CN113308440B - N7-methyltransferase defective coronavirus attenuated vaccine strain, and preparation method and application thereof - Google Patents

N7-methyltransferase defective coronavirus attenuated vaccine strain, and preparation method and application thereof Download PDF

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CN113308440B
CN113308440B CN202110577242.7A CN202110577242A CN113308440B CN 113308440 B CN113308440 B CN 113308440B CN 202110577242 A CN202110577242 A CN 202110577242A CN 113308440 B CN113308440 B CN 113308440B
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coronavirus
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CN113308440A (en
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陈宇
张贞
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Wuhan University WHU
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Abstract

The invention provides an N7-methyltransferase-deficient coronavirus attenuated vaccine strain, a preparation method and application thereof, which are obtained by mutating extremely conserved active sites D and/or Y in the N7-methyltransferase structural domain in a non-structural protein nsp14 in a wild coronavirus strain into A at fixed points. The attenuated live vaccine forward designed based on the N7-methyltransferase defect strategy is safe and stable, and simultaneously can induce the organism to generate powerful humoral immunity and T cell immune response by single-dose inoculation, thereby providing protection effect for the organism, and has good safety and strong immunogenicity.

Description

N7-methyltransferase defective coronavirus attenuated vaccine strain, and preparation method and application thereof
Technical Field
The invention belongs to the technical field of biology, and relates to an N7-methyltransferase defective coronavirus attenuated vaccine strain, a preparation method and application thereof.
Background
Coronaviruses are a widely distributed and diverse class of enveloped positive-strand RNA viruses that infect a wide variety of hosts, including humans, and cause varying degrees of respiratory, gastrointestinal, hepatic and nervous system disease, with significant threat to human health and production activity. Since 1931, a variety of coronaviruses have been discovered in humans, including avian-host Infectious Bronchitis Virus (IBV), swine-host transmissible gastroenteritis virus (TGEV), swine hemagglutinating encephalomyelitis virus (PHEV), porcine Epidemic Diarrhea Virus (PEDV), porcine Respiratory Coronavirus (PRCV), porcine delta coronavirus (PDCoV), porcine acute diarrhea syndrome coronavirus (SADS-CoV), rodent-host Murine Hepatitis Virus (MHV), and bovine coronavirus (BCoV), feline infectious peritonitis coronavirus (FIPV), canine coronavirus (CCoV), and the like, which cause infections and diseases that are generally fatal. In addition to the above-mentioned animal coronaviruses, several common low pathogenic human coronaviruses, HCoV-229E, HCoV-OC43, HCoV-NL63 and HCoV-HKU1, are also widely prevalent in the human population, but they usually only cause upper respiratory symptoms similar to common cold. However, this century has exploded in succession 3 highly pathogenic human coronaviruses, namely severe acute respiratory syndrome coronavirus (SARS-CoV), middle east respiratory syndrome coronavirus (MERS-CoV) and novel coronavirus (2019-nCoV, subsequently designated SARS-CoV-2). SARS-CoV and SARS-CoV-2 have very close relation in evolution, have very high similarity in disease symptoms, mainly include symptoms such as dry cough, fever, systemic debilitation, etc., develop to respiratory failure and acute pneumonia of different degrees in the early stage, the crowd suffering from basic disease and low immunity have higher fatality rate. In contrast, SARS-CoV-2 has a longer incubation period (about 14 days) and is available to asymptomatic patients, which gives them a superior spreading capacity and causes a worldwide pandemic in a short period of 2 months. By 2021, month 5, SARS-CoV-2 has caused about 1.5 hundred million infections worldwide, over 320 deaths. MERS-CoV infection causes symptoms similar to SARS, but may cause multiple complications such as cardiopulmonary dysfunction, diabetes, renal failure, etc. in the later stage of infection, the mortality rate is up to 40%, and the disease still presents a local sporadic state at present.
The frequent outbreaks of coronaviruses have attracted attention from scientists and, in their effort, the evolutionary processes, replication mechanisms, pathogenesis, etc. of coronaviruses are increasingly elucidated. Unfortunately, there are no specific drugs and universal vaccines for coronaviruses. The traditional means for controlling coronavirus mainly comprise physical isolation, biological safety control and the like, and the method for preventing partial transfer physical coronavirus infection mainly comprises a method for inoculating attenuated live vaccine. However, since these animal-applicable attenuated live vaccines are mainly obtained by serial passage of wild-type strains, the mechanism of attenuation is not clear, there is a risk of recombination with wild-type strains and restoration of virulence, and their abuse may instead exacerbate the transmission of coronaviruses. Prior to the covd-19 outbreak, research in the field of coronavirus vaccines for humans remained only in the laboratory phase without any approved SARS-CoV and MERS-CoV vaccines. After the outbreak of the novel coronavirus, in order to control the global spread situation of the covd-19, various SARS-CoV-2 vaccines including inactivated vaccines, RNA vaccines, recombinant subunit vaccines and the like have been rapidly developed and enter a clinical trial stage, and part of the vaccines have been urgently authorized for use or conditional on marketing. However, due to the lack of experience in success of coronavirus vaccines, coupled with the rapid mutation and immune escape capabilities of coronaviruses, the safety and efficacy of these vaccines also require longer-term observation and confirmation. Thus, the development of highly effective and specific anti-coronavirus drugs and universal vaccines remains a significant and arduous task worldwide.
Disclosure of Invention
In order to solve the technical problems, the invention provides an N7-methyltransferase defective coronavirus attenuated vaccine strain, a preparation method and application thereof, and the attenuated live vaccine forward designed based on the N7-methyltransferase defective strategy is safe and stable, and simultaneously can induce a body to generate powerful humoral immunity and T cell immune response by single-dose inoculation, thereby providing protection for the body; the novel attenuated live vaccine with good safety, strong immunogenicity and low cost is an ideal coronavirus vaccine candidate strain, and is expected to be applied to the development of general coronavirus vaccines.
In a first aspect of the invention, there is provided an attenuated N7-methyltransferase deficient coronavirus vaccine strain obtained by site-directed mutagenesis of an extremely conserved active site D and/or Y within the N7-methyltransferase domain of the nonstructural protein nsp14 of a wild-type coronavirus strain to A.
Further, the wild coronavirus strains include MHV, MERS-CoV, and SARS-CoV-2;
for the MHV, the 330 th site of the nonstructural protein nsp14 is mutated from D to A, or/and the 414 th site is mutated from Y to A;
for the MERS-CoV, the 331 st site of the nonstructural protein nsp14 is mutated from D to a, or/and the 416 st site is mutated from Y to a;
For SARS-CoV-2, the 331 st site of nonstructural protein nsp14 is mutated from D to A, or/and the 420 st site is mutated from Y to A.
In a second aspect of the invention, an N7-methyltransferase deficient coronavirus attenuated vaccine comprises the N7-methyltransferase deficient coronavirus attenuated vaccine strain.
In a third aspect of the present invention, there is provided a method of preparing an attenuated strain of a coronavirus deficient in N7-methyltransferase, the method comprising:
transfecting the host cell by using an expression vector to obtain a transfected host cell;
culturing the transfected host cells to obtain recombinant viruses containing the encoding nsp14 point mutation genes;
extracting and enzyme cutting the recombinant virus DNA to obtain full-length cDNA containing nsp14 point mutation genes, and obtaining RNA through in vitro transcription;
transfecting the RNA into a host cell to obtain an N7-methyltransferase defective coronavirus attenuated vaccine strain;
wherein the expression vector carries a nucleic acid molecule encoding a mutant protein, and the mutant protein has the following mutations: the extremely conserved active sites D and/or Y within the N7-methyltransferase domain of the nonstructural protein nsp14 are site-specific mutated to A.
Further, the mutant protein comprises one of the following:
MHV nsp14 mutant protein: the 330 th amino acid of the wild MHV nsp14 protein is mutated from aspartic acid D to alanine A, or/and the 414 th amino acid is mutated from tyrosine Y to alanine A;
MERS-CoVnsp14 mutant protein: mutation of the 331 st site of the wild-type MERS-CoV nsp14 protein from D to A, or/and mutation of the 416 st site from Y to A;
SARS-CoV-2nsp14 mutant protein: mutation of amino acid at 420 th site of wild SARS-CoV-2nsp14 protein from tyrosine Y to alanine A, or/and mutation of amino acid at 331 st site from aspartic acid D to alanine A; the amino acid at position 420 is mutated from tyrosine Y to alanine A.
In a fourth aspect of the present invention, there is provided a coronavirus nsp14 mutant protein comprising site-directed mutagenesis of extremely conserved active sites D and/or a within the N7-methyltransferase domain of the nonstructural protein nsp14 in a wild-type coronavirus strain.
Further, the coronavirus nsp14 mutant protein comprises one of the following:
MHV nsp14 mutant protein: the 330 th amino acid of the wild MHV nsp14 protein is mutated from aspartic acid D to alanine A, or/and the 414 th amino acid is mutated from tyrosine Y to alanine A;
SARS-CoV-2nsp14 mutant protein: mutation of amino acid at 420 th site of wild SARS-CoV-2nsp14 protein from tyrosine Y to alanine A, or/and mutation of amino acid at 331 st site from aspartic acid D to alanine A; the amino acid at position 420 is mutated from tyrosine Y to alanine A.
In a fifth aspect of the invention, there is provided a nucleic acid molecule encoding the mutant protein, the nucleic acid molecule comprising:
MHV nsp14 mutant nucleic acid molecules: the nucleotide sequence is shown in SEQ ID NO:3, or SEQ ID NO:4, or SEQ ID NO:5 is shown in the figure;
SARS-CoV-2nsp14 mutant nucleic acid molecule: nucleotide sequence SEQ ID NO:8, or SEQ ID NO:9, or SEQ ID NO: shown at 10.
In a sixth aspect of the invention, there is provided an expression vector comprising the nucleic acid molecule.
Further, the expression vector includes one of a prokaryotic expression vector and a viral vector.
In a seventh aspect of the invention, there is provided an engineered cell line or recombinant bacterium comprising the expression vector.
One or more technical solutions in the embodiments of the present invention at least have the following technical effects or advantages:
the invention provides an N7-methyltransferase defective coronavirus attenuated vaccine strain, a preparation method and application thereof, wherein mutations of key enzyme active sites of coronavirus N7-methyltransferase are introduced into MHV and SARS-CoV-2nsp14, and methyltransferase activities of different mutant proteins are tested; considering the biological safety risk of SARS-CoV-2, we selected MHV as model coronavirus, and tried to rescue N7-MTase defective recombinant MHV virus based on the above mutation point by reverse genetics system and evaluate its replication and stability, finally screening out point mutations D330A and Y414A which can partially inactivate MHV N7-MTase and point mutations D331A and Y420A which can partially inactivate SARS-CoV-2N 7-MTase; recombinant N7-methyltransferase-deficient viruses MHV-nsp14-D330A and MHV-nsp14-Y414A were obtained, and their replication characteristics, safety, attenuation mechanisms, pathogenic ability, immunogenicity and protective effects were subsequently explored at the cellular and mouse level. The result shows that the attenuated live vaccine forward designed based on the N7-methyltransferase deficiency strategy is safe and stable, and simultaneously, the single-dose inoculation can induce the organism to generate powerful humoral immunity and T cell immune response, thereby providing protection for the organism. The novel attenuated live vaccine with good safety, strong immunogenicity and low cost is an ideal coronavirus vaccine candidate strain, and is expected to be applied to the development of general coronavirus vaccines.
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In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for the description of the embodiments will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is the data result of example 1; wherein FIG. 1A is a schematic diagram of the coronavirus nsp14 domain and an N7-MTase amino acid sequence alignment assay; fig. 1B: based on 32 P isotope labeling method for detecting N7-methyltransferase activity of MHV nsp14 mutant proteins nsp14-D330A and nsp 14-Y414A; fig. 1C: based on 3 The H isotope labeling method detects the N7-methyltransferase activity of MHV nsp14 muteins nsp14-D330A and nsp 14-Y414A; fig. 1D: based on 3 Detecting N7-methyltransferase activity of SARS-CoV-2nsp14 muteins nsp14-D331A and nsp14-Y420A by an H isotope labeling method;
FIG. 2 is the data result of example 2; wherein, fig. 2A: plaque morphology of N7-MTase mutant viruses MHV-nsp14-D330A (P10) and MHV-nsp14-Y414A (P10);fig. 2B: genetic stability of the N7-MTase mutant viruses MHV-nsp14-D330A (P10) and MHV-nsp14-Y414A (P10); fig. 2C: dynamic replication curves of the N7-MTase mutant viruses MHV-nsp14-D330A (P10) and MHV-nsp14-Y414A (P10) in the cell line; fig. 2D: 3 Detecting the methylation state of the RNA cap of the N7-MTase mutant virus by an H isotope labeling method;
FIG. 3 is the data result of example 3; wherein, fig. 3A: the N7-MTase mutant virus significantly upregulates the expression level of type I interferon in host cells; fig. 3B: the N7-MTase mutant virus presents an attenuated phenotype in primary cell BMDC; fig. 3C: RNA-induced BMDC-produced IFN- β levels comprising different cap structures; fig. 3D: screening host pattern recognition receptors that mediate the attenuated phenotype of the N7-MTase mutant virus;
FIG. 4 is the data result of example 4; wherein, fig. 4A: weight change curve of C57BL/6 mice infected with N7-MTase mutant virus; fig. 4B: survival curve of C57BL/6 mice infected with N7-MTase mutant virus; fig. 4C: replication of N7-MTase mutant virus in liver of C57BL/6 mice; fig. 4D: liver tissue pathological changes after C57BL/6 mice are infected by N7-MTase mutant viruses;
FIG. 5 is the data result of example 5; wherein, fig. 5A: immunization of C57BL/6 mice with N7-MTase mutant virus induces the generation of neutralizing antibodies with high titers; fig. 5B: neutralizing antibodies induced by immunizing C57BL/6 mice with N7-MTase mutant viruses can exist for a long time; fig. 5C: N7-MTase mutant virus immunization of C57BL/6 mice can induce strong T cell immune response;
FIG. 6 is the data result of example 6; wherein, fig. 6A: body weight change curve of C57BL/6 mice after challenge; fig. 6B: survival curve of C57BL/6 mice after challenge; fig. 6C: liver viral load of C57BL/6 mice after challenge.
Detailed Description
The advantages and various effects of the present invention will be more clearly apparent from the following detailed description and examples. It will be understood by those skilled in the art that these specific embodiments and examples are intended to illustrate the invention, not to limit the invention.
Throughout the specification, unless specifically indicated otherwise, the terms used herein should be understood as meaning as commonly used in the art. Accordingly, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification will control.
Unless otherwise specifically indicated, the various raw materials, reagents, instruments, equipment and the like used in the present invention are commercially available or may be prepared by existing methods.
The technical scheme provided by the embodiment of the invention aims to solve the technical problems, and the overall thought is as follows:
Firstly, the invention selects a series of key enzyme active sites of coronavirus N7-methyltransferase conservation by comparing the coding sequences of different coronaviruses nsp14, constructs the nsp14 mutant proteins of MHV and SARS-CoV-2 and tests the methyltransferase activity thereof. Meanwhile, an N7-MTase recombinant mutant MHV virus is constructed by utilizing a reverse genetics system, mutation sites D330A and Y414A which can partially inactivate MHV N7-MTase and mutation sites D331A and Y420A which can partially inactivate SARS-CoV-2N7-MTase are finally screened, and N7-MTase recombinant mutant viruses MHV-nsp14-D330A and MHV-nsp14-Y414A are successfully rescued, and the genetic characters of the N7-MTase recombinant mutant viruses are stable and can be autonomously replicated in a cell line. Based on this, the cap methylation status of MHV N7-MTase mutant viruses was tested, and it was found that N7-MTase deficiency resulted in the lack of N7-methylation modification of the RNA cap structure of the viruses.
Subsequently, the present invention explores the replication phenotype and attenuation mechanism of N7-MTase mutant viruses in primary cells. The N7-MTase mutant virus was found to be capable of significantly up-regulating type I interferon while the replication capacity of the mutant virus was significantly reduced. Considering that the RNA cap structure of N7-MTase mutant viruses lacks N7-methylation modification, we examined the ability of RNA containing different cap structures to induce primary BMDCs to produce type I interferon, and as a result found that the GpppG-RNA lacking N7-methylation modification induced IFN- β levels significantly higher than m7GpppG-RNA with N7-methylation modification. A deeper mechanism search has found that this phenomenon is associated with the host pattern recognition receptors MDA5 and RIG-I. Because coronaviruses lose N7-MTase activity, the cap structure of RNA cannot be completely methylated, so that viral RNA is recognized by a host MDA5 and RIG-I and activates downstream antiviral natural immunity, and the viruses are accelerated to be cleared, namely, the viruses show an attenuated phenotype after being infected by BMDC. The mechanism provides a theoretical basis for forward designing a coronavirus attenuated live vaccine with low toxicity and high immunogenicity for targeting N7-methyltransferase.
Based on the results, the pathogenicity and safety of the N7-MTase mutant virus are evaluated in the C57BL/6 mice, the mutant virus is completely cleared by the organism after 2 days of infection, the mice do not show obvious disease symptoms, and the N7-MTase mutant virus is proved to have good safety.
Subsequently, the immunogenicity of the N7-MTase mutant virus is evaluated in C57BL/6 mice, and it is found that single-dose inoculation of the N7-MTase mutant virus can quickly induce the organism to generate neutralizing antibodies with high titer and long period, and can simultaneously induce the organism to generate helper T cell immune response killing T cell immune response equivalent to or even better than that of wild type virus.
In addition, the present invention evaluates the protective effect of N7-MTase mutant viruses on mice and finds that they can protect mice from lethal doses of wild-type virus infection.
In conclusion, the coronavirus attenuated live vaccine with good safety and strong immunogenicity based on the N7-methyltransferase defect strategy forward design has extremely strong feasibility and superiority, and can be applied to the design and production and use of existing or new coronavirus vaccines including IBV, TGEV, PEDV, SADS-CoV and the like. In example 3 of the present invention, the attenuation mechanism of the N7-MTase mutant virus was explored, and it was verified that the preparation of coronavirus attenuated live vaccine by using the "N7-methyltransferase deficiency" strategy was a general strategy.
Specifically, a coronavirus attenuated live vaccine forward designed based on an N7-methyltransferase deficiency strategy comprises an N7-methyltransferase mutant strain as a main component, wherein the mutant strain is obtained by mutating a nonstructural protein nsp14 of the mutant strain on the basis of a wild virus vector;
the wild virus strain comprises MHV, MERS-CoV, SARS-CoV-2 and other viruses;
the mutations occur at extremely conserved active sites D and A within the nsp 14N 7-methyltransferase domain, such as:
for the MHV, the 330 th site of the nonstructural protein nsp14 is mutated from D to A, or/and the 414 th site is mutated from Y to A;
for MERS-CoV, the 331 st site of nonstructural protein nsp14 is mutated from D to A, or/and the 416 st site is mutated from Y to A;
for SARS-CoV-2, the 331 st site of the nonstructural protein nsp14 is mutated from D to A, or/and the 420 st site is mutated from Y to A;
the N7-methyltransferase mutant strain has the advantages of good safety and strong immunogenicity.
In addition, for convenience of research, the invention also provides carrier tools: the prokaryotic expression vectors pET30A-SARS-CoV-2nsp14-D331A and pET30A-SARS-CoV-2nsp14-Y420A are described in example 1.
The MHV nsp14 protein sequence of the embodiment of the invention is shown as SEQ ID NO:1 is shown in the specification; the nucleic acid sequence of the MHV nsp14 coding region is shown as SEQ ID NO:2 is shown in the figure;
The SARS-CoV-2nsp14 protein sequence of the embodiment of the invention is shown in SEQ ID NO:6 is shown in the figure; the nucleic acid sequence of SARS-CoV-2nsp14 coding region is shown in SEQ ID NO: shown in figure 7;
the embodiment of the invention provides a nucleic acid molecule for encoding the mutant protein, which comprises the following components:
MHV nsp14 mutant nucleic acid molecules: the nucleotide sequence is shown in SEQ ID NO:3 (MHV nsp14-D330A coding region nucleic acid sequence), or SEQ ID NO:4 (MHV nsp14-Y414A coding region nucleic acid sequence), or SEQ ID NO:5 (MHV nsp14-D330A & Y414A coding region nucleic acid sequence);
SARS-CoV-2nsp14 mutant nucleic acid molecule: nucleotide sequence SEQ ID NO:8 (SARS-CoV-2 nsp14-D331A coding region nucleic acid sequence), or SEQ ID NO:9 (SARS-CoV-2 nsp14-Y420A coding region nucleic acid sequence), or SEQ ID NO:10 (SARS-CoV-2 nsp14-D331A & Y420A coding region nucleic acid sequence).
Specifically, the invention also provides a prokaryotic expression vector for expressing the MHV nsp14 mutant protein and a preparation method thereof, comprising the following steps: taking a gene fragment of the MHV nsp14 wild type protein as a template, and sequentially adopting the sequence shown as SEQ ID NO: 11. 14 and a primer set as set forth in SEQ ID NO: 12. 13, performing PCR on the primer pair shown in the figure to obtain a gene fragment; the gene fragment and the prokaryotic expression vector pET30A are respectively cut by BamHI and SalI and then connected to obtain prokaryotic expression vectors pET30A-MHV nsp14-D330A and pET30A-MHV nsp14-Y414A;
Taking a gene fragment of SARS-CoV-2nsp14 wild type protein as a template, and sequentially adopting the sequences shown as SEQ ID NO: 17. 20 and a primer set forth in SEQ ID NO: 18. 19, performing PCR on the primer pair shown in the figure to obtain a gene fragment; the gene fragment and the prokaryotic expression vector pET30A are respectively connected after being cut by BamHI and XhoI, and the prokaryotic expression vectors pET30A-SARS-CoV-2nsp14-D331A and pET30A-SARS-CoV-2nsp14-Y420A are obtained.
The invention also provides biological materials including expression vectors, engineering cell lines or recombinant bacteria, and the like, which are all within the protection scope of the invention.
In addition, the invention also provides a preparation method of the recombinant coronavirus for expressing the MHV nsp14 mutant protein, which comprises the following steps:
taking a gene fragment of the MHV nsp14 wild type protein as a template, and constructing a T vector for encoding the MHV nsp14 mutant protein through a bypass method PCR, enzyme digestion and connection;
infecting cells with a recombinant vaccinia virus comprising a full-length cDNA of MHV-A59 substituted by a Guanylate Phosphoribosyl Transferase (GPT) gene of Escherichia coli for a fragment of the MHV nsp14 gene, transfecting the T vector encoding the MHV nsp14 mutant protein, and performing intracellular homologous recombination to obtain the recombinant vaccinia virus comprising the gene encoding the MHV nsp14 point mutation;
Extracting vaccinia virus DNA and enzyme cutting to obtain MHV-A59 full-length cDNA containing MHV nsp14 point mutation gene, and obtaining RNA through in vitro transcription;
the RNA is transfected into host cells, and coronavirus recombinant strains MHV-nsp14-D330A and MHV-nsp14-Y414A which code MHV nsp14 mutant proteins are obtained by rescue.
The effects of the present application will be described in detail with reference to examples and experimental data. Unless otherwise specified, the molecular cloning methods, protein expression purification methods, cell culture methods, various detection methods, and the like mentioned in the following schemes are all conventional experimental methods, and can be obtained by querying documents; the relevant reagents used may be purchased from the corresponding reagent suppliers.
Example 1, detection of N7-methyltransferase Activity of MHV nsp14 and its muteins
The schematic diagram of coronavirus nsp14 domain and N7-MTase amino acid sequence alignment are shown in FIG. 1A; the coronaviruses selected were in order: beta coronavirus MHV-A59 (GenBank Accession: AY 700211), SARS-CoV (GenBank Accession: NC_ 004718.3), SARS-CoV-2 (GenBank Accession: NC_ 045512.2), MERS-CoV (GenBank Accession: NC_ 019843.3); alpha coronavirus TGEV (GenBank Accession: AJ 271965.2); gamma coronavirus IBV (GenBank Accession: NC_ 001451.1); delta coronavirus PDCoV (GenBank Accession: QFU 80839.1). From the results, it can be seen that the N7-MTase amino acid sequences of coronaviruses of different species are relatively conserved, and active sites D330 and Y414 are extremely conserved.
1. Construction of prokaryotic expression vectors for MHV nsp14 and SARS-CoV-2nsp14 muteins
1) The gene fragments of the MHV-A59 nsp14 wild-type proteins stored in the laboratory (the amino acid sequences of the MHV-A59 nsp14 wild-type proteins are shown in SEQ ID NO:1, the nucleotide sequence is shown as SEQ ID NO: 2) and a wild-type SARS-CoV-2nsp14 protein (the amino acid sequence of the wild-type SARS-CoV-2nsp14 protein is shown in SEQ ID NO:6, the nucleotide sequence is shown as SEQ ID NO: 7) as templates, and PCR amplification of gene fragments containing different point mutant proteins by a bypass method. The primer sequences were as follows:
TABLE 1
Figure BDA0003084858120000091
For MHV nsp14:
in the first round of PCR, a gene fragment of MHV-A59 nsp14 wild type protein is used as a template, and the sequence of the gene fragment is shown as SEQ ID NO: 11. 14 and a primer set as set forth in SEQ ID NO: 12. 13 to obtain an nsp14 upstream and downstream gene fragment containing D330A; simultaneously adopts the sequence shown as SEQ ID NO: 11. 16 and a primer set forth as SEQ ID NO: 12. 15 to obtain an nsp14 upstream and downstream gene fragment containing Y414A;
in the second round of PCR, the upstream and downstream products of the first round of PCR are used as templates, and the sequences shown in SEQ ID NO: 11. 12 to obtain the complete nsp14 gene fragment containing D330A and Y414A respectively.
For SARS-CoV-2nsp14:
in the first round of PCR, a gene fragment of SARS-CoV-2nsp14 wild type protein is used as a template, and the sequence of the gene fragment is shown as SEQ ID NO: 17. 20 and a primer set forth in SEQ ID NO: 18. 19 to obtain an nsp14 upstream and downstream gene fragment containing D331A; simultaneously adopts the sequence shown as SEQ ID NO: 17. 22 and a primer set as set forth in SEQ ID NO: 18. 21 to obtain an nsp14 upstream and downstream gene fragment containing Y420A;
in the second round of PCR, the upstream and downstream products of the first round of PCR are used as templates, and the sequences shown in SEQ ID NO: 17. 18 to obtain the complete nsp14 gene fragment containing D331A and Y420A respectively.
2) The obtained MHV nsp14 gene fragment containing D330A or Y414A mutation and a prokaryotic expression vector pET30A are respectively connected by BamHI and SalI enzyme digestion and T4 ligase, and are constructed on the prokaryotic expression vector pET30A to obtain prokaryotic expression vectors pET30A-MHV nsp14-D330A and pET30A-MHV nsp14-Y414A; the obtained SARS-CoV-2nsp14 gene fragment containing D331A or Y420A mutation and the prokaryotic expression vector pET30A are respectively connected by BamHI and XhoI enzyme digestion and T4 ligase, and are constructed on the prokaryotic expression vector pET30A to obtain the prokaryotic expression vector pET30A-SARS-CoV-2nsp14-D331A and pET30A-SARS-CoV-2nsp14-Y420A.
3) The above vectors were transformed into competent E.coli JM109, respectively, and plated on LB plates of the respective resistances. The culture is carried out upside down at 37 ℃ overnight (12 to 16 hours), and single colony is picked for PCR verification.
4) The correct products from colony PCR were sequenced by sequencing company. Amplifying the bacterial liquid with correct sequence, and extracting the plasmid by the rapid plasmid small extraction kit. The resulting plasmid can be frozen at-20℃for a long period.
2. Prokaryotic expression purification of MHV-A59 nsp14 and SARS-CoV-2nsp14 wild-type and mutant proteins
1) The mutant protein prokaryotic expression plasmid and the wild protein prokaryotic expression plasmid obtained in the last step are respectively transformed into BL21 strain competence, coated with corresponding resistance plates and cultured upside down at 37 ℃ for overnight. Colonies with good conditions were picked every morning and inoculated into 10ml LB-resistant tubes, and amplified by shaking culture at 37℃in a shaker at 220 rpm.
2) When the absorbance (OD 600) of the culture was 0.8-1.0, 10ml of the bacterial liquid was inoculated into 1L of LB-resistant medium, and shake-cultured at 37℃with shaking at 220 rpm. When the light absorption value (OD 600) reaches 0.6-0.8, isopropyl-beta-d-1-thiopyran galactoside (IPTG) with the final concentration of 100-400 mu M is added into the bacterial liquid, and shake culture is carried out for 12h at 220rpm at 16 ℃. And (5) centrifuging and collecting thalli.
3) After the cells were collected, they were resuspended in a suitable volume of Binding Buffer (containing 10mM imidazole) and disrupted by sonication. Centrifuging 8000g of the cracked bacterial liquid for 30min, and incubating the supernatant with a Ni filler gravity column for 30min. The packing was then rinsed three times with Wash Buffer (containing 30mM imidazole) and appropriate amount of Elutation Buffer (containing 250mM imidazole) to soak the packing for 5min, and the flow-through was collected.
4) SDS-PAGE gel electrophoresis, coomassie brilliant blue staining, and detection of the expression and purification of the target protein.
3. MHV nsp14 and N7-MTase activity detection of its mutein 32 P isotope labeling method
1) The 22nt RNA at the 5' end of the MHV genome is used as a template, a substrate is designed and is delivered to a double-stranded DNA template of a synthetic substrate of a company, and the single-stranded RNA which is not modified by capping is obtained through in vitro transcription.
2) Capping and methylation systems in vitro using poxviruses to 32 P-labeled GTP is used as a substrate to obtain cap precursor RNA (G.times.pppRNA) containing a cap core structure but not N7 methylation. Purifying the precursor RNA of the cap from the reaction system, quantifying, and then sub-packaging and freezing the precursor RNA80℃。
3) The nsp14 wild-type protein and mutein to be detected (0.5. Mu.g) were added to the methylation reaction system together with the cap precursor G ppp-RNA, respectively, and reacted at 37℃for 1.5h.
4) Exonuclease P1 digests the reaction product, and the digestions are then subjected to thin layer chromatography on a polyethyleneimine plate.
5) The typhoon instrument scans the laminated board, 32 the amount of cap structure of the P-tag can be detected. The results are shown in FIG. 1B. From FIG. 1B, it is understood that the D330A and Y414A mutations result in the loss of N7-methylation function of MHV nsp 14.
4. Detection of N7-MTase Activity of wild-type and mutant proteins of MHV nsp14 and SARS-CoV-2nsp14 3 H isotope labeling method
1) The substrate template was synthesized as described above.
2) Cap precursor RNA (Gppp-RNA) containing the cap core structure but not N7 methylated was obtained using the poxvirus in vitro capping and methylation system. Purifying the cap precursor RNA from the reaction system, quantifying, split charging and freezing at-80 ℃.
3) The nsp14 wild-type protein and the mutein to be detected (0.5. Mu.g) and the prepared cap precursor RNA (2. Mu.g) were added to a methylation reaction system (containing 50mM Tris-HCl,6mM KCl,2mM DTT,1.25mM MgCl) 2 10U RNase inhibitor), and finally 1. Mu. Ci was added to the system 3 H-labeled SAM substrate and reacted at 37℃for 1.5H.
4) Separation by low-volume DEAE-Sephadex chromatographic column 3 H-labeled products and detection by liquid scintillation 3 H incorporation amount.
5) MHV nsp14 3 The H incorporation reading was set to 100% and the relative methylation activities of the mutant and control were calculated. The methylation activity of MHV nsp14 and its mutants is shown in figure 1C. As can be seen from FIG. 1C, when the N7-MTase activity of nsp14-WT was 100%, the activity of nsp14-D330A was only 15% and the activity of nsp14-Y414A was 20%, indicating that the D330A and Y414A mutations could result in partial loss of the N7-MTase activity. Similarly, the methylation activity of SARS-CoV-2nsp14 and its mutants is shown in FIG. 1D when N7-MTase of nsp14-WT is active At 100%, the activity of nsp14-D331A was only 17% and the activity of nsp14-Y420A was 24%, indicating that the mutations of D331A and Y420A could result in partial loss of SARS-CoV-2N7-MTase activity.
EXAMPLE 2 construction and replication characterization of N7-MTase mutant viruses
1. Construction of N7-MTase mutant Virus
Based on the in vitro N7-methyltransferase activity result, 2 strains of N7-MTase mutant viruses MHV-nsp14-D330A and MHV-nsp14-Y414A and 1 strain of exonuclease mutant virus MHV-nsp14-D89A & E91A are successfully constructed by using a recombinant vaccinia virus reverse genetics system by taking the murine hepatitis virus MHV-59 as a model virus, and meanwhile, the recombinant wild type virus MHV-WT is constructed as a control.
(1) The gene fragment upstream and downstream of MHV-A59 nsp14, which contains the D330A, Y414A, D A & E91A mutation, was cloned into the pMD18-T vector according to the method for constructing point mutations in example 1. Taking a gene fragment of the MHV nsp14 wild protein as a template, and constructing by a bypass method PCR, enzyme digestion and connection to obtain the pMD18-T vector for encoding the MHV nsp14 mutant protein.
In the first round of PCR, cDNA comprising the MHV nsp14 coding gene and upstream and downstream gene fragments thereof is used as a template, and the cDNA is sequentially expressed by SEQ ID NO: 23. 14 and a primer set as set forth in SEQ ID NO: 13. 24 to obtain an nsp14 upstream and downstream gene fragment containing D330A; simultaneously adopts the sequence shown as SEQ ID NO: 23. 16 and a primer set forth as SEQ ID NO: 15. 24 to obtain an nsp14 upstream and downstream gene fragment containing Y414A; the sequence shown in SEQ ID NO: 23. 26 and a primer set forth in SEQ ID NO: 25. 24 to obtain an nsp14 upstream and downstream gene fragment containing D89A & E91A double mutation;
In the second round of PCR, the upstream and downstream products of the first round of PCR are used as templates, and the sequences shown in SEQ ID NO: 23. 24 to obtain the complete nsp14 gene fragment containing D330A, Y414A and D89A & E91A respectively.
Primers used to construct the pMD18-T vector and the double mutant D89A & E91A were complemented as follows:
TABLE 2
Figure BDA0003084858120000121
(2) CV-1 cells were infected with a recombinant vaccinia virus containing a full-length cDNA of the MHV-A59 strain substituted with the gene fragment of MHV nsp14 by the gpt gene of E.coli, and the above-mentioned pMD18-T vector encoding the MHV nsp14 mutant protein was transfected after virus adsorption for 1 hour, so that a recombinant vaccinia virus comprising the gene encoding the MHV nsp14 point mutation was obtained by intracellular homologous recombination.
The transfection solution was discarded 6 hours after transfection and fresh DMEM medium was replaced. After culturing for 2-3 days, the cell culture is collected after obvious cytopathy is observed. Inoculating the cell culture to CV-1 cells containing GPT positive screening medicaments, and screening to obtain recombinant vaccinia virus culture containing GPT genes; then the culture is further inoculated into D980R cells containing GTP negative screening medicines, and the recombinant vaccinia virus containing the gene for encoding MHV nsp14 point mutation is obtained through intracellular homologous recombination.
(3) The recombinant vaccinia virus obtained in the last step is amplified in BHK-21 cells, and after 100% cytopathy is reached, cell sediment containing the virus is collected, and freeze thawing is repeated for three times. Viral DNA is then extracted and quantified. PCR, sequencing and identifying whether the obtained DNA contains a gene fragment encoding a mutant protein.
(4) The recombinant vaccinia virus identified as correct in the previous step was reamplified, and viral pellet was collected and recombinant vaccinia virus DNA was extracted. The DNA was digested with EagI to obtain the full-length cDNA of the MHV genome containing the desired gene mutation. The digested product was recovered and purified, and the full-length cDNA of the MHV genome was subjected to in vitro transcription (see example 1) to obtain full-length RNA of the MHV genome.
(5) BHK-21 cells were transfected with the above RNA, and after 24 hours the transfected cells were mixed with 4-fold amounts of 17-CL1 cells, followed by further culture and observation of cytopathy. After obvious cytopathy appears, collecting cell supernatant and sediment, and freezing for later use.
(6) The virus solution obtained in the previous step is re-infected with 17-CL1 cells, and the obtained recombinant MHV virus is purified by plaque method. After sequencing and identification, the virus is frozen and stored, and is marked as P0 generation virus.
2. Amplification of MHV virus
(1) All of the recombinant MHV viruses described above were amplified in Neuro 2a cells. Neuro 2a cells were resuscitated and passaged in DMEM medium containing 10% fbs. After the cell growth state is good, the cells are inoculated in a 15cm cell culture dish.
(2) After the cell density reaches 80-90%, virus mother liquor is added into the cells and adsorbed for 1 hour. And then removing the adsorption liquid, replacing the fresh culture medium, and culturing for 16-20 hours in a carbon dioxide incubator at 37 ℃.
(3) After 70% or more CPE appears on the cells, collecting cell supernatant, centrifuging to remove cell fragments, and obtaining the amplified virus liquid. Sub-packaging and freezing at-80 ℃.
3. Titer detection of MHV virus (plaque method)
(1) The MHV virus described above detects viral titers in L2 cells. L2 cells were resuscitated and passaged in DMEM medium containing 10% fbs. After the cell growth state is good, inoculating the cells into a 24-hole cell culture plate.
(2) After the cell density reached 90%, titer was measured. Taking out the virus liquid to be tested, respectively carrying out 10-time gradient dilution, and covering the dilution times by 10 1 -10 8 . The cells were removed, the medium was discarded, and the diluted virus was added to a 24-well plate and adsorbed for 1 hour. The adsorption solution was then discarded, and the 1% methylcellulose semisolid culture medium was replaced, and incubated in a carbon dioxide incubator at 37℃for about 48 hours.
(3) After visible plaques were observed, the plates were removed and the paraformaldehyde was fixed and stained with crystal violet stain for 4 hours. Cells were then washed, plaque counted, and viral titers were calculated as fold dilutions, measured in PFU/ml.
4. Continuous passage and stability detection of N7-MTase mutant viruses
(1) Serial passaging of the N7-MTase mutant virus was performed in Neuro 2a cells. Neuro 2a was inoculated into 6cm dishes, and after the density was 80%, the P0 generation mutant viruses were infected and grown for 16 hours, respectively. The same Neuro 2a cells were prepared at the same time for infection with the next generation virus.
(2) The P1 generation virus is harvested, one part is split and frozen, and the other part is used for the next passage. And sequentially completing 10 times of virus passage to obtain the P10 generation mutant virus. The plaque forms of the N7-MTase mutant viruses MHV-nsp14-D330A (P10) and MHV-nsp14-Y414A (P10) are shown in FIG. 2A, and the plaque forms and sizes of the N7-MTase mutant viruses and the wild-type viruses are not obviously different.
(3) Viral RNA (TIANGEN) was extracted from wild-type virus MHV-WT and P10 generation N7-MTase mutant virus supernatants according to the instructions of the reagent manufacturer.
(4) Reverse transcription is carried out on the viral RNA obtained in the last step, cDNA is obtained as a template, and the MHV nsp14 fragment is amplified by PCR. Fragments were sent to the company for one generation of sequencing.
(5) The sequences of the wild type virus and the P10 generation N7-MTase mutant virus were aligned, respectively, to confirm whether the introduced mutation sites (D330A and Y414A) were stable and whether other mutations were introduced. The sequence alignment results containing the mutation sites are shown in FIG. 2B, and it is known that in the mutant virus MHV-nsp14-D330A, the nucleotide encoding 330 amino acids is converted from GAC (D) to GCA (A); in the mutant virus MHV-nsp14-Y414A, the nucleotide encoding the 414 th amino acid is converted into GCA (A) from TAT (Y), the sequence is single, and the mutation exists stably.
5. Replication curve assay for N7-MTase mutant viruses
(1) The present invention measures the replication profile of mutant viruses in Neuro 2a cells and L2 cells, respectively. First, two kinds of cells were seeded in 24-well plates, respectively.
(2) After the cell density reached about 80%, the cells were counted, and virus was added at a multiplicity of infection (MOI) of 1, followed by adsorption for 1 hour. The adsorption solution was then discarded, and the fresh medium was replaced and incubated in an incubator at 37 ℃. Supernatants were harvested 0-32 hours (Neuro 2a cells) or 0-24 hours (L2 cells) after infection, every 4 hours, for virus titer detection.
(3) Plaque assay virus titer in supernatant samples, prism7 software plots virus growth curves. The growth curves are shown in FIG. 2C, and it is known that the N7-MTase mutant viruses can complete autonomous replication in the cell line and only have weak attenuation phenotype.
Hat methylation status detection of N7-MTase mutant Virus
(1) Neuro 2a was inoculated into 10cm dishes and grown for 24 hours after reaching 80% density, and infected with wild-type virus MHV-WT and N7-MTase mutant virus, respectively.
(2) Total polyA-RNA was enriched in cells and the procedure was performed according to the instructions of the kit manufacturer (Invitrogen).
(3) The enriched total polyA-RNA is added to the methylation reaction system together with the nsp14 protein or the nsp10/nsp16 protein, respectively (same as in example 1), followed by the addition of 3 H-labeled SAM substrate, at 37℃for 1.5H. Detection by liquid flashing instrument 3 H incorporation (same as in example 1) and the results are shown in FIG. 2D, it is understood that N7-MTase of the N7-MTase mutant virus is inactivated, and the cap structure of RNA lacks N7-methylation instead of 2' -O-methylation.
Example 3, attenuation mechanism exploration of N7-MTase mutant Virus
1. Preparation of dendritic cells (BMDCs) derived from mouse bone marrow
Male C57BL/6 mice with age of 6-8 weeks are taken, and cervical dislocation is euthanized. Bone marrow cells in the femur and tibia of the hindlimb of the mouse were isolated to DMEM medium in a sterile environment, filtered to remove tissue debris, and erythrocytes were lysed. Cells were washed 1 pass with PBS, resuspended and counted. According to 1.5X10 7 cell/dish density cells were seeded on 10cm dishes and induced by the addition of GM-CSF (Peprotech) cytokine for 7 days, during which time the medium was changed 1 time. After the cells are induced to mature, the cells are digested by pancreatin and plated according to experimental requirements.
Viral infection of BMDC
Mature BMDCs were plated onto 24-well plates. After cell attachment, the MHV-WT or N7-MTase mutant virus was added for infection (MOI=0.1). After 1 hour, the virus adsorption solution was discarded and the fresh medium was replaced. Cell supernatants were collected 4, 8, 12, 16, and 20 hours post-infection and split into two parts, one for detecting IFN- β amounts and one for detecting viral loads. IFN- β detection was performed according to the instructions provided by the kit (Biolegend), and viral loads were detected by plaque assay (same as in example 2). The results are shown in fig. 3A and 3B. From FIG. 3A, it is suggested that the deletion of N7-methylated cap structure could activate the natural immune signaling pathway earlier and faster, up-regulating the level of type I interferon expression. As can be seen from fig. 3B, in the primary cells with perfect immune system, the wild type virus with complete cap structure has strong immune escape ability and can still replicate relatively efficiently; whereas coronaviruses lacking the N7-methylated cap structure have reduced immune escape capacity, reduced replication capacity and obvious attenuated phenotype.
3. Detection of IFN- β levels induced by RNA of different cap structures to BMDC production
(1) The DNA fragment containing 50kb nucleotide was designed and synthesized using the nucleotide sequence at the 5' -end of the MHV-A59 genome as a template. The DNA fragment was dissolved in water, transcribed into single stranded RNA (i.e., pppG-RNA) using the T7 in vitro transcription kit (Invitrogen), and the RNA was purified by phenol chloroform extraction for further use. The resulting RNA was processed with poxvirus capping and methylation system (NEB) to GpppG-RNA, m7GpppG-RNA, m7GpppGm-RNA, respectively. Commercial PolyI: C (MCE) was also purchased as a positive control stimulator.
(2) BMDCs were prepared and plated as described above. Cells were transfected with the above RNA and positive control stimulus using lipo3000 (Invitrogen). Cells were lysed with Trizol (Life) 20 hours after transfection, total cellular RNA was extracted, and randomly reverse transcribed to give cDNA (TAKARA). Using cDNA as a template, the mRNA level of IFN- β was detected by q-PCR quantification.
4. Screening for host pattern recognition receptors that mediate the attenuated phenotype of N7-MTase mutant viruses
(1) BMDCs were prepared and plated as described above. Pattern recognition receptors that recognize RNA in cells mainly include RLR receptors such as RIG-I, MDA 5. In vitro siRNA targeting RIG-I and MDA5 was synthesized and transfected into BMDC, and after 24 hours of transfection, it was infected with MHV-WT or N7-MTase mutant virus (MOI=0.1). 16 hours after infection, cell supernatants were harvested for detection of viral load (plaque method). The level of viral replication is shown in FIG. 3C. From FIG. 3C, it can be seen that RNA with different cap structures induces BMDC to up-regulate IFN- β levels in the order of from high to low: pppG-RNA without cap structure, gpppG-RNA without N7-methylation modified cap structure, m7GpppG-RNA with N7-methylation modified cap structure, m7GpppGm-RNA with N7-methylation and 2' -O-methylation double modified cap structure. Poly I: C is a double-stranded nucleotide analog, and is a type I interferon inducer.
(2) Screening for host pattern recognition receptors that mediate the attenuated phenotype of the N7-MTase mutant virus as shown in FIG. 4, knocking down the intracellular RIG-I expression level increased the MHV-nsp14-Y414A viral load by about 2-fold, while knocking down the MDA5 expression level increased the MHV-nsp14-Y414A viral load by about 6-fold, but the changes in the expression levels of these two molecules did not significantly affect replication of the wild-type virus. From the results of FIG. 3C, it is assumed that RNA lacking the N7-methylated cap structure is considered as a pathogen-associated molecular pattern, and is likely to be recognized by pattern recognition receptors MDA5 and RIG-I, thereby activating the host's antiviral natural immune response and accelerating the viral clearance, and thus the N7-MTase mutant virus exhibits an attenuated phenotype. The mechanism provides a theoretical basis for forward designing of N7-MTase defective coronavirus attenuated live vaccine.
Example 4 evaluation of pathogenicity and safety of N7-MTase mutant Virus
Mouse infection experiment with MHV-WT and its mutant Virus
56C 57BL/6 mice of 3 to 4 weeks of age were randomly grouped according to the following table.
TABLE 3 Table 3
Figure BDA0003084858120000161
Figure BDA0003084858120000171
Mice were injected intrahepatically with MHV-WT or N7-MTase mutant virus at a dose of 2X 10 according to the group 6 PFU, while injecting an equal amount of viral solvent as a control. At 1, 2 and 5 days after infection, 3 mice were euthanized at random per group B, D, F, H, livers were isolated, and half frozen for detection of viral load; half was fixed in 4% paraformaldehyde for pathological section analysis. A. C, E, G group of mice are connected The mice were continuously observed for 8 days, the survival status and weight change were recorded, and the survival rate curve and the weight change curve were plotted, and the results are shown in fig. 4A and 4B.
As can be seen from fig. 4A, mice infected with MHV wild type virus continue to lose weight from day 2 until death; whereas mice infected with the N7-MTase mutant virus developed a short body weight plateau only on day 1 of infection, followed by a gradual increase in body weight, and eventually recovered to levels similar to those of the control mice.
As can be seen from FIG. 4B, the mice infected with the wild-type virus all died on days 4 to 8, while the mice infected with the N7-MTase mutant virus and the control mice all survived. As can be seen from a combination of the results of FIG. 4A, the pathogenic ability of the N7-MTase mutant virus was significantly decreased.
2. Mouse liver viral load detection
Livers were taken, weighed and recorded. 1ml of PBS was added to the liver, the homogenate was ground, centrifuged at 10000rpm for 5min, and the supernatant was taken. The supernatant of the homogenate was subjected to gradient dilution, followed by detection of virus titer by plaque assay. The liver viral load was calculated from the resulting viral titer, dilution fold and liver weight in PFU/g. The results are shown in fig. 4C. As can be seen from FIG. 4C, the N7-MTase mutant virus was detected only on day 1 after inoculation, the virus had been completely cleared on day 2, while the wild-type virus load was continuously increased and was at peak on day 5. The N7-MTase mutant virus is rapidly cleared in a body with complete immune system, and continuous and efficient pathogenic infection cannot be established.
3. Mouse liver pathology analysis
The liver with the paraformaldehyde fixed is delivered to a company, embedded in paraffin, sliced and then HE stained for judging the degree of inflammation and pathological changes. The results are shown in fig. 4D. As can be seen from FIG. 4D, on day 5 after virus infection, the liver of the MHV-WT mice showed severe inflammatory cell infiltration, tissue fibrosis, local hemorrhage, etc., while the infection with the N7-MTase mutant virus caused only very slight inflammatory cell infiltration, again demonstrating a severe reduction in pathogenicity of the N7-MTase mutant virus.
EXAMPLE 5 immunogenicity evaluation of N7-MTase mutant Virus
In this part of the experiment we selected a reported attenuated live vaccine designed based on the coronavirus nsp14 exonuclease active site (D89A & E91A), i.e.MHV-nsp 14-D89A & E91A as control.
Mouse immunity experiment of MHV-WT and its mutant virus
50C 57BL/6 mice aged 3-4 weeks were randomly grouped according to the following table.
TABLE 4 Table 4
Figure BDA0003084858120000181
Mice were subcutaneously injected with MHV-WT, N7-MTase mutant virus or exonuclease mutant virus in groups at a dose of 5X 10 5 PFU, while injecting an equal amount of viral solvent as a control. A. C, E, G, I groups of mice were bled at weeks 2, 4, 8, 12, 24, 36, 48 post immunization for detection of neutralizing antibodies; B. group D, F, H, J mice were euthanized 4 weeks after immunization and spleens were taken for detection of T cell immune responses.
2. Neutralizing antibody detection (plaque reduction method)
(1) After the blood of the mice is collected, the mice are kept stand for 1 to 2 hours at room temperature. And then centrifuging at 2000rpm for 20min, separating serum, inactivating at 56 ℃ for 30min, and sub-packaging for freezing.
(2) And (3) taking wild MHV virus with known virus titer, diluting to 1000PFU/ml, and sub-packaging for freezing. Meanwhile, L2 cells are inoculated in a 24-well plate, and a neutralization experiment can be started after the density reaches 90%.
(3) Serum was removed and serial 2-fold gradient dilutions were performed on serum with 1:5 as initial dilution. 100ul of serum was then mixed with an equal volume of 100ul of virus (about 100 PFU) and incubated for 1 hour at 37 ℃.
(4) After the incubation, L2 cells were taken, the medium was discarded, and the virus-serum mixture was transferred to the cells and adsorbed for 1 hour. Afterwards, the adsorption solution is discarded, and the culture solution of 1% methyl cellulose is replaced, and the culture is carried out for 48 hours at 37 ℃ until plaque is formed which is visible to naked eyes. Fixing, staining, and counting the number of plaques.
(5) Calculation of plaquesReducing the rate. Plaque reduction = (1-number of plaques in sample wells/number of plaques in control Kong Shiban) ×100%. Calculating PRNT according to Reed-Muech method 50 This value represents the corresponding serum dilution that reduces the viral plaque number by half, and can characterize the neutralizing antibody titer. Neutralizing antibody titers are shown in figures 5A and 5B.
In FIG. 5A, two N7-MTase mutant viruses rapidly induced mice to produce high titers of neutralizing antibodies at levels comparable to MHV-WT; unexpectedly, the level of neutralizing antibodies induced by the exonuclease mutant virus was very low, suggesting that attenuated live vaccines designed based on N7-MTase have significant advantages.
As can be seen from FIG. 5B, the neutralizing antibodies induced by the two N7-MTase mutant viruses were able to exist for about 1 year, but remained at a higher level (69.18 to 131.83) despite the reduced antibody titers.
T cell immune response assessment
(1) 4 weeks after immunization, mice were euthanized and spleens isolated. Gently rolling spleen in a sterile environment, filtering the extruded spleen cells into RPMI 1640 medium, centrifuging to remove tissue fragments, and lysing erythrocytes. Spleen cells were washed, resuspended, counted and plated in 96-well plates at a density of 5X 10 6 cell/well.
(2) After cells were plated, 5×10 cells were added to each well at moi=0.1 5 PFU MHV was restimulated. After 19 hours of stimulation, the golgi inhibitor BFA was added to block protein transport. Cells were harvested 24 hours after stimulation for detection.
(3) Collecting spleen cells, arranging blank tube (x 1), single-dyeing tube (x 4), and experiment tube (several times), each tube respectively taking 2 x 10 6 Cells were centrifuged at 350g for 5min in a 1.5mL centrifuge tube and the supernatant discarded. Cells were washed 1 pass with PBS. The cells were stained with surface antibody PerCP/Cy5.5-CD3, FITC-CD4, APC-CD8 for 30min at room temperature (except for blank tubes) and washed 2 times with PBS.
(4) Adding a Fixation Buffer, and fixing at room temperature for 30min in a dark place. The PBS was washed 1 pass.
(5) Cells were washed 3 times with Permeabilization Buffer.
(6) Adding an intracellular cytokine antibody PE-IFN-gamma and an isotype control antibody PE-IgG1, and staining at room temperature for 30min in a dark place. The cells were then washed 2 times.
(7) Cells were resuspended in PBS, filtered through a 200 mesh screen to flow tubes, and checked on an engine. Removal of dead cells by forward and side scattered light, gating to CD3 + CD4 + IFN-γ + T cell populations and CD3 + CD8 + IFN-γ + T cell populations, the positive proportion of which was counted. The results obtained are shown in fig. 5C.
As can be seen from FIG. 5C, the N7-MTase mutant virus can induce a strong helper T cell response (CD 4) + ) And killer T cells (CD 8) + ) Reaction in which MHV-nsp14-Y414A strain induced CD8 + T cell responses are even stronger than wild-type viruses; in contrast, exonuclease mutant viruses are not able to induce significant CD4 + And CD8 + T cell response.
EXAMPLE 6 evaluation of protective Effect of N7-MTase mutant Virus
44C 57BL/6 mice aged 3-4 weeks were randomly grouped as shown in the following table and immunized as in example 4. 30 days after immunization, intrahepatic injection is carried out by using wild MHV, and the toxin counteracting dosage is 2 multiplied by 10 7 PFU。
TABLE 5
Figure BDA0003084858120000201
A. C, E, G mice were continuously observed for 8 days, the survival status and weight change of the mice were recorded, and the survival rate curve and the weight change curve were plotted, and the results are shown in fig. 6A and 6B.
As can be seen from FIG. 6A, all mice immunized with the N7-MTase mutant virus and the WT virus were challenged, the body weight remained stable and the growth state was good, while the mice of the control group immunized with the solvent showed a cliff-like decrease in body weight until death.
As can be seen from FIG. 6B, all mice survived 100% (5/5) after challenge with the N7-MTase mutant virus and WT virus; and after the mice of the control group which are immunized by the solvent attack the virus, only 20 percent (1/5) of the mice survive, which proves that the N7-MTase serving as the attenuated live vaccine can provide the vaccinated animals with the protection effect similar to that of the wild type virus, and the protection effect is obvious.
On days 2 and 5 after challenge, group B, D, F, H randomly received 3 mice euthanized, and livers were isolated for detection of viral load, in the same manner as in example 4. The results are shown in FIG. 6C.
As can be seen from FIG. 6C, on day 2 after challenge, live virus could not be detected in the liver of 100% (3/3) MHV-WT immunized mice, 67% (2/3) MHV-nsp14-Y414A immunized mice, 33% (1/3) MHV-nsp14-D330A immunized mice, and the liver viral load of control mice was at a higher level; on day 5 after challenge, no live virus could be detected in the livers of all virus immunized mice, and the viral load of the control mice remained at peak. The inoculation of the N7-MTase attenuated live vaccine can timely block the reinfection and replication of viruses. From the figure, it can be seen that the mice inoculated with N7-MTase minus strain completely clear the re-infected virus within 5 days, providing significant protective effect for the mice.
Finally, it is also noted that the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. It is therefore intended that the following claims be interpreted as including the preferred embodiments and all such alterations and modifications as fall within the scope of the invention.
It will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention also include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.
Sequence listing
<110> university of Wuhan
<120> an N7-methyltransferase-deficient coronavirus attenuated vaccine strain, and preparation method and application thereof
<160> 26
<170> SIPOSequenceListing 1.0
<210> 1
<211> 521
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 1
Cys Thr Thr Asn Leu Phe Lys Asp Cys Ser Arg Ser Tyr Val Gly Tyr
1 5 10 15
His Pro Ala His Ala Pro Ser Phe Leu Ala Val Asp Asp Lys Tyr Lys
20 25 30
Val Gly Gly Asp Leu Ala Val Cys Leu Asn Val Ala Asp Ser Ala Val
35 40 45
Thr Tyr Ser Arg Leu Ile Ser Leu Met Gly Phe Lys Leu Asp Leu Thr
50 55 60
Leu Asp Gly Tyr Cys Lys Leu Phe Ile Thr Arg Asp Glu Ala Ile Lys
65 70 75 80
Arg Val Arg Ala Trp Val Gly Phe Asp Ala Glu Gly Ala His Ala Ile
85 90 95
Arg Asp Ser Ile Gly Thr Asn Phe Pro Leu Gln Leu Gly Phe Ser Thr
100 105 110
Gly Ile Asp Phe Val Val Glu Ala Thr Gly Met Phe Ala Glu Arg Asp
115 120 125
Gly Tyr Val Phe Lys Lys Ala Ala Ala Arg Ala Pro Pro Gly Glu Gln
130 135 140
Phe Lys His Leu Ile Pro Leu Met Ser Arg Gly Gln Lys Trp Asp Val
145 150 155 160
Val Arg Ile Arg Ile Val Gln Met Leu Ser Asp His Leu Val Asp Leu
165 170 175
Ala Asp Ser Val Val Leu Val Thr Trp Ala Ala Ser Phe Glu Leu Thr
180 185 190
Cys Leu Arg Tyr Phe Ala Lys Val Gly Arg Glu Val Val Cys Ser Val
195 200 205
Cys Thr Lys Arg Ala Thr Cys Phe Asn Ser Arg Thr Gly Tyr Tyr Gly
210 215 220
Cys Trp Arg His Ser Tyr Ser Cys Asp Tyr Leu Tyr Asn Pro Leu Ile
225 230 235 240
Val Asp Ile Gln Gln Trp Gly Tyr Thr Gly Ser Leu Thr Ser Asn His
245 250 255
Asp Pro Ile Cys Ser Val His Lys Gly Ala His Val Ala Ser Ser Asp
260 265 270
Ala Ile Met Thr Arg Cys Leu Ala Val His Asp Cys Phe Cys Lys Ser
275 280 285
Val Asn Trp Asn Leu Glu Tyr Pro Ile Ile Ser Asn Glu Val Ser Val
290 295 300
Asn Thr Ser Cys Arg Leu Leu Gln Arg Val Met Phe Arg Ala Ala Met
305 310 315 320
Leu Cys Asn Arg Tyr Asp Val Cys Tyr Asp Ile Gly Asn Pro Lys Gly
325 330 335
Leu Ala Cys Val Lys Gly Tyr Asp Phe Lys Phe Tyr Asp Ala Ser Pro
340 345 350
Val Val Lys Ser Val Lys Gln Phe Val Tyr Lys Tyr Glu Ala His Lys
355 360 365
Asp Gln Phe Leu Asp Gly Leu Cys Met Phe Trp Asn Cys Asn Val Asp
370 375 380
Lys Tyr Pro Ala Asn Ala Val Val Cys Arg Phe Asp Thr Arg Val Leu
385 390 395 400
Asn Lys Leu Asn Leu Pro Gly Cys Asn Gly Gly Ser Leu Tyr Val Asn
405 410 415
Lys His Ala Phe His Thr Ser Pro Phe Thr Arg Ala Ala Phe Glu Asn
420 425 430
Leu Lys Pro Met Pro Phe Phe Tyr Tyr Ser Asp Thr Pro Cys Val Tyr
435 440 445
Met Glu Gly Met Glu Ser Lys Gln Val Asp Tyr Val Pro Leu Arg Ser
450 455 460
Ala Thr Cys Ile Thr Arg Cys Asn Leu Gly Gly Ala Val Cys Leu Lys
465 470 475 480
His Ala Glu Glu Tyr Arg Glu Tyr Leu Glu Ser Tyr Asn Thr Ala Thr
485 490 495
Thr Ala Gly Phe Thr Phe Trp Val Tyr Lys Thr Phe Asp Phe Tyr Asn
500 505 510
Leu Trp Asn Thr Phe Thr Arg Leu Gln
515 520
<210> 2
<211> 1563
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 2
tgtactacaa atttgtttaa ggattgtagc aggagctatg taggatatca cccagcccat 60
gcaccatcct ttttggcagt tgatgacaaa tataaggtag gcggtgattt agccgtttgc 120
cttaatgttg ctgattctgc tgtcacttat tcgcggctta tatcactcat gggattcaag 180
cttgacttga cccttgatgg ttattgtaag ctgtttataa ctagagatga agctatcaaa 240
cgtgttagag cctgggttgg cttcgatgca gaaggtgccc atgcgatacg tgatagcatt 300
gggacaaatt tcccattaca attaggcttt tcgactggaa ttgattttgt tgtcgaagcc 360
actggaatgt ttgctgagag agatggttat gtctttaaaa aggcagccgc acgagctcct 420
cctggcgaac aatttaaaca ccttatccca cttatgtcaa gagggcagaa atgggatgtg 480
gttcgcatta gaatagtaca aatgttgtca gaccacctag tggatttggc agacagtgtt 540
gtacttgtga cgtgggctgc cagctttgag ctcacatgtt tgcgatattt cgctaaagtt 600
ggaagagaag ttgtgtgtag tgtctgcacc aagcgtgcga catgttttaa ttctagaact 660
ggatactatg gatgctggcg acatagttat tcctgtgatt acctgtacaa cccactaata 720
gttgacattc aacagtgggg atatacagga tctttaacta gcaatcatga tcctatttgc 780
agcgtgcata agggtgctca tgttgcatca tctgatgcta tcatgacccg gtgtctagct 840
gttcatgatt gcttttgtaa gtctgttaat tggaatttag aataccccat tatttcaaat 900
gaggtcagtg ttaatacctc ctgcaggtta ttgcagcgcg taatgtttag ggctgcgatg 960
ctatgcaata ggtatgatgt gtgttatgac attggcaacc ctaaaggtct tgcctgtgtc 1020
aaaggatatg attttaagtt ttatgatgcc tcccctgttg ttaagtctgt taaacagttt 1080
gtttataaat acgaggcaca taaagatcaa tttttagatg gtttgtgtat gttttggaac 1140
tgcaatgtgg ataagtatcc agcgaatgca gttgtgtgta ggtttgacac gcgtgtgttg 1200
aacaaattaa atctccctgg ctgtaatggt ggcagtttgt atgttaacaa acatgcattc 1260
cacaccagtc cctttacccg ggctgccttc gagaatttga agcctatgcc tttcttttat 1320
tattcagata cgccctgtgt gtatatggaa ggcatggaat ctaagcaggt cgattatgtc 1380
ccattgagaa gcgctacatg catcacaaga tgcaatttag gtggcgctgt ttgtttaaaa 1440
catgctgagg agtatcgtga gtaccttgag tcttacaata cggcaaccac agcgggtttt 1500
actttttggg tctataagac ttttgatttt tataaccttt ggaatacttt tactaggctc 1560
caa 1563
<210> 3
<211> 1563
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 3
tgtactacaa atttgtttaa ggattgtagc aggagctatg taggatatca cccagcccat 60
gcaccatcct ttttggcagt tgatgacaaa tataaggtag gcggtgattt agccgtttgc 120
cttaatgttg ctgattctgc tgtcacttat tcgcggctta tatcactcat gggattcaag 180
cttgacttga cccttgatgg ttattgtaag ctgtttataa ctagagatga agctatcaaa 240
cgtgttagag cctgggttgg cttcgatgca gaaggtgccc atgcgatacg tgatagcatt 300
gggacaaatt tcccattaca attaggcttt tcgactggaa ttgattttgt tgtcgaagcc 360
actggaatgt ttgctgagag agatggttat gtctttaaaa aggcagccgc acgagctcct 420
cctggcgaac aatttaaaca ccttatccca cttatgtcaa gagggcagaa atgggatgtg 480
gttcgcatta gaatagtaca aatgttgtca gaccacctag tggatttggc agacagtgtt 540
gtacttgtga cgtgggctgc cagctttgag ctcacatgtt tgcgatattt cgctaaagtt 600
ggaagagaag ttgtgtgtag tgtctgcacc aagcgtgcga catgttttaa ttctagaact 660
ggatactatg gatgctggcg acatagttat tcctgtgatt acctgtacaa cccactaata 720
gttgacattc aacagtgggg atatacagga tctttaacta gcaatcatga tcctatttgc 780
agcgtgcata agggtgctca tgttgcatca tctgatgcta tcatgacccg gtgtctagct 840
gttcatgatt gcttttgtaa gtctgttaat tggaatttag aataccccat tatttcaaat 900
gaggtcagtg ttaatacctc ctgcaggtta ttgcagcgcg taatgtttag ggctgcgatg 960
ctatgcaata ggtatgatgt gtgttatgca attggcaacc ctaaaggtct tgcctgtgtc 1020
aaaggatatg attttaagtt ttatgatgcc tcccctgttg ttaagtctgt taaacagttt 1080
gtttataaat acgaggcaca taaagatcaa tttttagatg gtttgtgtat gttttggaac 1140
tgcaatgtgg ataagtatcc agcgaatgca gttgtgtgta ggtttgacac gcgtgtgttg 1200
aacaaattaa atctccctgg ctgtaatggt ggcagtttgt atgttaacaa acatgcattc 1260
cacaccagtc cctttacccg ggctgccttc gagaatttga agcctatgcc tttcttttat 1320
tattcagata cgccctgtgt gtatatggaa ggcatggaat ctaagcaggt cgattatgtc 1380
ccattgagaa gcgctacatg catcacaaga tgcaatttag gtggcgctgt ttgtttaaaa 1440
catgctgagg agtatcgtga gtaccttgag tcttacaata cggcaaccac agcgggtttt 1500
actttttggg tctataagac ttttgatttt tataaccttt ggaatacttt tactaggctc 1560
caa 1563
<210> 4
<211> 1563
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 4
tgtactacaa atttgtttaa ggattgtagc aggagctatg taggatatca cccagcccat 60
gcaccatcct ttttggcagt tgatgacaaa tataaggtag gcggtgattt agccgtttgc 120
cttaatgttg ctgattctgc tgtcacttat tcgcggctta tatcactcat gggattcaag 180
cttgacttga cccttgatgg ttattgtaag ctgtttataa ctagagatga agctatcaaa 240
cgtgttagag cctgggttgg cttcgatgca gaaggtgccc atgcgatacg tgatagcatt 300
gggacaaatt tcccattaca attaggcttt tcgactggaa ttgattttgt tgtcgaagcc 360
actggaatgt ttgctgagag agatggttat gtctttaaaa aggcagccgc acgagctcct 420
cctggcgaac aatttaaaca ccttatccca cttatgtcaa gagggcagaa atgggatgtg 480
gttcgcatta gaatagtaca aatgttgtca gaccacctag tggatttggc agacagtgtt 540
gtacttgtga cgtgggctgc cagctttgag ctcacatgtt tgcgatattt cgctaaagtt 600
ggaagagaag ttgtgtgtag tgtctgcacc aagcgtgcga catgttttaa ttctagaact 660
ggatactatg gatgctggcg acatagttat tcctgtgatt acctgtacaa cccactaata 720
gttgacattc aacagtgggg atatacagga tctttaacta gcaatcatga tcctatttgc 780
agcgtgcata agggtgctca tgttgcatca tctgatgcta tcatgacccg gtgtctagct 840
gttcatgatt gcttttgtaa gtctgttaat tggaatttag aataccccat tatttcaaat 900
gaggtcagtg ttaatacctc ctgcaggtta ttgcagcgcg taatgtttag ggctgcgatg 960
ctatgcaata ggtatgatgt gtgttatgac attggcaacc ctaaaggtct tgcctgtgtc 1020
aaaggatatg attttaagtt ttatgatgcc tcccctgttg ttaagtctgt taaacagttt 1080
gtttataaat acgaggcaca taaagatcaa tttttagatg gtttgtgtat gttttggaac 1140
tgcaatgtgg ataagtatcc agcgaatgca gttgtgtgta ggtttgacac gcgtgtgttg 1200
aacaaattaa atctccctgg ctgtaatggt ggcagtttgg cagttaacaa acatgcattc 1260
cacaccagtc cctttacccg ggctgccttc gagaatttga agcctatgcc tttcttttat 1320
tattcagata cgccctgtgt gtatatggaa ggcatggaat ctaagcaggt cgattatgtc 1380
ccattgagaa gcgctacatg catcacaaga tgcaatttag gtggcgctgt ttgtttaaaa 1440
catgctgagg agtatcgtga gtaccttgag tcttacaata cggcaaccac agcgggtttt 1500
actttttggg tctataagac ttttgatttt tataaccttt ggaatacttt tactaggctc 1560
caa 1563
<210> 5
<211> 1563
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 5
tgtactacaa atttgtttaa ggattgtagc aggagctatg taggatatca cccagcccat 60
gcaccatcct ttttggcagt tgatgacaaa tataaggtag gcggtgattt agccgtttgc 120
cttaatgttg ctgattctgc tgtcacttat tcgcggctta tatcactcat gggattcaag 180
cttgacttga cccttgatgg ttattgtaag ctgtttataa ctagagatga agctatcaaa 240
cgtgttagag cctgggttgg cttcgatgca gaaggtgccc atgcgatacg tgatagcatt 300
gggacaaatt tcccattaca attaggcttt tcgactggaa ttgattttgt tgtcgaagcc 360
actggaatgt ttgctgagag agatggttat gtctttaaaa aggcagccgc acgagctcct 420
cctggcgaac aatttaaaca ccttatccca cttatgtcaa gagggcagaa atgggatgtg 480
gttcgcatta gaatagtaca aatgttgtca gaccacctag tggatttggc agacagtgtt 540
gtacttgtga cgtgggctgc cagctttgag ctcacatgtt tgcgatattt cgctaaagtt 600
ggaagagaag ttgtgtgtag tgtctgcacc aagcgtgcga catgttttaa ttctagaact 660
ggatactatg gatgctggcg acatagttat tcctgtgatt acctgtacaa cccactaata 720
gttgacattc aacagtgggg atatacagga tctttaacta gcaatcatga tcctatttgc 780
agcgtgcata agggtgctca tgttgcatca tctgatgcta tcatgacccg gtgtctagct 840
gttcatgatt gcttttgtaa gtctgttaat tggaatttag aataccccat tatttcaaat 900
gaggtcagtg ttaatacctc ctgcaggtta ttgcagcgcg taatgtttag ggctgcgatg 960
ctatgcaata ggtatgatgt gtgttatgca attggcaacc ctaaaggtct tgcctgtgtc 1020
aaaggatatg attttaagtt ttatgatgcc tcccctgttg ttaagtctgt taaacagttt 1080
gtttataaat acgaggcaca taaagatcaa tttttagatg gtttgtgtat gttttggaac 1140
tgcaatgtgg ataagtatcc agcgaatgca gttgtgtgta ggtttgacac gcgtgtgttg 1200
aacaaattaa atctccctgg ctgtaatggt ggcagtttgg cagttaacaa acatgcattc 1260
cacaccagtc cctttacccg ggctgccttc gagaatttga agcctatgcc tttcttttat 1320
tattcagata cgccctgtgt gtatatggaa ggcatggaat ctaagcaggt cgattatgtc 1380
ccattgagaa gcgctacatg catcacaaga tgcaatttag gtggcgctgt ttgtttaaaa 1440
catgctgagg agtatcgtga gtaccttgag tcttacaata cggcaaccac agcgggtttt 1500
actttttggg tctataagac ttttgatttt tataaccttt ggaatacttt tactaggctc 1560
caa 1563
<210> 6
<211> 527
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 6
Ala Glu Asn Val Thr Gly Leu Phe Lys Asp Cys Ser Lys Val Ile Thr
1 5 10 15
Gly Leu His Pro Thr Gln Ala Pro Thr His Leu Ser Val Asp Thr Lys
20 25 30
Phe Lys Thr Glu Gly Leu Cys Val Asp Ile Pro Gly Ile Pro Lys Asp
35 40 45
Met Thr Tyr Arg Arg Leu Ile Ser Met Met Gly Phe Lys Met Asn Tyr
50 55 60
Gln Val Asn Gly Tyr Pro Asn Met Phe Ile Thr Arg Glu Glu Ala Ile
65 70 75 80
Arg His Val Arg Ala Trp Ile Gly Phe Asp Val Glu Gly Cys His Ala
85 90 95
Thr Arg Glu Ala Val Gly Thr Asn Leu Pro Leu Gln Leu Gly Phe Ser
100 105 110
Thr Gly Val Asn Leu Val Ala Val Pro Thr Gly Tyr Val Asp Thr Pro
115 120 125
Asn Asn Thr Asp Phe Ser Arg Val Ser Ala Lys Pro Pro Pro Gly Asp
130 135 140
Gln Phe Lys His Leu Ile Pro Leu Met Tyr Lys Gly Leu Pro Trp Asn
145 150 155 160
Val Val Arg Ile Lys Ile Val Gln Met Leu Ser Asp Thr Leu Lys Asn
165 170 175
Leu Ser Asp Arg Val Val Phe Val Leu Trp Ala His Gly Phe Glu Leu
180 185 190
Thr Ser Met Lys Tyr Phe Val Lys Ile Gly Pro Glu Arg Thr Cys Cys
195 200 205
Leu Cys Asp Arg Arg Ala Thr Cys Phe Ser Thr Ala Ser Asp Thr Tyr
210 215 220
Ala Cys Trp His His Ser Ile Gly Phe Asp Tyr Val Tyr Asn Pro Phe
225 230 235 240
Met Ile Asp Val Gln Gln Trp Gly Phe Thr Gly Asn Leu Gln Ser Asn
245 250 255
His Asp Leu Tyr Cys Gln Val His Gly Asn Ala His Val Ala Ser Cys
260 265 270
Asp Ala Ile Met Thr Arg Cys Leu Ala Val His Glu Cys Phe Val Lys
275 280 285
Arg Val Asp Trp Thr Ile Glu Tyr Pro Ile Ile Gly Asp Glu Leu Lys
290 295 300
Ile Asn Ala Ala Cys Arg Lys Val Gln His Met Val Val Lys Ala Ala
305 310 315 320
Leu Leu Ala Asp Lys Phe Pro Val Leu His Asp Ile Gly Asn Pro Lys
325 330 335
Ala Ile Lys Cys Val Pro Gln Ala Asp Val Glu Trp Lys Phe Tyr Asp
340 345 350
Ala Gln Pro Cys Ser Asp Lys Ala Tyr Lys Ile Glu Glu Leu Phe Tyr
355 360 365
Ser Tyr Ala Thr His Ser Asp Lys Phe Thr Asp Gly Val Cys Leu Phe
370 375 380
Trp Asn Cys Asn Val Asp Arg Tyr Pro Ala Asn Ser Ile Val Cys Arg
385 390 395 400
Phe Asp Thr Arg Val Leu Ser Asn Leu Asn Leu Pro Gly Cys Asp Gly
405 410 415
Gly Ser Leu Tyr Val Asn Lys His Ala Phe His Thr Pro Ala Phe Asp
420 425 430
Lys Ser Ala Phe Val Asn Leu Lys Gln Leu Pro Phe Phe Tyr Tyr Ser
435 440 445
Asp Ser Pro Cys Glu Ser His Gly Lys Gln Val Val Ser Asp Ile Asp
450 455 460
Tyr Val Pro Leu Lys Ser Ala Thr Cys Ile Thr Arg Cys Asn Leu Gly
465 470 475 480
Gly Ala Val Cys Arg His His Ala Asn Glu Tyr Arg Leu Tyr Leu Asp
485 490 495
Ala Tyr Asn Met Met Ile Ser Ala Gly Phe Ser Leu Trp Val Tyr Lys
500 505 510
Gln Phe Asp Thr Tyr Asn Leu Trp Asn Thr Phe Thr Arg Leu Gln
515 520 525
<210> 7
<211> 1581
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 7
gcggagaacg tgaccggtct gtttaaggat tgcagcaaag ttatcaccgg cctgcacccg 60
acccaggcgc cgacccacct gagcgtggac accaagttca aaaccgaagg tctgtgcgtt 120
gacatcccgg gcattccgaa agatatgacc taccgtcgtc tgatcagcat gatgggtttt 180
aagatgaact accaagtgaa cggctatccg aacatgttca tcacccgtga ggaagcgatt 240
cgtcacgtgc gtgcgtggat tggttttgat gttgaaggtt gccatgcgac ccgtgaagcg 300
gttggtacca acctgccgct gcagctgggt ttcagcaccg gcgtgaacct ggtggcggtt 360
ccgaccggct acgttgacac cccgaacaac accgatttta gccgtgtgag cgcgaagccg 420
ccgccgggtg accagttcaa acacctgatc ccgctgatgt ataagggcct gccgtggaac 480
gtggttcgta tcaaaattgt tcaaatgctg agcgacaccc tgaagaacct gagcgatcgt 540
gtggtttttg tgctgtgggc gcacggtttc gagctgacca gcatgaagta ctttgttaaa 600
attggcccgg aacgtacctg ctgcctgtgc gaccgtcgtg cgacctgctt cagcaccgcg 660
agcgatacct atgcgtgctg gcaccacagc atcggttttg actacgtgta taacccgttc 720
atgattgatg ttcagcaatg gggttttacc ggcaacctgc agagcaacca cgacctgtac 780
tgccaagtgc acggcaacgc gcacgttgcg agctgcgatg cgatcatgac ccgttgcctg 840
gcggtgcacg agtgcttcgt gaaacgtgtt gactggacca tcgagtaccc gatcattggt 900
gatgaactga agattaacgc ggcgtgccgt aaagtgcagc acatggtggt taaggcggcg 960
ctgctggcgg acaaatttcc ggttctgcac gatatcggca acccgaaggc gattaaatgc 1020
gtgccgcagg cggacgttga atggaaattt tacgacgcgc aaccgtgcag cgataaggcg 1080
tataaaatcg aggaactgtt ctacagctat gcgacccaca gcgacaagtt taccgatggt 1140
gtgtgcctgt tctggaactg caacgttgac cgttacccgg cgaacagcat tgtgtgccgt 1200
ttcgataccc gtgttctgag caacctgaac ctgccgggtt gcgatggtgg cagcctgtat 1260
gtgaacaaac acgcgttcca caccccggcg tttgataaga gcgcgttcgt taacctgaaa 1320
cagctgccgt tcttttacta tagcgacagc ccgtgcgaga gccacggtaa acaagtggtt 1380
agcgacatcg attacgtgcc gctgaagagc gcgacctgca ttacccgttg caacctgggt 1440
ggcgcggttt gccgtcacca cgcgaacgaa taccgtctgt atctggatgc gtataacatg 1500
atgattagcg cgggttttag cctgtgggtt tacaagcagt tcgacaccta taacctgtgg 1560
aacaccttca cccgtctgca a 1581
<210> 8
<211> 1581
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 8
gcggagaacg tgaccggtct gtttaaggat tgcagcaaag ttatcaccgg cctgcacccg 60
acccaggcgc cgacccacct gagcgtggac accaagttca aaaccgaagg tctgtgcgtt 120
gacatcccgg gcattccgaa agatatgacc taccgtcgtc tgatcagcat gatgggtttt 180
aagatgaact accaagtgaa cggctatccg aacatgttca tcacccgtga ggaagcgatt 240
cgtcacgtgc gtgcgtggat tggttttgat gttgaaggtt gccatgcgac ccgtgaagcg 300
gttggtacca acctgccgct gcagctgggt ttcagcaccg gcgtgaacct ggtggcggtt 360
ccgaccggct acgttgacac cccgaacaac accgatttta gccgtgtgag cgcgaagccg 420
ccgccgggtg accagttcaa acacctgatc ccgctgatgt ataagggcct gccgtggaac 480
gtggttcgta tcaaaattgt tcaaatgctg agcgacaccc tgaagaacct gagcgatcgt 540
gtggtttttg tgctgtgggc gcacggtttc gagctgacca gcatgaagta ctttgttaaa 600
attggcccgg aacgtacctg ctgcctgtgc gaccgtcgtg cgacctgctt cagcaccgcg 660
agcgatacct atgcgtgctg gcaccacagc atcggttttg actacgtgta taacccgttc 720
atgattgatg ttcagcaatg gggttttacc ggcaacctgc agagcaacca cgacctgtac 780
tgccaagtgc acggcaacgc gcacgttgcg agctgcgatg cgatcatgac ccgttgcctg 840
gcggtgcacg agtgcttcgt gaaacgtgtt gactggacca tcgagtaccc gatcattggt 900
gatgaactga agattaacgc ggcgtgccgt aaagtgcagc acatggtggt taaggcggcg 960
ctgctggcgg acaaatttcc ggttctgcac gcaatcggca acccgaaggc gattaaatgc 1020
gtgccgcagg cggacgttga atggaaattt tacgacgcgc aaccgtgcag cgataaggcg 1080
tataaaatcg aggaactgtt ctacagctat gcgacccaca gcgacaagtt taccgatggt 1140
gtgtgcctgt tctggaactg caacgttgac cgttacccgg cgaacagcat tgtgtgccgt 1200
ttcgataccc gtgttctgag caacctgaac ctgccgggtt gcgatggtgg cagcctgtat 1260
gtgaacaaac acgcgttcca caccccggcg tttgataaga gcgcgttcgt taacctgaaa 1320
cagctgccgt tcttttacta tagcgacagc ccgtgcgaga gccacggtaa acaagtggtt 1380
agcgacatcg attacgtgcc gctgaagagc gcgacctgca ttacccgttg caacctgggt 1440
ggcgcggttt gccgtcacca cgcgaacgaa taccgtctgt atctggatgc gtataacatg 1500
atgattagcg cgggttttag cctgtgggtt tacaagcagt tcgacaccta taacctgtgg 1560
aacaccttca cccgtctgca a 1581
<210> 9
<211> 1581
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 9
gcggagaacg tgaccggtct gtttaaggat tgcagcaaag ttatcaccgg cctgcacccg 60
acccaggcgc cgacccacct gagcgtggac accaagttca aaaccgaagg tctgtgcgtt 120
gacatcccgg gcattccgaa agatatgacc taccgtcgtc tgatcagcat gatgggtttt 180
aagatgaact accaagtgaa cggctatccg aacatgttca tcacccgtga ggaagcgatt 240
cgtcacgtgc gtgcgtggat tggttttgat gttgaaggtt gccatgcgac ccgtgaagcg 300
gttggtacca acctgccgct gcagctgggt ttcagcaccg gcgtgaacct ggtggcggtt 360
ccgaccggct acgttgacac cccgaacaac accgatttta gccgtgtgag cgcgaagccg 420
ccgccgggtg accagttcaa acacctgatc ccgctgatgt ataagggcct gccgtggaac 480
gtggttcgta tcaaaattgt tcaaatgctg agcgacaccc tgaagaacct gagcgatcgt 540
gtggtttttg tgctgtgggc gcacggtttc gagctgacca gcatgaagta ctttgttaaa 600
attggcccgg aacgtacctg ctgcctgtgc gaccgtcgtg cgacctgctt cagcaccgcg 660
agcgatacct atgcgtgctg gcaccacagc atcggttttg actacgtgta taacccgttc 720
atgattgatg ttcagcaatg gggttttacc ggcaacctgc agagcaacca cgacctgtac 780
tgccaagtgc acggcaacgc gcacgttgcg agctgcgatg cgatcatgac ccgttgcctg 840
gcggtgcacg agtgcttcgt gaaacgtgtt gactggacca tcgagtaccc gatcattggt 900
gatgaactga agattaacgc ggcgtgccgt aaagtgcagc acatggtggt taaggcggcg 960
ctgctggcgg acaaatttcc ggttctgcac gatatcggca acccgaaggc gattaaatgc 1020
gtgccgcagg cggacgttga atggaaattt tacgacgcgc aaccgtgcag cgataaggcg 1080
tataaaatcg aggaactgtt ctacagctat gcgacccaca gcgacaagtt taccgatggt 1140
gtgtgcctgt tctggaactg caacgttgac cgttacccgg cgaacagcat tgtgtgccgt 1200
ttcgataccc gtgttctgag caacctgaac ctgccgggtt gcgatggtgg cagcctggca 1260
gtgaacaaac acgcgttcca caccccggcg tttgataaga gcgcgttcgt taacctgaaa 1320
cagctgccgt tcttttacta tagcgacagc ccgtgcgaga gccacggtaa acaagtggtt 1380
agcgacatcg attacgtgcc gctgaagagc gcgacctgca ttacccgttg caacctgggt 1440
ggcgcggttt gccgtcacca cgcgaacgaa taccgtctgt atctggatgc gtataacatg 1500
atgattagcg cgggttttag cctgtgggtt tacaagcagt tcgacaccta taacctgtgg 1560
aacaccttca cccgtctgca a 1581
<210> 10
<211> 1581
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 10
gcggagaacg tgaccggtct gtttaaggat tgcagcaaag ttatcaccgg cctgcacccg 60
acccaggcgc cgacccacct gagcgtggac accaagttca aaaccgaagg tctgtgcgtt 120
gacatcccgg gcattccgaa agatatgacc taccgtcgtc tgatcagcat gatgggtttt 180
aagatgaact accaagtgaa cggctatccg aacatgttca tcacccgtga ggaagcgatt 240
cgtcacgtgc gtgcgtggat tggttttgat gttgaaggtt gccatgcgac ccgtgaagcg 300
gttggtacca acctgccgct gcagctgggt ttcagcaccg gcgtgaacct ggtggcggtt 360
ccgaccggct acgttgacac cccgaacaac accgatttta gccgtgtgag cgcgaagccg 420
ccgccgggtg accagttcaa acacctgatc ccgctgatgt ataagggcct gccgtggaac 480
gtggttcgta tcaaaattgt tcaaatgctg agcgacaccc tgaagaacct gagcgatcgt 540
gtggtttttg tgctgtgggc gcacggtttc gagctgacca gcatgaagta ctttgttaaa 600
attggcccgg aacgtacctg ctgcctgtgc gaccgtcgtg cgacctgctt cagcaccgcg 660
agcgatacct atgcgtgctg gcaccacagc atcggttttg actacgtgta taacccgttc 720
atgattgatg ttcagcaatg gggttttacc ggcaacctgc agagcaacca cgacctgtac 780
tgccaagtgc acggcaacgc gcacgttgcg agctgcgatg cgatcatgac ccgttgcctg 840
gcggtgcacg agtgcttcgt gaaacgtgtt gactggacca tcgagtaccc gatcattggt 900
gatgaactga agattaacgc ggcgtgccgt aaagtgcagc acatggtggt taaggcggcg 960
ctgctggcgg acaaatttcc ggttctgcac gcaatcggca acccgaaggc gattaaatgc 1020
gtgccgcagg cggacgttga atggaaattt tacgacgcgc aaccgtgcag cgataaggcg 1080
tataaaatcg aggaactgtt ctacagctat gcgacccaca gcgacaagtt taccgatggt 1140
gtgtgcctgt tctggaactg caacgttgac cgttacccgg cgaacagcat tgtgtgccgt 1200
ttcgataccc gtgttctgag caacctgaac ctgccgggtt gcgatggtgg cagcctggca 1260
gtgaacaaac acgcgttcca caccccggcg tttgataaga gcgcgttcgt taacctgaaa 1320
cagctgccgt tcttttacta tagcgacagc ccgtgcgaga gccacggtaa acaagtggtt 1380
agcgacatcg attacgtgcc gctgaagagc gcgacctgca ttacccgttg caacctgggt 1440
ggcgcggttt gccgtcacca cgcgaacgaa taccgtctgt atctggatgc gtataacatg 1500
atgattagcg cgggttttag cctgtgggtt tacaagcagt tcgacaccta taacctgtgg 1560
aacaccttca cccgtctgca a 1581
<210> 11
<211> 30
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 11
cgcggatcct gtactacaaa tttgtttaag 30
<210> 12
<211> 34
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 12
gcgtgtcgac ttattggagc ctagtaaaag tatt 34
<210> 13
<211> 26
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 13
gatgtgtgtt atgcaattgg caaccc 26
<210> 14
<211> 26
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 14
gggttgccaa ttgcataaca cacatc 26
<210> 15
<211> 26
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 15
ggtggcagtt tggcagttaa caaaca 26
<210> 16
<211> 26
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 16
tgtttgttaa ctgccaaact gccacc 26
<210> 17
<211> 29
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 17
cgcggatccg cggagaacgt gaccggtct 29
<210> 18
<211> 32
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 18
ccgctcgagt tattgcagac gggtgaaggt gt 32
<210> 19
<211> 27
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 19
ccggttctgc acgcaatcgg caacccg 27
<210> 20
<211> 27
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 20
cgggttgccg attgcgtgca gaaccgg 27
<210> 21
<211> 27
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 21
ggtggcagcc tggcagtgaa caaacac 27
<210> 22
<211> 27
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 22
gtgtttgttc actgccaggc tgccacc 27
<210> 23
<211> 26
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 23
accaagctaa tgtgttgttt gggtcc 26
<210> 24
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 24
ttacgcacag caaaccaaaa ttcc 24
<210> 25
<211> 38
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 25
tgggttggct tcgcagcagc aggtgcccat gcgatacg 38
<210> 26
<211> 38
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 26
cgcatgggca cctgctgctg cgaagccaac ccaggctc 38

Claims (8)

1. An attenuated strain of an N7-methyltransferase deficient coronavirus obtainable by site-directed mutagenesis of an extremely conserved active site D or Y within the N7-methyltransferase domain of the nonstructural protein nsp14 of a wild-type coronavirus strain, said wild-type coronavirus strain being MHV having the sequence set forth in SEQ ID NO:1 from D to A or from Y to A at position 414.
2. An attenuated vaccine for an N7-methyltransferase deficient coronavirus comprising the attenuated vaccine strain for an N7-methyltransferase deficient coronavirus of claim 1.
3. A method of preparing an attenuated strain of N7-methyltransferase deficient coronavirus as claimed in claim 1, comprising:
transfecting the host cell by using a cloning vector to obtain a transfected host cell;
culturing the transfected host cell to obtain a recombinant virus containing a gene encoding the nsp14 point mutation;
extracting and enzyme cutting the recombinant virus DNA to obtain full-length cDNA containing nsp14 point mutation genes, and obtaining RNA through in vitro transcription;
Transfecting the RNA into a host cell to obtain an N7-methyltransferase defective coronavirus attenuated vaccine strain;
wherein the cloning vector carries a nucleic acid molecule encoding a mutant protein having the following mutations: the extremely conserved active site D or Y within the N7-methyltransferase domain of the nonstructural protein nsp14 is site-directed mutated to A.
4. A method for preparing an attenuated vaccine strain of N7-methyltransferase deficient coronavirus as claimed in claim 3, wherein the mutant protein is:
MHV nsp14 mutant protein: as set forth in SEQ ID NO:1 from aspartic acid D to alanine a or from tyrosine Y to alanine a at amino acid position 330 of the wild-type MHV nsp14 protein.
5. A coronavirus nsp14 mutant protein, comprising site-directed mutagenesis of an extremely conserved active site D or Y within the N7-methyltransferase domain of the nonstructural protein nsp14 of a wild-type coronavirus strain, said coronavirus nsp14 mutant protein being:
MHV nsp14 mutant protein: as set forth in SEQ ID NO:1 from aspartic acid D to alanine a or from tyrosine Y to alanine a at amino acid position 330 of the wild-type MHV nsp14 protein.
6. A nucleic acid molecule encoding the coronavirus nsp14 mutant protein of claim 5, characterized in that said nucleic acid molecule is:
MHV nsp14 mutant nucleic acid molecules: the nucleotide sequence is shown in SEQ ID NO:3 or SEQ ID NO: 4.
7. An expression vector comprising the nucleic acid molecule of claim 6.
8. A biological material, characterized in that it is an engineered cell line or recombinant bacterium comprising the expression vector of claim 7.
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GB201601498D0 (en) * 2016-01-27 2016-03-09 Pirbright Inst The Coronavirus
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