CN116814562A - Human 5-type replication defective recombinant adenovirus based on target gene S1, preparation method and vaccine for preventing SARS-CoV-2 infection - Google Patents

Abstract

The invention belongs to the technical field of bioengineering, and particularly discloses a human type 5 replication defective recombinant adenovirus based on a target gene S1, wherein the nucleotide sequence of the target gene S1 of the human type 5 replication defective recombinant adenovirus is shown as SEQ ID NO. 1, and the target gene S1 can express S1 protein of SARS-CoV-2 in human cells or human bodies. The human type 5 replication-defective recombinant adenovirus shows good immunogenicity in a cell model, can effectively induce organism to generate humoral and cellular immune response, and has good safety. In addition, the preparation method of the human 5-type replication-defective recombinant adenovirus is simple and easy to operate, has extremely high application value, and can be used for mass production and application to possibly sudden epidemic situations in a short time. And the expression level of the target gene S1 in transfected cells is obviously improved after optimization.

Description

Human 5-type replication defective recombinant adenovirus based on target gene S1, preparation method and vaccine for preventing SARS-CoV-2 infection

Technical Field

The invention relates to the technical field of bioengineering, in particular to a human 5 type replication defective recombinant adenovirus based on a target gene S1, a preparation method and a vaccine for preventing SARS-CoV-2 infection.

Background

At present, epidemic caused by novel coronaviruses forms a great threat to global public safety, and designing and developing vaccines against the novel coronaviruses has great significance for preventing and controlling epidemic spread. Novel coronaviruses belonging to the genus beta, single-stranded positive strand RNA viruses with a envelope. The genome contains at least 10 ORFs, ORF1ab is converted to a multimeric protein, processed into 16 nonstructural proteins (NSPs). This NSP plays an important role in the viral life cycle. The novel coronavirus has 5 essential genes encoding the 4 structural proteins of the translation nucleus protein (N), the viral envelope protein (E), the matrix protein (M) and the spike protein (S) and the RNA-dependent RNA polymerase (RdRp), respectively. Wherein, the S protein is a multifunctional transmembrane protein, and plays an important role in the processes of virus adsorption, fusion and host cell injection. The S protein has three domains, the S1 domain at its top layer being the most important surface antigen. When the novel coronavirus enters a human body, the novel coronavirus can avoid inherent immune killing by combining an S1 structural domain with angiotensin converting enzyme II; can also induce organism immune response, so that the lung cells expressing Human Leukocyte Antigen (HLA) -E inhibit natural killer cell activation and IFN-gamma secretion, thereby being complicated with a series of serious clinical critical serious symptoms, such as inflammatory factor storm, idiopathic pulmonary fibrosis and the like, and causing different degrees of damage to important organs of human body. Thus, the development of vaccines against the S1 domain of the S protein (i.e. the S1 protein) will become one of the main prophylactic therapeutic strategies.

Meanwhile, human adenovirus serotype 5 (HAdV-C5) is widely used in basic virology as a vector for gene therapy and vaccine delivery. HAdV-C5 has natural diversity and can be parasitic to most hosts, which is the basis of animal experimental development. The recombinant adenovirus with infectivity and replication defect constructed by HAdV-C5 has good application prospect in the fields of vaccine delivery and gene therapy as a vector. Currently, adenoviruses have been approved as vaccines against acute respiratory infections, and there have been many trials of such vaccines, such as malaria and HIV-1. In addition, antigens delivered by the adenovirus-corresponding vector can induce intense cellular and humoral immunity after a single immunization, making them useful as an urgent preventative tool for pandemic. Thus, adenovirus-based vaccine strategies are an important component of the strategy to combat new coronavirus infections.

Disclosure of Invention

In order to overcome the defects in the prior art, the invention provides a human type 5 replication-defective recombinant adenovirus based on a target gene S1, a preparation method and a vaccine for preventing SARS-CoV-2 infection.

The technical scheme adopted for solving the technical problems is as follows: the nucleotide sequence of the target gene S1 of the human type 5 replication-defective recombinant adenovirus is shown as SEQ ID NO. 1, and the target gene S1 can express the S1 protein of SARS-CoV-2 in human cells or human bodies.

As a further improvement, the human type 5 replication defective recombinant adenovirus lacks the E1 gene and the E3 gene.

As a further improved scheme, the amino acid sequence of the S1 protein is shown as SEQ ID NO. 2.

Also provided is a method for preparing the human type 5 replication-defective recombinant adenovirus, which is used for preparing the human type 5 replication-defective recombinant adenovirus, and comprises the following steps:

(1) Synthesizing a nucleotide sequence of a target gene S1 and cloning the target gene into a pUC18 plasmid;

(2) Constructing a recombinant adenovirus shuttle plasmid pAdeno-CMV-S1 of a target gene S1;

(3) Transfecting a backbone plasmid of the virus into a host cell together with the shuttle plasmid of step (2);

(4) Culturing the host cell of step (3);

(5) Obtaining a human replication-defective recombinant adenovirus type 5 released by the host cell of step (4);

(6) Purifying and amplifying the strain of the human type 5 replication-defective recombinant adenovirus in the step (5);

(7) And (3) identifying the amplified strain of the human type 5 replication-defective recombinant adenovirus in the step (6).

Also provides a vaccine for preventing SARS-CoV-2 infection, which is prepared by adopting the human type 5 replication defective recombinant adenovirus.

The invention has at least one of the following beneficial effects:

1. the human type 5 replication-defective recombinant adenovirus shows good immunogenicity in a cell model, can effectively induce organism to generate humoral and cellular immune response, and has good safety.

2. The preparation method of the human 5-type replication-defective recombinant adenovirus has simple and easy operation steps, has extremely high application value, and can be used for mass production and application to possibly sudden epidemic situations in a short time.

3. After the target gene S1 is optimized, the expression level in transfected cells is obviously improved.

Drawings

FIG. 1 is a schematic diagram showing the construction of a cloning plasmid pUC18-S1 into which a target gene S1 has been inserted in the examples of the present invention;

FIG. 2 is a schematic diagram of the recombinant adenovirus shuttle plasmid pAdeno-CMV-S1 into which the target gene S1 is inserted in the examples of the present invention;

FIG. 3 shows the result of gel electrophoresis (M: DNA Marker,1-4 positive clone, 5 negative control) of PCR assay identification gel of recombinant adenovirus shuttle plasmid pAdeno-CMV-S1 of target gene S1 in the examples of the present invention;

FIG. 4 is a graph showing quantitative values and statistical analysis results of plasmid identification qPCR in the examples of the present invention;

FIG. 5 is a schematic diagram of a human replication-defective recombinant adenovirus type 5 according to an embodiment of the invention;

FIG. 6 is a graph showing quantitative values and statistical analysis results of qPCR of human type 5 replication defective recombinant adenovirus in the examples of the present invention;

FIG. 7 is a graph showing the result of Western Blot virus expression in the examples of the present invention;

FIG. 8 shows the result of cellular immunofluorescence assay in an embodiment of the present invention;

FIG. 9 is a graph showing quantitative values and statistical analysis results of qPCR for adenovirus purification and identification in the examples of the present invention;

FIG. 10 shows ELISA S1 antibody titers in examples of the invention;

FIG. 11 is a diagram showing neutralizing antibodies against SARS-COV-2 pseudovirus in the examples of the present invention;

FIG. 12 is a neutralizing antibody against SARS-COV-2 in an embodiment of the invention;

FIG. 13 is a graph showing the results of immunological evaluation of human replication-defective recombinant adenovirus type 5 on a mouse model in the examples of the present invention.

Detailed Description

The following describes the embodiments of the present invention further with reference to the drawings. The description of these embodiments is provided to assist understanding of the present invention, but is not intended to limit the present invention. In addition, the technical features of the embodiments of the present invention described below may be combined with each other as long as they do not collide with each other.

In the examples, the human replication-defective recombinant adenovirus type 5 was modified so that the E1 gene and E3 gene were removed, but the adenovirus still had the ability to infect cells.

Example 1

The purpose of this example is to prepare a human replication-defective recombinant adenovirus type 5 based on the gene of interest S1, the preparation method comprising the steps of:

1. construction of recombinant adenovirus shuttle plasmid

1.1 optimization and Synthesis of the Gene of interest S1

The coding region sequence of the target gene S1 is utilized to obtain the amino acid sequence encoded by the gene, and then the optimal codon nucleotide sequence suitable for mammalian cell expression is designed on the premise that the amino acid sequence of the encoded product protein is kept unchanged according to the degeneracy of the gene. Next, the first amino acid of the S1 protein is removed, and the tissue plasminogen activator signal peptide tPA is connected before the second amino acid to further increase the expression level of the S1 protein. The nucleotide obtained by the optimization operation is named as a target gene S1, and the sequence of the nucleotide is specifically shown as SEQ ID NO. 1; the protein of the product is named as S1 protein, and the sequence of the protein is specifically shown in SEQ ID NO. 2.

1.2 construction of recombinant adenovirus shuttle plasmid corresponding to the Gene S1 of interest

The plasmid pUC18 and the optimized target gene S1 are subjected to enzyme digestion and are connected through DNA ligase, so that the nucleotide sequence of the target gene S1 is cloned in pUC18 to obtain a cloning plasmid containing the target gene S1, which is named pUC18-S1, and the plasmid spectrum is shown in the attached figure 1.

The target gene S1 synthesized and cloned in the above experiment on pUC18 was amplified by PCR, and then the target gene S1 fragment was recovered by agarose gel recovery kit, and pUC18-S1 and adenovirus shuttle plasmid pAdeno-CMV-MCS were digested with endonucleases (SpeI/XbaI) in double. Then DNA ligase is used for connection to obtain the recombinant adenovirus shuttle plasmid with correct insertion, which is named pAdeno-CMV-S1, and the structure diagram is shown in figure 2. The obtained pAdeno-CMV-S1 was transformed into DH 5-alpha competent, coated on AmprLB plates, and the monoclonal was selected for PCR assay, and the results are shown in FIG. 3. The shuttle plasmid after the PCR bacteria authentication is right is sequenced, and the primer sequences are MCMV-F ggtataagaggcgcgaccag and S1-R acaataagtagggactgggtc. The results showed that the sequences were further confirmed to be consistent with the design.

1.3 Identification of Gene of interest S1

1.3.1 Real-time PCR detection (qPCR detection)

Transfecting recombinant adenovirus shuttle plasmid pAdeno-CMV-S1 into HEK293 cells, extracting total RNA from the transfected cells, centrifuging the cells and removing cell supernatant, adding 1000 mul Trizol into a hole, blowing and mixing uniformly, standing and transferring to a new centrifuge tube. 200 μl of chloroform is added into each tube, the mixture is stirred, mixed and kept stand, and supernatant is sucked into a new centrifuge tube after centrifugation. Adding an equal volume of isopropanol, uniformly mixing and precipitating, centrifuging, discarding supernatant, adding at least 1ml of precooled 75% ethanol, washing the precipitate and the tube wall, centrifuging, discarding supernatant, centrifuging again to remove residual liquid, drying at room temperature, adding 30-40 μl of RNase-free water until the RNA is completely dissolved, and measuring the concentration of the extracted RNA by ultraviolet analysis. The extracted RNA is subjected to reverse transcription to obtain cDNA, and qPCR detection is carried out on the extracted cDNA.

Primer sequence of target gene S1:

XQ3389-1：GGTGATTCTTCTTCAGGTTGGA

XQ3389-2：GTTTCTGAGAGAGGGTCAAGTG

reference Gene (GAPDH) primer sequences:

QCprimer002-F：GTCTTCACCACCATGGAGAA

QCprimer002-R：TAAGCAGTTGGTGGTGCAG

the experimental results are shown in fig. 4 and table 1. The results showed that the expression level of the target gene S1 in the virus ADV supernatant-pAdeno-CMV-S1 infected cell group was 15374300.53% compared with the control virus ADV supernatant-pAeno-CMV-MCS infected cell group, indicating that the recombinant adenovirus shuttle plasmid pAdeno-CMV-N can express higher content of S1 protein.

TABLE 1 quantitative values and analysis (2-DeltaCt assay)

2. Packaging and identification of human replication defective recombinant adenoviruses

2.1 packaging human type 5 replication defective recombinant adenoviruses

Firstly, preparing solution A (prepared by mixing recombinant adenovirus shuttle plasmid pAdeno-CMV-S1 3 mu g and skeleton plasmid pBHGlox delta E1,3Cre 6 mu g into 250 mu l of Opti-MEM culture medium) and solution B (prepared by mixing 10 mu l LipofectamineTM2000 into 240 mu l of Opti-MEM culture medium), standing at room temperature for 5min, adding solution A into solution B, mixing uniformly, and standing at room temperature for 20min. 293 cells cultured on DMEM medium without antibiotic in 5% fcs the day before transfection were taken and subjected to pipetting. Then, the A+B mixture was added and incubated at 37℃under 5% CO 2. 7-10 days after transfection, cells were taken and centrifuged at 800rpm for 5min, and the supernatant was discarded. The pellet was resuspended and freeze-thawed 4 times repeatedly. Centrifuging at 2000rpm for 10min, and collecting supernatant to obtain primary virus liquid (first generation) P1, wherein the obtained recombinant adenovirus is named as human replication defective recombinant adenovirus 5 (rAD 5-S1), and storing at-80deg.C.

The structural diagram of the human type 5 replication defective recombinant adenovirus (rAD 5-S1) obtained in the above way is shown in the figure 5.

2.2 identification of human replication defective recombinant adenoviruses of type 5

2.2.1RT-qPCR detection

The total RNA of the human type 5 replication defective recombinant adenovirus is extracted, the extracted RNA is reversely transcribed into cDNA, and the extracted cDNA is subjected to qPCR detection, and the specific detection method is described in 1.3.1.

The experimental results are shown in fig. 6 and table 2. The result shows that compared with the control virus ADV supernatant-pAeno-CMV-MCS infected cell group, the expression level of the target gene S1 in the virus ADV supernatant-pAdeno-CMV-S1 infected cell group is 417806.62%, which indicates that the human type 5 replication defective recombinant adenovirus can express higher S1 protein.

TABLE 2 quantitative values and analysis (2-DeltaCt assay)

2.2.2 Western Blot (WB) detection

a) The transfected HEK293 cells were subjected to total RNA extraction by first mixing 990. Mu.l of cell lysate with 10. Mu.l of PMSF and placing on ice at 4 ℃. Taking out cells, removing a cell culture solution, washing for 1-2 times by using PBS, adding a proper amount of precooled Western and IP cell lysate (containing PMSF), and lysing the cells on ice. After 30min the cells were scraped off and placed in a sterile EP tube which was taken out of the pre-chilled. Centrifuging to collect cells;

b) The protein concentration of the protein is required to be measured, and a cell protein solution (3 mu l of PBS is extracted from the stock solution, and then 27 mu l of PBS is added for dilution by 10 times) and a BCA working solution are prepared. 2000. Mu.g/ml BSA standard was then diluted to 0, 25, 125, 250, 500, 750, 1000, 1500, 2000. Mu.g/ml. 25 μl of each of the standard and sample was added to a 96-well plate; 200 μl of BCA working solution is added into each hole, and the holes are placed for 30min at 37 ℃; measuring the absorbance of A562 by using an enzyme-labeled instrument; calculating the protein concentration of the sample according to the standard curve and the volume of the sample;

c) And (3) preparing a sample required by WB, expanding and diluting the calculated data of each hole according to the actual volume of the sample mother solution, and adding a loading buffer according to a ratio of 1:4 (loading buffer: cell sap), mixing, decocting at 95deg.C for 5min, placing on ice chest, and centrifuging;

d) Performing SDS-PAGE gel electrophoresis and membrane transfer on the sample;

e) An immune response experiment was performed.

Antibody information:

resistance information: SARS-CoV-2 (2019-nCoV) Spike Antibody (1:5000, cat# 40591-T62, sino Biological)

Secondary antibody information: goat anti-rabbit immunoglobulin G (IgG) H & L (HRP) (1:10,000, cat.#ab 6721, abcam)

The membranes were first transferred to TBST incubators containing 5% nonfat milk powder and blocked by shaking on a decolorizing shaker at room temperature. TBST containing 5% nonfat milk powder was diluted to an appropriate concentration for use as a primary antibody, the membrane was removed from the blocking solution, the protein side of the membrane was placed face up on the antibody liquid surface, residual air bubbles were removed, and the membrane was shaken on a decolorizing shaker overnight. The secondary TBST is washed three times on a decolorization shaking table at room temperature, the secondary antibody is diluted by the same method and contacted with a membrane, and after incubation for 1-2 hours at room temperature, the secondary antibody is washed three times on the decolorization shaking table at room temperature and then subjected to chemiluminescence reaction.

The experimental results are shown in figure 7. The result shows that the predicted protein size of the target gene S1 is about 75.6 kDa. The experimental results showed that protein bands were detected near Marker 75 kDa.

2.2.3 immunofluorescence detection experiments

The experimental method is as follows: penetration was first performed using Triton X-100 and then washed 3 times with PBS. After removing PBS, BSA blocking solution was added, and incubated at room temperature for blocking. After the end of the blocking, the blocking solution was discarded and a primary antibody was used: SARS-CoV-2 (2019-nCoV) Spike Antibody (1:2000, cat# 40591-T62, sino Biological) was incubated overnight at 4℃and after the primary Antibody incubation was completed, the primary Antibody was discarded and washed 3 times with PBS. After PBS removal, secondary antibodies were added: the coat anti-rabit IgG H & L (Alexa Fluor 488) (1:1000, cat.# 550037, zenBio) was incubated at room temperature in the absence of light. After the secondary antibody incubation was completed, the secondary antibody was discarded, washed 3 times with PBS to 1: hoechst staining was performed at 500 dilution ratio and incubated for 5min, after which incubation the incubation was washed 3 times with PBS.

The experimental results are shown in FIG. 8, which shows that the human replication-defective recombinant adenovirus type 5 can effectively express a protein with S1 protein characteristics.

3. Amplification, purification and identification of human 5-type replication-defective recombinant adenoviruses

3.1 Amplifying and purifying human 5-type replication defective recombinant adenovirus

10 μl of virus (about 107-108 PFU/ml) of appropriate titer was taken and spread on 30-40 10cm dish HEK293 cells. After 2-3 days (total lesions of cells), nonidet P40 (NP 40) was added to lyse the cells. Lysates were centrifuged at 12000rpm for 10min, the supernatant was collected and virus pellet (20% PEG8000,2.5M NaCl) was added to pellet virus (1 h on ice). The mixture was centrifuged at 12000rpm for 20min, the pellet was suspended in a CsCl solution (solvent 20 mM Tris-HCl, pH 8.0) at a density of 1.10g/ml, and the virus suspension was collected by centrifugation at 7000rpm at 4℃for 5min.

1.40g/ml CsCl solution (solvents as above), 1.30. 1.30 g/ml CsCl solution and virus suspension were added sequentially to an ultracentrifuge tube and centrifuged at 22800 rpm at 4℃for 2.5h. The virus bands with a density between 1.30 and 1.40g/ml were collected into dialysis bags. In dialysis buffer, dialysis was performed overnight at 4 ℃. Collecting virus, and storing at-80deg.C for use.

3.2 Identification of purified human type 5 replication defective recombinant adenoviruses

The target gene sequence is amplified by PCR, data is quantified and the result is analyzed, and the experimental method is the same as 1.3.1. The experimental results are shown in fig. 9 and table 3. The result shows that compared with the control virus ADV purified-pAeno-CMV-MCS infected cell group, the expression level of the target gene S1 in the virus ADV purified-CMV-S1 infected cell group is 4615284.26%, which indicates that the purified human type 5 replication defective recombinant adenovirus can express N protein with higher content.

TABLE 3 quantitative values and analysis (2-DeltaCt assay)

4. Titer detection human replication-defective recombinant adenovirus 5

Resuspension of cells with complete medium to form a cell suspension, seeding in 24-well plates and incubating at 37℃with 5% CO ₂ Culturing. Take 10 ^-5 To 10 ^-8 Adding diluted virus solution into 24-well plate, and adding 5% CO at 37deg.C ₂ Infection was performed for 48h. After removal of the medium, 500. Mu.l of pre-chilled methanol was slowly added along the side wall of the 24-well plate and fixed at-20℃for 20min. After blocking with PBS 3 times and 1% BSA at 37℃for 1 hour, primary and secondary antibody solutions were added to each well, respectively, and incubated at 37℃for 1 hour (3 times between the addition of primary and secondary antibody solutions, the cells were washed with PBS). After the incubation is completed, the secondary antibody is washed off by PBS, and then the newly prepared working solution is added, and the incubation is carried out for 5-10min at room temperature. After washing with PBS 2 times, 1000. Mu.l of PBS was added to each well and the mixture was observed. Randomly selecting 5 visual fields from each hole, and calculating the number of positive cells under a 10X objective lens of an optical microscope; m) the average number of positive cells per well and the viral titer were calculated.

The average number of positive cells calculated in 5 fields under a microscope in the experiment is 6, the virus in the hole is diluted 108 times, and the positive cells are obtained according to the formula:

the result of the measurement of the number of the viral titer shows that the titer of the recombinant adenovirus after purification can reach 2.37X10 ¹¹ (pfu/ml), demonstrating that the purified human replication-defective recombinant adenovirus type 5 has higher titers.

Example 2

Immunological evaluation of different constructed human type 5 replication defective recombinant adenoviruses on cell and animal models

1. Binding antibody assay

The corresponding S1 protein (1. Mu.g/mL) of SARS-CoV-2 was coated with 0.05M bicarbonate buffer over night on a 96-well plate (100. Mu.L/well). With PBST (0.2 g KH) ₂ PO ₄ , 2.9 g Na ₂ HPO ₄ •12H ₂ O, 8.0 g NaCl, 0.2 g KCl, 0.5mL Tween-20, water to 1000 mL) was added and after 5 washes, blocking solution was added and incubated at 37℃for 1 h. Serum samples were diluted and added to each well (100 μl/well), incubated at 37 ℃ for 1h, and washed 5 times with PBST. Adding primary Antibody [ SARS-CoV-2 (2019-nCoV) Spike Antibody (1:2000, cat# 40591-T62, sino Biological)]Incubation 1H followed by 5 washes with PBST followed by addition of secondary antibody [ coat anti-rabit IgG H ]&L (HRP) (1:10,000, cat. #ab6721, abcam)]. After washing 5 times with PBST, 3,30,5,50-Tetramethylbenzidine (TMB) was added and reacted for 5min. After stopping the reaction with 2M sulfuric acid, the optical densities were measured at 450 nm and 630 nm with an enzyme marker (Molecular Devices, spectraMax 190) and then fitted to a standard curve.

The experimental results are shown in fig. 10. The results show that specific IgG is detected in serum of two doses of human type 5 replication defective recombinant adenovirus (rAD 5-S1) injected mice, and the highest titer is 1:2 ^9.1 Average titer 1:2 ⁹ And no specific IgG was detected in the serum of mice in PBS/Ad injection group, indicating that the injection of two rAD5-S1 doses can smoothly perform humoral immunity.

2. Neutralizing antibody assay

2.1 Pseudovirus neutralization assay

The neutralizing activity of mouse serum was assessed using a Vesicular Stomatitis Virus (VSV) based pseudoviral system. 293T cells were plated in 96-well plates and diluted serum samples were mixed with pseudovirus liquid diluted in 2% FBS DMEM medium. After incubation at 37 ℃ for 1h, the mixture was added to a 96-well plate. After further incubation at 37℃for 48h, luciferase (relative light unit, RLU) expression was detected. The serum antibody which can achieve an infection suppression ratio of 50% or more after dilution is considered to have a neutralizing activity [ infection suppression ratio (%) = (1-serogroup RLU/virus control group RLU) ×100% ].

The experimental results are shown in the figure11. The results showed that the average neutralization titers were 1:2 on fourteen days after the first injection and fourteen days after the second injection, respectively ^8.1 And 1:2 ^9.6 It was demonstrated that the serum of mice injected with two doses of rAd5-S1 had neutralizing activity against VSV virus.

2.2 Live virus neutralization assay

The neutralization activity of the serum of the mice was evaluated by the in vivo neutralization test. The diluted two-fold serum was mixed with SARS-CoV-2, incubated at 37℃for 1 hour, and then, vero E6 cells were infected by adding a 96-well plate. After further incubation at 37 ℃ for 72 h, the virus cytopathic effect (CPE) was observed under a x 40 microscope and the neutralization titers (the reciprocal of the 50% serum dilution required to neutralize the virus infection) were calculated. All of the above steps are performed in a biosafety level 3 environment.

The experimental results are shown in fig. 12. The results show that the serum of mice injected with two doses of human replication-defective recombinant adenovirus type 5 (rAd 5-S1) can protect Vero-E6 cells in vitro, and the average titer is 1:2 on fourteen days after the first injection and fourteen days after the second injection, respectively ^4.3 And 1:2 ^5.0 It was demonstrated that the serum of mice injected with two doses of rAd5-S1 can protect Vero-E6 cells in vitro.

3. Human 5-type replication-defective recombinant adenovirus vaccine immunogenicity experiments

40 SPF-class female mice (6-8 weeks old) were randomly divided into 4 groups of 10. Mice were immunized with rAd5-S1 according to the groupings shown in table 1.

TABLE 1 recombinant adenovirus vaccine immunogenicity protocol

The experimental results are shown in FIG. 13. It was found that the vaccine was safe and had no adverse reaction found.

Example 3

The present example discloses a vaccine for preventing SARS-CoV-2 infection, which is prepared by using the human type 5 replication defective recombinant adenovirus.

The nucleotide sequence SEQ ID NO 1 corresponding to the target gene S1 and the amino acid sequence SEQ ID NO 2 corresponding to the S1 protein in the examples of the present invention are as follows.

Sequence listing

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Artificial sequence (Artificial Sequence)

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ATGTTCTTGT TAACAACTAA ACGAACAATG TTTGTTTTTC TTGTTTTATT GCCACTAGTC 60

TCTAGTCAGT GTGTTAATCT TACAACCAGA ACTCAATTAC CCCCTGCATA CACTAATTCT 120

TTCACACGTG GTGTTTATTA CCCTGACAAA GTTTTCAGAT CCTCAGTTTT ACATTCAACT 180

CAGGACTTGT TCTTACCTTT CTTTTCCAAT GTTACTTGGT TCCATGCTAT ACATGTCTCT 240

GGGACCAATG GTACTAAGAG GTTTGATAAC CCTGTCCTAC CATTTAATGA TGGTGTTTAT 300

TTTGCTTCCA CTGAGAAGTC TAACATAATA AGAGGCTGGA TTTTTGGTAC TACTTTAGAT 360

TCGAAGACCC AGTCCCTACT TATTGTTAAT AACGCTACTA ATGTTGTTAT TAAAGTCTGT 420

GAATTTCAAT TTTGTAATGA TCCATTTTTG GGTGTTTATT ACCACAAAAA CAACAAAAGT 480

TGGATGGAAA GTGAGTTCAG AGTTTATTCT AGTGCGAATA ATTGCACTTT TGAATATGTC 540

TCTCAGCCTT TTCTTATGGA CCTTGAAGGA AAACAGGGTA ATTTCAAAAA TCTTAGGGAA 600

TTTGTGTTTA AGAATATTGA TGGTTATTTT AAAATATATT CTAAGCACAC GCCTATTAAT 660

TTAGTGCGTG ATCTCCCTCA GGGTTTTTCG GCTTTAGAAC CATTGGTAGA TTTGCCAATA 720

GGTATTAACA TCACTAGGTT TCAAACTTTA CTTGCTTTAC ATAGAAGTTA TTTGACTCCT 780

GGTGATTCTT CTTCAGGTTG GACAGCTGGT GCTGCAGCTT ATTATGTGGG TTATCTTCAA 840

CCTAGGACTT TTCTATTAAA ATATAATGAA AATGGAACCA TTACAGATGC TGTAGACTGT 900

GCACTTGACC CTCTCTCAGA AACAAAGTGT ACGTTGAAAT CCTTCACTGT AGAAAAAGGA 960

ATCTATCAAA CTTCTAACTT TAGAGTCCAA CCAACAGAAT CTATTGTTAG ATTTCCTAAT 1020

ATTACAAACT TGTGCCCTTT TGGTGAAGTT TTTAACGCCA CCAGATTTGC ATCTGTTTAT 1080

GCTTGGAACA GGAAGAGAAT CAGCAACTGT GTTGCTGATT ATTCTGTCCT ATATAATTCC 1140

GCATCATTTT CCACTTTTAA GTGTTATGGA GTGTCTCCTA CTAAATTAAA TGATCTCTGC 1200

TTTACTAATG TCTATGCAGA TTCATTTGTA ATTAGAGGTG ATGAAGTCAG ACAAATCGCT 1260

CCAGGGCAAA CTGGAAAGAT TGCTGATTAT AATTATAAAT TACCAGATGA TTTTACAGGC 1320

TGCGTTATAG CTTGGAATTC TAACAATCTT GATTCTAAGG TTGGTGGTAA TTATAATTAC 1380

CTGTATAGAT TGTTTAGGAA GTCTAATCTC AAACCTTTTG AGAGAGATAT TTCAACTGAA 1440

ATCTATCAGG CCGGTAGCAC ACCTTGTAAT GGTGTTGAAG GTTTTAATTG TTACTTTCCT 1500

TTACAATCAT ATGGTTTCCA ACCCACTAAT GGTGTTGGTT ACCAACCATA CAGAGTAGTA 1560

GTACTTTCTT TTGAACTTCT ACATGCACCA GCAACTGTTT GTGGACCTAA AAAGTCTACT 1620

AATTTGGTTA AAAACAAATG TGTCAATTTC AACTTCAATG GTTTAACAGG CACAGGTGTT 1680

CTTACTGAGT CTAACAAAAA GTTTCTGCCT TTCCAACAAT TTGGCAGAGA CATTGCTGAC 1740

ACTACTGATG CTGTCCGTGA TCCACAGACA CTTGAGATTC TTGACATTAC ACCATGTTCT 1800

TTTGGTGGTG TCAGTGTTAT AACACCAGGA ACAAATACTT CTAACCAGGT TGCTGTTCTT 1860

TATCAGGATG TTAACTGCAC AGAAGTCCCT GTTGCTATTC ATGCAGATCA ACTTACTCCT 1920

ACTTGGCGTG TTTATTCTAC AGGTTCTAAT GTTTTTCAAA CACGTGCAGG CTGTTTAATA 1980

GGGGCTGAAC ATGTCAACAA CTCATATGAG TGTGACATAC CCATTGGTGC AGGTATATGC 2040

TAA 2043

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Amino acids

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MFLLTTKRTM FVFLVLLPLV SSQCVNLTTR TQLPPAYTNS FTRGVYYPDK VFRSSVLHST 60

QDLFLPFFSN VTWFHAIHVS GTNGTKRFDN PVLPFNDGVY FASTEKSNII RGWIFGTTLD 120

SKTQSLLIVN NATNVVIKVC EFQFCNDPFL GVYYHKNNKS WMESEFRVYS SANNCTFEYV 180

SQPFLMDLEG KQGNFKNLRE FVFKNIDGYF KIYSKHTPIN LVRDLPQGFS ALEPLVDLPI 240

GINITRFQTL LALHRSYLTP GDSSSGWTAG AAAYYVGYLQ PRTFLLKYNE NGTITDAVDC 300

ALDPLSETKC TLKSFTVEKG IYQTSNFRVQ PTESIVRFPN ITNLCPFGEV FNATRFASVY 360

AWNRKRISNC VADYSVLYNS ASFSTFKCYG VSPTKLNDLC FTNVYADSFV IRGDEVRQIA 420

PGQTGKIADY NYKLPDDFTG CVIAWNSNNL DSKVGGNYNY LYRLFRKSNL KPFERDISTE 480

IYQAGSTPCN GVEGFNCYFP LQSYGFQPTN GVGYQPYRVV VLSFELLHAP ATVCGPKKST 540

NLVKNKCVNF NFNGLTGTGV LTESNKKFLP FQQFGRDIAD TTDAVRDPQT LEILDITPCS 600

FGGVSVITPG TNTSNQVAVL YQDVNCTEVP VAIHADQLTP TWRVYSTGSN VFQTRAGCLI 660

GAEHVNNSYE CDIPIGAGIC 680

The embodiments of the present invention have been described in detail above with reference to the accompanying drawings, but the present invention is not limited to the described embodiments. It will be apparent to those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, and yet fall within the scope of the invention.

Claims (5)

1. A human type 5 replication defective recombinant adenovirus based on gene of interest S1, characterized in that: the nucleotide sequence of the target gene S1 of the human type 5 replication-defective recombinant adenovirus is shown as SEQ ID NO. 1, and the target gene S1 can express the S1 protein of SARS-CoV-2 in human cells or human bodies.

2. The human replication-defective recombinant adenovirus type 5 based on gene S1 of interest according to claim 1, wherein: the human type 5 replication defective recombinant adenovirus lacks the E1 gene and the E3 gene.

3. The human replication-defective recombinant adenovirus type 5 based on gene S1 of interest according to claim 1, wherein: the amino acid sequence of the S1 protein is shown as SEQ ID NO. 2.

4. The preparation method of the human 5-type replication-defective recombinant adenovirus is characterized by comprising the following steps: the preparation method is used for preparing the human type 5 replication-defective recombinant adenovirus according to any one of claims 1-3, and comprises the following steps:

(1) Synthesizing a nucleotide sequence of a target gene S1 and cloning the target gene into a pUC18 plasmid;

(2) Constructing a recombinant adenovirus shuttle plasmid pAdeno-CMV-S1 of a target gene S1;

(3) Transfecting a backbone plasmid of the virus into a host cell together with the shuttle plasmid of step (2);

(4) Culturing the host cell of step (3);

(5) Obtaining a human replication-defective recombinant adenovirus type 5 released by the host cell of step (4);

(6) Purifying and amplifying the strain of the human type 5 replication-defective recombinant adenovirus in the step (5);

(7) And (3) identifying the amplified strain of the human type 5 replication-defective recombinant adenovirus in the step (6).

5. A vaccine for preventing SARS-CoV-2 infection, characterized in that: the recombinant adenovirus of human type 5 replication-defective type according to any one of claims 1 to 4.