CN114150005B

CN114150005B - Adenovirus vector vaccine for prevention of SARS-CoV-2 Oncuronjorn strain

Info

Publication number: CN114150005B
Application number: CN202210120315.4A
Authority: CN
Inventors: 陈凌; 杨臣臣; 汪乾; 关素华
Original assignee: Guangzhou N Biomed Ltd
Current assignee: Guangzhou N Biomed Ltd
Priority date: 2022-02-09
Filing date: 2022-02-09
Publication date: 2022-04-22
Anticipated expiration: 2042-02-09
Also published as: CN114150005A; WO2023151172A1

Abstract

The present invention relates to an adenovirus vector vaccine for the prevention of SARS-CoV-2 Onckhorden strain. The invention adopts codon preference to optimize to obtain a new S gene sequence, which can efficiently express in human cells, can efficiently express S antigen after immunizing organisms, generates a neutralizing antibody aiming at the Onck Ron strain SARS-CoV-2, and can effectively protect the organisms from being infected by the Onck Ron strain.

Description

Adenovirus vector vaccine for prevention of SARS-CoV-2 Oncuronjorn strain

Technical Field

The invention relates to the technical field of biological medicine, in particular to an adenovirus vector vaccine for preventing SARS-CoV-2 Oncorks strain.

Background

In the face of new coronary pneumonia epidemic situation, prevention is well achieved, and blocking of virus transmission is the key to controlling the epidemic situation. Vaccines are the most cost-effective intervention to prevent and control new types of coronavirus infection. In the virus particle structure of SARS-CoV-2 coronavirus, the S-protein constituting "crown" is an obvious target, and becomes the focus of most research teams. Research teams have successfully revealed the relationship of the S protein to its receptor ACE2 in invading cells by computer modeling the three-dimensional structure of the S protein. Thus, the S protein plays an important role in mediating binding of virions to host cell receptors and in inducing neutralizing antibodies. According to research reports, the S protein has a pre-fusion conformation and a post-fusion conformation, the S protein is combined with an ACEII receptor of a host cell, the protein is divided into S1 and S2 by cutting with furin of the host cell, fusion of a virus envelope and a host cell membrane is promoted to realize infection invasion of the virus, most of antibodies generated after fusion are combined with the antibodies, and no neutralization effect exists, so how to maintain the pre-fusion conformation of the S protein in vaccine development and design is the key for successful vaccine development.

According to the report of the world health organization, there is a recent mutant strain (Ormcken strain) whose pathogenicity and transmission are greatly enhanced. And the S gene of the Ormcken mutant strain has at least 27 mutations, the prior vaccine has extremely poor immune protection effect on the Ormcken mutant strain, the neutralization function of the neutralizing antibody of the prior vaccine can be easily escaped, the immune protection function of the prior vaccine is greatly weakened, and the Delta mutant strain is replaced. The vaccines currently marketed are inactivated vaccines, subunit protein vaccines, mRNA vaccines and adenovirus vector vaccines, and the vaccines mainly aim at the original strain SARS-CoV-2, the protection effect of the Onckrojon mutant strain is reduced, and the Oncojon strain can not produce ideal immune effect after immunization. In addition, the natural SARS-CoV-2 spike protein S gene has very low expression level in human kidney cell HEK293, so if expressed as antigen by the S codon of SARS-CoV-2 Onckrosn strain, the S gene sequence protein of SARS-CoV-2 Onckrosn strain is not effective to be expressed in cell efficiently, and the vaccine may not be effective or has very low potency, which is not enough to resist virus infection.

Disclosure of Invention

The invention aims to provide an optimized S protein nucleotide sequence and a novel vaccine thereof, wherein a carrier is adopted to carry an S gene of an Onckhorden strain (Omicron) optimized by codons, the S antigen can be efficiently expressed after the organism is immunized, a neutralizing antibody aiming at the Onckhorden strain SARS-CoV-2 is generated, and the organism can be effectively protected from being infected by the Onckhorden strain.

The technical scheme adopted by the invention is as follows:

in a first aspect of the invention, there is provided a vaccine comprising:

a) SEQ ID NO: 2; or

b) And SEQ ID NO: 2 having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence homology.

In some examples, the vaccine is used to prevent or treat infection caused by SARS-CoV-2. In some examples, the vaccine is used to prevent or treat infection caused by a SARS-CoV-2 Ormcken mutant strain. In some examples, the vaccine is used to prevent or treat infection by one or more of a SARS-CoV-2 original strain, a SARS-CoV-2 Delta mutant strain, a SARS-CoV-2 Alpha mutant strain, a SARS-CoV-2 Beta mutant strain, and a SARS-CoV-2 Gamma mutant strain.

In some examples, the nucleic acid sequence is used to express a protein that causes immunogenicity to SARS-CoV-2. In some examples, the nucleic acid sequence is used to express a protein that is immunogenic for a SARS-CoV-2 Ormkjon mutant. In some examples, the nucleic acid sequence is used to express a protein that causes immunogenicity to one or more of a SARS-CoV-2 original strain, a SARS-CoV-2 Delta mutant strain, a SARS-CoV-2 Alpha mutant strain, a SARS-CoV-2 Beta mutant strain, and a SARS-CoV-2 Gamma mutant strain.

In some examples, the nucleic acid sequences are used to express proteins in humans or in cells of human origin.

In some examples, the protein may be in a human:

inducing an immune response; and/or

Producing a biological reporter molecule; and/or

Generating a molecule for detection; and/or

Modulating gene function; and/or

Becoming a therapeutic molecule.

The induced immune response includes antibody and cell-mediated immune responses.

In some examples, the vector of the vaccine is a DNA plasmid, an RNA expression plasmid, or a viral vector.

In some examples, the vaccine is an adenoviral vector vaccine. In some examples, the vaccine comprises a polypeptide loaded with SEQ ID NO: 2 or a homologous sequence thereof. In some examples, the adenoviral vector is at least one of an Ad vector, or Ad vector. In some examples, the vector is an empty vector. In some examples, the adenoviral vector is a replication-defective vector. In some examples, the replication deficient vector is a replication deficient adenovirus vector lacking the genes of the E1 and E3 regions. In some examples, the vaccine comprises a polypeptide loaded with SEQ ID NO: 2 or a homologous sequence thereof. In some examples, the vector is an Ad5 empty vector. In some examples, the adenovirus vector is a replication-defective Ad5 vector. In some examples, the replication-deficient Ad5 vector is a replication-deficient Ad5 vector lacking the genes of the E1 and E3 regions. In some examples, the vaccine comprises a polypeptide loaded with SEQ ID NO: 2 or a homologous sequence thereof. In some examples, the vector is an Ad35 empty vector. In some examples, the adenovirus vector is a replication-defective Ad35 vector. In some examples, the replication-deficient Ad35 vector is a replication-deficient Ad35 vector lacking the genes of the E1 and E3 regions.

In some examples, the vaccine further comprises at least one of a pharmaceutically acceptable adjuvant, carrier, diluent, or excipient. At least one of a suitable adjuvant, carrier, diluent or excipient may be selected according to particular needs.

In some examples, the formulation of the adenoviral vector vaccine includes, but is not limited to, oral, injectable, aerosol inhalant and the like common vaccine formulations.

In some examples, the adenoviral vector vaccine can also be used in combination with other drugs. In some examples, the vaccine further comprises at least one agent that has a prophylactic and/or therapeutic effect on COVID-19.

To further improve the purity and safety of the product, in some examples, the vector (e.g., an adenovirus vector such as Ad5 vector, Ad35 vector, etc.) can regulate the expression of the nucleic acid sequence.

To ensure that SEQ ID NO: 2 or a homologous sequence thereof, and in some instances, the nucleic acid sequence of SEQ ID NO: 2 or homologous sequences thereof, and the transcription direction of other genes of the vector (such as an adenovirus vector such as an Ad5 vector and an Ad35 vector).

In a second aspect of the invention, there is provided an expression vector comprising a nucleic acid sequence according to the first aspect of the invention (the nucleic acid sequence shown in SEQ ID NO: 2 or a homologous sequence thereof).

In some examples, the vector may regulate expression of a nucleic acid sequence according to the first aspect of the invention. In some examples, the direction of transcription of the nucleic acid sequence according to the first aspect of the invention is opposite to the direction of transcription of other genes of the vector. The protein obtained by expression can be further ensured to have higher purity and safety.

In some examples, the vector is a DNA plasmid, an RNA expression plasmid, or a viral vector.

In some examples, the viral vector is an adenoviral vector. In some examples, the adenoviral vector is at least one of the foregoing Ad1-Ad52 vectors. In some examples, the vector is an empty vector. In some examples, the adenoviral vector is a replication-defective adenoviral vector. In some examples, the replication-defective adenovirus vector is a replication-defective adenovirus vector lacking genes of E1 and E3 regions. In some examples, the adenoviral vector is an Ad5 vector. In some examples, the vector is an Ad5 empty vector. In some examples, the adenovirus vector is a replication-defective Ad5 vector. In some examples, the replication-deficient Ad5 vector is a replication-deficient Ad5 vector lacking the genes of the E1 and E3 regions. In some examples, the adenoviral vector is an Ad35 vector. In some examples, the vector is an Ad35 empty vector. In some examples, the adenovirus vector is a replication-defective Ad35 vector. In some examples, the replication-deficient Ad35 vector is a replication-deficient Ad35 vector lacking the genes of the E1 and E3 regions.

In a third aspect of the present invention, there is provided: use of a vaccine according to the first aspect of the invention or an expression vector according to the second aspect of the invention, the use comprising:

preparing the medicine for preventing or treating SARS-CoV-2 caused infection.

Preparing a COVID-19 detection reagent; or

Preparing gene function regulator.

In some examples, the infection is caused by a SARS-CoV-2 Oncuronk mutant.

In some examples, the infection is caused by one or more of a SARS-CoV-2 original strain, a SARS-CoV-2 Delta mutant strain, a SARS-CoV-2 Alpha mutant strain, a SARS-CoV-2 Beta mutant strain, and a SARS-CoV-2 Gamma mutant strain.

In a fourth aspect of the invention, there is provided a method of preventing or treating infection by the SARS-CoV-2 strain comprising administering to a subject in need thereof a prophylactically effective amount or a therapeutically effective amount of a vaccine according to the first aspect of the invention.

In some examples, the infection is caused by a SARS-CoV-2 Oncuronk mutant.

According to the foregoing aspects of the invention, in some examples, the amino acid sequence of the Spike protein (S) of the SARS-CoV-2 original strain of the invention is set forth in NCBI accession number YP _ 009724390.1. In some examples, the complete genomic sequence of the SARS-CoV-2 original strain of the invention is shown in NCBI accession number NC-045512.2.

The invention has the beneficial effects that:

1) the original S gene sequence protein of SARS-CoV-2 Oncken strain can not be effectively expressed in cell, and we adopt codon preference to make optimization to obtain new S gene sequence, and said S gene sequence can be effectively expressed in human source cell, and can effectively express S antigen after immunizing organism.

2) After the optimized sequence is expressed in human body or human-derived cells, a neutralizing antibody aiming at the Onckhun strain SARS-CoV-2 with higher titer can be induced to be generated, and the organism can be effectively protected from being infected by the Onckhun strain; can also induce specific antibody aiming at SARS-CoV-2 original strain and other types of mutant strains to play multiple protection roles.

Drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings that are needed to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present application, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without exceeding the scope of the claimed application.

FIG. 1 shows the expression of S protein after transfection of equal amounts of pGA1-S-Ori, PGA1-S50, PGA261-S50 and PGA351-S50, respectively.

FIG. 2 is a flow chart of the construction of pAd 5-S50.

FIG. 3 is a diagram of the virus purification of pAd 5-S50.

FIG. 4 is a flow chart of the construction of pAd 35-S50.

FIG. 5 is a diagram of the virus purification of pAd 35-S50.

FIG. 6 is a graph showing the expression of S protein after pAd5-S50 and Ad35-S50 infected A549 cells. 1 is blank control of A549 cells, 2 is Ad35-S50 sample, 3 is Ad5-S50 sample, 4 is positive control of S protein, and M is Marker.

FIG. 7 is the serum binding antibody levels of Ad5-S50 immunized mice against the novel coronavirus Omicron strain.

FIG. 8 is the serum binding antibody levels of Ad5-S50 immunized mice against the original strain of the novel coronavirus.

FIG. 9 is serum binding antibody levels of Ad5-S50 immunized mice against the novel coronavirus Delta strain.

FIG. 10 is serum neutralizing antibody levels of Ad5-S50 immunized mice.

FIG. 11 is serum binding antibody levels of Ad35-S50 immunized mice against the novel coronavirus Omicron strain.

FIG. 12 is serum binding antibody levels of Ad35-S50 immunized mice against the original strain of the novel coronavirus.

FIG. 13 is serum binding antibody levels of Ad35-S50 immunized mice against the novel coronavirus Delta strain.

FIG. 14 is serum neutralizing antibody levels of Ad35-S50 immunized mice.

Detailed Description

The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application, and it is obvious that the described embodiments are some, but not all, embodiments of the present application. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.

It should be noted that the embodiments and features of the embodiments in the present application may be combined with each other without conflict. The present invention will be described in detail with reference to examples.

Nucleic acid sequences of Spike protein (S) of SARS-CoV-2 such as GISAID: EPI _ ISL _6640916, designated SEQ ID NO: 1. the process by which mRNA precursors transcribed by eukaryotic cells can produce different mRNA splice isoforms by different splicing patterns (different splice site combinations are selected), ultimately resulting in different proteins produced from the same gene sequence. This is very disadvantageous for the expression of the protein. The inventor ensures the uniqueness of protein expression and reduces the occurrence of protein expression miscut by performing codon optimization on a wild-type natural nucleic acid sequence and removing potential variable shearing sites based on an own technology. The optimized nucleic acid sequence is shown as SEQ ID NO: 2 (hereinafter the vector is designated S50).

The present invention is described in further detail below with reference to specific examples, which are not to be construed as limiting the scope of the invention as claimed herein.

Example 1 analysis of codon optimization Effect of Spike Gene

1. Construction of shuttle plasmid pGA1-S50 for the S Gene of the Ormckh mutant

The target fragment S50 was obtained by PCR amplification using pcDNA3.1-S50 (synthesized by Nanjing King-Shirui Biotech Co., Ltd., S50 being SEQ ID NO: 2) plasmid as a template, S50-F and S50-R as primers, and Primer Star Mix (TaKaRa).

S50 amplification primer sequences:

S50-F：gtaccgagctcggatccgccaccatgttcgtgttcctggtcctactgcc（SEQ ID NO：3）；

S50-R：agaatagggccctctagactagtttatcaggtgtagtgcagcttt（SEQ ID NO：4）。

PCR procedure: 3min at 98 ℃; 28 cycles of 98 ℃ for 10 s, 60 ℃ for 5 s, 72 ℃ for 30 s; preserving at 72 deg.C for 5min and 4 deg.C.

PGA1 was obtained by PCR amplification of the target fragment using PGA1-EGFP plasmid (stored by Guangzhou Enbao Biopharmaceutical science and technology Co., Ltd.) as a template and CMV-R and BGH-F as primers, using Primer Star Mix (TaKaRa).

pGA1 backbone amplification primer sequences:

CMV-R：ggatccgagctcggtaccaagcttaagtttaaacgctagagtccgg（SEQ ID NO：5）；

BGH-F：tctagagggccctattctatagtgtc（SEQ ID NO：6）。

The desired fragment S50 and the vector backbone pGA1 were recombined using a homologous recombinase (Vazyme) to give a shuttle plasmid pGA1-S50 carrying the S gene of the Ormichken mutant strain.

2. Construction of shuttle plasmid pGA261-S50 for the S Gene of the Ormckh mutant

Using pcDNA3.1-S50 (synthesized by Nanjing Kingsrie Biotech Co., Ltd., S50 is SEQ ID NO: 2) plasmid as template, S50-F (SEQ ID NO: 3) and S50-R (SEQ ID NO: 4) as primers, and PCR amplification with Primer Star Mix (TaKaRa) was performed to obtain the target fragment S50.

The PGA261-EGFP plasmid (saved by Guangzhou Enbao biomedical science and technology Co., Ltd.) was used as a template, CMV-R (SEQ ID NO: 5) and BGH-F (SEQ ID NO: 6) were used as primers, and the Primer Star Mix (TaKaRa) was used to perform PCR amplification to obtain the target fragment PGA 261.

The desired fragment S50 and the vector backbone pGA261 were recombined with a homologous recombinase (Vazyme) to give a shuttle plasmid pGA261-S50 carrying the S gene of the Ormichken mutant strain.

3. Construction of shuttle plasmid pGA351-S50 for the S Gene of the Ormckh mutant

PGA351-EGFP plasmid (carrying Ad35E1 region homologous recombination arm plasmid, saved by Guangzhou Enbao biological medicine science and technology Co., Ltd.) is used as a template, CMV-R (SEQ ID NO: 5) and BGH-F (SEQ ID NO: 6) are used as primers, and Primer Star Mix (TaKaRa) is adopted for PCR amplification to obtain the target fragment PGA 351.

The desired fragment S50 and the vector backbone pGA351 were recombined using a homologous recombinase (Vazyme) to give a shuttle plasmid pGA351-S50 carrying the S gene of the Ormichken mutant strain.

4. Construction of shuttle plasmid pGA1-S-Ori for the original S Gene of the Ormckh mutant

The target fragment S-Ori was obtained by PCR amplification using pcDNA3.1-S-Ori (available from Nanjing King Spiri Biotechnology Ltd., load S-Ori, SEQ ID NO: 1) plasmid as a template, SOri-F and SOri-R as primers, and Primer Star Mix (TaKaRa).

S-Ori amplification primer sequence:

SOri-F：gtaccgagctcggatccgccaccatgtttgtttttcttgttttattgccact（SEQ ID NO：7）；

SOri-R：tagaatagggccctctagactagtttattatgtgtaatgtaatttgactcctt（SEQ ID NO：8）。

The target fragment PGA1 was obtained by PCR amplification using PGA1-EGFP plasmid (stored by Guangzhou Enbao Biopharmaceutical science and technology Co., Ltd.) as a template and CMV-R (SEQ ID NO: 5) and BGH-F (SEQ ID NO: 6) as primers using Primer Star Mix (TaKaRa).

The desired fragment S-Ori and the vector backbone pGA1 were recombined using a homologous recombinase (Vazyme) to obtain a shuttle plasmid pGA1-S-Ori carrying the original S gene of the Ormichjon mutant.

5. Analysis of codon optimization effect of Spike gene

HEK293 cells were transfected with 2.5g of pGA1-S-Ori, PGA1-S50, PGA261-S50 and PGA351-S50 prepared in steps 1 to 4 of example 1 using cationic liposomes according to a conventional method, respectively, and after 48 hours of transfection, the cells were collected, and the samples were treated according to a conventional WesternBlot method and protein detection was performed. As can be seen from FIG. 1, the expression of the S protein was not detected in the pGA1-S-Ori samples, while the expression of the S protein was observed in the codon-optimized PGA1-S50, PGA261-S50 and PGA351-S50 samples, indicating that the optimized S50 sequence had unexpected effect.

Example 2 construction of the S antigen vector pAd5-S50 carrying the Ornkejon mutant

1. Construction of shuttle plasmid pGA1-S50 for the S Gene of the Ormckh mutant

PGA1-EGFP plasmid (carrying Ad5E1 region homologous recombination arm plasmid, preserved by Guangzhou Enbao biological medicine science and technology Co., Ltd.) is used as a template, CMV-R (SEQ ID NO: 5) and BGH-F (SEQ ID NO: 6) are used as primers, and Primer Star Mix (TaKaRa) is adopted for PCR amplification to obtain the target fragment PGA 1.

2. Construction of pAd5-S50 carrying the S Gene of the Ornkerng mutant

And (3) carrying the CMV-S50-BGH target segment of the homologous recombination arm by PCR amplification by taking pGA1-S50 plasmid as a template, and recovering the gel.

CMV-S50-BGH target fragment amplification primer sequence:

Ad5-SB-F：TTGGATTGAAGCCAATATGATAATGAGGGGGTGG（SEQ ID NO：9）；

Ad5-SB-R：GCATCGGTCGAGGACAGGCCTCTCAAGTCTGTATAC（SEQ ID NO：10）。

PCR procedure: 3min at 98 ℃; 28 cycles of 98 ℃ for 10 s, 60 ℃ for 5 s, 72 ℃ for 50 s; recovering target fragment with gel recovery kit at 72 deg.C for 5min, and storing at 4 deg.C.

pAd5 delta E1 delta E3 was recovered by ethanol precipitation after linearization with ClaI; the CMV-S50-BGH target fragment carrying the homologous recombination arm and the linearized PAd5 delta E1 delta E3 are co-transformed into BJ5183, homologous recombination is carried out to obtain pAd5-S50 plasmid carrying the S50 gene, and the technical flow is shown in figure 2.

3. Rescue and production of Ad5-S50 vector

1) According to the conventional method, Ad5-S50 is linearized by PacI, ethanol precipitation recovery and transfection of 293 cells by a cationic liposome transfection method; 2) 4 hours after transfection, 2 ml of DMEM medium containing 5% fetal calf serum is added, incubation is carried out for 7-10 days, and cytopathic effect is observed; 3) after toxin is discharged, collecting cells and culture supernatant, repeatedly freezing and thawing for 3 times in 37-degree water bath and liquid nitrogen, centrifuging to remove cell debris, and infecting the supernatant into a 10 cm dish; 4) collecting cells and culture supernatant after 2-3 days, repeatedly freezing and thawing for 3 times and centrifuging to remove cell debris, wherein the supernatant is infected into 3-5 15 cm dishes; 5) after 2-3 days, collecting cells, repeatedly freezing and thawing for 3 times and centrifuging to remove cell debris; 6) after the supernatant fluid is infected into 30 15 cm dishes for 2 to 3 days, collecting cells, repeatedly freezing and thawing for 3 times and centrifuging to remove cell debris; 7) adding the supernatant into a cesium chloride density gradient centrifuge tube; centrifuging at 4 deg.C and 40000 rpm for 4 hr; sucking out virus bands, desalting and subpackaging; 8) the titer of the virus particles is determined by OD260 absorbance, and the calculation formula is as follows: viral concentration = OD260 × dilution factor × 36/genome length (Kb); the virus stock was frozen at-80 ℃. Viral purification is shown in figure 3.

Example 3 construction of the S antigen vector pAd35-S50 carrying the Ornkejon mutant

1. Construction of shuttle plasmid pGA351-S50 for the S Gene of the Ormckh mutant

2. Construction of pAd35-S50 carrying the S Gene of the Ornkerng mutant Strain

And (3) carrying a CMV-S50-BGH target fragment of a homologous recombination arm by PCR amplification by taking pGA351-S50 plasmid as a template, and recovering glue.

CMV-S50-BGH target fragment amplification primer sequence:

Ad35-SB-F：agaattggatccgaattcgcggccgcgcgatcgccatcatcaataatatacctt（SEQ ID NO：11）；

Ad35-SB-R：gcgtcgcagatccgaattcgtatacccatccaagctgcacgataa（SEQ ID NO：12）。

pAd35 delta E1 delta E3 was recovered by ethanol precipitation after linearization with PmeI; the CMV-S50-BGH target fragment carrying the homologous recombination arm and the linearized PAd35 delta E1 delta E3 (5E 4) are co-transformed into BJ5183, and homologous recombination is carried out to obtain pAd35-S50 plasmid carrying the S50 gene, and the technical flow is shown in figure 4.

3. Rescue and production of Ad35-S50 vector

1) According to a conventional method, Ad35-S50 is linearized by AsisiI, ethanol is precipitated and recovered, and 293 cells are transfected by a cationic liposome transfection method; 2) 4 hours after transfection, 2 ml of DMEM medium containing 5% fetal calf serum is added, incubation is carried out for 7-10 days, and cytopathic effect is observed; 3) after toxin is discharged, collecting cells and culture supernatant, repeatedly freezing and thawing for 3 times in 37-degree water bath and liquid nitrogen, centrifuging to remove cell debris, and infecting the supernatant into a 10 cm dish; 4) collecting cells and culture supernatant after 2-3 days, repeatedly freezing and thawing for 3 times and centrifuging to remove cell debris, wherein the supernatant is infected into 3-5 15 cm dishes; 5) after 2-3 days, collecting cells, repeatedly freezing and thawing for 3 times and centrifuging to remove cell debris; 6) after the supernatant fluid is infected into 30 15 cm dishes for 2 to 3 days, collecting cells, repeatedly freezing and thawing for 3 times and centrifuging to remove cell debris; 7) adding the supernatant into a cesium chloride density gradient centrifuge tube; centrifuging at 4 deg.C and 40000 rpm for 4 hr; sucking out virus bands, desalting and subpackaging; 8) the titer of the virus particles is determined by OD260 absorbance, and the calculation formula is as follows: viral concentration = OD260 × dilution factor × 36/genome length (Kb); the virus stock was frozen at-80 ℃. The results of virus purification are shown in FIG. 5.

Example 4 detection of Spike Gene expression

A549 cells are infected by Ad5-S50 and Ad35-S50 viruses, and the cells are collected after 24 hours. Samples were processed according to the conventional WesternBlot method and protein detection was performed, the results are shown in FIG. 6. It can be seen that the expression of the S protein of the Ormichjon mutant strain can be observed in the samples of the vaccine candidate strains Ad5-S50 and Ad35-S50, which shows that the Ad5-S50 and Ad35-S50 vaccine candidate strains are correctly constructed and can successfully express the S antigen protein.

Example 5 evaluation of Ad5-S50 animal immunogenicity

Balb/c mice (purchased from Beijing Wittingle laboratory animal technology Co., Ltd.) 6-8 weeks old were divided into 2 groups of 10 mice each; day 0, vaccine group (i.e., sample group) intramuscular immunization of Ad5-S50 dose prepared in example 2: 5X 10⁹vp/body, control group (i.e., negative group) intramuscular injection Ad5-empty dose: 5X 10⁹vp/only; on day 9, blood was drawn from the orbit and serum was isolated.

1. Binding antibodies

The antibody level in serum was determined by enzyme-linked immunosorbent assay (ELISA) using RBD proteins of the Oncuronte mutant, original strain, Delta mutant of New crown Virus (purchased from Beijing Yiwangshizhou technologies, Ltd.) as antigens.

The specific operation is as follows:

1) adding 50ng of RBD protein into each 96-well plate, and standing overnight at 4 ℃;

2) the supernatant was aspirated off, washed 3 times with PBST, 200. mu.l of 5% BSA was added to each well and blocked for 2h at room temperature;

3) PBST washing 3 times; mouse sera diluted 1:400, and 1:800, and 1:1600, and 1:3200, and 1:6400, and 1:12800, and 1:25600, and 1:51200 with PBS were added to each well and incubated at 37 ℃ for 2 h;

4) adding an enzyme-labeled antibody: adding 100 mu l of diluted HRP-labeled IgG secondary antibody, and incubating for 2h at 37 ℃;

5) PBST washing for 6-8 times;

6) adding a substrate solution for color development: adding 100 mul TMB for color development;

7) and (3) terminating the reaction: adding 50 μ l of 1M sulfuric acid to terminate the reaction;

8) and (4) judging a result: measuring OD value, and controlling the OD value to be 0.1-4;

9) the results of the experiments are shown in figure 7, figure 8 and figure 9, figure 7 showing that Ad5-S50 is capable of eliciting specific binding antibodies against the ormoxaden mutant RBD protein in mice; furthermore, FIGS. 8 and 9 show that Ad5-S50 can also produce specific binding antibodies against original strain RBD protein and Delta mutant RBD protein.

2. Neutralizing antibodies

The specific operation of the determination of the pseudovirus neutralizing antibody is as follows:

1) inactivating the serum to be detected in 56 ℃ water bath for 30min, and centrifuging at 6000g for 3 min; performing 30-fold gradient dilution on the serum;

2) ormickjon pseudovirus (purchased from south kyoto kezan biotechnology limited, cat no: DD 1768-02) to 1.3X 10⁴TCID50/ml was mixed well with the above diluted serum and placed in a cell culture incubator (37 ℃, 5% CO)₂) Incubating for 1 hour;

3) adding the incubated serum into ACEII cells of 96-well plate, placing in cell culture box, and culturing at 37 deg.C with 5% CO₂Culturing for 72 hours;

4) after culturing for 72 hours, taking out the 96-well plate from the cell culture box, sucking 100 mu l of supernatant from each sample loading hole by using a multi-channel pipette, then adding 100 mu l of luciferase detection reagent, and reacting for 2min at room temperature in a dark place;

5) calculating the neutralization inhibition rate: the inhibition ratio was [ 1- (mean value of luminescence intensity of sample group-mean value of luminescence intensity of blank control)/(mean value of luminescence intensity of negative group-mean value of luminescence intensity of blank control value) ] × 100%; blank refers to background value of 96-well cells; the negative group refers to Ad5-empty immune group;

6) from the results of the neutralization inhibition ratio, IC50 was calculated by the Reed-Muench method.

The results are shown in FIG. 10: the vaccine can stimulate the body of mice to produce high-titer specific neutralizing antibodies aiming at the Ormcken new coronavirus.

Example 6 evaluation of Ad35-S50 animal immunogenicity

The 6-8 week old Balb/c mice (Beijing Wittingle laboratory animal technology Co., Ltd.) were divided into 2 groups of 10 mice each; 0 thDay, immunization of vaccine groups (i.e., sample groups) intramuscular immunization example 3 Ad35-S50 doses were prepared: 5X 10⁹vp/body, control group (i.e., negative group) intramuscular injection Ad35-empty dose: 5X 10⁹vp/only; on day 14, blood was drawn from the orbit and serum was isolated.

1. Binding antibodies

The antibody level in serum was determined by enzyme-linked immunosorbent assay (ELISA) using RBD proteins of the Oncork mutant, original strain, and Delta mutant (purchased from Beijing Yiqiao Shenzhou technologies, Ltd.) as antigens.

The specific procedure for the ELISA-conjugated antibody assay was:

3) PBST washing 3 times; adding mouse serum diluted with PBS at a ratio of 1:400, 1:800, 1:1600, 1:3200, 1:6400, 1:12800, 1:25600 and, 1:51200 into each well, and incubating at 37 ℃ for 2 h;

5) PBST washing for 6-8 times;

9) the results of the experiments are shown in FIG. 11, FIG. 12 and FIG. 13, FIG. 11 showing that Ad35-S50 is able to elicit the production of specific binding antibodies against the Ormckhrn mutant RBD protein in mice. FIGS. 12 and 13 show that Ad35-S50 can also produce specific binding antibodies against original strain RBD protein and Delta mutant RBD protein.

2. Neutralizing antibodies

5) calculating the neutralization inhibition rate: the inhibition ratio was [ 1- (mean value of luminescence intensity of sample group-mean value of luminescence intensity of blank control)/(mean value of luminescence intensity of negative group-mean value of luminescence intensity of blank control value) ] × 100%; blank refers to background value of 96-well cells; the negative group refers to Ad35-empty immune group;

The results are shown in FIG. 14: the vaccine can stimulate the body of mice to produce high-titer specific neutralizing antibodies aiming at the Ormcken new coronavirus.

The foregoing detailed description of the embodiments of the present application has been presented to illustrate the principles and implementations of the present application, and the description of the embodiments is only intended to facilitate the understanding of the methods and their core concepts of the present application. Meanwhile, a person skilled in the art should, according to the idea of the present application, change or modify the embodiments and applications of the present application based on the scope of the present application. In view of the above, the description should not be taken as limiting the application.

SEQUENCE LISTING

<110> Guangzhou Enbao biomedical science and technology Co., Ltd

<120> adenovirus vector vaccine for prevention of SARS-CoV-2 Oncork strain

<160> 12

<170> PatentIn version 3.5

<210> 1

<211> 3813

<212> DNA

<213> SARS-CoV-2

<220>

<221> misc_feature

<222> (2932)..(2932)

<223> n is a, c, g, t

<400> 1

atgtttgttt ttcttgtttt attgccacta gtctctagtc agtgtgttaa tcttacaacc 60

agaactcaat taccccctgc atacactaat tctttcacac gtggtgttta ttaccctgac 120

aaagttttca gatcctcagt tttacattca actcaggact tgttcttacc tttcttttcc 180

aatgttactt ggttccatgt tatctctggg accaatggta ctaagaggtt tgataaccct 240

gtcctaccat ttaatgatgg tgtttatttt gcttccattg agaagtctaa cataataaga 300

ggctggattt ttggtactac tttagattcg aagacccagt ccctacttat tgttaataac 360

gctactaatg ttgttattaa agtctgtgaa tttcaatttt gtaatgatcc atttttggac 420

cacaaaaaca acaaaagttg gatggaaagt gagttcagag tttattctag tgcgaataat 480

tgcacttttg aatatgtctc tcagcctttt cttatggacc ttgaaggaaa acagggtaat 540

ttcaaaaatc ttagggaatt tgtgtttaag aatattgatg gttattttaa aatatattct 600

aagcacacgc ctattatagt gcgtgagcca gaagatctcc ctcagggttt ttcggcttta 660

gaaccattgg tagatttgcc aataggtatt aacatcacta ggtttcaaac tttacttgct 720

ttacatagaa gttatttgac tcctggtgat tcttcttcag gttggacagc tggtgctgca 780

gcttattatg tgggttatct tcaacctagg acttttctat taaaatataa tgaaaatgga 840

accattacag atgctgtaga ctgtgcactt gaccctctct cagaaacaaa gtgtacgttg 900

aaatccttca ctgtagaaaa aggaatctat caaacttcta actttagagt ccaaccaaca 960

gaatctattg ttagatttcc taatattaca aacttgtgcc cttttgatga agtttttaac 1020

gccaccagat ttgcatctgt ttatgcttgg aacaggaaga gaatcagcaa ctgtgttgct 1080

gattattctg tcctatataa tctcgcacca tttttcactt ttaagtgtta tggagtgtct 1140

cctactaaat taaatgatct ctgctttact aatgtctatg cagattcatt tgtaattaga 1200

ggtgatgaag tcagacaaat cgctccaggg caaactggaa atattgctga ttataattat 1260

aaattaccag atgattttac aggctgcgtt atagcttgga attctaacaa gcttgattct 1320

aaggttagtg gtaattataa ttacctgtat agattgttta ggaagtctaa tctcaaacct 1380

tttgagagag atatttcaac tgaaatctat caggccggta acaaaccttg taatggtgtt 1440

gcaggtttta attgttactt tcctttacga tcatatagtt tccgacccac ttatggtgtt 1500

ggtcaccaac catacagagt agtagtactt tcttttgaac ttctacatgc accagcaact 1560

gtttgtggac ctaaaaagtc tactaatttg gttaaaaaca aatgtgtcaa tttcaacttc 1620

aatggtttaa aaggcacagg tgttcttact gagtctaaca aaaagtttct gcctttccaa 1680

caatttggca gagacattgc tgacactact gatgctgtcc gtgatccaca gacacttgag 1740

attcttgaca ttacaccatg ttcttttggt ggtgtcagtg ttataacacc aggaacaaat 1800

acttctaacc aggttgctgt tctttatcag ggtgttaact gcacagaagt ccctgttgct 1860

attcatgcag atcaacttac tcctacttgg cgtgtttatt ctacaggttc taatgttttt 1920

caaacacgtg caggctgttt aataggggct gaatatgtca acaactcata tgagtgtgac 1980

atacccattg gtgcaggtat atgcgctagt tatcagactc agactaagtc tcatcggcgg 2040

gcacgtagtg tagctagtca atccatcatt gcctacacta tgtcacttgg tgcagaaaat 2100

tcagttgctt actctaataa ctctattgcc atacccacaa attttactat tagtgttacc 2160

acagaaattc taccagtgtc tatgaccaag acatcagtag attgtacaat gtacatttgt 2220

ggtgattcaa ctgaatgcag caatcttttg ttgcaatatg gcagtttttg tacacaatta 2280

aaacgtgctt taactggaat agctgttgaa caagacaaaa acacccaaga agtttttgca 2340

caagtcaaac aaatttacaa aacaccacca attaaatatt ttggtggttt taatttttca 2400

caaatattac cagatccatc aaaaccaagc aagaggtcat ttattgaaga tctacttttc 2460

aacaaagtga cacttgcaga tgctggcttc atcaaacaat atggtgattg ccttggtgat 2520

attgctgcta gagacctcat ttgtgcacaa aagtttaaag gccttactgt tttgccacct 2580

ttgctcacag atgaaatgat tgctcaatac acttctgcac tgttagcggg tacaatcact 2640

tctggttgga cctttggtgc aggtgctgca ttacaaatac catttgctat gcaaatggct 2700

tataggttta atggtattgg agttacacag aatgttctct atgagaacca aaaattgatt 2760

gccaaccaat ttaatagtgc tattggcaaa attcaagact cactttcttc cacagcaagt 2820

gcacttggaa aacttcaaga tgtggtcaac cataatgcac aagctttaaa cacgcttgtt 2880

aaacaactta gctccaaatt tggtgcaatt tcaagtgttt taaatgatat cntttcacgt 2940

cttgacaaag ttgaggctga agtgcaaatt gataggttga tcacaggcag acttcaaagt 3000

ttgcagacat atgtgactca acaattaatt agagctgcag aaatcagagc ttctgctaat 3060

cttgctgcta ctaaaatgtc agagtgtgta cttggacaat caaaaagagt tgatttttgt 3120

ggaaagggct atcatcttat gtccttccct cagtcagcac ctcatggtgt agtcttcttg 3180

catgtgactt atgtccctgc acaagaaaag aacttcacaa ctgctcctgc catttgtcat 3240

gatggaaaag cacactttcc tcgtgaaggt gtctttgttt caaatggcac acactggttt 3300

gtaacacaaa ggaattttta tgaaccacaa atcattacta cagacaacac atttgtgtct 3360

ggtaactgtg atgttgtaat aggaattgtc aacaacacag tttatgatcc tttgcaacct 3420

gaattagatt cattcaagga ggagttagat aaatatttta agaatcatac atcaccagat 3480

gttgatttag gtgacatctc tggcattaat gcttcagttg taaacattca aaaagaaatt 3540

gaccgcctca atgaggttgc caagaattta aatgaatctc tcatcgatct ccaagaactt 3600

ggaaagtatg agcagtatat aaaatggcca tggtacattt ggctaggttt tatagctggc 3660

ttgattgcca tagtaatggt gacaattatg ctttgctgta tgaccagttg ctgtagttgt 3720

ctcaagggct gttgttcttg tggatcctgc tgcaaatttg atgaagacga ctctgagcca 3780

gtgctcaaag gagtcaaatt acattacaca taa 3813

<210> 2

<211> 3821

<212> DNA

<213> SARS-CoV-2

<400> 2

atgttcgtgt tcctggtcct actgccactg gtcagcagcc agtgcgtgaa tctgacgact 60

aggacccaac tgcctccagc ctataccaac agcttcacca gaggagtcta ctaccccgac 120

aaggtgtttc ggtcttctgt gctgcattct acacaggacc tgttcctgcc cttcttcagc 180

aatgtcacct ggttccacgt gatctccggc accaacggaa ccaaacgatt tgataatcct 240

gtgctgcctt tcaacgacgg agtgtacttc gcctctatcg agaagagcaa tatcatccgg 300

ggctggatct tcggcacaac gctggacagc aagacccaga gcctgctgat cgttaacaat 360

gctaccaacg ttgttatcaa ggtgtgcgag ttccagtttt gcaacgaccc tttcctggac 420

cacaagaaca acaagagttg gatggaaagc gagttcagag tgtactctag cgctaataac 480

tgcacattcg agtacgtctc tcagcctttc ctgatggacc tggaaggcaa acagggaaat 540

ttcaaaaatc tgagagaatt cgtgttcaag aacatcgacg gctactttaa gatctactct 600

aagcacacac ccatcatcgt gcgggaacca gaggacctgc cccagggctt cagcgctctg 660

gagccactgg ttgacctgcc catcggcatc aacattacaa gattccaaac tctgcttgca 720

ctgcatagat cctatctgac ccctggcgat tcctcaagcg gatggaccgc cggcgccgct 780

gcctactacg tgggatacct gcaacctcgg acctttctgc tgaagtataa cgagaacggc 840

accattaccg acgccgtgga ctgcgccctg gaccccctga gcgagacaaa gtgcaccctg 900

aaaagcttca ccgtggaaaa gggcatctac caaaccagca actttcgggt gcagcctacc 960

gaatctatcg tgcggttccc caacatcaca aacctgtgcc ctttcgacga ggtgttcaac 1020

gccaccagat tcgccagcgt gtatgcctgg aacagaaaga gaatctcgaa ttgcgtggcc 1080

gattactccg tgctctataa cctcgcccct ttcttcacat tcaagtgcta cggcgtgagc 1140

cccaccaagc tcaacgacct gtgttttacc aacgtgtacg ccgacagctt tgtgatcaga 1200

ggtgacgagg tgcggcagat cgcaccagga cagacaggca acattgctga ctacaactac 1260

aaactgcctg acgatttcac cggctgcgtg atcgcctgga attctaacaa gctggatagc 1320

aaggtgtctg gcaattacaa ctacctgtac cggctgttta gaaagagcaa cctgaagcct 1380

ttcgagagag acatctctac cgagatatac caggccggca acaaaccttg taacggcgtt 1440

gcgggattca actgctactt ccctctgaga agctacagct ttcggcctac atacggcgtc 1500

ggccaccagc cctaccgggt ggtggtactg agcttcgagt tactgcacgc tcctgcgacc 1560

gtctgcggcc ctaagaagag caccaatctg gtgaagaaca agtgcgtcaa cttcaacttt 1620

aacggcctga agggcacagg tgtgctgacc gagagcaaca agaaattcct cccattccaa 1680

caattcggta gagatatcgc cgacaccact gatgcagtta gggaccccca gaccctggaa 1740

atcctggata tcaccccttg ctcattcggc ggtgtgagcg tcatcacccc tggcaccaac 1800

acctccaacc aggtggccgt cctgtaccag ggcgttaatt gtaccgaggt gcctgtggcc 1860

atccacgccg accagctcac ccctacgtgg agagtgtaca gcacaggcag taacgtgttt 1920

cagactcggg ccggctgcct catcggtgcc gagtacgtga ataatagtta tgagtgtgac 1980

attcccattg gcgccggcat ctgcgccagc taccagaccc agacaaagag tcacggcagc 2040

gctagctctg tggccagcca gagcattatc gcctacacca tgtctctggg cgctgaaaac 2100

agcgtggcct actctaacaa ctccatcgcc atccctacca acttcacaat ctccgtgacc 2160

acagagattc tgcccgtgtc tatgaccaag acctctgtgg actgtacaat gtacatctgc 2220

ggcgatagca ccgaatgcag caacctgctc ctgcaatacg gcagcttctg cacccagctg 2280

aaaagagctc tgaccggtat cgctgtggaa caggacaaga acacacagga ggtgttcgcc 2340

caggttaagc agatctacaa gacccctcct atcaaatact tcggcggctt caacttcagc 2400

cagatcctgc ctgatccaag caaacctagc aagcgcagct tcatcgagga ccttctgttt 2460

aataaagtta ccctggccga tgccggattt atcaagcaat acggagattg cttaggcgat 2520

atcgctgcca gagatctgat ctgtgctcag aaattcaagg gcctgaccgt cctgcctcct 2580

ctcctgaccg acgagatgat cgctcagtac acctctgccc tgctggccgg cacaatcaca 2640

tcaggctgga ccttcggagc cggagccgct ctgcagatcc cctttgcaat gcaaatggcc 2700

tacagattca acggcattgg cgtcacacag aacgtgctgt acgagaatca gaagctgata 2760

gccaaccagt tcaactccgc tatcggcaag atccaggaca gcctgagctc caccgcctcc 2820

gccctcggaa aactgcagga cgtggtgaac cataatgccc aggctctgaa caccctggtg 2880

aagcaactga gcagcaagtt cggcgccatc agctctgtcc tgaacgacat cttctcaaga 2940

ttggatcctc ccgaagccga agtccagatc gatagactga taaccggcag gctgcaaagc 3000

ctccagacat acgtgacaca gcaactgatc agagccgctg agatccgagc cagcgctaac 3060

ctggccgcca ccaagatgtc agagtgcgtc ctggggcaga gcaaaagagt ggacttctgt 3120

ggcaagggct atcacctgat gagcttccct cagagcgccc cgcacggagt ggtgttcctg 3180

cacgtgacct acgtgcccgc tcaggaaaaa aacttcacca cagccccagc tatctgtcac 3240

gacggcaagg cccacttccc aagggaaggc gtgttcgtga gcaatggcac acactggttt 3300

gtgacccaga gaaacttcta cgagcctcag atcatcacaa ccgacaacac ctttgtgagc 3360

ggcaattgcg atgtggtgat cggcatcgtg aacaacaccg tgtacgaccc cctgcagcct 3420

gaactcgata gtttcaaaga agagctggac aagtacttca aaaaccacac gagccctgac 3480

gtggacctcg gcgacatcag cggtatcaac gccagcgtcg tcaacatcca aaaagagatc 3540

gacagactga acgaggtggc caagaacctg aatgagagtc tgatcgacct gcaggagctg 3600

ggaaagtacg aacagtacat caagtggccc tggtacatct ggctgggatt catcgccggc 3660

ctgatcgcta tcgtcatggt tactattatg ctgtgctgta tgacatcatg ttgtagctgt 3720

ctcaaaggct gctgcagctg tggcagctgc tgcaagttcg acgaagatga ctctgagcca 3780

gtgctcaagg gcgtaaagct gcactacacc tgataaacta g 3821

<210> 3

<211> 49

<212> DNA

<213> Artificial sequence

<400> 3

gtaccgagct cggatccgcc accatgttcg tgttcctggt cctactgcc 49

<210> 4

<211> 45

<212> DNA

<213> Artificial sequence

<400> 4

agaatagggc cctctagact agtttatcag gtgtagtgca gcttt 45

<210> 5

<211> 46

<212> DNA

<213> Artificial sequence

<400> 5

ggatccgagc tcggtaccaa gcttaagttt aaacgctaga gtccgg 46

<210> 6

<211> 26

<212> DNA

<213> Artificial sequence

<400> 6

tctagagggc cctattctat agtgtc 26

<210> 7

<211> 52

<212> DNA

<213> Artificial sequence

<400> 7

gtaccgagct cggatccgcc accatgtttg tttttcttgt tttattgcca ct 52

<210> 8

<211> 53

<212> DNA

<213> Artificial sequence

<400> 8

tagaataggg ccctctagac tagtttatta tgtgtaatgt aatttgactc ctt 53

<210> 9

<211> 34

<212> DNA

<213> Artificial sequence

<400> 9

ttggattgaa gccaatatga taatgagggg gtgg 34

<210> 10

<211> 36

<212> DNA

<213> Artificial sequence

<400> 10

gcatcggtcg aggacaggcc tctcaagtct gtatac 36

<210> 11

<211> 54

<212> DNA

<213> Artificial sequence

<400> 11

agaattggat ccgaattcgc ggccgcgcga tcgccatcat caataatata cctt 54

<210> 12

<211> 45

<212> DNA

<213> Artificial sequence

<400> 12

gcgtcgcaga tccgaattcg tatacccatc caagctgcac gataa 45

Claims

1. A vaccine, comprising: SEQ ID NO: 2.

2. The vaccine of claim 1, wherein: the vaccine is an adenovirus vector vaccine.

3. The vaccine of claim 2, comprising a polypeptide loaded with SEQ ID NO: 2, or an Ad5 vector loaded with the nucleic acid sequence shown in SEQ ID NO: 2, or a nucleic acid sequence thereof.

4. The vaccine of any one of claims 1-3, characterized in that: further comprising at least one of a pharmaceutically acceptable adjuvant, carrier, diluent or excipient.

5. The vaccine of any one of claims 1-3, characterized in that: also comprises at least one drug which has preventive and/or therapeutic effects on COVID-19.

6. An expression vector comprising the nucleotide sequence of SEQ ID NO: 2.

7. The expression vector of claim 6, wherein: the vector is a DNA plasmid, an RNA expression plasmid or a virus vector.

8. The expression vector according to claim 6 or 7, characterized in that: the vector can regulate the expression of the nucleic acid sequence.

9. Use of the vaccine of any one of claims 1-5 or the expression vector of any one of claims 6-8, comprising:

preparing a medicament for preventing infection caused by SARS-CoV-2; and/or

Preparing the detection reagent of COVID-19.