CN117210496A - Novel immunogen of coronavirus and application thereof - Google Patents
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Abstract
The application belongs to the technical field of biology, and discloses a novel immunogen of coronavirus and application thereof. The immunogen of the novel coronavirus comprises a vector and a nucleic acid molecule of a novel coronavirus M protein coding sequence; the M protein coding sequence is inserted into a pVAX1 vector through HindIII and XhoI cleavage sites to form a plasmid pVAX1-M. Pharmaceutical compositions comprising the immunogens of the novel coronaviruses of the present application can be derived from the immunogens. According to the application, the novel coronavirus M protein is taken as an immunogen main component, and an immune experiment shows that the plasmid pVAX1-M can be taken as the immunogen to induce an organism to generate a specific cellular immune response, so that the application has the potential of preparing vaccines and/or medicaments for treating or preventing novel coronavirus infection.
Description
Technical Field
The application belongs to the technical field of biology, and particularly relates to a novel immunogen of coronavirus and application thereof.
Background
The novel coronavirus (severe acute respiratory syndrome coronavirus type 2, SARS-CoV-2) is abbreviated as novel coronavirus, and is a novel respiratory pathogen. SARS-CoV-2 is a beta coronavirus, the genome is single-stranded positive strand RNA, the full length is about 29.9kb, the envelope is round or oval, and the diameter is about 100nm. The novel coronavirus particles mainly comprise 4 structural proteins: spike protein (S), envelope protein (E), membrane protein (M), and nucleocapsid protein (N).
The novel coronavirus has stronger infectivity, and the genes are frequently mutated and recombined in the process of transmission in the crowd, so that the infectivity is enhanced and the immunity is escaped, and breakthrough infection and a certain proportion of reinfection are caused. By the end of 2022, WHO has published 5 VOC strains (variants of interest) in succession, including Alpha (Alpha), beta (Beta), gamma (Gamma), delta (delta), and omicon (omicon). The Omicron almost completely escapes the antibody response induced by the original strain, rapidly replacing the Delta variant strain in the early 2022, becoming a popular strain with absolute global dominance.
The novel coronavirus recognizes and binds host cell surface ACE2 receptor mediated infection via its receptor binding domain (receptor binding domain, RBD) on the S protein, making the S protein or its RBD region a major target for vaccine development. In 2023, china approves the use of 15 new coronavirus vaccines at least, including inactivated vaccine, recombinant protein vaccine, viral vector vaccine and mRNA vaccine 4, wherein other vaccines except the inactivated vaccine all take virus S or RBD region thereof as the effective components of the vaccine. However, viruses can escape vaccine-induced neutralizing antibodies by mutation, significantly reducing the protective effect of the vaccine, resulting in having to re-develop a targeted vaccine after the emergence of new dominant mutants. Thus, it is particularly important to develop vaccines that protect against infection by different mutants, even future mutants.
Disclosure of Invention
The application aims to provide a novel immunogen of coronavirus and application thereof. The novel coronavirus M protein is the most abundant structural protein on the viral envelope and plays a central role in viral assembly and morphogenesis. A large number of researches show that the M-specific CD8T cells exist in the body of the patient in the convalescence period, so that the virus in the body can be removed, and the convalescence is promoted. Furthermore, the M protein sequence is highly conserved, further highlighting its potential as a novel vaccine component. The vaccine based on the M protein can induce human bodies to generate broad-spectrum virus-specific CD8T cells, and has a protective effect on infection of different mutant strains and even future mutant strains.
In order to achieve the above purpose, the present application adopts the following technical scheme:
in a first aspect the application provides a novel coronavirus immunogen comprising a vector and a nucleic acid molecule of a novel coronavirus M protein coding sequence.
In one implementation mode of the application, the amino acid sequence of the M protein is shown as SEQ ID NO. 1. The novel coronavirus M protein is an M protein sequence of OmicrionBA.5, and comprises D3N, Q E and A63T mutation compared with the original strain of Wuhan strain. The latest popular XBB series strains, including XBB.1.5, XBB.1.9, XBB.1.16, XBB.2.3, and the like, all contain the mutations.
In one implementation mode of the application, the coding sequence of the M protein is shown as SEQ ID NO. 2.
In one implementation of the application, the vector is pVAX1.pVAX1 is a eukaryotic expression vector, 3.0kb in size, which is the only vector plasmid recommended by the U.S. Food and Drug Administration (FDA) that can be used in human experiments. The pVAX1 contains CMV enhancer, CMV promoter, T7 promoter, chimeric intron and BGHpoly (A) tailing signal, and the region of the multiple cloning site has HindIII, xhoI and other enzyme cutting sites.
In one embodiment of the application, the M protein coding sequence is inserted into the pVAX1 vector through HindIII and XhoI cleavage sites to form plasmid pVAX1-M.
In one implementation mode of the application, the nucleotide sequence of the plasmid pVAX1-M is shown as SEQ ID NO. 3.
In a second aspect the application provides a pharmaceutical composition comprising an immunogen according to the first aspect of the application.
In one implementation of the application, the pharmaceutical composition further comprises pharmaceutically acceptable adjuvants and/or adjuvants.
In a third aspect the present application provides the use of an immunogen according to the first aspect of the present application, or a pharmaceutical composition according to the second aspect of the present application, in the manufacture of a vaccine and/or medicament for the treatment or prophylaxis of a novel coronavirus infection.
It should be noted that the above specific nucleotide sequence is only a nucleotide sequence specifically employed in one implementation of the present application, and it is understood that there may be a plurality of codons for one amino acid; thus, depending on the degeneracy of the codons, there may be several nucleotide sequences encoding the same protein, in addition to the nucleotide sequences defined above, while ensuring that the coding sequence is unchanged, all within the scope of the present application.
Due to the adoption of the technical scheme, the application has the beneficial effects that:
according to the application, the novel coronavirus M protein is taken as an immunogen main component, and an immune experiment shows that the plasmid pVAX1-M can be taken as the immunogen to induce an organism to generate a specific cellular immune response, so that the application has the potential of preparing vaccines and/or medicaments for treating or preventing novel coronavirus infection.
Drawings
FIG. 1 is a schematic diagram showing the structure of a pVAX1-M recombinant plasmid according to example 1 of the present application.
FIG. 2 shows the results of double enzyme electrophoresis for identifying pVAX1-M recombinant plasmid in example 1 of the present application.
FIG. 3 shows the expression of M protein after transient transfection of 293T cells by flow cytometry in example 2 of the present application.
FIG. 4 shows the results of detection of M-specific cellular immune responses in immunized mice by ELISPot in example 3 of the present application.
Detailed Description
The application will be described in further detail below with reference to the drawings by means of specific embodiments. The following examples are merely illustrative of the present application and should not be construed as limiting the application. Unless otherwise indicated, the instruments and materials used in the examples below are those conventionally used in laboratories, and the technical solutions are conventional in the art.
Example 1: construction and identification of recombinant plasmid pVAX1-M
In this example, pVAX1, into which no foreign gene was inserted, was selected as a vector for the recombinant plasmid pVAX1-M. pVAX1 is a eukaryotic expression vector, 3.0kb in size, which is the only vector plasmid recommended by the U.S. Food and Drug Administration (FDA) that can be used in human experiments. The pVAX1 contains CMV enhancer, CMV promoter, T7 promoter, chimeric intron and BGHpoly (A) tailing signal, and the region of the multiple cloning site has HindIII, xhoI and other enzyme cutting sites. In this example, the M protein coding sequence was inserted into the pVAX1 vector through HindIII and XhoI cleavage sites to construct the recombinant plasmid pVAX1-M shown in FIG. 1.
The construction of the pVAX1-M recombinant plasmid of the embodiment specifically comprises the following steps:
step 1, carrying out codon optimization based on a novel coronavirus M protein sequence (M protein sequence of OmicrionBA.5, which contains D3N, Q E and A63T mutation compared with an original strain of Wuhan strain) shown in SEQ ID NO.1 to obtain an M protein coding sequence shown in SEQ ID NO.2, respectively adding HindIII and XhoI enzyme cleavage sites at two ends of the M protein coding sequence, and carrying out gene synthesis to obtain SARS-CoV-2M gene fragments.
Step 2, performing double enzyme digestion on the SARS-CoV-2M gene fragment and the pVAX1 vector synthesized in the step 1 by using HindIII and XhoI restriction enzymes; after the complete digestion is detected by 1% agarose gel electrophoresis, SARS-CoV-2M target gene fragment with 675bp length and 2920bp pVAX1 carrier skeleton fragment are recovered. Wherein, the operation of recovering the target gene fragment after double enzyme digestion is implemented according to the operation instruction of the gel recovery kit.
Step 3, mixing the SARS-CoV-2M target gene fragment obtained in the step 2 with the pVAX1 vector skeleton fragment, and connecting at 16 ℃ for overnight under the action of T4 ligase to obtain the pVAX1-M connecting product.
And 4, converting the pVAX1-M connection product obtained in the step 3 into competent cells of escherichia coli TOP10, coating the competent cells on an LB plate containing 100 mug/mL kanamycin resistance, and standing and culturing overnight at 37 ℃ to obtain a plurality of single colonies. Wherein the conversion is carried out in a heat shock conversion manner conventional in the art.
Step 5, single colonies in the step 4 are picked and inoculated into LB liquid medium containing 100 mug/mL kanamycin resistance, and shake-cultured overnight at 37 ℃.
And 6, carrying out plasmid extraction operation on the bacterial liquid cultured overnight in the step 5, carrying out double digestion on the extracted pVAX1-M plasmid and pVAX1 vector by using HindIII and XhoI restriction enzymes, and identifying digestion products by using 1% agarose gel electrophoresis. As a result of the identification, the bands of the pVAX1 vector and the M fragment were seen at the expected positions, respectively, as shown in FIG. 2. Wherein the plasmid extraction procedure was performed according to the instructions of the plasmid extraction kit.
And 7, sequencing the plasmid identified in the step 6, wherein the plasmid with the correct sequencing result is the pVAX1-M recombinant plasmid, and the nucleotide sequence of the plasmid is shown as SEQ ID NO. 3.
The source information for the reagents and materials used in this example are shown below:
pVAX1 (Invitrogen, cat# V260-20), hindIII (ThermoFisher, cat# FD 0504), xhoI (ThermoFisher, cat# FD 0694), gel recovery kit (Qiagen, cat# 28704), DNA molecular weight marker (Tiangen, cat# MD 113-02), T4 ligase (Invitrogen, cat# 15224017), TOP10 competent cells (Bio, cat# B528412-0020), plasmid extraction kit (Qiagen, cat# 27206).
Example 2: identification of M protein expression in eukaryotic cells by recombinant plasmid pVAX1-M
In this example, 293T cells were selected as the expression cells, and expression of M protein in 293T cells was examined by flow cytometry after transient transfection of pVAX1-M. The method specifically comprises the following steps:
step 1, 293T cells were cultured according to a protocol of 6X 10 5 The inoculation amount of each hole is inoculated into the hole of a six-hole plate and placed in a carbon dioxide cell incubator at 37 ℃ and 5 percent CO 2 Culturing overnight.
Step 2, when the confluence rate of the expression system in the six-hole plate in the step 1 reaches 70-90%, the method comprises the following steps of2000 kit instructions, pVAX1-M plasmid and pVAX1 vector were transfected into 293T cells, respectively.
And step 3, after 48 hours of transfection, cells are harvested after digestion with 0.25% pancreatin, and are fixed, broken and washed according to the operation instructions of a fixed film breaking kit, and are centrifuged at 600 Xg for 5 minutes.
Step 4, centrifuging, discarding supernatant, re-suspending cells by using M protein rabbit polyclonal antibody (1:200 dilution), and incubating at 4 ℃ for 30 minutes.
Step 5, after washing the cells 2 times by centrifugation, the cells were resuspended with AF488 fluorescence-labeled goat anti-rabbit secondary antibody (1:800 dilution) and incubated at 4℃for 30 minutes in the absence of light.
And 6, after the cells are centrifugally washed for 2 times, the cells are resuspended in PBS, and the expression condition of the M protein is analyzed by a flow cytometer. As shown in FIG. 3, about 30% of the 293T cells were positive for M protein 2 days after transfection with the pVAX1-M plasmid, indicating that the M protein was correctly transcribed and translated in eukaryotic cells.
The source information for the reagents and materials used in this example are shown below:
293T cells (ATCC, cat# CRL-3216),2000 (Invitrogen, cat# 11668-027), 0.25% pancreatin (Gibco, cat# 25200056), immobilized membrane-disrupting reagent (BD, cat# 554714), M protein rabbit polyclonal antibody (proteontech, cat# 28882-1-AP), goat anti-rabbit fluorescent secondary antibody (Invitrogen, cat# A11034).
Example 3: immunogenicity detection of recombinant plasmid pVAX1-M in mice
The immunogenicity of recombinant plasmid pVAX1-M in mice is tested in this example, comprising the steps of:
step 1, respectively transforming recombinant plasmids pVAX1-M and pVAX1 vector plasmids into competent cells of escherichia coli TOP10, plating and culturing overnight, respectively picking single clones, and culturing in 500ml LB culture medium for 18-24 hours.
And 2, respectively extracting recombinant plasmids pVAX1-M and pVAX1 vector plasmids according to the specification of the large endotoxin removal plasmid extraction kit.
Step 3, the recombinant plasmid pVAX1-M is subjected to enzyme digestion identification and sequencing identification by the method of reference example 1.
Step 4, 10 BALB/c test mice are taken, females and animals with the age of about 7 weeks are randomly divided into an experimental group and a control group after inspection and quarantine, and recombinant plasmids pVAX1-M and pVAX1 vector plasmids are respectively immunized.
Step 5, intramuscular injection and electroporation are carried out on the test mice, the electroporation is carried out through a TERESA-EPTI type drug introducer, 100 mug of plasmid is injected each time, 3 times of continuous immunization are carried out, and each time of immunization interval is 3 weeks.
Step 6, killing the mice after 2 weeks of the last immunization, taking spleens, and separating the spleen lymphocytes of the mice according to the reagent specifications of the lymphocyte separating liquid of the mice.
Step 7, taking spleen lymphocytes of mice, stimulating the spleen lymphocytes (each polypeptide concentration is 2 mug/ml) by using an M protein peptide library (each polypeptide is 15 amino acids in length, and 2 adjacent polypeptides are overlapped by 11 amino acids), and detecting the M specific cellular immune response by an ELISPot test. The ELISpot assay was performed according to the mouse IFNg pre-coated ELISpot kit instructions. The test results are shown in FIG. 4, and the average value of the number of the IFN gamma secretion spleen lymphocytes in the experimental group (pVAX 1-M) is significantly higher than that in the control group (pVAX 1), which indicates that the recombinant plasmid pVAX1-M can induce the mice to generate specific cellular immune responses.
The source information for the reagents and materials used in this example are shown below:
TOP10 competent cells (Bio, cat# B528412-0020), endotoxin-free plasmid large extraction reagent (Qiagen, cat# 12381), BALB/c test mice (Guangdong Kangdong Biotechnology Co., ltd.), TERESA-EPTI type drug introducer (Shanghai Taruisha Biotechnology Co., ltd.), mouse lymphocyte separation solution (Daidae, cat# DKW 33-R0100), M protein peptide library (synthesized by Nanjing gold Biotechnology Co., ltd.), mouse IFNg pre-coated ELISPot kit (Daidae, cat# 2210007).
According to the application, the novel coronavirus M protein is taken as an immunogen main component, and an immune experiment shows that the plasmid pVAX1-M can be taken as the immunogen to induce an organism to generate a specific cellular immune response, so that the application has the potential of preparing vaccines and/or medicaments for treating or preventing novel coronavirus infection.
The foregoing is a further detailed description of the application in connection with specific embodiments, and it is not intended that the application be limited to such description. It will be apparent to those skilled in the art that several simple deductions or substitutions can be made without departing from the spirit of the application.
Claims (9)
1. A novel coronavirus immunogen comprising a vector and a nucleic acid molecule of novel coronavirus M protein coding sequence.
2. The immunogen of claim 1, wherein the amino acid sequence of the M protein is shown in SEQ ID No. 1.
3. The immunogen of the novel coronavirus of claim 1, wherein the M protein coding sequence is shown in SEQ ID No. 2.
4. The immunogen of the novel coronavirus of claim 1, wherein the vector is pVAX1.
5. The immunogen of the novel coronavirus of claim 4, wherein the M protein coding sequence is inserted into the pVAX1 vector via HindIII and XhoI cleavage sites to form plasmid pVAX1-M.
6. The immunogen of claim 5, wherein the nucleotide sequence of plasmid pVAX1-M is shown in SEQ ID No. 3.
7. A pharmaceutical composition comprising an immunogen of the novel coronavirus of any one of claims 1 to 6.
8. The pharmaceutical composition of claim 7, further comprising a pharmaceutically acceptable adjuvant and/or adjuvant.
9. Use of an immunogen of a novel coronavirus according to any one of claims 1 to 6, or a pharmaceutical composition according to any one of claims 7 to 8, for the preparation of a vaccine and/or medicament for the treatment or prevention of a novel coronavirus infection.
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