CN115216452A - SARS-CoV-2 virus replicon and its construction method and use - Google Patents
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Abstract
The invention belongs to the technical field of biological medicine, and relates to a SARS-CoV-2 virus replicon, a construction method and application thereof. The SARS-CoV-2 virus replicon of the invention removes a new coronavirus structural protein gene S, M, E and auxiliary protein genes ORF3a, ORF6 and ORF7a, or removes the nucleotide sequences from 21563 to 28259 of the genome sequence of SARS-CoV-2 strain SH-01 based on the genome sequence of SARS-CoV-2 strain SH-01, and inserts a target gene into the TRS downstream of the S gene. After the replicon is transcribed in vitro, RNA is transfected into mammalian cells, and the replicon can be used for researching the functional relationship between SARS-CoV-2 gene and protein, screening and verifying antiviral drugs and researching the interaction between viruses and host factors.
Description
Technical Field
The invention belongs to the technical field of biological medicine, and particularly relates to a SARS-CoV-2 virus replicon and a construction method and application thereof.
Background
A viral replicon refers to a viral subgenome with the removal of viral structural protein genes and the retention of replication-associated non-structural protein genes, which has the ability to autonomously replicate and translationally express the non-structural proteins and the inserted proteins of interest. Therefore, the virus replicon is not only a technical platform for researching virus gene structure and function, but also a novel means for expressing foreign protein and developing novel replicon vaccines.
SARS-CoV-2 belongs to coronavirus, the coronavirus virion is irregular shape, about 60-220nm in diameter, and is wrapped by fat membrane. There are three glycoproteins on the membrane surface: spike glycoprotein (S, spike Protein, which is the receptor binding site, cytolytic and major antigenic site); small Envelope glycoprotein (E, envelope Protein, smaller, envelope-bound Protein); membrane glycoproteins (M, membrane proteins, responsible for transmembrane transport of nutrients, budding release of nascent viruses and formation of viral envelope). A few classes also have hemagglutinin-esterases (HE proteins). The nucleic acid of coronavirus is non-segmented single-stranded (+) RNA, has the length of 27-31kb, is the longest RNA nucleic acid chain in RNA virus, and has important structural characteristics specific to positive-strand RNA: namely, the 5 'end of the RNA chain is provided with a methylated cap, and the 3' end is provided with a polyA tail structure. This structure is very similar to eukaryotic mRNA and is an important structural basis for the genomic RNA itself to function as a translation template, and the RNA-DNA-RNA transcription process is omitted. The rate of recombination between the RNA of coronaviruses and RNA is very high, and it is this high rate of recombination that causes the virus to mutate. After recombination, the RNA sequence is changed, and the amino acid sequence encoded by the nucleic acid is also changed, and the protein composed of amino acids is changed, so that the antigenicity is changed. The result of the change of antigenicity is that the original vaccine is ineffective and the immunity fails.
RNA polymerase (Viral RNA polymerase) required for RNA virus replication is not present in the mature coronavirus particles, and after entering a host cell, viral RNA polymerase is expressed directly by using Viral genomic RNA as a translation template. This enzyme is then used to complete the transcription and synthesis of sub-genomic RNA (sub-genomic RNA), the synthesis of mRNAs of various structural proteins, and the replication of viral genomic RNA. The mature mRNA of each structural protein of coronavirus is synthesized, there is no post-transcriptional modification and cleavage process, but directly through RNA polymerase and some transcription factors, in a 'discontinuous transcription' mechanism, by recognizing specific TRS), the whole components of a mature mRNA are obtained by one-time transcription selectively from the negative strand RNA. After the replication of structural proteins and genomic RNA is completed, new coronavirus particles will be assembled (assembly) at the endoplasmic reticulum of the host cell and secreted out of the cell by the golgi apparatus, completing its life cycle.
It is recorded that among the known 7 human coronaviruses, HCoV-229E, HCoV-NL63, HCoV-HKU1 and HCoV-OC43 are common cold and respiratory tract infection in human and are less pathogenic and infectious. The new SARS-CoV, MERS-CoV and SARS-CoV-2 belong to beta type coronavirus, which can cause fatal respiratory system diseases, especially SARS-CoV-2 is more contagious. The results of the study showed that the novel coronaviruses SARS-CoV-2 and HCoV-NL63, SARS-CoV both enter cells using the same cellular receptor ACE2 (angiotensin converting enzyme 2), which suggests that they have similar infection target cells and tissues. However, the pathogenicity of the three is quite obvious. The common human coronaviruses (including 229E, NL, OC43 and HKU1 types), often cause mild or moderate upper respiratory tract diseases, such as the common cold. The symptoms mainly comprise rhinorrhea, headache, cough, sore throat, fever and the like, sometimes cause pneumonia, bronchitis and other lower respiratory diseases, and are common in patients with cardiopulmonary diseases, people with low immunity, infants and the elderly. MERS-CoV and SARS-CoV often cause more severe symptoms. MERS symptoms typically include fever, cough and shortness of breath, even with the development of pneumonia, with a mortality rate of about 34.4%. SARS symptoms usually include fever, chills and body aches, even with the development of pneumonia, with a mortality rate of about 9.6%, while SARS-CoV-2 is slow in onset, low in fever or even asymptomatic, with recurrent episodes, long latency periods, or can suddenly cause severe acute respiratory disease.
At present, the virology characteristics of SARS-CoV-2 are still little recognized, and lack of specific medicine, and in addition, due to the limitation of BSL-3, a tool which can develop the functions of SARS-CoV-2 gene and protein, the interaction of virus and host factor, and the screening and verification of antiviral medicine in common laboratory is urgently needed, and the replicon has the above functions, so the construction and application of SARS-CoV-2 replicon are particularly urgent and important.
Disclosure of Invention
Aiming at the problems in the prior art, the invention aims to provide a SARS-CoV-2 virus replicon and a construction method thereof, wherein the construction method is simple, a large amount of single homogeneous replicon RNA can be prepared by in vitro transcription, and the replicon RNA can be quantitatively transfected to be used for screening antiviral drugs, verifying host genes of antagonistic viruses and the like.
The invention also aims to provide the application of the SARS-CoV-2 virus replicon, which comprises the application as a basic material for screening antiviral drugs and verifying host genes of antagonistic viruses.
In one aspect, the invention provides a SARS-CoV-2 virus replicon, which takes the genome sequence of SARS-CoV-2 strain SH-01 as the basis to remove the new coronavirus structural protein gene S, M, E and auxiliary protein genes ORF3a, ORF6 and ORF7a.
Preferably, the nucleotide sequence of 21563 to 28259 of the genome sequence of SARS-CoV-2 strain SH-01 is deleted on the basis of the genome sequence of SARS-CoV-2 strain SH-01.
Preferably, the target gene is inserted into the TRS downstream of the S gene of the structural protein gene of the novel coronavirus.
In another aspect, the invention provides a method for constructing a SARS-CoV-2 virus replicon, comprising the following steps:
synthesizing SARS-CoV-2SH01 nucleic acid fragment and splicing to form complete ARS-CoV-2SH01 nucleic acid; and
removing the structural protein S, E, M of the virus and removing the auxiliary proteins ORF3a, ORF6, ORF7a and ORF8.
Preferably, the above replicon expression vector containing the SARS-CoV-2 virus replicon is obtained.
Preferably, the method comprises the step of inserting Nanoluc-P2A-Puromycin or a gene of interest downstream of the S protein TRS.
Preferably, the SARS-CoV-2SH01 nucleic acid fragment is synthesized and spliced based on the genome sequence of SARS-CoV-2 strain SH-01, and the nucleic acid fragment with the structural protein S, E, M of the virus removed and the auxiliary proteins ORF3a, ORF6, ORF7a and ORF8 removed is synthesized and then spliced.
Preferably, the nucleic acid fragment from which the structural protein S, E, M of the virus is removed and the helper proteins ORF3a, ORF6, ORF7a, ORF8 are removed includes six fragments of ABCDEF as shown in fig. 1:
fragment A: start to B fragment in ORF1 a;
fragment B: the ending end of the A fragment in ORF1a is connected to the beginning end of the C fragment;
c fragment: in ORF1a, the end of the B fragment is from the beginning of the D fragment;
d, fragment: the end of the C fragment in ORF1b is close to the beginning of the E fragment;
e fragment: ORF1b, from the middle rear end to the end, but with the original S protein gene sequence deleted;
fragment F is a fragment close to 3' UTR, except for the sequence of S, ORF3a, E, M, ORF6, ORF7a, ORF8 in the SARS-CoV-2SH01 nucleic acid fragment.
Preferably, the construction method comprises the following steps:
1) A SARS-CoV-2SH01 nucleic acid fragment is synthesized and divided into six fragments ABCDEF, the restriction enzyme cutting sites and cohesive ends at both ends of the 6 fragments are designed according to the whole virus genome and are respectively connected to pSMART-LCAmp plasmids which are named as pSMART-A, pSMART-B, pSMART-C, pSMART-D, pSMART-E and pSMART-F.
2) Primers were designed to PCR amplify pSMART-A, and T7 promoter was introduced into the front end of the A fragment, pSMART-T7p A, which was named. pSMART-E was PCR amplified to delete the nucleic acid sequence (S protein gene), which was named pSMART-E Sdel. Primers were designed to PCR amplify pSMART-F to delete S, ORF3a, E, M, ORF6, ORF7a, ORF8 (21563 nt to 28259 nt), while primers were designed to PCR amplify Nanoluc-P2A-Puromycin by SOE, which was inserted into pSMART-F and named pSMART-F-Nanoluc.
3) Primers containing HDVr and T7T are designed to amplify pSMART-F-Nanoluc to obtain pSMART-F-Nanoluc-HDVr T7T.
4) The pSMART-T7p A, pSMART-B, pSMART-C, pSMART-D, pSMART-ESdel were digested with BsaI to give T7p A, B, C, D, E Sdel fragments. The pSMART-F-Nanoluc-HDVr T7T was digested with Bsembi to give F-Nanoluc-HDVr T7T.
5) The pBeloBAC11 skeleton plasmid is modified by designing a primer, a single BsaI enzyme cutting site in the pBeloBAC11 is subjected to point mutation, a new enzyme cutting site (BsaI-BsaI) is introduced into a multiple cloning site of the pBeloBAC11, and the pBeloBAC11-BsaI is named. pBeloBAC11-BsaI was digested with BsaI to give Linear-BeloBAC11-BsaI.
6) T7p A, B, C, D, E Sdel, F-Nanoluc-HDVr T7T and Linear-BeloBAC11-BsaI are connected by T4 ligase to obtain pBAC-delS to orf8-Nanoluc, and then the SARS-CoV-2 virus replicon is obtained.
The SARS-CoV-2 virus replicon constructing process includes eliminating virus structural proteins S, E and M and auxiliary proteins ORF3a, ORF6, ORF7a and ORF8 based on SARS-CoV-2SH01 virus genome sequence, and maintaining the replicon expressing vector pBAC-delS to ORF8-Nanoluc of other virus sequences.
After the Nanoluc-P2A-Puromycin or a target gene is inserted into the downstream of S protein TRS, RNA is obtained through in vitro transcription and then mammalian cells are transfected or the recombinant replicon is directly transfected into the mammalian cells expressing T7 polymerase.
The SARS-CoV-2 virus replicon has the application in the functional relationship between SARS-CoV-2 gene and protein, antiviral drug screening and verification and the interaction between virus and host factor.
Preferably, after the Nanoluc-P2A-Puromycin or the target gene is inserted into the downstream of the S protein TRS, RNA is obtained through in vitro transcription and then mammal cells are transfected, or the recombinant replicon is directly transfected into the mammal cells expressing T7 polymerase.
In still another aspect, the present invention provides the use of the SARS-CoV-2 virus replicon, for example, the use of the SARS-CoV-2 virus replicon in the preparation of reagents for studying the functional relationship between SARS-CoV-2 gene and protein, screening and verifying antiviral drugs, or the interaction between virus and host factor.
Preferably, the application comprises the application of the SARS-CoV-2 virus replicon in the preparation of medicaments for detecting or treating new coronavirus.
The invention provides a SARS-CoV-2 virus replicon and a construction method and application thereof. The construction method in the technical scheme of the invention is simple, effective, convenient to operate, safe and accurate, the obtained virus replicon maintains the basic structure but has obviously reduced toxicity and infectivity, a large amount of single and homogeneous replicon RNA can be prepared by in vitro transcription, and the replicon RNA can be quantitatively transfected to screen antiviral drugs, verify host genes of antagonistic viruses and the like.
Drawings
FIG. 1 is a schematic diagram of replicon and associated vector construction, wherein,
a: ORF1a begins at the start of the fragment B, begins with AGCA, and ends with CAAC;
b: the end of the A fragment in ORF1a is connected with the beginning of the C fragment, starting with GTTG and ending with TTCG;
c: in ORF1a, the end of the B fragment is from the beginning of the D fragment, beginning with AAGC and ending with AAAG;
d: the end of the C fragment in ORF1b is close to the beginning of the E fragment, and the end is started by TTTC and ended by ACTA;
e: ORF1b begins with TGAT and ends with ATCT from the middle rear end to the end;
f is near 3' UTR, ending with TTCC.
FIG. 2 shows the chemiluminescence values of the different time-point detection replicon BAC-delS to orf8-Nanoluc reporter (Remdesivir is a control group).
Detailed Description
The advantageous effects of the present invention will now be further described with reference to examples of the present invention.
Experimental materials: a fragment of synthetic SARS-CoV-2; plasmids pSMART-LCAmp, pNL1.1, plentiCRISPRV2-puro, pBeloBAC11, BHK21-ACE2,
Luciferase Assay System、Lipofectamine TM MessengerMAX、mMESSAGE mMACHINE TM The T7 Transcription Kit was provided and prepared by the department of molecular virology education of Shanghai medical college of the university of Compound Dan/Wei Jian, the focal laboratory.
Example 1
A SARS-CoV-2SH01 nucleic acid fragment is synthesized and divided into six fragments ABCDEF, the restriction enzyme cutting sites and cohesive ends at both ends of the 6 fragments are designed according to the whole virus genome and are respectively connected to pSMART-LCAmp plasmids which are named as pSMART-A, pSMART-B, pSMART-C, pSMART-D, pSMART-E and pSMART-F. Primers were designed to PCR amplify pSMART-A, and T7 promoter was introduced at the front of the A fragment, which was named pSMART-T7p A. pSMART-E was PCR amplified to delete the nucleic acid sequence (S protein gene), which was named pSMART-E Sdel. Primers were designed to PCR amplify pSMART-F to delete S, ORF3a, E, M, ORF6, ORF7a, ORF8 (21563 nt to 28259 nt), while primers were designed to PCR amplify Nanoluc-P2A-Puromycin by SOE, which was inserted into pSMART-F and named pSMART-F-Nanoluc. Primers containing HDVr and T7T are designed to amplify pSMART-F-Nanoluc to obtain pSMART-F-Nanoluc-HDVr T7T. The pSMART-T7p A, pSMART-B, pSMART-C, pSMART-D, pSMART-E Sdel was digested with BsaI to yield T7p A, B, C, D, E Sdel fragments. The pSMART-F-Nanoluc-HDVr T7T was digested with Bsembi to give F-Nanoluc-HDVr T7T. The pBeloBAC11 skeleton plasmid is modified by designing a primer, a single BsaI enzyme cutting site in the pBeloBAC11 is subjected to point mutation, a new enzyme cutting site (BsaI-BsaI) is introduced into a multiple cloning site of the pBeloBAC11, and the pBeloBAC11-BsaI is named. pBeloBAC11-BsaI was digested with BsaI to give Linear-BeloBAC11-BsaI. T7p A, B, C, D, E Sdel, F-Nanoluc-HDVr T7T and Linear-BeloBAC11-BsaI are connected by T4 ligase to obtain pBAC-delS to orf8-Nanoluc, and then the SARS-CoV-2 virus replicon is obtained.
Example 2
A SARS-CoV-2SH01 nucleic acid fragment is synthesized and divided into six fragments ABCDEF, the restriction enzyme cutting sites and cohesive ends at both ends of the 6 fragments are designed according to the whole virus genome and are respectively connected to pSMART-LCAmp plasmids which are named as pSMART-A, pSMART-B, pSMART-C, pSMART-D, pSMART-E and pSMART-F. Primers were designed to PCR amplify pSMART-A, and T7 promoter was introduced into the front end of the A fragment, pSMART-T7p A, which was named. pSMART-E was PCR amplified to delete the nucleic acid sequence (S protein gene), which was named pSMART-E Sdel. Primers were designed to PCR amplify pSMART-F to delete S, ORF3a, E, M, ORF6, ORF7a, ORF8 (21563 nt to 28259 nt), while primers were designed to PCR amplify Nanoluc-P2A-Puromycin by SOE, which was inserted into pSMART-F and named pSMART-F-Nanoluc. Primers containing HDVr and T7T are designed to amplify pSMART-F-Nanoluc to obtain pSMART-F-Nanoluc-HDVr T7T. The pSMART-T7p A, pSMART-B, pSMART-C, pSMART-D, pSMART-E Sdel was digested with BsaI to yield T7p A, B, C, D, E Sdel fragments. The pSMART-F-Nanoluc-HDVr T7T was digested with Bsembi to give F-Nanoluc-HDVr T7T. The pBeloBAC11 skeleton plasmid is modified by designing a primer, a single BsaI enzyme cutting site in the pBeloBAC11 is subjected to point mutation, a new enzyme cutting site (BsaI-BsaI) is introduced into a multiple cloning site of the pBeloBAC11, and the pBeloBAC11-BsaI is named. pBeloBAC11-BsaI was digested with BsaI to obtain Linear-BeloBAC11-BsaI. T7p A, B, C, D, E Sdel, F-Nanoluc-HDVr T7T and Linear-BeloBAC11-BsaI are connected by T4 ligase to obtain pBAC-delS to orf8-Nanoluc, and then the SARS-CoV-2 virus replicon is obtained.
Example 3
BHK21-ACE2 cells were transfected. The plasmid pBAC-delS to orf8-Nanoluc is greatly extracted by using an external transcription kit mMESSAGE mMACHINE TM The T7 Transcription Kit is used for preparing RNA, and the specific method refers to mMESSAGE mMACHINE TM T7 Transcription Kit Specification. Using Lipofectamine TM Transfecting the BHK21-ACE2 cells by MessengerMAX, wherein the specific method refers to Lipofectamine TM MessengerMAX specification. The optimal ratio of the plasmid to the liposome dosage is determined by a square matrix experiment, and the cell density during transfection is determined. The plasmid was then transfected into 96-well monolayers of BHK21-ACE2 cells, using the SARS-CoV-2 replication inhibitor Remdesivir as a control.
Example 4
And (5) detecting a reporter gene. Chemiluminescence values of Nanoluc in cells were measured at various time points and may reflect replicon replication in BHK21-ACE 2. Adding Nano-Glo Luciferase reagent with the same volume as the supernatant at the corresponding time point, incubating for 15 minutes at room temperature, and transferring all liquid in the hole to a white opaque plate
Luciferase Assay System instructions. The reading was done using Flex Station 3.
Example 5
A method for constructing SARS-CoV-2 virus replicon. The T7 promoter is introduced into the front end of the replicon, single and stable replicon RNA can be prepared in a large amount by using an in vitro transcription kit, or the replicon RNA can be prepared by directly transfecting a cell expressing the T7 polymerase with a plasmid. The Nanoluc is inserted into the downstream of the S gene TRS, so that stronger transcription expression can be obtained, the Nanoluc is expressed intracellularly, and after the Nanoluc reacts with a corresponding substrate, a signal is stronger than that of the traditional Firefly and Renilla, and in addition, the Nanoluc is not secreted to the outside of the cell, so that the detection is more convenient and accurate. In addition, the presence of puromycin selection tag makes it possible to obtain cell lines with stably replicated replicons. Elements such as HDVr and T7T are introduced, so that T7 transcription can be terminated in time, and cutting is carried out, so that the 3' UTR end of the replicon conforms to the structure in the natural state, and finally self-replication of the replicon is realized (see figure 1).
Example 6
Transfection of BHK21-ACE2 cells and monitoring of chemiluminescence values. Using Lipofectamine TM After transfection of BHK21-ACE2 cells with MessengerMAX, chemiluminescence of Nanoluc was measured every 12 h. The results show that: the chemiluminescence value of Nanoluc reaches the maximum value at 24h, and a stronger signal can be detected at 48h (see figure 2).
The above description is only for the specific embodiments of the present application, but the scope of the present application is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present disclosure should be covered within the scope of the present application. Therefore, the protection scope of the present application shall be subject to the protection scope of the claims.
Claims (11)
1. A SARS-CoV-2 virus replicon is characterized in that based on the genome sequence of SARS-CoV-2 strain SH-01, the new coronavirus structural protein gene S, M, E and auxiliary protein genes ORF3a, ORF6 and ORF7a are removed.
2. The SARS-CoV-2 virus replicon of claim 1 wherein the nucleotide sequence of from 21563 to 28259 of the SARS-CoV-2 strain SH-01 genome sequence is deleted based on the SARS-CoV-2 strain SH-01 genome sequence.
3. The SARS-CoV-2 virus replicon according to claim 1 or 2, wherein the desired gene is inserted downstream of TRS of the novel coronavirus structural protein gene S gene.
4. A method for constructing SARS-CoV-2 virus replicon, comprising the following steps:
synthesizing SARS-CoV-2SH01 nucleic acid fragment and splicing to form complete ARS-CoV-2SH01 nucleic acid; and
removing the structural protein S, E, M of the virus, and removing the auxiliary proteins ORF3a, ORF6, ORF7a and ORF8;
obtaining a replicon expression vector containing a SARS-CoV-2 viral replicon.
5. The method of claim 4, comprising the step of inserting Nanoluc-P2A-Puromycin or a gene of interest downstream of the S protein TRS.
6. The method of claim 4, wherein the nucleic acid fragment of SARS-CoV-2SH01 is synthesized and spliced based on the genome sequence of SH-01 of SARS-CoV-2 strain, and the nucleic acid fragments from which the structural protein S, E, M of the virus and the auxiliary proteins ORF3a, ORF6, ORF7a and ORF8 are removed are synthesized and then spliced.
7. The method of claim 6, wherein the nucleic acid fragment from which the structural protein S, E, M of the virus and the helper proteins ORF3a, ORF6, ORF7a, ORF8 are removed comprises six fragments ABCDEF:
fragment A: start to B fragment in ORF1 a;
fragment B: the ending end of the A fragment in ORF1a is connected to the beginning end of the C fragment;
c fragment: in ORF1a, the end of the B fragment is from the beginning of the D fragment;
d, fragment: the ending end of the C fragment in ORF1b is close to the beginning end of the E fragment;
e fragment: ORF1b, from the middle rear end to the end, but with the original S protein gene sequence deleted;
fragment F close to 3' UTR but deleting positions 21563 to 28259 in the nucleic acid sequence, or
The sequence of S, ORF a, E, M, ORF6, ORF7a, ORF8 in the SARS-CoV-2SH01 nucleic acid fragment was deleted.
8. The method of construction according to claim 4, comprising the steps of:
1) Synthesizing a SARS-CoV-2SH01 nucleic acid fragment, dividing the nucleic acid fragment into six fragments ABCDEF, designing enzyme cutting sites and cohesive ends at two ends of the 6 fragments according to the whole virus genome, respectively connecting the fragments to pSMART-LCAmp plasmids, and naming the fragments as pSMART-A, pSMART-B, pSMART-C, pSMART-D, pSMART-E and pSMART-F;
2) Designing a primer for PCR amplification of pSMART-A, introducing a T7 promoter to the front end of the A fragment, and naming the fragment as pSMART-T7p A; PCR-amplifying pSMART-E to delete the nucleic acid sequence S protein gene sequence, which was named pSMART-E Sdel; designing primers, carrying out PCR amplification on pSMART-F to delete S, ORF3a, E, M, ORF6, ORF7a and ORF8, and simultaneously designing primers to obtain Nanoluc-P2A-Puromycin through SOE PCR amplification, inserting the Nanoluc-P2A-Puromycin into pSMART-F, and naming the Nanoluc-P2A-Puromycin;
3) Designing a primer containing HDVr and T7T to amplify pSMART-F-Nanoluc to obtain pSMART-F-Nanoluc-HDVr T7T;
4) Cutting pSMART-T7p A, pSMART-B, pSMART-C, pSMART-D and pSMART-E Sdel by BsaI to obtain T7p A, B, C, D and E Sdel fragments, cutting pSMART-F-Nanoluc-HDVr T7T by Bsambi to obtain F-Nanoluc-HDVr T7T;
5) Designing a primer to modify pBeloBAC11 skeleton plasmid, point-mutating a single BsaI restriction site in the pBeloBAC11, introducing a new restriction site BsaI-BsaI at a multiple cloning site, and naming the new restriction site BsaI-BsaI as pBeloBAC11-BsaI; cutting pBeloBAC11-BsaI by BsaI to obtain Linear-BeloBAC11-BsaI;
6) T7p A, B, C, D, E Sdel, F-Nanoluc-HDVr T7T and Linear-BeloBAC11-BsaI are connected by T4 ligase to obtain pBAC-delS to orf8-Nanoluc, and then the SARS-CoV-2 virus replicon is obtained.
9. The method for constructing a SARS-CoV-2 virus replicon according to claim 4, wherein after Nanoluc-P2A-Puromycin or a target gene is inserted downstream of S protein TRS, RNA is obtained by in vitro transcription and then mammalian cells are transfected or the recombinant replicon is directly transfected into mammalian cells expressing T7 polymerase.
10. Use of the SARS-CoV-2 viral replicon of claim 1 in the preparation of reagents for functional relationship between SARS-CoV-2 gene and protein, screening and verification of antiviral drugs, or interaction between virus and host factor.
11. The use of claim 10, wherein the SARS-CoV-2 viral replicon is used in the manufacture of a medicament for detecting or treating a novel coronavirus.
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