CN117210496A - Novel immunogen of coronavirus and application thereof - Google Patents

Abstract

The invention belongs to the field of biotechnology and discloses an immunogen of a new coronavirus and its application. The immunogen of the novel coronavirus of the present invention includes a vector and a nucleic acid molecule of the novel coronavirus M protein coding sequence; the M protein coding sequence is inserted into the pVAX1 vector through the HindIII and XhoI restriction sites to form plasmid pVAX1-M. Pharmaceutical compositions including the immunogen of the novel coronavirus of the present invention can be derived from the immunogen. The present invention uses the M protein of the new coronavirus as the main component of the immunogen. It is found through immune experiments that the plasmid pVAX1-M, as the immunogen, can induce the body to produce a specific cellular immune response, and has the potential to be used to prepare vaccines for treating or preventing new coronavirus infections. /or the potential of the drug.

Description

A novel coronavirus immunogen and its application

Technical field

The invention belongs to the field of biotechnology, and specifically relates to a novel coronavirus immunogen and its application.

Background technique

The new coronavirus (severe acute respiratory syndrome coronavirus 2, SARS-CoV-2), referred to as the new coronavirus, is a new respiratory pathogen. SARS-CoV-2 is a beta coronavirus with a genome of single-stranded positive-sense RNA, about 29.9kb in length, enveloped, round or oval in shape, and about 100nm in diameter. The new coronavirus particles mainly contain four kinds of structural proteins: spike protein (S protein), envelope protein (E protein), membrane protein (membraneprotein (M protein)) and nucleocapsid protein (nucleocapsid, N protein).

The new coronavirus is highly contagious. During the process of spreading among the population, genes frequently mutate and recombine, leading to increased infectivity and immune escape, causing breakthrough infections and a certain proportion of reinfections. As of the end of 2022, WHO has announced 5 VOC strains (mutants of concern), including Alpha, Beta, Gamma, Delta and Omicron. ). Omicron almost completely escaped the antibody response induced by the original strain and quickly replaced the Delta mutant strain in early 2022, becoming the overwhelmingly dominant epidemic strain in the world.

The new coronavirus recognizes and binds to the ACE2 receptor on the host cell surface through the receptor binding domain (RBD) on its S protein to mediate infection, making the S protein or its RBD region the main target for vaccine development. In 2023, my country has approved the use of at least 15 new coronavirus vaccines, including 4 categories: inactivated vaccines, recombinant protein vaccines, viral vector vaccines and mRNA vaccines. Except for inactivated vaccines, other vaccines are based on the virus S or its RBD region. Vaccine active ingredients. However, the virus can escape the neutralizing antibodies induced by the vaccine through mutation, significantly reducing the protective effect of the vaccine. As a result, when new dominant mutant strains appear, targeted vaccines have to be developed again. Therefore, it is particularly important to develop vaccines that protect against infection by different mutant strains and even future mutant strains.

Contents of the invention

The purpose of the present invention is to provide an immunogen of a new coronavirus and its application. The M protein of the new coronavirus is the most abundant structural protein on the virus envelope and plays a central role in virus assembly and morphogenesis. A large number of studies have found that M-specific CD8 T cells exist in convalescent patients, which can remove viruses from the body and promote recovery. In addition, the M protein sequence is highly conserved, further highlighting its potential as a new vaccine component. Vaccines based on M protein can induce the body to produce a broad spectrum of virus-specific CD8 T cells, which can protect against infection by different mutant strains and even future mutant strains.

In order to achieve the above objects, the present invention adopts the following technical solutions:

The first aspect of the present invention provides an immunogen of a new coronavirus, which includes a vector and a nucleic acid molecule encoding the M protein coding sequence of the new coronavirus.

In one implementation of the present invention, the amino acid sequence of the M protein is shown in SEQ ID NO. 1. The novel coronavirus M protein of the present invention is the M protein sequence of OmicrionBA.5, which contains D3N, Q19E and A63T mutations compared with the original strain Wuhan strain. The latest popular XBB series strains, including XBB.1.5, XBB.1.9, XBB.1.16, XBB.2.3, etc., all contain the above mutations.

In one implementation of the present invention, the M protein coding sequence is shown in SEQ ID NO. 2.

In an implementation manner of the present invention, the vector is pVAX1. pVAX1 is a eukaryotic expression vector with a size of 3.0kb. It is the only vector plasmid recommended by the U.S. Food and Drug Administration (FDA) that can be used in human experiments. pVAX1 contains CMV enhancer, CMV promoter, T7 promoter, chimeric intron and BGHpoly(A) tailing signal, and its multiple cloning site region has HindIII and XhoI enzyme cutting sites.

In one implementation of the present invention, the M protein coding sequence is inserted into the pVAX1 vector through HindIII and XhoI restriction sites to form plasmid pVAX1-M.

In one implementation of the present invention, the nucleotide sequence of the plasmid pVAX1-M is shown in SEQ ID NO. 3.

A second aspect of the present invention provides a pharmaceutical composition comprising the immunogen described in the first aspect of the present invention.

In one implementation of the present invention, the pharmaceutical composition further includes pharmaceutically acceptable adjuvants and/or excipients.

The third aspect of the present invention provides the immunogen described in the first aspect of the present invention, or the pharmaceutical composition described in the second aspect of the present invention in the preparation of vaccines and/or medicines for treating or preventing novel coronavirus infection. application.

It should be noted that the above specific nucleotide sequence is only a nucleotide sequence specifically used in one implementation of the present invention. It can be understood that there can be multiple codons for one amino acid; therefore, according to the codon sequence Degeneracy, while ensuring that the coding sequence remains unchanged, in addition to the nucleotide sequences defined above, there can also be several nucleotide sequences encoding the same protein, which are all within the scope of the present invention.

Due to the adoption of the above technical solutions, the beneficial effects of the present invention are:

The present invention uses the M protein of the new coronavirus as the main component of the immunogen. It is found through immune experiments that the plasmid pVAX1-M, as the immunogen, can induce the body to produce a specific cellular immune response, and has the potential to prepare vaccines for treating or preventing new coronavirus infections. /or the potential of the drug.

Description of drawings

Figure 1 is a schematic structural diagram of the pVAX1-M recombinant plasmid in Example 1 of the present invention.

Figure 2 is the result of identification of pVAX1-M recombinant plasmid by double enzyme digestion electrophoresis in Example 1 of the present invention.

Figure 3 is a flow cytometric analysis of M protein expression after transient transfection of 293T cells in Example 2 of the present invention.

Figure 4 is the result of detecting M-specific cellular immune response in immunized mice by ELISpot in Example 3 of the present invention.

Detailed ways

The present invention will be further described in detail below through specific embodiments in conjunction with the accompanying drawings. The following examples only further illustrate the present invention and should not be construed as limitations of the present invention. Unless otherwise specified, the instruments and materials used in the following examples are all equipment commonly used in laboratories, and the technical solutions described are conventional techniques in this field.

Example 1: Construction and identification of recombinant plasmid pVAX1-M

In this example, pVAX1 with no foreign gene inserted was selected as the vector of the recombinant plasmid pVAX1-M. pVAX1 is a eukaryotic expression vector with a size of 3.0kb. It is the only vector plasmid recommended by the U.S. Food and Drug Administration (FDA) that can be used in human experiments. pVAX1 contains CMV enhancer, CMV promoter, T7 promoter, chimeric intron and BGHpoly(A) tailing signal, and its multiple cloning site region has HindIII and XhoI enzyme cutting sites. In this example, the M protein coding sequence was inserted into the pVAX1 vector through the HindIII and XhoI restriction sites to construct the recombinant plasmid pVAX1-M as shown in Figure 1.

The construction of pVAX1-M recombinant plasmid in this example specifically includes the following steps:

Step 1. Codon optimization is performed based on the M protein sequence of the new coronavirus shown in SEQ ID NO.1 (the M protein sequence of OmicrionBA.5, which contains D3N, Q19E and A63T mutations compared with the original strain Wuhan strain), Obtain the M protein coding sequence as shown in SEQ IDNO.2, add HindIII and XhoI restriction sites at both ends of the M protein coding sequence, and then perform gene synthesis to obtain the SARS-CoV-2M gene fragment.

Step 2. Use HindIII and XhoI restriction endonucleases to double-digest the SARS-CoV-2M gene fragment and pVAX1 vector synthesized in step 1; use 1% agarose gel electrophoresis to detect that the enzyme digestion is complete. The gel recovered a 675bp SARS-CoV-2M target gene fragment and a 2920bp pVAX1 vector backbone fragment. Among them, the operation of recovering the target gene fragment after double enzyme digestion was carried out according to the instructions of the gel recovery kit.

Step 3. Mix the SARS-CoV-2M target gene fragment obtained in Step 2 and the pVAX1 vector backbone fragment, and ligate overnight at 16°C under the action of T4 ligase to obtain the pVAX1-M ligation product.

Step 4. Transform the pVAX1-M ligation product obtained in step 3 into E. coli TOP10 competent cells, spread it on an LB plate containing 100 μg/mL kanamycin resistance, and incubate it overnight at 37°C to obtain multiple cells. A single colony. Among them, the transformation is carried out according to the conventional heat shock transformation method in this field.

Step 5: Pick a single colony from step 4 and inoculate it into LB liquid medium containing 100 μg/mL kanamycin resistance, and culture it overnight at 37°C with shaking.

Step 6. Extract the plasmid from the bacteria cultured overnight in step 5. Use HindIII and XhoI restriction endonucleases to double-digest the extracted pVAX1-M plasmid and pVAX1 vector respectively, and use 1% agarose gel. Identification of enzyme digestion products by electrophoresis. The identification results are shown in Figure 2. The bands of pVAX1 vector and M fragment can be seen at the expected positions respectively. The plasmid extraction operation was performed according to the plasmid extraction kit operating instructions.

Step 7. Sequence the plasmid identified correctly in step 6. The plasmid with correct sequencing results is the pVAX1-M recombinant plasmid, and its nucleotide sequence is shown in SEQ ID NO.3.

The source information of the reagents and materials used in this example is as follows:

pVAX1 (Invitrogen, Cat. No. V260-20), HindIII (ThermoFisher, Cat. No. FD0504), XhoI (ThermoFisher, Cat. No. FD0694), gel recovery kit (Qiagen, Cat. No. 28704), DNA molecular weight marker (Tiangen Biotech, Cat. No. MD113-02) , T4 ligase (Invitrogen, Cat. No. 15224017), TOP10 competent cells (Sangon Biotech, Cat. No. B528412-0020), plasmid extraction kit (Qiagen, Cat. No. 27206).

Example 2: Identification of M protein expression of recombinant plasmid pVAX1-M in eukaryotic cells

In this example, 293T cells were selected as expression cells. After transient transfection with pVAX1-M, the expression of M protein in 293T cells was detected by flow cytometry. Specifically, it includes the following steps:

Step 1. Inoculate 293T cells into the wells of a six-well plate at an inoculation volume of 6×10 ⁵ cells/well, place them in a carbon dioxide cell incubator, and culture overnight at 37°C and 5% CO ₂ .

Step 2. When the confluence rate of the expression system in the wells of the six-well plate in step 1 reaches 70-90%, proceed as follows According to the operating instructions of the 2000 kit, pVAX1-M plasmid and pVAX1 vector are transfected into 293T cells respectively.

Step 3. 48 hours after transfection, harvest the cells after digestion with 0.25% trypsin. Fix, rupture and wash the cells according to the instructions of the fixation and rupture kit, and centrifuge at 600×g for 5 minutes.

Step 4. Discard the supernatant after centrifugation, resuspend the cells with M protein rabbit polyclonal antibody (1:200 dilution), and incubate at 4°C for 30 minutes.

Step 5. After centrifuging and washing the cells twice, resuspend the cells with AF488 fluorescently labeled goat anti-rabbit secondary antibody (1:800 dilution), and incubate at 4°C in the dark for 30 minutes.

Step 6: After centrifuging and washing the cells twice, resuspend the cells in PBS and analyze the M protein expression with flow cytometry. The results are shown in Figure 3. Two days after transfection with pVAX1-M plasmid, approximately 30% of 293T cells were positive for M protein, indicating that M protein can be correctly transcribed and translated in eukaryotic cells.

The source information of the reagents and materials used in this example is as follows:

293T cells (ATCC, Cat. No. CRL-3216), 2000 (Invitrogen, Cat. No. 11668-027), 0.25% trypsin (Gibco, Cat. No. 25200056), fixed membrane-breaking reagent (BD, Cat. No. 554714), M protein rabbit polyclonal antibody (proteintech, Cat. No. 28882-1-AP), goat antibody Rabbit fluorescent secondary antibody (Invitrogen, Cat. No. A11034).

Example 3: Immunogenicity detection of recombinant plasmid pVAX1-M in mice

This example tests the immunogenicity of the recombinant plasmid pVAX1-M in mice, specifically including the following steps:

Step 1. Take the recombinant plasmid pVAX1-M and pVAX1 vector plasmid and transform them into E. coli TOP10 competent cells respectively. Plate them and culture them overnight. Pick individual clones and culture them in 500 ml LB medium for 18 to 24 hours.

Step 2. Extract the recombinant plasmid pVAX1-M and pVAX1 vector plasmid respectively according to the instructions of the endotoxin-removing plasmid maximal extraction kit.

Step 3. Refer to the method in Example 1 to perform restriction enzyme digestion and sequencing identification on the recombinant plasmid pVAX1-M.

Step 4. Take 10 BALB/c test mice, female, about 7 weeks old. After inspection and quarantine, the animals are randomly divided into experimental groups and control groups, and immunized with the recombinant plasmid pVAX1-M and pVAX1 vector plasmid respectively.

Step 5: Immunize the test mice with intramuscular injection + electroporation. The electroporation is implemented through a TERESA-EPTI drug introduction device. Each injection of 100 μg of plasmid is performed for 3 consecutive immunizations, with an interval of 3 weeks between each immunization.

Step 6: Kill the mice 2 weeks after the last immunization, remove the spleen, and isolate mouse spleen lymphocytes according to the instructions of the mouse lymphocyte separation solution reagent.

Step 7. Take mouse spleen lymphocytes, use the M protein peptide library (each polypeptide is 15 amino acids in length, and two adjacent polypeptides overlap by 11 amino acids) to stimulate the spleen lymphocytes (each polypeptide concentration is 2 μg/ml), and use ELISpot The test detects M-specific cellular immune responses. The ELISpot test was performed according to the instructions of the mouse IFNg pre-coated ELISpot kit. The test results are shown in Figure 4. The average number of spleen lymphocytes secreting IFNγ in the experimental group (pVAX1-M) was significantly higher than that in the control group (pVAX1), indicating that the recombinant plasmid pVAX1-M can induce specific cellular immune responses in mice.

The source information of the reagents and materials used in this example is as follows:

TOP10 competent cells (Sangon Biotech, Cat. No. B528412-0020), endotoxin-removing plasmid maximal extraction reagent (Qiagen, Cat. No. 12381), BALB/c test mice (Guangdong Yaokang Biotechnology Co., Ltd.), TERESA-EPTI type drug Introduction instrument (Shanghai Tarisa Biotechnology Co., Ltd.), mouse lymphocyte isolation medium (Dakewei, Cat. No. DKW33-R0100), M protein peptide library (synthesized by Nanjing Genscript Biotechnology Co., Ltd.), mouse IFNg Precoated ELISpot kit (Dakewi, Cat. No. 2210007).

The present invention uses the M protein of the new coronavirus as the main component of the immunogen. It is found through immune experiments that the plasmid pVAX1-M, as the immunogen, can induce the body to produce a specific cellular immune response, and has the potential to prepare vaccines for treating or preventing new coronavirus infections. /or the potential of the drug.

The above content is a further detailed description of the present application in combination with specific implementation modes, and it cannot be concluded that the specific implementation of the present application is limited to these descriptions. For those of ordinary skill in the technical field to which this application belongs, several simple deductions or substitutions can be made without departing from the concept of this 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.