CN112618707B

CN112618707B - SARS-CoV-2 coronavirus vaccine and its preparation method

Info

Publication number: CN112618707B
Application number: CN202011103983.3A
Authority: CN
Inventors: 黄文林; 田烁; 周晓鸿
Original assignee: Guangzhou Doublink Biological Products Co
Current assignee: Guangzhou Doublink Biological Products Co
Priority date: 2020-10-15
Filing date: 2020-10-15
Publication date: 2023-07-04
Anticipated expiration: 2040-10-15
Also published as: CN112618707A; WO2022077593A1

Abstract

The invention discloses a vaccine of SARS-CoV-2 coronavirus and its preparing process, wherein the S gene of SARS-CoV-2 coronavirus is codon optimized, the truncated and mutant sequences of the S gene are led into secretory defective adenovirus carrier, and the corresponding recombinant adenovirus is obtained through packaging. The recombinant adenovirus can express SARS-CoV-2 virus related protein in vivo, and can complete modification such as processing, folding, glycosylation, etc., basically maintain the natural conformation of S protein, and has the characteristics of high biological activity, long half-life, durable immunogenicity, etc. The defective adenovirus vector carrying the secretion peptide adopted by the product can enable the recombinant adenovirus vaccine to be secreted outside cells after being expressed in vivo, thereby activating humoral immunity.

Description

SARS-CoV-2 coronavirus vaccine and its preparation method

Technical Field

The invention belongs to the field of biological genetic engineering, and in particular relates to a SARS-CoV-2 coronavirus vaccine.

Background

The novel coronavirus has extremely strong transmission ability in humans and has a transmission index R0 of about 2.5. The virus is mainly transmitted to infected people through respiratory tract or contact, can cause acute pneumonic lesions, and can cause serious damage to other systems such as urinary system, digestive system and nervous system, and no effective drug treatment means exists at present. Therefore, it is very important to design and develop a vaccine against the virus effectively.

In addition, SARS-CoV-2 coronavirus is a single-stranded RNA virus, which is very unstable and is subject to immune supervision and rejection in somatic cells, and is extremely prone to mutation. Therefore, the vaccine designed according to the antigenic determinant with the relative conserved sequence can induce to generate a relatively stable antibody, effectively reduce the failure phenomenon of the vaccine caused by virus mutation, and achieve the effect of long-term protection.

Different scientists worldwide have isolated several strains of coronavirus, and the analysis result of the viral gene sequence suggests that the DNA sequence of the novel coronavirus has 80% homology with the genes of the novel coronavirus and MERS, wherein the homology of spike protein genes and the novel coronavirus reaches 74.2%, the RBD region of spike protein and angiotensin transferase receptor 2 have 4 binding domains, and the World Health Organization (WHO) has been published for 10 days in 1 month, and the pathogen causing the pneumonia of the novel coronavirus is a coronavirus subtype variant. SARS-CoV-2 virus is a non-segmented single-stranded positive-strand RNA virus, and has a molecular weight of up to 32kb and consists of 5 genomes. SARS-CoV-2 virus particles are about 100nm in size and have a plurality of sparse coryneform proteins on their surface. The electron microscope photograph shows that the virus shape is like a coronal shape, and belongs to coronaviruses. The genome of SARS-CoV-2 virus mainly encodes four structural proteins, spike protein (S protein), nucleoprotein (N protein), membrane protein (M protein), and envelope protein (E protein). The result of serum antibody detection for SARS-CoV-2 virus infected population shows that there are a large amount of antibodies for S protein and N protein in serum of new coronaries in convalescence. Wherein the spike protein/S protein is a membrane-penetrating glycoprotein and contains important virus neutralization epitopes. The SARS-CoV-2 virus S protein, when bound to a host cell receptor (e.g., angiotensin converting enzyme 2[angiotensin I coverting enzyme,ACE2), mediates viral invasion by endocytosis. The related scholars have proved through experiments that the S protein is combined with ACE2 receptor mainly through RBD region. Structural biology studies have shown that this region has 4 ACE2 binding sites. The binding domain is structurally conserved and is also the dominant antigen region for the generation of immunologically neutralizing antibodies.

Isolation of SARS-CoV-2 virus provides the original material for vaccine development. Currently, there are mainly several types of inactivated, subunit and DNA vaccines in development or entry into the clinical stage. The inactivated vaccine is easy to prepare in technical angle, can efficiently induce humoral immunity, and is the most classical vaccine form. However, the mass production process of inactivated vaccines may present a risk of leakage. Furthermore, inactivated vaccines against SARS-CoV-2 virus may produce virus-induced reactivation in immunized humans, causing localized pandemics. In addition, the cellular immune response caused by the inactivated vaccine against SARS and MERS in the early stage is low, and researches show that the MERS inactivated vaccine causes allergic pathological reaction in the lung of mice. In addition, vaccinia virus carrying the full length of the S gene, in rhesus challenge experiments, instead exacerbates lung injury. Meanwhile, many studies have also shown that the full-length S gene may cause a serious antibody-enhancing effect (ADE, antibody dependent enhancement). However, in the novel vaccine route, researchers can utilize bioinformatics analysis and combine technical methods such as molecular biology, molecular cloning, reverse genetic recoding, immunology and the like to screen out vaccines with strong immunogenicity and good biosafety. Furthermore, vaccines constructed with adenovirus vectors also have the potential to induce a combined humoral and cellular immune response, thereby improving the protective capacity of the vaccine. Thus, the development of new vaccines facilitates a comprehensive assessment of different vaccine routes and helps us choose the optimal vaccine from a safety and effectiveness perspective.

Adenoviruses are a class of non-integrating viruses with high affinity for the upper respiratory tract. It is able to enter epithelial cells via CAR receptor (coxsackie/adenovirus receptor ) and is more likely to elicit respiratory mucosal immune responses, producing IgM antibodies. In addition, adenovirus with E1 and E3 genes deleted cannot be amplified in ordinary cells and can be used as a carrier carrying macromolecules.

An ebola virus vaccine based on chimpanzee adenovirus vectors is disclosed in chinese patent 201610696322.3. The replication-defective chimpanzee adenovirus vectors disclosed therein include engineered chimpanzee adenovirus AdC68 genomic sequences in which E1 is deleted. The chimpanzee adenovirus adopted by the patent has high immunogenicity, can induce specific humoral immune response and specific cellular immune response, and is not influenced by pre-existing neutralizing antibodies against human serum adenovirus in human body, thus being an ideal vaccine carrier.

Chinese patent 201811262788.8 discloses an adenovirus vector system and a recombinant adenovirus construction method, which comprises the following steps: PCR amplification to obtain target gene fragment with homologous overlapping region on two sides, DNA assembly with adenovirus plasmid linearized by PmeI to obtain adenovirus plasmid with exogenous target gene; or cloning a plurality of gene fragments into a shuttle plasmid, cutting fragments containing all target genes by using restriction endonuclease, and carrying out DNA assembly with the PmeI-linearized pKAd5f11pES-PmeI to obtain adenovirus plasmid containing exogenous target genes.

There is no report on the preparation of S protein truncated vaccine against SARS-CoV-2 virus by adenovirus vector.

Disclosure of Invention

The applicant analyzed each genome of SARS-CoV-2 virus by the result of SARS-CoV-2 virus gene sequencing, and cloned the codon-optimized Spike gene sequence (Spike) into the shuttle plasmid. The plasmid with spike protein gene and adenovirus skeleton plasmid are transfected into 293 cell for plaque selection. After visual plaque selection, cells were lysed and transfected into 293 cells of pre-cultured 24 well plates and expanded to 1X 10 ⁶ The pfu/mL concentration is identified by PCR and sequencing, an adenovirus 5 type carrier carrying SARS-CoV-2 spike gene is constructed, and the recombinant adenovirus carrier vaccine aiming at SARS-CoV-2 is obtained by amplifying, purifying and cross-protecting test.

The invention optimizes the codon of S gene of SARS-CoV-2 coronavirus, introduces the truncated body or mutant sequence of S gene into secretion type defective adenovirus vector, and packages to obtain the corresponding recombinant adenovirus. The recombinant adenovirus can express different truncations or mutants of SARS-CoV-2 virus S protein in vivo, complete modification such as processing, folding, glycosylation and the like, basically maintain the natural conformation of the S protein, and has the characteristics of high biological activity, long half-life, lasting immunogenicity and the like. In addition, the product adopts a defective adenovirus vector carrying secretory peptide. Thus, the recombinant adenovirus vaccine can be secreted outside cells after in vivo expression, thereby activating humoral immunity.

In one aspect, the invention provides a SARS-CoV-2 coronavirus vaccine.

The SARS-CoV-2 coronavirus vaccine comprises a fragment from the S gene of SARS-CoV-2 coronavirus and a replication defective adenovirus sequence.

The SARS-CoV-2 coronavirus S gene fragment is the full-length sequence or truncated sequence of the S gene after wild type or codon optimization or the mutant sequence thereof; the full-length sequence of the SARS-CoV-2 coronavirus S gene is SEQ ID NO:9.

the truncated sequence of SARS-CoV-2 coronavirus S gene includes full-length sequence or partial sequence of S1 and/or S2 structural domain of S gene.

The truncated sequence of SARS-CoV-2 coronavirus S gene includes one or several of S1 gene N-terminal sequence, S1 gene C-terminal sequence, S1 gene intermediate fragment sequence, S2 gene N-terminal sequence, S2 gene C-terminal sequence and S2 gene intermediate fragment sequence.

The fragment from SARS-CoV-2 coronavirus S gene includes the sequence of S gene with amino acid number 319-685.

The replication defective adenovirus is a type 5 adenovirus of a C subclass in which an E1 and/or E3 region is completely deleted and/or partially deleted.

Preferably, the replication defective adenovirus is an adenovirus type 5 of a C subclass in which the E3 region is completely deleted, an adenovirus type 5 of a C subclass in which the E3 region is partially deleted, an adenovirus type 5 of a C subclass in which the E1 region is completely deleted, or an adenovirus type 5 of a C subclass in which the E1 region is partially deleted.

Further, the replication defective adenovirus is internally provided with a secretory peptide sequence.

Further, the replication defective adenovirus is internally provided with a CMV promoter and a BGH gene polyA sequence.

Preferably, the vaccine also comprises thymus pentapeptide; the adding amount of the thymus pentapeptide in the vaccine is 1-2mg/mL, preferably 1.6mg/mL.

As some preferred embodiments, the SARS-CoV-2 coronavirus vaccine comprises a fragment from the S gene of SARS-CoV-2 coronavirus; the segment of the SARS-CoV-2 coronavirus S gene is the 1 st-2055 th gene segment, the 1 st-1827 th gene segment, the 955 th-2055 th gene segment or the 955 th-1827 th gene segment of the full-length sequence of the SARS-CoV-2 coronavirus S gene; or the 454 th amino acid of S protein of S gene aiming at the above gene fragment is mutated from R to A and/or the 466 rd amino acid is mutated from R to A.

On the other hand, the present invention also provides a preparation method of SARS-CoV-2 coronavirus vaccine.

The preparation method comprises the following steps:

(1) Obtaining the sequence of S gene from SARS-CoV-2 coronavirus;

(2) Recombinant binding of the sequence of the S gene from SARS-CoV-2 coronavirus to the defective adenovirus;

(3) Transfecting the packaging cells;

(4) Amplifying, separating and purifying to obtain the final product.

The sequence of S gene from SARS-CoV-2 coronavirus in step (1) is full-length or partial sequence of S gene.

The sequence of the S gene from SARS-CoV-2 coronavirus is obtained by PCR; the PCR primer is V1-V4, wherein the sequence of V1 is SEQ ID NO:1, v2 is SEQ ID NO:2, V3 is SEQ ID NO:3, v4 is SEQ ID NO:4.

V1(SEQ ID NO：1)：TCCCCCGGGATGTTCGTCTTCCTGGTCCT

V2(SEQ ID NO：2)：TCCCCCGGGATGAGGGTGCAGCCAACCGAG

V3(SEQ ID NO：3)：CCCAAGCTTTTAGGCCACCTGGTTGCTTGTAT

V4(SEQ ID NO：4)：CCCAAGCTTTTACCGGGCTCTTCTGGGAGAGT

the underlined portion of each primer is the cleavage site. The cleavage sites of V1 and V2 are SmaI, and the cleavage sites of V3 and V4 are HindIII.

V1 and V4 are a pair of primers, and the amplified S1 gene is named Ad/S1, and is 1-2055 of the S gene.

V1 and V3 are a pair, and the N-terminal fragment of the S1 gene is amplified and named Ad/S1N, and is 1 st-1827 th site of the S gene.

V2 and V4 are a pair of primers, and the C-terminal fragment of the S1 gene is amplified, named Ad/S1C, and is 955-2055 of the S gene.

V2 and V3 are a pair of primers, and the intermediate fragment of the S1 gene, named Ad/S1M, is the 955 th-1827 th site of the S gene.

The fragment amplification schematic of the S gene is shown in FIG. 1.

Further, according to the earlier research result of SARS vaccine, designing mutation primer to reduce possible risk of SARS-CoV-2, the mutation primer is V5-V8, wherein the sequence of V5 is SEQ ID NO: 5, V6 is SEQ ID NO:6, v7 is SEQ ID NO:7, v8 is SEQ ID NO:8.

in the mutation primer, V5 and V6 are a pair, and the 454 th amino acid of the S gene can be mutated from R to A; v7 and V8 are a pair, and the 466 th amino acid of the S gene can be mutated from R to A; the mutation primers were used to mutate Ad/S1, ad/S1N, ad/S1C and Ad/S1M, respectively.

Preferably, the step (2) is: the sequence of S gene from SARS-CoV-2 coronavirus was cloned into pShuttle plasmid and recombined with adenovirus backbone plasmid.

Preferably, the adenovirus backbone plasmid is pBHGlox (delta) E1,3Cre.

Preferably, the step (2) is: the purified target S gene fragment is connected to pShuttle plasmid after being cut by SmaI/HindIII, transformed and screened by ampicillin resistance, and then recombined and combined with adenovirus skeleton plasmid pBHGlox (delta) E1,3Cre in HEK293 cells.

Further, the packaging cell in the step (3) is a cell line or a cell strain integrated with a gene of an E1 region of a subtype C5 adenovirus (Ad 5), preferably HEK293 cells integrated with a gene of an E1 region of a subtype C5 adenovirus (Ad 5).

Specifically, the purification method in the step (4) is plaque purification.

Preferred vaccine preparation techniques of the invention are illustrated in circuit form in figure 2.

Drawings

FIG. 1 is a schematic diagram of fragment amplification of S gene.

Figure 2 is a technical scheme of the vaccine preparation of the present invention.

FIG. 3 shows the sequencing result of the recombinant adenovirus of the step (3) of example 1.

FIG. 4 shows the sequencing result of the recombinant adenovirus of the step (3) of example 2.

FIG. 5 shows the sequencing result of the recombinant adenovirus of the step (3) of example 3.

FIG. 6 shows the sequencing result of the recombinant adenovirus of the step (3) of example 4.

FIG. 7 shows the results of RT-PCR detection of SARS-CoV-2 vaccine prepared in examples 1-12.

FIG. 8 shows the results of cross-reaction of the expressed antigen of each SARS-CoV-2 vaccine with the serum of patients with convalescence of new coronaries.

FIG. 9 shows the results of cross-reaction of mutant expressed antigens of each SARS-CoV-2 vaccine with serum of patients with convalescence of new coronavirus.

FIG. 10 shows the results of the animal immune neutralizing antibody titer detection for SARS-CoV-2 vaccine.

FIG. 11 shows the results of animal immune specific antibody titer detection for SARS-CoV-2 vaccine.

Detailed Description

The present invention will be described in further detail with reference to the following examples, which are not intended to limit the present invention, but are merely illustrative of the present invention. The experimental methods used in the following examples are not specifically described, but the experimental methods in which specific conditions are not specified in the examples are generally carried out under conventional conditions, and the materials, reagents, etc. used in the following examples are commercially available unless otherwise specified.

In the following examples:

pShuttle vector was purchased from Microbix Biosystems under the accession number PD-01-64 with viral backbone plasmid pBHGlox (delta) E1,3Cre.

293 cells were purchased from ATCC under the designation CRL-1573.

Balb/C mice were purchased from the laboratory animal center in Guangdong province.

Basic example optimization of S Gene of SARS-CoV-2 coronavirus

Optimizing S gene of SARS-CoV-2 coronavirus, and the sequence of the optimized S gene is SEQ ID NO:9, carrying out total gene synthesis on the gene, and taking the total gene as an amplification template for subsequent vaccine construction.

EXAMPLE 1 construction of SARS-CoV-2 coronavirus vaccine Ad/S1

(1) Amplification of S Gene fragment:

the S gene obtained in the basic example was used as a template and amplified with the following primers:

V1(SEQ ID NO：1)：TCCCCCGGGATGTTCGTCTTCCTGGTCCT

V4(SEQ ID NO：4)：CCCAAGCTTTTACCGGGCTCTTCTGGGAGAGT

the S1 gene, i.e., positions 1-2055 of the S gene of the basic example, was amplified, and the amplified product was designated as S1.

(2) Constructing a recombinant plasmid:

the S1 fragment of the PCR amplification product obtained above is subjected to gel running identification and gel cutting recovery, and then subjected to enzyme cutting by SmaI and HindIII under the condition of 37 ℃. The pShuttle was simultaneously digested with both enzymes. The digested PCR product was then ligated with pShuttle overnight at 16℃using T4 ligase. The ligation product was transformed into E.coli DH 5. Alpha. And positive clones were screened using ampicillin resistance and selected for colony PCR identification. The positive colonies were cultured and plasmids were extracted.

(3) Co-transfection:

the sequenced recombinant plasmid was co-transfected into 293 cells with viral backbone plasmids pBHGlox (delta) E1,3Cre to package recombinant adenovirus. The virus is collected by adopting a plaque-picking mode: the addition of low melting point agarose to the culture medium, small plaques were visible under the microscope, typically on days 10-21 after transfection. After plaque formation, the plaques were picked up with agarose and placed in 1mL fresh medium overnight. Typically 3-6 plaques are picked and then compared for titer, and the one plaque with the highest titer is used for subsequent experiments. The virus in the culture medium is added into fresh 293 cell culture liquid to carry out small-scale virus amplification. And (3) collecting cells and supernatant until the cells are subjected to plaque again, repeatedly freezing and thawing for three times to collect viruses, taking the viruses as P1 generation viruses, infecting 293 cells with the P1 generation viruses, continuously carrying out third generation infection, carrying out mass amplification of the viruses until the P4 generation viruses, collecting the viruses after the plaque is formed, and carrying out in vitro purification and concentration on the viruses. The virus obtained after purification is SARS-CoV-2 vaccine. The SARS-CoV-2 vaccine comprises SARS-CoV-2 virus S gene and defective adenovirus. Defective adenovirus is type 5 adenovirus of the C subclass with the E1 region completely deleted, and cannot replicate in common human cells; the recombinant adenovirus is named as Ad/S1 vaccine, and the inserted gene is sequenced, and the sequencing result is shown in figure 3.

EXAMPLE 2 construction of SARS-CoV-2 coronavirus vaccine Ad/S1N

The difference from example 1 is that: the PCR amplification primers used in the step (1) are as follows:

V1(SEQ ID NO：1)：TCCCCCGGGATGTTCGTCTTCCTGGTCCT

V3(SEQ ID NO：3)：CCCAAGCTTTTAGGCCACCTGGTTGCTTGTAT

the remainder being identical.

The amplified product was designated as S1N fragment, the final SARS-CoV-2 vaccine was designated as Ad/S1N vaccine, and the inserted gene was sequenced, the sequencing result being shown in FIG. 4.

EXAMPLE 3 construction of SARS-CoV-2 coronavirus vaccine Ad/S1C

V2(SEQ ID NO：2)：TCCCCCGGGATGAGGGTGCAGCCAACCGAG

V4(SEQ ID NO：4)：CCCAAGCTTTTACCGGGCTCTTCTGGGAGAGT

the remainder being identical.

The amplified product was designated as S1C fragment, the final SARS-CoV-2 vaccine was designated as Ad/S1C vaccine, and the inserted gene was sequenced, the sequencing result being shown in FIG. 5.

EXAMPLE 4 construction of SARS-CoV-2 coronavirus vaccine Ad/S1M

V2(SEQ ID NO：2)：TCCCCCGGGATGAGGGTGCAGCCAACCGAG

V3(SEQ ID NO：3)：CCCAAGCTTTTAGGCCACCTGGTTGCTTGTAT

the remainder being identical.

The amplified product was designated as S1M fragment, the final SARS-CoV-2 vaccine was designated as Ad/S1M vaccine, and the inserted gene was sequenced, the sequencing result being shown in FIG. 6.

EXAMPLE 5 construction of SARS-CoV-2 coronavirus vaccine Ad/S1R454A

The Ad/S1R454A vaccine is different from the Ad/S1 vaccine in that the 454 th amino acid of S protein corresponding to the S gene is mutated from R to A; mutation was achieved by PCR using PCR primers:

V5(SEQ ID NO：5)：CTACAATTATCTGTACCGGCTGTTTAGAAAGAGCA

V6(SEQ ID NO：6)：TGCTCTTTCTAAACAGCCGGTACAGATAATTGTAG

construction of the recombinant plasmid and cotransfection were as in example 1.

The final SARS-CoV-2 vaccine was designated as Ad/S1R454A vaccine.

EXAMPLE 6 construction of SARS-CoV-2 coronavirus vaccine Ad/S1R466A

The Ad/S1R466A vaccine is different from the Ad/S1 vaccine in that the 466 th amino acid of S protein corresponding to the S gene is mutated from R to A; mutation was achieved by PCR using PCR primers:

V7(SEQ ID NO：7)：CTGAAGCCCTTCGAGAGGGACATCTCTACAGAA

V8(SEQ ID NO：8)：TTCTGTAGAGATGTCCCTCTCGAAGGGCTTCAG

The final SARS-CoV-2 vaccine was designated as Ad/S1R466A vaccine.

EXAMPLE 7 construction of SARS-CoV-2 coronavirus vaccine Ad/S1NR454A

The Ad/S1NR454A vaccine is different from the Ad/S1N vaccine in that the 454 th amino acid of S protein corresponding to the S gene is mutated from R to A; mutation was achieved by PCR using PCR primers:

V5(SEQ ID NO：5)：CTACAATTATCTGTACCGGCTGTTTAGAAAGAGCA

V6(SEQ ID NO：6)：TGCTCTTTCTAAACAGCCGGTACAGATAATTGTAG

The final SARS-CoV-2 vaccine was designated as Ad/S1NR454A vaccine.

Example 8 construction of SARS-CoV-2 coronavirus vaccine Ad/S1NR466A

The Ad/S1NR466A vaccine differs from the Ad/S1N vaccine in that the 466 th amino acid of the S protein corresponding to the S gene is mutated from R to A; mutation was achieved by PCR using PCR primers:

V7(SEQ ID NO：7)：CTGAAGCCCTTCGAGAGGGACATCTCTACAGAA

V8(SEQ ID NO：8)：TTCTGTAGAGATGTCCCTCTCGAAGGGCTTCAG

The final SARS-CoV-2 vaccine was designated as Ad/S1NR466A vaccine.

EXAMPLE 9 construction of SARS-CoV-2 coronavirus vaccine Ad/S1CR454A

The Ad/S1CR454A vaccine is different from the Ad/S1C vaccine in that the 454 th amino acid of S protein corresponding to the S gene is mutated from R to A; mutation was achieved by PCR using PCR primers:

V5(SEQ ID NO：5)：CTACAATTATCTGTACCGGCTGTTTAGAAAGAGCA

V6(SEQ ID NO：6)：TGCTCTTTCTAAACAGCCGGTACAGATAATTGTAG

The final SARS-CoV-2 vaccine was designated as Ad/S1CR454A vaccine.

EXAMPLE 10 construction of SARS-CoV-2 coronavirus vaccine Ad/S1CR466A

The Ad/S1CR466A vaccine is different from the Ad/S1C vaccine in that the 466 th amino acid of S protein corresponding to the S gene is mutated from R to A; mutation was achieved by PCR using PCR primers:

V7(SEQ ID NO：7)：CTGAAGCCCTTCGAGAGGGACATCTCTACAGAA

V8(SEQ ID NO：8)：TTCTGTAGAGATGTCCCTCTCGAAGGGCTTCAG

The final SARS-CoV-2 vaccine was designated as Ad/S1CR466A vaccine.

EXAMPLE 11 construction of SARS-CoV-2 coronavirus vaccine Ad/S1MR454A

The Ad/S1MR454A vaccine is different from the Ad/S1M vaccine in that the 454 th amino acid of S protein corresponding to the S gene is mutated from R to A; mutation was achieved by PCR using PCR primers:

V5(SEQ ID NO：5)：CTACAATTATCTGTACCGGCTGTTTAGAAAGAGCA

V6(SEQ ID NO：6)：TGCTCTTTCTAAACAGCCGGTACAGATAATTGTAG

The final SARS-CoV-2 vaccine was designated as Ad/S1MR454A vaccine.

EXAMPLE 12 construction of SARS-CoV-2 coronavirus vaccine Ad/S1MR466A

The Ad/S1MR466A vaccine is different from the Ad/S1M vaccine in that the 466 th amino acid of S protein corresponding to the S gene is mutated from R to A; mutation was achieved by PCR using PCR primers:

V7(SEQ ID NO：7)：CTGAAGCCCTTCGAGAGGGACATCTCTACAGAA

V8(SEQ ID NO：8)：TTCTGTAGAGATGTCCCTCTCGAAGGGCTTCAG

The final SARS-CoV-2 vaccine was designated as Ad/S1MR466A vaccine.

Test example 1 in vitro expression validation of SARS-CoV-2 vaccine

Each SARS-CoV-2 vaccine purified from examples 1-12 was purified at 1X 10 ⁷ After pfu/mL infection of A549 cells for 2h, the virus suspension was discarded, and the cell culture solution was added at 37℃with 5% CO ₂ Culturing under the condition. Cells were collected 48h after infection, RNA was extracted, and the expression level of mRNA of each inserted gene was detected by RT-PCR, and a blank control (template-free) and a negative control (template-RNA extracted from cells not vaccinated) were set. The results are shown in FIG. 7.

The results show that: SARS-CoV-2 vaccine at 1X 10 ⁷ After pfu/mL infection of A549 cells, mRNA of the corresponding S protein antigen could be detected (FIG. 7).

Test example 2 immunogenicity detection of SARS-CoV vaccine carrying antigen sequence

The individual SAR's obtained by purification of examples 1-4S-CoV-2 vaccine was used in 1X 10 vaccine ⁷ After pfu/mL infection of A549 cells for 2h, the virus suspension was discarded, and the cell culture solution was added at 37℃with 5% CO ₂ Culturing under the condition. Cell lysates were collected 48h after infection, and cross-reactions of antigens expressed by the respective SARS-CoV-2 vaccine with convalescence serum of the patient with COVID-19 were detected by indirect ELISA, and the specific results are shown in FIG. 8.

The results show that: SARS-CoV-2 vaccine at 1X 10 ⁷ After pfu/mL infection of A549 cells, the expressed antigen was able to cross-react with serum from convalescence of the patient with COVID-19 (FIG. 8). This indicates that the SARS-CoV-2 vaccine in this application has good immunogenicity.

Test example 3 immunogenicity detection of SARS-CoV vaccine carrying mutant antigen sequence

The SARS-CoV-2 vaccine purified from each of examples 5-12 was used in a 1X 10 vaccine ⁷ After pfu/mL infection of A549 cells for 2h, the virus suspension was discarded, and the cell culture solution was added at 37℃with 5% CO ₂ Culturing under the condition. Cell lysates were collected 48h after infection, and cross-reactions of antigens expressed by the respective SARS-CoV-2 vaccine with convalescence serum of the patient with COVID-19 were detected by indirect ELISA, and the specific results are shown in FIG. 9.

The results show that: SARS-CoV-2 vaccine at 1X 10 ⁷ After pfu/mL infection of A549 cells, the expressed mutant antigen was still able to cross-react with serum from the convalescence of the patient with COVID-19 (FIG. 9). This shows that the mutant SARS-CoV-2 vaccine in this application has good immunogenicity on the basis of ensuring higher safety.

Test example 4SARS-CoV-2 vaccine induced neutralizing antibody titre

Each SARS-CoV-2 vaccine obtained by purification in examples 1-13 was validated.

The test subjects Balb/C mice were grouped as follows:

vaccine group and physiological saline control group.

The administration mode is as follows: intramuscular injection of the inner thigh.

Vaccine stock solution concentration: 1X 10 ¹¹ vp/mL。

Mouse dose: 1X 10 ⁹ vp/v.

Immunization procedure: immunization was performed 3 times every 6 days; mouse serum was taken on day 12, day 18, and day 24, respectively.

Euthanized blood sampling procedure: mouse serum was taken on day 12, day 18, and day 24, respectively.

Neutralizing antibody titer detection method: neutralization of SARS-CoV-2 pseudovirus.

The results are shown in FIG. 10.

The results are experimental results of SARS-CoV-2 vaccine obtained in example 3, and indicate that: after diluting the mouse immune serum by 25 times, the serum of the mouse immunized for 12 days can neutralize 59% of SARS-CoV-2 pseudovirus, the serum of the mouse immunized for 18 days can neutralize 65% of SARS-CoV-2 pseudovirus, and the serum of the mouse immunized for 24 days can neutralize 91% of SARS-CoV-2 pseudovirus. In general, the Ad/S1C vaccine is capable of eliciting high titers of SARS-CoV-2 neutralizing antibodies in mice, and the production of neutralizing antibodies can be detected on day 12.

Test example 5SARS-CoV-2 vaccine antibody titre test

The SARS-CoV-2 vaccine obtained in example 3 was used in this example.

The test subjects Balb/C mice were grouped as follows:

low dose vaccine + thymopentin group: vaccine stock solution 2×10 ⁸ vp/v+thymopentin only;

high dose vaccine + thymopentin group: vaccine stock solution 1 x 10 ⁹ vp/v+thymopentin only;

low dose vaccine group: vaccine stock solution 2×10 ⁸ vp/only;

high dose vaccine group: vaccine stock solution 1 x 10 ⁹ vp/only;

saline control group: saline was injected in the same volume as the other groups of reagents.

Vaccine stock solution concentration: 1X 10 ¹¹ vp/mL。

The concentration of the thymus pentapeptide in the vaccine injection is 1.6mg/mL.

Immunization procedure: immunization was performed 3 times every 6 days; the sera of the mice on day 18 and day 24 were taken separately.

Euthanized blood sampling procedure: the sera of the mice on day 18 and day 24 were taken separately.

Immunogenicity detection method: the specific binding antibody titers of mice against the RBD region of S protein were determined by ELISA and the results are shown in figure 11.

The results show that:

(1) Serum titers against new crown Spike RBD: serum titers were approximately 2500-3400 at 18 days post immunization. Serum titers were about 8000-32000 24 days post immunization.

(2) The addition of thymopentin greatly enhances the titer of antibodies in mice. The serum titers of mice increased from 2500 to 3400 18 days post immunization; 24 days after immunization, the serum titers increased from 8000 to 32000.

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<211> 33

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 8

ttctgtagag atgtccctct cgaagggctt cag 33

<210> 9

<211> 3822

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 9

atgttcgtct tcctggtcct gctgcctctg gtctcctcac agtgcgtcaa tctgacaact 60

cggactcagc tgccacctgc ttatactaat agcttcacca gaggcgtgta ctatcctgac 120

aaggtgttta gaagctccgt gctgcactct acacaggatc tgtttctgcc attctttagc 180

aacgtgacct ggttccacgc catccacgtg agcggcacca atggcacaaa gcggttcgac 240

aatcccgtgc tgccttttaa cgatggcgtg tacttcgcct ctaccgagaa gagcaacatc 300

atcagaggct ggatctttgg caccacactg gactccaaga cacagtctct gctgatcgtg 360

aacaatgcca ccaacgtggt catcaaggtg tgcgagttcc agttttgtaa tgatcccttc 420

ctgggcgtgt actatcacaa gaacaataag agctggatgg agtccgagtt tagagtgtat 480

tctagcgcca acaactgcac atttgagtac gtgagccagc ctttcctgat ggacctggag 540

ggcaagcagg gcaatttcaa gaacctgagg gagttcgtgt ttaagaatat cgacggctac 600

ttcaaaatct actctaagca cacccccatc aacctggtgc gcgacctgcc tcagggcttc 660

agcgccctgg agcccctggt ggatctgcct atcggcatca acatcacccg gtttcagaca 720

ctgctggccc tgcacagaag ctacctgaca cccggcgact cctctagcgg atggaccgcc 780

ggcgctgccg cctactatgt gggctacctc cagccccgga ccttcctgct gaagtacaac 840

gagaatggca ccatcacaga cgcagtggat tgcgccctgg accccctgag cgagacaaag 900

tgtacactga agtcctttac cgtggagaag ggcatctatc agacatccaa tttcagggtg 960

cagccaaccg agtctatcgt gcgctttcct aatatcacaa acctgtgccc atttggcgag 1020

gtgttcaacg caacccgctt cgccagcgtg tacgcctgga ataggaagcg gatcagcaac 1080

tgcgtggccg actatagcgt gctgtacaac tccgcctctt tcagcacctt taagtgctat 1140

ggcgtgtccc ccacaaagct gaatgacctg tgctttacca acgtctacgc cgattctttc 1200

gtgatcaggg gcgacgaggt gcgccagatc gcccccggcc agacaggcaa gatcgcagac 1260

tacaattata agctgccaga cgatttcacc ggctgcgtga tcgcctggaa cagcaacaat 1320

ctggattcca aagtgggcgg caactacaat tatctgtacc ggctgtttag aaagagcaat 1380

ctgaagccct tcgagaggga catctctaca gaaatctacc aggccggcag caccccttgc 1440

aatggcgtgg agggctttaa ctgttatttc ccactccagt cctacggctt ccagcccaca 1500

aacggcgtgg gctatcagcc ttaccgcgtg gtggtgctga gctttgagct gctgcacgcc 1560

ccagcaacag tgtgcggccc caagaagtcc accaatctgg tgaagaacaa gtgcgtgaac 1620

ttcaacttca acggcctgac cggcacaggc gtgctgaccg agtccaacaa gaagttcctg 1680

ccatttcagc agttcggcag ggacatcgca gataccacag acgccgtgcg cgacccacag 1740

accctggaga tcctggacat cacaccctgc tctttcggcg gcgtgagcgt gatcacaccc 1800

ggcaccaata caagcaacca ggtggccgtg ctgtatcagg acgtgaattg taccgaggtg 1860

cccgtggcta tccacgccga tcagctgacc ccaacatggc gggtgtacag caccggctcc 1920

aacgtcttcc agacaagagc cggatgcctg atcggagcag agcacgtgaa caattcctat 1980

gagtgcgaca tcccaatcgg cgccggcatc tgtgcctctt accagaccca gacaaactct 2040

cccagaagag cccggagcgt ggcctcccag tctatcatcg cctataccat gtccctgggc 2100

gccgagaaca gcgtggccta ctctaacaat agcatcgcca tcccaaccaa cttcacaatc 2160

tctgtgacca cagagatcct gcccgtgtcc atgaccaaga catctgtgga ctgcacaatg 2220

tatatctgtg gcgattctac cgagtgcagc aacctgctgc tccagtacgg cagcttttgt 2280

acccagctga atagagccct gacaggcatc gccgtggagc aggataagaa cacacaggag 2340

gtgttcgccc aggtgaagca aatctacaag acccccccta tcaaggactt tggcggcttc 2400

aatttttccc agatcctgcc tgatccatcc aagccttcta agcggagctt tatcgaggac 2460

ctgctgttca acaaggtgac cctggccgat gccggcttca tcaagcagta tggcgattgc 2520

ctgggcgaca tcgcagccag ggacctgatc tgcgcccaga agtttaatgg cctgaccgtg 2580

ctgccacccc tgctgacaga tgagatgatc gcacagtaca caagcgccct gctggccggc 2640

accatcacat ccggatggac cttcggcgca ggagccgccc tccagatccc ctttgccatg 2700

cagatggcct ataggttcaa cggcatcggc gtgacccaga atgtgctgta cgagaaccag 2760

aagctgatcg ccaatcagtt taactccgcc atcggcaaga tccaggacag cctgtcctct 2820

acagccagcg ccctgggcaa gctccaggat gtggtgaatc agaacgccca ggccctgaat 2880

accctggtga agcagctgag cagcaacttc ggcgccatct ctagcgtgct gaatgacatc 2940

ctgagccggc tggacaaggt ggaggcagag gtgcagatcg accggctgat caccggccgg 3000

ctccagagcc tccagaccta tgtgacacag cagctgatca gggccgccga gatcagggcc 3060

agcgccaatc tggcagcaac caagatgtcc gagtgcgtgc tgggccagtc taagagagtg 3120

gacttttgtg gcaagggcta tcacctgatg tccttccctc agtctgcccc acacggcgtg 3180

gtgtttctgc acgtgaccta cgtgcccgcc caggagaaga acttcaccac agcccctgcc 3240

atctgccacg atggcaaggc ccactttcca agggagggcg tgttcgtgtc caacggcacc 3300

cactggtttg tgacacagcg caatttctac gagccccaga tcatcaccac agacaacacc 3360

ttcgtgagcg gcaactgtga cgtggtcatc ggcatcgtga acaataccgt gtatgatcca 3420

ctccagcccg agctggacag ctttaaggag gagctggata agtatttcaa gaatcacacc 3480

tcccctgacg tggatctggg cgacatcagc ggcatcaatg cctccgtggt gaacatccag 3540

aaggagatcg accgcctgaa cgaggtggct aagaatctga acgagagcct gatcgacctc 3600

caggagctgg gcaagtatga gcagtacatc aagtggccct ggtacatctg gctgggcttc 3660

atcgccggcc tgatcgccat cgtgatggtg accatcatgc tgtgctgtat gacatcctgc 3720

tgttcttgcc tgaagggctg ctgtagctgt ggctcctgct gtaagtttga cgaggatgac 3780

tctgaacctg tgctgaaggg cgtgaagctg cattacacct aa 3822

Claims

1. A SARS-CoV-2 coronavirus vaccine comprising a fragment of the S gene of SARS-CoV-2 coronavirus and a gene sequence of a replication defective adenovirus; the segment of SARS-CoV-2 coronavirus S gene is S gene truncated sequence after codon optimization; the truncated sequence of the SARS-CoV-2 coronavirus S gene is SEQ ID NO: positions 955-2055 of the C-terminal sequence of 9; the replication defective adenovirus is a type 5 adenovirus of a C subclass with completely deleted E1 and E3 regions.

2. The SARS-CoV-2 coronavirus vaccine of claim 1, further comprising thymopentin, wherein the amount of thymopentin added to the vaccine is 1-2mg/mL.

3. A method for preparing a SARS-CoV-2 coronavirus vaccine according to any one of claims 1 or 2, comprising the steps of:

(1) Obtaining the sequence of the truncated S gene from SARS-CoV-2 coronavirus after codon optimization;

(2) Recombining the sequence of the truncated S gene from SARS-CoV-2 coronavirus after codon optimization with the defective adenovirus; the sequence of the truncated S gene from SARS-CoV-2 coronavirus after codon optimization is obtained by PCR; the PCR primers are V2 and V4, wherein the sequence of V2 is SEQ ID NO:2, v4 is SEQ ID NO:4, a step of; the cleavage site of V2 is SmaI, and the cleavage site of V4 is HindIII;

(3) Transfecting the packaging cells;

(4) Amplifying, separating and purifying to obtain the final product.