CN113186223A

CN113186223A - Expression vector of novel coronavirus vaccine, construction method and application thereof, and vaccine

Info

Publication number: CN113186223A
Application number: CN202110673867.3A
Authority: CN
Inventors: 苏小平; 李锐; 李伟迎; 张齐
Original assignee: Zhejiang Geyuan Zhizhen Biomedical Technology Co ltd
Current assignee: Zhejiang Geyuan Zhizhen Biomedical Technology Co ltd
Priority date: 2021-06-17
Filing date: 2021-06-17
Publication date: 2021-07-30

Abstract

The invention is applicable to the technical field of biology, and provides an expression vector of a novel coronavirus vaccine, a construction method, application and a vaccine thereof, wherein the construction method of the expression vector comprises the following steps: connecting nucleotide sequences expressing the new coronavirus S protein and NP protein by using 2A peptide to synthesize a fusion gene; two ends of the fusion gene respectively contain two enzyme cutting sites and are loaded to plasmids to obtain recombinant plasmids; carrying out double enzyme digestion on the recombinant plasmid, cutting the gel and recovering a target gene fragment; carrying out double enzyme digestion on the original plasmid, cutting the gel and recovering a carrier fragment; and connecting the target gene fragment with the vector fragment to obtain the expression vector. According to the embodiment of the invention, the coronavirus S protein receptor binding region and the NP protein are simultaneously expressed, so that the cells infected by the expression vector can induce not only antibody reaction but also T cell reaction, thereby effectively inducing humoral immunity and cellular immunity and providing stronger immune protection for a subject.

Description

Expression vector of novel coronavirus vaccine, construction method and application thereof, and vaccine

Technical Field

The invention belongs to the technical field of biology, and particularly relates to an expression vector of a novel coronavirus vaccine, a construction method, application and the vaccine thereof.

Background

The biggest problem facing global public health today is new and recurrent infections. Coronavirus is a type of infectious virus that causes serious diseases, is widely present in human, mammalian and avian hosts, is an enveloped RNA virus, and causes diseases of the respiratory tract, intestinal tract, liver and nervous system. The zoonotic infectious disease virus which can be fatal in the existing coronavirus strain is severe acute respiratory syndrome coronavirus (SARS-CoV) and middle east respiratory syndrome virus (MERS-CoV). The fulminant infectious diseases caused by coronavirus have the characteristics of strong infectivity, long incubation period, inconsistent disease symptoms, lethality and the like, and cause heavy striking and fatal threat to national economy and life safety of people. Among them, the new coronavirus, 2019 novel coronavirus (SARS-CoV-2), is a new coronavirus which is newly developed at present.

There is currently no approved therapy for the treatment or prevention of coronaviruses. Several national and international research groups are working on various preventive and therapeutic interventions. Potential approaches being explored include drug therapy, convalescent plasma therapy, interferon-based therapy, small molecule drugs, vaccine therapy. The antiviral drugs applied clinically to the current medicine treatment have few types and similar action mechanisms, and are mostly effective to actively replicating viruses but cannot clear latent viruses or viruses in non-replicating stages. Restorative plasma therapy is a classical adaptive immunotherapy that has been used for over a century for the prevention and treatment of many infectious diseases, but there are some challenges related to donor identification and repeated swabs testing for new coronary pneumonia. Vaccines are an effective means of combating the virus, but there is currently no vaccine that is effective in preventing SARS-CoV-2 infection or any other human coronavirus infection.

Various new corona vaccines are being researched and developed by multiple enterprises and scientific research institutions at home and abroad, the main technical route comprises six types of RNA, DNA, adenovirus vectors, inactivation, attenuation and recombinant proteins, and each type of vaccine comprises a plurality of candidate vaccines which are researched and developed in a saturated mode in parallel. The preparation of purified virus strain inactivated vaccine by using the published novel coronavirus strain is a good idea for preparing the vaccine. However, the preparation process of the fire-fighting vaccine is complex and has extremely high requirements on production environment. In addition, more importantly, because the inactivated vaccine has infection risk, the mutation speed of the coronavirus is high, the protective effect is limited, only the preventive effect can be achieved, and the effect on asymptomatic infected persons is not achieved. Other vaccine designs mostly target the S protein of the new coronavirus, mainly aiming at inducing antibodies to prevent the binding of the virus S protein and the cellular ACE2 receptor to block the invasion of the virus.

If one vaccine, after vaccination, is capable of both generating neutralizing antibodies in the subject and eliciting a better T cell immune response, the vaccine will provide greater short-term protection to the vaccinated population than other vaccines. If a certain degree of immunological memory (T cells, B cells) can also be stimulated, there is a greater probability of long-term protection. Therefore, there is a need to develop a vaccine that simultaneously expresses both humoral and cellular immunity.

Disclosure of Invention

The purpose of the embodiments of the present invention is to provide an expression vector of a novel coronavirus vaccine, aiming at solving the problems proposed in the background art.

The embodiment of the invention is realized by the expression vector of the novel coronavirus vaccine, and the expression vector can simultaneously express the S protein and the NP protein of the novel coronavirus.

As another preferable scheme of the embodiment of the invention, the amino acid sequence of the protein expressed by the expression vector is shown as SEQ ID NO. 1 of the sequence table.

Another object of the embodiments of the present invention is to provide a method for constructing an expression vector of the novel coronavirus vaccine, which comprises the following steps:

connecting nucleotide sequences expressing the new coronavirus S protein and NP protein by using 2A peptide to synthesize a fusion gene;

two ends of the fusion gene respectively contain two enzyme cutting sites and are loaded to plasmids to obtain recombinant plasmids;

carrying out double enzyme digestion on the recombinant plasmid, cutting the gel and recovering a target gene fragment;

carrying out double enzyme digestion on the original plasmid, cutting the gel and recovering a carrier fragment;

and connecting the target gene fragment with the vector fragment to obtain the expression vector.

As another preferred scheme of the embodiment of the invention, the amino acid sequence of the 2A peptide is shown in a sequence table SEQ ID NO. 2.

As another preferable embodiment of the present invention, the two sites are XbaI and SalI sites, respectively.

As another preferred embodiment of the present invention, the two sites are NheI and HindIII sites, respectively.

As another preferable embodiment of the present invention, in the step, a DNA ligase is used to join the target gene fragment and the vector fragment.

Another object of the present invention is to provide an expression vector obtained by the above-described construction method.

Another object of the embodiments of the present invention is to provide an application of the above expression vector in the preparation of a medicament for treating and/or preventing a novel coronavirus.

It is another object of embodiments of the present invention to provide a vaccine prepared from the above expression vector.

The expression vector of the novel coronavirus vaccine provided by the embodiment of the invention gives consideration to humoral immunity and cellular immunity, integrates a S protein region of the novel coronavirus and an NP protein region which is easy to induce T cell immune response, performs special sequence design, and further provides the capability of inducing humoral immunity and cellular immunity, thereby being a brand-new virus vaccine design; specifically, the embodiment of the invention simultaneously expresses the binding region of the coronavirus S protein receptor and the NP protein, so that the cells infected by the expression vector can induce not only antibody reaction but also T cell reaction, thereby effectively inducing humoral immunity and cellular immunity and providing stronger immune protection for a subject.

Drawings

FIG. 1 is a diagram showing the construction of an expression vector of a novel coronavirus vaccine provided in the embodiment of the present invention; in fig. 1, S: a novel coronavirus S protein sequence; NP: a novel coronavirus NP protein sequence; 2A: 2A peptide sequence.

FIG. 2 is a graph showing the results of the antibody-inducing ability test of the vaccine according to the embodiment of the present invention; in fig. 2, PBS: PBS buffer control group; DC: empty DC cells; DC + S, DC cell transfection S protein RNA vaccine; DC + S/NP: DC cell transfection S protein and NP protein RNA vaccine; a in FIG. 2 is a graph showing the results of experiments in C57 mice, and B in FIG. 2 is a graph showing the results of experiments in BALB/C mice.

FIG. 3 is a graph showing the results of the ability test of the vaccine provided in the example of the present invention to induce T cells; in fig. 3, PBS: PBS buffer control group; DC: empty DC cells; DC + S, DC cell transfection S protein RNA vaccine; DC + S/NP: DC cell transfection S protein and NP protein RNA vaccine; a in FIG. 3 is a graph showing the results of experiments in C57 mice, and B in FIG. 3 is a graph showing the results of experiments in BALB/C mice.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

In one embodiment of the present invention, an expression vector capable of simultaneously expressing the S protein and the NP protein of the novel coronavirus is provided. The amino acid sequence of the protein expressed by the expression vector can be as follows: MLLLVTSLLLCELPHPAFLLIPRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGSGEGRGSLLTCGDVEENPGPMRVTAPRTLILLLSGALALTETWAGSMSDNGPQNQRNAPRITFGGPSDSTGSNQNGERSGARSKQRRPQGLPNNTASWFTALTQHGKEDLKFPRGQGVPINTNSSPDDQIGYYRRATRRIRGGDGKMKDLSPRWYFYYLGTGPEAGLPYGANKDGIIWVATEGALNTPKDHIGTRNPANNAAIVLQLPQGTTLPKGFYAEGSRGGSQASSRSSSRSRNSSRNSTPGSSRGTSPARMAGNGGDAALALLLLDRLNQLESKMSGKGQQQQGQTVTKKSAAEASKKPRQKRTATKAYNVTQAFGRRGPEQTQGNFGDQELIRQGTDYKHWPQIAQFAPSASAFFGMSRIGMEVTPSGTWLTYTGAIKLDDKDPNFKDQVILLNKHIDAYKTFPPTEPKKDKKKKADETQALPQRQKKQQTVTLLPAADLDDFSKQLQQSMSSADSTQAIVGIVAGLAVLAVVVIGAVVATVMCRRKSSGGKGGSYSQAASSDSAQGSDVSLTA, which is specifically shown in SEQ ID NO 1 of the sequence table.

Specifically, the embodiment of the invention screens and transforms a new coronavirus S protein sequence by applying a genetic engineering technology, so that the S protein expressed by cells can be secreted and then captured by B cells to induce corresponding antibodies.

Further, in order to promote the vaccine to induce the cellular immune response, the embodiment of the invention adds and optimizes a new coronavirus NP sequence, and selects the NP sequence with the strongest induction capability of the T cell activity from a plurality of sequences.

More specifically: the expression vector carries two target genes, RNA vaccine is prepared through in vitro transcription, and then subcutaneous re-transfusion is carried out on the mouse by combining with the liposome vector, and B cell humoral immune response and T cell immune response are induced in the mouse.

The specific construction method and application of the above expression vector can refer to the following examples, but are not limited to the following examples.

Example 1

As shown in FIG. 1, the embodiment provides a method for constructing an expression vector of a novel coronavirus vaccine, which comprises the following steps:

s1, connecting the nucleotide sequences expressing the new coronavirus S protein and the NP protein by using 2A peptide to synthesize a fusion gene; wherein, the nucleotide sequence of the expression S protein is as follows: ATGCTTCTCCTGGTGACAAGCCTTCTGCTCTGTGAGTTACCACACCCAGCATTCCTCCTGATCCCAAGAGTCCAACCAACAGAATCTATTGTTAGATTTCCTAATATTACAAACTTGTGCCCTTTTGGTGAAGTTTTTAACGCCACCAGATTTGCATCTGTTTATGCTTGGAACAGGAAGAGAATCAGCAACTGTGTTGCTGATTATTCTGTCCTATATAATTCCGCATCATTTTCCACTTTTAAGTGTTATGGAGTGTCTCCTACTAAATTAAATGATCTCTGCTTTACTAATGTCTATGCAGATTCATTTGTAATTAGAGGTGATGAAGTCAGACAAATCGCTCCAGGGCAAACTGGAAAGATTGCTGATTATAATTATAAATTACCAGATGATTTTACAGGCTGCGTTATAGCTTGGAATTCTAACAATCTTGATTCTAAGGTTGGTGGTAATTATAATTACCTGTATAGATTGTTTAGGAAGTCTAATCTCAAACCTTTTGAGAGAGATATTTCAACTGAAATCTATCAGGCCGGTAGCACACCTTGTAATGGTGTTGAAGGTTTTAATTGTTACTTTCCTTTACAATCATATGGTTTCCAACCCACTAATGGTGTTGGTTACCAACCATACAGAGTAGTAGTACTTTCTTTTGAACTTCTACATGCACCAGCAACTGTTTGTGGACCTAAAAAGTCTACTAATTTGGTTAAAAACAAATGTGTCAATTTCAACTTCAATGGTTAA, specifically shown in SEQ ID NO of sequence Listing 4; the nucleotide sequence of the NP protein is expressed as follows: ATGCGGGTCACGGCACCCCGAACCCTGATCCTGCTGCTCTCGGGGGCCCTGGCCCTGACCGAGACCTGGGCCGGCTCCATGTCTGATAATGGACCCCAAAATCAGCGAAATGCACCCCGCATTACGTTTGGTGGACCCTCAGATTCAACTGGCAGTAACCAGAATGGAGAACGCAGTGGGGCGCGATCAAAACAACGTCGGCCCCAAGGTTTACCCAATAATACTGCGTCTTGGTTCACCGCTCTCACTCAACATGGCAAGGAAGACCTTAAATTCCCTCGAGGACAAGGCGTTCCAATTAACACCAATAGCAGTCCAGATGACCAAATTGGCTACTACCGAAGAGCTACCAGACGAATTCGTGGTGGTGACGGTAAAATGAAAGATCTCAGTCCAAGATGGTATTTCTACTACCTAGGAACTGGGCCAGAAGCTGGACTTCCCTATGGTGCTAACAAAGACGGCATCATATGGGTTGCAACTGAGGGAGCCTTGAATACACCAAAAGATCACATTGGCACCCGCAATCCTGCTAACAATGCTGCAATCGTGCTACAACTTCCTCAAGGAACAACATTGCCAAAAGGCTTCTACGCAGAAGGGAGCAGAGGCGGCAGTCAAGCCTCTTCTCGTTCCTCATCACGTAGTCGCAACAGTTCAAGAAATTCAACTCCAGGCAGCAGTAGGGGAACTTCTCCTGCTAGAATGGCTGGCAATGGCGGTGATGCTGCTCTTGCTTTGCTGCTGCTTGACAGATTGAACCAGCTTGAGAGCAAAATGTCTGGTAAAGGCCAACAACAACAAGGCCAAACTGTCACTAAGAAATCTGCTGCTGAGGCTTCTAAGAAGCCTCGGCAAAAACGTACTGCCACTAAAGCATACAATGTAACACAAGCTTTCGGCAGACGTGGTCCAGAACAAACCCAAGGAAATTTTGGGGACCAGGAACTAATCAGACAAGGAACTGATTACAAACATTGGCCGCAAATTGCACAATTTGCCCCCAGCGCTTCAGCGTTCTTCGGAATGTCGCGCATTGGCATGGAAGTCACACCTTCGGGAACGTGGTTGACCTACACAGGTGCCATCAAATTGGATGACAAAGATCCAAATTTCAAAGATCAAGTCATTTTGCTGAATAAGCATATTGACGCATACAAAACATTCCCACCAACAGAGCCTAAAAAGGACAAAAAGAAGAAGGCTGATGAAACTCAAGCCTTACCGCAGAGACAGAAGAAACAGCAAACTGTGACTCTTCTTCCTGCTGCAGATTTGGATGATTTCTCCAAACAATTGCAACAATCCATGAGCAGTGCTGACTCAACTCAGGCCATCGTGGGCATTGTTGCTGGCCTGGCTGTCCTAGCAGTTGTGGTCATCGGAGCTGTGGTCGCTACTGTGATGTGTAGGAGGAAGAGCTCAGGTGGAAAAGGAGGGAGCTACTCTCAGGCTGCGTCCAGCGACAGTGCCCAGGGCTCTGATGTGTCTCTCACAGCTTAA, specifically shown in SEQ ID NO of sequence Listing 5; the amino acid sequence of the 2A peptide is: GSGEGRGSLLTCGDVEENPGP, specifically shown in SEQ ID NO of the sequence table.

S2, NheI and HindIII enzyme cutting sites are respectively contained at two ends of the fusion gene, and the fusion gene is loaded to pcDNA3.1+ plasmid to obtain recombinant plasmid.

S3, performing double enzyme digestion on the recombinant plasmid by using NheI and HindIII endonucleases, and cutting gel to recover a target gene fragment.

S4, carrying out double digestion on the original pcDNA3.1+ plasmid by utilizing NheI and HindIII endonucleases, and cutting gel to recover a 2.3kb vector fragment.

S5, connecting the target gene fragment recovered in the step S3 and the vector fragment recovered in the step S4 by using DNA ligase to obtain the expression vector of the novel coronavirus vaccine.

Example 2

This embodiment provides a method for constructing an expression vector for a novel coronavirus vaccine, comprising the steps of:

s1, connecting the nucleotide sequences of the expressed new coronavirus S protein (shown as SEQ ID NO:4 in the sequence table) and the NP protein (shown as SEQ ID NO:5 in the sequence table) by using 2A peptide to synthesize a fusion gene; wherein the amino acid sequence of the 2A peptide is: GSGEGRGSLLTCGDVEENPGP, specifically shown in SEQ ID NO of the sequence table.

S2, XbaI and SalI enzyme cutting sites are respectively contained at two ends of the fusion gene, and the XbaI and SalI enzyme cutting sites are loaded on pcDNA3.1+ plasmid to obtain recombinant plasmid.

And S3, performing double digestion on the recombinant plasmid by using XbaI and SalI endonucleases, and cutting gel to recover a target gene fragment.

S4, carrying out double digestion on the original pcDNA3.1+ plasmid by using XbaI and SalI endonucleases, and cutting the gel to recover a vector fragment.

Comparative example 1

The comparative example provides a method for constructing an expression vector for a novel coronavirus vaccine, comprising the steps of:

s1, respectively containing NheI and HindIII enzyme cutting sites at two ends of a nucleotide sequence (shown as a sequence table SEQ ID NO: 4) only expressing a new coronavirus S protein (the amino acid sequence is RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFN, and is specifically shown as the sequence table SEQ ID NO: 3), and loading the nucleotide sequence into pcDNA3.1+ plasmid to obtain the recombinant plasmid.

S2, performing double enzyme digestion on the recombinant plasmid by using NheI and HindIII endonucleases, and cutting gel to recover a target gene fragment.

S3, carrying out double digestion on the original pcDNA3.1+ plasmid by utilizing NheI and HindIII endonucleases, and cutting gel to recover a 2.3kb vector fragment.

S4, connecting the target gene fragment recovered in the step S2 and the vector fragment recovered in the step S3 by using DNA ligase, and obtaining the comparative expression vector.

Example 3

This example, for comparing the antibody response-inducing ability of the expression vectors obtained in example 1 and comparative example 1 above, specifically includes the following steps:

1. preparation of RNA vaccine by in vitro transcription with expression vector:

(1) (ii) linearization of the plasmid using Xhol enzyme;

(2) linearized DNA recovery using a DNA purification kit;

(3) in vitro transcription of RNA using an in vitro transcription kit;

(4) RNA purification and recovery were carried out using an RNA purification kit, and S protein and NP protein RNA vaccines prepared from the expression vector obtained in example 1 and S protein RNA vaccines prepared from the expression vector obtained in comparative example 1 were obtained, respectively.

2. Acquisition of mouse DCs:

(1) killing the mice by a cervical dislocation method for 6-10 weeks, taking out all thighbones and shinbones by an operation, and removing muscle tissues around the bones as clean as possible by scissors and tweezers;

(2) cutting off two ends of a bone by using scissors, extracting PBS (phosphate buffered saline) by using a syringe, respectively inserting a needle into a marrow cavity from two ends of the bone, and repeatedly washing out the marrow in a culture dish;

(3) collecting bone marrow suspension, and filtering with 200 mesh nylon net to remove small pieces and muscle tissue;

(4) centrifuging at 1200rpm for 5min, and removing the supernatant;

(5) cell concentration was adjusted to 2 × 10 using 10% FBS (fetal bovine serum) in RPMI640 complete medium after cell counting⁵/mL；

(6) The mouse DC medium was added, half of the medium was changed for 2 days, and the culture was continued until day 8.

3. DC electrotransformation of RNA vaccine:

(1) preparing electrotransformation liquid and a required culture medium;

(2) resuspending the cells, centrifuging, discarding the supernatant, and adding a transfer solution;

(3) cells were harvested by electroporation and incubated overnight.

4. Vaccination of mice:

(1) selecting BALB/C and C57 mice;

(2) subcutaneous injection electrotransfer, grouping of PBS, DC cells without electrotransfer RNA, DC cells of the electrotransfer S protein RNA vaccine (comparative example 1), DC cells of the electrotransfer S protein and NP protein RNA vaccine (example 1);

5. blood collection and serum separation, ELISA (enzyme linked immunosorbent assay) detection of antibody response:

(1) 7 days after inoculation, blood is taken from the orbit of the mouse, after 1-2 hours of room temperature coagulation, 3000 r/min is separated, and the supernatant is collected;

(2) preparing a recombinant RBD or S2 protein solution by using a recombinant RBD or S2 protein as a control and a carbonate buffer solution, coating a 96-well plate, and standing overnight at 4 ℃;

(3) washing 3-time well plates with PBS containing 0.1% tween 20, preparing a blocking solution by adding 1% BSA to PBS containing 0.1% tween 20, and incubating at 37 ℃ for 1 hour;

(4) serum was added and incubated at 37 ℃ for 1 hour, and the plates were washed 3 times with PBS containing 0.1% Tween 20. Adding a goat anti-mouse secondary antibody, incubating at room temperature for 1 hour, washing the well plate for 5 times by using PBS containing 0.1% Tween 20, adding TMB (3,3',5,5' -tetramethylbenzidine) for 10 minutes, and adding a stop solution to stop the reaction;

(5) measuring absorbance after 450 nm;

(6) serum samples were serially diluted with recombinant protein to determine the titer of induced RBD specific antibodies.

The results of the comparison are shown in FIG. 2, and it can be seen from FIG. 2 that the RNA vaccine prepared by using the expression vector provided by the embodiment of the present invention has stronger antibody-inducing ability.

Example 4

This example, for comparing the T cell response-inducing ability of the expression vectors obtained in example 1 and comparative example 1 above, specifically includes the following steps:

1. preparation of RNA vaccine by in vitro transcription with expression vector:

(1) (ii) linearization of the plasmid using Xhol enzyme;

(2) linearized DNA recovery using a DNA purification kit;

(3) in vitro transcription of RNA using an in vitro transcription kit;

2. Acquisition of mouse DCs:

(2) shearing two ends of a bone by using scissors, extracting PBS by using an injector, respectively inserting a needle into a marrow cavity from two ends of the bone, and repeatedly washing out the marrow in a culture dish;

(4) centrifuging at 1200rpm for 5min, and removing the supernatant;

(5) cell concentration was adjusted to 2 × 10 using 10% FBS in RPMI640 complete medium after cell counting⁵/mL；

3. DC electrotransformation of RNA vaccine:

(1) preparing electrotransformation liquid and a required culture medium;

(3) cells were harvested by electroporation and incubated overnight.

4. Vaccinated mice

(1) Selecting BALB/C and C57 mice;

5. taking mouse splenocytes to separate T cells, taking mouse DC cells:

(1) preparing the hourly spleen tissue into a single cell suspension for later use by using a Meitian whirlwind single cell suspension preparation system;

(2) taking mouse DC cells, and performing the method as 2;

(3) after the mouse DC cells are incubated with the inactivated viruses, the mouse DC cells are incubated with the T cells;

(4) elispot detects new coronavirus reactive T cell status using PBS and DC cells without transfected RNA vaccine as negative controls.

The results of the comparison are shown in FIG. 3, and it can be seen from FIG. 3 that the RNA vaccine prepared by using the expression vector provided by the embodiment of the present invention has stronger T cell induction capability.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Sequence listing

<110> Zhejiang Geyuan Zhen Zhi Zhen Zhi Zhen Zhi Zhen Zhi Zhen Zhi Zhen Zhi Zhen Zhi Zhen Zhi Zhen Zhi Zhen Zhi Zhen Zhi Zhen Zhi Zhen Zhi Zhen Zhi Zhen Zhi Zhen Zhi Zhen Zhi Zhen Zhe

<120> expression vector of novel coronavirus vaccine, construction method and application thereof, and vaccine

<160> 5

<170> SIPOSequenceListing 1.0

<210> 1

<211> 769

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro

1 5 10 15

Ala Phe Leu Leu Ile Pro Arg Val Gln Pro Thr Glu Ser Ile Val Arg

20 25 30

Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala

35 40 45

Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn

50 55 60

Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr

65 70 75 80

Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe

85 90 95

Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg

100 105 110

Gln Ile Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys

115 120 125

Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn

130 135 140

Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe

145 150 155 160

Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile

165 170 175

Tyr Gln Ala Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys

180 185 190

Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly

195 200 205

Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala

210 215 220

Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn

225 230 235 240

Lys Cys Val Asn Phe Asn Phe Asn Gly Ser Gly Glu Gly Arg Gly Ser

245 250 255

Leu Leu Thr Cys Gly Asp Val Glu Glu Asn Pro Gly Pro Met Arg Val

260 265 270

Thr Ala Pro Arg Thr Leu Ile Leu Leu Leu Ser Gly Ala Leu Ala Leu

275 280 285

Thr Glu Thr Trp Ala Gly Ser Met Ser Asp Asn Gly Pro Gln Asn Gln

290 295 300

Arg Asn Ala Pro Arg Ile Thr Phe Gly Gly Pro Ser Asp Ser Thr Gly

305 310 315 320

Ser Asn Gln Asn Gly Glu Arg Ser Gly Ala Arg Ser Lys Gln Arg Arg

325 330 335

Pro Gln Gly Leu Pro Asn Asn Thr Ala Ser Trp Phe Thr Ala Leu Thr

340 345 350

Gln His Gly Lys Glu Asp Leu Lys Phe Pro Arg Gly Gln Gly Val Pro

355 360 365

Ile Asn Thr Asn Ser Ser Pro Asp Asp Gln Ile Gly Tyr Tyr Arg Arg

370 375 380

Ala Thr Arg Arg Ile Arg Gly Gly Asp Gly Lys Met Lys Asp Leu Ser

385 390 395 400

Pro Arg Trp Tyr Phe Tyr Tyr Leu Gly Thr Gly Pro Glu Ala Gly Leu

405 410 415

Pro Tyr Gly Ala Asn Lys Asp Gly Ile Ile Trp Val Ala Thr Glu Gly

420 425 430

Ala Leu Asn Thr Pro Lys Asp His Ile Gly Thr Arg Asn Pro Ala Asn

435 440 445

Asn Ala Ala Ile Val Leu Gln Leu Pro Gln Gly Thr Thr Leu Pro Lys

450 455 460

Gly Phe Tyr Ala Glu Gly Ser Arg Gly Gly Ser Gln Ala Ser Ser Arg

465 470 475 480

Ser Ser Ser Arg Ser Arg Asn Ser Ser Arg Asn Ser Thr Pro Gly Ser

485 490 495

Ser Arg Gly Thr Ser Pro Ala Arg Met Ala Gly Asn Gly Gly Asp Ala

500 505 510

Ala Leu Ala Leu Leu Leu Leu Asp Arg Leu Asn Gln Leu Glu Ser Lys

515 520 525

Met Ser Gly Lys Gly Gln Gln Gln Gln Gly Gln Thr Val Thr Lys Lys

530 535 540

Ser Ala Ala Glu Ala Ser Lys Lys Pro Arg Gln Lys Arg Thr Ala Thr

545 550 555 560

Lys Ala Tyr Asn Val Thr Gln Ala Phe Gly Arg Arg Gly Pro Glu Gln

565 570 575

Thr Gln Gly Asn Phe Gly Asp Gln Glu Leu Ile Arg Gln Gly Thr Asp

580 585 590

Tyr Lys His Trp Pro Gln Ile Ala Gln Phe Ala Pro Ser Ala Ser Ala

595 600 605

Phe Phe Gly Met Ser Arg Ile Gly Met Glu Val Thr Pro Ser Gly Thr

610 615 620

Trp Leu Thr Tyr Thr Gly Ala Ile Lys Leu Asp Asp Lys Asp Pro Asn

625 630 635 640

Phe Lys Asp Gln Val Ile Leu Leu Asn Lys His Ile Asp Ala Tyr Lys

645 650 655

Thr Phe Pro Pro Thr Glu Pro Lys Lys Asp Lys Lys Lys Lys Ala Asp

660 665 670

Glu Thr Gln Ala Leu Pro Gln Arg Gln Lys Lys Gln Gln Thr Val Thr

675 680 685

Leu Leu Pro Ala Ala Asp Leu Asp Asp Phe Ser Lys Gln Leu Gln Gln

690 695 700

Ser Met Ser Ser Ala Asp Ser Thr Gln Ala Ile Val Gly Ile Val Ala

705 710 715 720

Gly Leu Ala Val Leu Ala Val Val Val Ile Gly Ala Val Val Ala Thr

725 730 735

Val Met Cys Arg Arg Lys Ser Ser Gly Gly Lys Gly Gly Ser Tyr Ser

740 745 750

Gln Ala Ala Ser Ser Asp Ser Ala Gln Gly Ser Asp Val Ser Leu Thr

755 760 765

Ala

<210> 2

<211> 21

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Gly Ser Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu

1 5 10 15

Glu Asn Pro Gly Pro

20

<210> 3

<211> 226

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn

1 5 10 15

Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val

20 25 30

Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser

35 40 45

Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val

50 55 60

Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp

65 70 75 80

Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln

85 90 95

Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr

100 105 110

Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly

115 120 125

Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys

130 135 140

Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr

145 150 155 160

Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser

165 170 175

Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val

180 185 190

Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly

195 200 205

Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn

210 215 220

Phe Asn

225

<210> 4

<211> 750

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

atgcttctcc tggtgacaag ccttctgctc tgtgagttac cacacccagc attcctcctg 60

atcccaagag tccaaccaac agaatctatt gttagatttc ctaatattac aaacttgtgc 120

ccttttggtg aagtttttaa cgccaccaga tttgcatctg tttatgcttg gaacaggaag 180

agaatcagca actgtgttgc tgattattct gtcctatata attccgcatc attttccact 240

tttaagtgtt atggagtgtc tcctactaaa ttaaatgatc tctgctttac taatgtctat 300

gcagattcat ttgtaattag aggtgatgaa gtcagacaaa tcgctccagg gcaaactgga 360

aagattgctg attataatta taaattacca gatgatttta caggctgcgt tatagcttgg 420

aattctaaca atcttgattc taaggttggt ggtaattata attacctgta tagattgttt 480

aggaagtcta atctcaaacc ttttgagaga gatatttcaa ctgaaatcta tcaggccggt 540

agcacacctt gtaatggtgt tgaaggtttt aattgttact ttcctttaca atcatatggt 600

ttccaaccca ctaatggtgt tggttaccaa ccatacagag tagtagtact ttcttttgaa 660

cttctacatg caccagcaac tgtttgtgga cctaaaaagt ctactaattt ggttaaaaac 720

aaatgtgtca atttcaactt caatggttaa 750

<210> 5

<211> 1503

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

atgcgggtca cggcaccccg aaccctgatc ctgctgctct cgggggccct ggccctgacc 60

gagacctggg ccggctccat gtctgataat ggaccccaaa atcagcgaaa tgcaccccgc 120

attacgtttg gtggaccctc agattcaact ggcagtaacc agaatggaga acgcagtggg 180

gcgcgatcaa aacaacgtcg gccccaaggt ttacccaata atactgcgtc ttggttcacc 240

gctctcactc aacatggcaa ggaagacctt aaattccctc gaggacaagg cgttccaatt 300

aacaccaata gcagtccaga tgaccaaatt ggctactacc gaagagctac cagacgaatt 360

cgtggtggtg acggtaaaat gaaagatctc agtccaagat ggtatttcta ctacctagga 420

actgggccag aagctggact tccctatggt gctaacaaag acggcatcat atgggttgca 480

actgagggag ccttgaatac accaaaagat cacattggca cccgcaatcc tgctaacaat 540

gctgcaatcg tgctacaact tcctcaagga acaacattgc caaaaggctt ctacgcagaa 600

gggagcagag gcggcagtca agcctcttct cgttcctcat cacgtagtcg caacagttca 660

agaaattcaa ctccaggcag cagtagggga acttctcctg ctagaatggc tggcaatggc 720

ggtgatgctg ctcttgcttt gctgctgctt gacagattga accagcttga gagcaaaatg 780

tctggtaaag gccaacaaca acaaggccaa actgtcacta agaaatctgc tgctgaggct 840

tctaagaagc ctcggcaaaa acgtactgcc actaaagcat acaatgtaac acaagctttc 900

ggcagacgtg gtccagaaca aacccaagga aattttgggg accaggaact aatcagacaa 960

ggaactgatt acaaacattg gccgcaaatt gcacaatttg cccccagcgc ttcagcgttc 1020

ttcggaatgt cgcgcattgg catggaagtc acaccttcgg gaacgtggtt gacctacaca 1080

ggtgccatca aattggatga caaagatcca aatttcaaag atcaagtcat tttgctgaat 1140

aagcatattg acgcatacaa aacattccca ccaacagagc ctaaaaagga caaaaagaag 1200

aaggctgatg aaactcaagc cttaccgcag agacagaaga aacagcaaac tgtgactctt 1260

cttcctgctg cagatttgga tgatttctcc aaacaattgc aacaatccat gagcagtgct 1320

gactcaactc aggccatcgt gggcattgtt gctggcctgg ctgtcctagc agttgtggtc 1380

atcggagctg tggtcgctac tgtgatgtgt aggaggaaga gctcaggtgg aaaaggaggg 1440

agctactctc aggctgcgtc cagcgacagt gcccagggct ctgatgtgtc tctcacagct 1500

taa 1503

Claims

1. An expression vector of a novel coronavirus vaccine, wherein the expression vector can simultaneously express an S protein and an NP protein of the novel coronavirus.

2. The expression vector of the novel coronavirus vaccine of claim 1, wherein the amino acid sequence of the protein expressed by the expression vector is shown as SEQ ID NO. 1 of the sequence table.

3. A method for constructing the expression vector of the novel coronavirus vaccine as described in any one of claims 1-2, comprising the steps of:

4. The method for constructing the expression vector of the novel coronavirus vaccine as claimed in claim 3, wherein the amino acid sequence of the 2A peptide is shown as SEQ ID NO. 2 of the sequence table.

5. The method for constructing an expression vector of a novel coronavirus vaccine as claimed in claim 3, wherein the two cleavage sites are XbaI cleavage site and SalI cleavage site.

6. The method for constructing an expression vector of a novel coronavirus vaccine as claimed in claim 3, wherein the two sites are NheI and HindIII sites.

7. The method for constructing an expression vector of a novel coronavirus vaccine as claimed in claim 3, wherein the step of ligating the target gene fragment with the vector fragment is carried out by using DNA ligase.

8. An expression vector obtained by the construction method according to any one of claims 3 to 7.

9. Use of the expression vector of any one of claims 1-2, 8 in the manufacture of a medicament for the treatment and/or prevention of a neocoronavirus.

10. A vaccine prepared from the expression vector of any one of claims 1 to 2 or 8.