTR II reagent kit to each group, and let it stand for 15 minutes to form a transfection reagent mixture. Then, the transfection reagent mixture of each group was added to 293T cells for transfection. The transfected cells were cultured in a 37 ^° C/5% _CO2 cell incubator for 24 hours, and then analyzed by flow cytometry.

Flow cytometry analysis . Same as described in Example 1.

Statistical analysis . Same as described in Example 1.

result

The active regions of several coronavirus nucleocapsid proteins increase reporter expression in expression plasmids . As shown in Figure 10 and Table 5, compared with the control group (cells transfected with the EGFP gene only, the average proportion of cells expressing green fluorescence was 24.7%), the average proportion of cells expressing green fluorescence in the AE group of the test group was 24.7%. significantly improved, and there was a statistically significant difference ( p < 0.05). Among them, experimental group B (cells transfected with EGFP gene + 1000 ng SARS-CoV-2 nucleocapsid protein 241-419 amino acids (SEQ ID NO: 15) gene plasmid) and experimental group A (transfected with EGFP gene + 1000 ng SARS-CoV-1 nucleocapsid protein 241-422 amino acids (SEQ ID NO: 28) gene plasmid cells) have the most significant effect on improving green fluorescence performance. In addition, it is worth noting that according to the results of Example 5, the effect of HCoV OC43 nucleocapsid protein (SEQ ID NO: 24) on improving gene expression is not significant; however, according to the results of this example, HCoV OC43 nucleocapsid protein Amino acids 246-448 (SEQ ID NO: 31) can significantly improve the expression of the target gene (as shown in the results of test group E in Table 5).

Table 5 Proportions of fluorescent cells in each group in Example 6 Group Average proportion of fluorescent cells (n=3) standard deviation P value control group pEGFP-N2 plasmid only 24.7% 0.3 - Experimental group A pEGFP-N2 + SARSCoV1_NP(241-422aa) 72.6% 5.9 <0.05 Experimental group B pEGFP-N2 + SARSCoV2_NP(241-419aa) 85.7% 1.1 <0.05 Experimental group C pEGFP-N2 + MERSCoV_NP(232-413aa) 53.1% 1.6 <0.05 Experimental group D pEGFP-N2 + HCoVHKU1_NP(245-441aa) 63.6% 1.3 <0.05 Experimental group E pEGFP-N2 + HCoVOC43_NP(246-448aa) 56.4% 9.4 <0.05

The results of this example show that the active regions (including dimerization domains and CTDs) of several coronavirus nucleocapsid proteins can significantly increase the expression of target genes in the expression plasmid, especially SARS-CoV-1 and SARS-CoV- 2 nucleocapsid protein active region has the best effect on improving the expression of target genes.

Example 7 SARS-CoV-1 and SARS-CoV-2 Effects of mutation sequences in the active region of nucleocapsid protein on gene expression

Materials and methods

Preparation of plasmids containing genes encoding mutant sequences in the active regions of SARS-CoV-1 and SARS-CoV-2 nucleocapsid proteins. Mutagenesis reagents (GeneMorph II Random Mutagenesis Kit, Agilent Technologies, Santa Clara, California, USA) were used to mutate amino acids 241-422 of the SARS-CoV-1 nucleocapsid protein (SEQ ID NO: 28). And the gene sequence of amino acids 241-419 of SARS-CoV-2 nucleocapsid protein (SEQ ID NO: 15) was randomly mutated, and a mutation gene database was established. These mutated sequences were then cloned into the pCAGGS vector to generate plasmids with various mutations. These plasmids (1000 ng) were co-transfected into cells with the pEGFP-N2 plasmid (500 ng) carrying the EGFP reporter gene, and analyzed by flow cytometry to screen for mutations that could increase the expression of fluorescent proteins. strain.

Cell transfection . Same as described in Example 3.

Flow cytometry analysis . Same as described in Example 1.

Statistical analysis . Same as described in Example 1.

result

Several SARS-CoV-1 and SARS-CoV-2 nucleocapsid protein active region mutation sequences increase the expression of reporters in expression plasmids . As shown in Table 6, compared with the control group (cells transfected with the EGFP gene only, the average proportion of cells expressing green fluorescence was 21%), in the experimental group AE, pCAGGS_Rho100 co-transfected with pEGFP-N2 plasmid (Expressing the mutation sequence of the active region of the SARS-CoV-2 nucleocapsid protein, SEQ ID NO: 32), pCAGGS_Rho103 (Expressing the mutation sequence of the active region of the SARS-CoV-2 nucleocapsid protein, SEQ ID NO: 33), pCAGGS_Rho110 (Expressing the mutation sequence of the active region of the SARS-CoV-2 nucleocapsid protein, SEQ ID NO: 33), pCAGGS_Rho110 (Expressing the mutation sequence of the active region of the SARS-CoV-2 nucleocapsid protein, SEQ ID NO: 32) 2 Nucleocapsid protein active region mutation sequence SEQ ID NO: 34), pCAGGS_RhoA (expressing SARS-CoV-2 nucleocapsid protein active region mutation sequence SEQ ID NO: 35), and pCAGGS_RhoB (expressing SARS-CoV-1 nucleocapsid protein activity The region mutation sequence SEQ ID NO: 36) plastid can increase the proportion of cells showing green fluorescence by approximately 2.7-3.8 times.

Table 6 Proportions of fluorescent cells in each group in Example 7 Group plastid Fluorescent cell ratio The fold increase of fluorescent cells* control group pEGFP-N2 plasmid only twenty one% 1 times Experimental group A pEGFP-N2 + pCAGGS_Rho100 76% 3.62 times Experimental group B pEGFP-N2 + pCAGGS_Rho103 77% 3.67 times Experimental group C pEGFP-N2 + pCAGGS_Rho110 79% 3.76 times Experimental group D pEGFP-N2 + pCAGGS_RhoA 56% 2.66 times Experimental group E pEGFP-N2 + pCAGGS_RhoB 58% 2.76 times * Compared with the control group, the fold increase of fluorescent cells

Of course, many changes and modifications can be made to the above-described embodiments of the invention without departing from the scope of the invention. Accordingly, in order to promote the advancement of science and useful fields, this invention is disclosed and is intended to be limited only by the scope of the appended claims.

EBOV-GFP vRNA: Ebola virus minigenome with green fluorescent protein (GFP) gene NP: Ebola virus nucleocapsid protein L: Ebola virus L protein 30: Ebola virus VP30 protein 35: Ebola virus VP35 protein SARS2-NP: SARS-CoV-2 nucleocapsid protein

The foregoing summary, as well as the following detailed description of the invention, will be better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the invention, the accompanying drawings illustrate some, but not all, alternative embodiments. It should be understood, however, that this invention is not limited to the precise arrangements and instrumentalities shown. The drawings, which are incorporated in and constitute a part of this specification, help explain the principles of the invention.

Figure 1 shows the commonly known mode of action of the Ebola virus minigenome system. EBOV-GFP vRNA: Ebola virus minigenome with green fluorescent protein (GFP) gene; NP: Ebola virus nucleocapsid protein; L: Ebola virus L protein; 30: Ebola virus VP30 protein; 35: Ebola virus Pull virus VP35 protein.

Figure 2 shows the mode of action of the Ebola virus minigenome system containing the coronavirus nucleocapsid protein fragment of the present invention. EBOV-GFP vRNA: Ebola virus minigenome with green fluorescent protein (GFP) gene; NP: Ebola virus nucleocapsid protein; L: Ebola virus L protein; 30: Ebola virus VP30 protein; 35: Ebola virus Virus VP35 protein; SARS2-NP: SARS-CoV-2 nucleocapsid protein.

Figure 3 shows cells in the control group (293T cells transfected with the Ebola virus minigenome system only) and experimental group 1 cells (293T cells transfected with the Ebola virus minigenome system + 1000 ng SARS-CoV-2 nucleocapsid proteosome cells), 24 and 48 hours after transfection, the results of reporter gene (GFP gene) expression were analyzed using fluorescence microscopy.

Figure 4 shows cells in the control group (293T cells transfected with the Ebola virus minigenome system only) and experimental group 1 cells (293T cells transfected with the Ebola virus minigenome system + 1000 ng SARS-CoV-2 nucleocapsid proteosome cells), 24 and 48 hours after transfection, the results of reporter gene (GFP gene) expression were analyzed by flow cytometry. au (arbitrary unit): arbitrary unit.

Figure 5 shows cells in the control group (293T cells transfected with the Ebola virus minigenome system only) and test group 1 cells (293T cells transfected with the Ebola virus minigenome system + 1000 ng SARS-CoV-2 nucleocapsid proteosome). cells), and experimental group 2 cells (293T cells transfected with Ebola virus minigenome system + 500 ng SARS-CoV-2 nucleocapsid protein), the percentage of cells expressing the GFP gene at 24 and 48 hours after transfection The statistical results (n=6). The Mann-Whitney U test was used to compare differences between each experimental group and the control group. Compared with the control group, * indicates p < 0.05, and ** indicates p < 0.01.

Figure 6A shows cells in the control group (293T cells transfected with the Ebola virus minigenome system only) and experimental group 1 cells (293T cells transfected with the Ebola virus minigenome system + 1000 ng SARS-CoV-2 nucleocapsid proteosomes cells), statistical results of total RNA expression of cells at 0, 12, 24, 36, and 48 hours after transfection (n=4). The Mann-Whitney U test was used to compare differences between each experimental group and the control group. Compared with the control group, * indicates p < 0.05.

Figure 6B shows the cells of test group 1 (293T cells transfected with Ebola virus minigenome system + 1000 ng SARS-CoV-2 nucleocapsid proteosome) at 0, 12, 24, 36, and 48 hours after transfection. Statistics on the proportion of expression amounts of viral RNA (vRNA), mRNA, and complementary RNA (cRNA) relative to the expression amounts of vRNA, mRNA, and cRNA in control cells (293T cells transfected only with the Ebola virus minigenome system) Results (n=4). The Mann-Whitney U test was used to compare differences between each experimental group and the control group. Compared with the 0 hour data, * indicates p < 0.05.

Figure 7 shows the use of the Ebola virus minigenome system (control group) or the Ebola virus minigenome system + 1000 ng SARS-CoV-2 nucleocapsid proteosome (test group) for the treatment of Remdesivir antiviral drugs Statistical results of screening (n=3). The Mann-Whitney U test was used to compare differences between each experimental group and the control group. Compared with the control group, * indicates p < 0.05.

Figure 8A shows cells in the negative control group (293T cells not transfected with any plastids), G1 cells in the experimental group (293T cells transfected with only EGFP gene), and G2 cells in the experimental group (transfected with EGFP gene + 1000 ng SARS-CoV-2 nucleocapsid protein gene plasmid 239T cells), 24 hours after transfection, the results of the expression of the reporter gene (EGFP gene) were analyzed by flow cytometry. au (arbitrary unit): arbitrary unit.

Figure 8B shows cells in the negative control group (293T cells not transfected with any plastid), R1 cells in the test group (293T cells transfected with only the RFP gene), and R2 cells in the test group (293T cells transfected with the RFP gene). + 1000 ng SARS-CoV-2 nucleocapsid protein gene plasmid in 239T cells), 24 hours after transfection, the results of reporter gene (RFP gene) expression were analyzed by flow cytometry. au (arbitrary unit): arbitrary unit.

Figure 9 shows a schematic diagram of the domains of the SARS-CoV-2 nucleocapsid protein and the domains contained in each recombinant protein fragment (left picture), and control group 1 cells (only 293T transfected with the Ebola virus minigenome system cells), control group 2 cells (293T cells transfected with Ebola virus minigenome system + SARS-CoV-2 nucleocapsid proteosome), and experimental group AI cells (transfected with Ebola virus minigenome system + each SARS-CoV -293T cells containing the recombinant protein fragment of nucleocapsid protein, 48 hours after transfection, statistical results of the percentage of cells expressing the GFP gene (n=3). NTD: amine terminal domain; CTD: carboxyl terminal domain. The Mann-Whitney U test was used to compare differences between each experimental group and the control group. Compared to control group 1, * indicates p < 0.05 (right panel).

Figure 10 shows a schematic diagram of the domains of coronavirus nucleocapsid proteins and the domains contained in recombinant protein fragments of different coronavirus nucleocapsid proteins (left picture), and control cells (293T cells transfected with only the EGFP gene ) and the experimental group AE cells (293T cells transfected with EGFP gene + plasmids of recombinant protein fragments of each coronavirus nucleocapsid protein), 24 hours after transfection, the statistical results of the percentage of cells expressing the EGFP gene (n= 3). The Mann-Whitney U test was used to compare differences between each experimental group and the control group. Compared with the control group, * indicates p < 0.05 (right panel).

EBOV-GFP vRNA: Ebola virus minigenome with green fluorescent protein (GFP) gene NP: Ebola virus nucleocapsid protein L: Ebola virus L protein 30: Ebola virus VP30 protein 35: Ebola virus VP35 protein SARS2-NP: SARS-CoV-2 nucleocapsid protein

Claims (15)

A protein fragment is used to increase the expression of a target gene, wherein the protein fragment includes a protein selected from severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1), novel coronavirus (SARS-CoV- 2) Dimerization of nucleocapsid proteins of coronaviruses from the group consisting of Middle East respiratory syndrome-related coronavirus (MERS-CoV) and human coronavirus HKU1 (HCoV HKU1) dimerization domain (dimerization domain) and/or the carboxyl-terminal domain (CTD) of the nucleocapsid protein of the coronavirus, or dimerization of the nucleocapsid protein of a human coronavirus OC43 (HCoV OC43) domain and carboxyl-terminal domain. The use as described in claim 1, wherein the nucleocapsid protein of a coronavirus selected from the group consisting of severe acute respiratory syndrome coronavirus, novel coronavirus, Middle East respiratory syndrome coronavirus, and human coronavirus HKU1 The dimerization domain and/or the protein fragment of the carboxyl-terminal domain of the nucleocapsid protein of the coronavirus further comprises at least one domain selected from the group consisting of: the amino terminus of the nucleocapsid protein of the coronavirus The N-terminal domain (NTD), the RNA binding domain (RBD) of the nucleocapsid protein of the coronavirus, and the linker of the nucleocapsid protein of the coronavirus. The use as described in claim 1, wherein the protein fragment contains at least 90%, 91%, 92%, 93%, 94%, 95%, 96% of an amino acid sequence selected from the group consisting of: , 97%, 98%, 99%, or 100% similar amino acid sequence: SEQ ID NO: 1, 12, 13, 15, 17, 18, 19, 20, 21, 22, 23, 28, 29 , 30, 31, 32, 33, 34, 35, and 36. A protein fragment for increasing the expression of a target gene, comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, Amino acid sequences with 97%, 98%, 99%, or 100% similarity: SEQ ID NO: 19 and SEQ ID NO: 20. A polynucleotide encoding a protein fragment as described in claim 4. A plasmid comprising a nucleotide sequence encoding a protein fragment as described in claim 4, and a promoter sequence at the 5' end of the nucleotide sequence. A cell comprising a protein fragment as described in claim 4, a polynucleotide as described in claim 5, or a plastid as described in claim 6. A method for increasing the expression of a target gene, comprising the following steps: (1) providing a plasmid, wherein the plasmid contains a nucleotide sequence encoding a protein fragment and a promoter at the 5' end of the nucleotide sequence Sequence, wherein the protein fragment includes a dimerization domain of a nucleocapsid protein of a coronavirus selected from the group consisting of severe acute respiratory syndrome coronavirus, novel coronavirus, Middle East respiratory syndrome coronavirus, and human coronavirus HKU1 and/or the carboxyl-terminal domain (CTD) of the nucleocapsid protein of the coronavirus, or consisting of the dimerization domain and the carboxyl-terminal domain of the nucleocapsid protein of human coronavirus OC43, (2) providing a For the expression vector of the target gene, (3) co-transfect the plasmid of step (1) and the expression vector of step (2) into an expression host cell, and (4) provide an appropriate condition to express the target gene. A method for increasing the expression of a target gene, comprising the following steps: (1) providing a cell, wherein the cell contains a protein fragment, a polynucleotide encoding the protein fragment, or a plasmid, wherein the plasmid contains the encoding A nucleotide sequence of the protein fragment and a promoter sequence at the 5' end of the nucleotide sequence, wherein the protein fragment includes a protein selected from the group consisting of severe acute respiratory syndrome coronavirus The dimerization domain of the nucleocapsid protein of the coronavirus, the novel coronavirus, the Middle East respiratory syndrome coronavirus, and the human coronavirus HKU1 and/or the carboxyl-terminal domain of the nucleocapsid protein of the coronavirus (CTD), or consisting of the dimerization domain and carboxyl-terminal domain of the nucleocapsid protein of human coronavirus OC43, (2) providing an expression vector containing the target gene, (3) combining step (2) The expression vector is transfected into the cells in step (1), and (4) an appropriate condition is provided to express the target gene. A method for screening drugs that inhibit viruses, comprising the following steps: (1) providing a plasmid, wherein the plasmid contains a nucleotide sequence encoding a protein fragment and a promoter at the 5' end of the nucleotide sequence Sequence, wherein the protein fragment includes a dimerization domain of a nucleocapsid protein of a coronavirus selected from the group consisting of severe acute respiratory syndrome coronavirus, novel coronavirus, Middle East respiratory syndrome coronavirus, and human coronavirus HKU1 and/or the carboxyl-terminal domain (CTD) of the nucleocapsid protein of the coronavirus, or consisting of the dimerization domain and the carboxyl-terminal domain of the nucleocapsid protein of human coronavirus OC43, (2) providing a A reporter gene viral minigenome system (minigenome system), (3) co-transfecting the plasmid of step (1) and the viral minigenome system of step (2) into a expressing host cell, (4) Add a candidate drug to the cells, and (5) observe the expression of the reporter gene. If the reporter gene does not express, the candidate drug has the effect of inhibiting the virus. A method for screening drugs that inhibit viruses, comprising the following steps: (1) providing a cell, wherein the cell contains a protein fragment, a polynucleotide encoding the protein fragment, or a plasmid, wherein the plasmid contains the encoding A nucleotide sequence of the protein fragment and a promoter sequence at the 5' end of the nucleotide sequence, wherein the protein fragment includes a virus selected from the group consisting of severe acute respiratory syndrome coronavirus, novel coronavirus, and Middle East respiratory syndrome coronavirus, and the dimerization domain of the nucleocapsid protein of the coronavirus of the group consisting of human coronavirus HKU1 and/or the nucleocapsid protein of the coronavirus The carboxyl-terminal domain (CTD) of white may be composed of the dimerization domain and the carboxyl-terminal domain of the nucleocapsid protein of human coronavirus OC43, (2) provide a viral minigenome system containing a reporter gene, (3) Transfect the viral minigenome system of step (2) into the cells of step (1), (4) add a candidate drug to the cells, (5) observe the expression of the reporter gene, if the reporter gene does not performance, the candidate drug has the effect of inhibiting the virus. The method according to claim 10 or 11, wherein the reporter gene is a fluorescent protein gene. The method of claim 10 or 11, wherein the viral minigenome system is an Ebola virus minigenome system (EBOV minigenome system), and the Ebola virus minigenome system consists of the following: an EBOV minigenome system containing a reporter gene An expression vector for Ebola virus minigenome, an expression vector containing a gene encoding Ebola virus nucleocapsid protein (NP), an expression vector containing a gene encoding Ebola virus polymerase protein (L), and an expression vector containing An expression vector encoding Ebola virus VP30 protein gene, and an expression vector containing a gene encoding Ebola virus VP35 protein. The method of any one of claims 8 to 11, wherein the coronavirus is selected from the group consisting of severe acute respiratory syndrome coronavirus, novel coronavirus, Middle East respiratory syndrome coronavirus, and human coronavirus HKU1 The dimerization domain of the nucleocapsid protein and/or the protein fragment of the carboxyl-terminal domain of the nucleocapsid protein of the coronavirus further comprises at least one domain selected from the group consisting of: the nucleocapsid of the coronavirus The amino terminal domain (NTD) of the protein, the RNA binding domain (RBD) of the nucleocapsid protein of the coronavirus, and the linker of the nucleocapsid protein of the coronavirus. The method according to any one of claims 8 to 11, wherein the protein fragment comprises at least 90%, 91%, 92%, 93%, 94% of an amino acid sequence selected from the group consisting of: Amino acid sequence with 95%, 96%, 97%, 98%, 99%, or 100% similarity: SEQ ID NO: 1, 12, 13, 15, 17, 18, 19, 20, 21, 22, 23, 28, 29, 30, 31, 32, 33, 34, 35, and 36.