Recombinant protein and application thereof in preparation of monkey pox vaccine

Abstract

The invention relates to a recombinant protein and application thereof in preparation of a monkey pox vaccine. The recombinant protein is a fusion protein of the monkey pox virus and can be obtained by a specific construction method. The monkey pox virus antibody exists in a 2-linked or 3-linked fusion protein form, and comprises 2 or 3 monkey pox virus surface antigens. The fusion protein is used for preparing monkey pox vaccines and can induce efficient immune response. Experiments show that the vaccine has a remarkable prevention and protection effect on monkey pox virus infection in an animal model, a very potential vaccine solution is provided for monkey pox prevention, and public health challenges caused by monkey pox epidemic situations are expected to be coped with.

Description

Technical Field

[0001] The present invention relates to in vitro diagnostic techniques, and in particular to a recombinant protein and its application in preparing monkeypox vaccines. Background Art

[0002] Monkeypox is an acute infectious disease caused by the monkeypox virus (MPXV). In recent years, the monkeypox virus has shown a spreading trend globally, attracting widespread attention.

[0003] Currently, the prevention and control of monkeypox face many challenges. Traditional monkeypox vaccines are mostly based on modified smallpox vaccines, such as the live attenuated vaccinia virus (Modified Vaccinia Ankara, MVA) vaccine or the vaccinia vaccine, but they have certain limitations. On the one hand, these vaccines may cause adverse reactions in some vaccinated individuals, especially for those with weakened immune function, the risk may be higher. On the other hand, smallpox vaccines are used based on partial cross-protection between smallpox and the monkeypox virus, and it is difficult to accurately guarantee the specific immune effect against monkeypox.

[0004] From the characteristics of the virus itself, the genome and antigenic structure of the monkeypox virus are relatively complex. There are multiple antigenic proteins on its surface, and different antigens play different roles in the processes of virus infection, immune escape, etc. Developing more precise and efficient vaccines against the monkeypox virus requires an in-depth understanding of its antigenic characteristics and using modern biotechnology means to construct reasonable immunogens.

[0005] In modern vaccine research and development, recombinant protein vaccines have the advantages of high safety, controllable quality, easy production and storage, etc. By reasonably designing and constructing recombinant proteins that can express effective antigens of the monkeypox virus, it is expected to prepare ideal monkeypox vaccines. For example, combining multiple effective antigens in a single recombinant protein can mimic the situation where multiple antigens act together during natural virus infection, thereby stimulating a more comprehensive and effective immune response. However, constructing such recombinant proteins and ensuring their effectiveness in vaccines is not easy, and many problems need to be solved, such as antigen selection, fusion methods, protein expression and purification, the intensity and persistence of inducing immune responses in organisms, etc.

[0006] CN116603060A discloses a monkeypox virus subunit vaccine, its preparation method and application, which relates to the field of biotechnology. It includes an antigen and an adjuvant. The antigen component is the monkeypox virus antigen proteins M1R, A29L, A35R, and B6R, and His tags are added to the tails of the monkeypox virus antigen proteins M1R, A29L, A35R, or B6R.

[0007] The present invention provides a dual recombinant protein for preventing monkeypox, and a construction method and application thereof, so as to solve the problems existing in current inactivated vaccines and attenuated vaccines, such as unstable protective effects, easy secondary infections, and reversion of virulence. Summary of the Invention

[0008] In some embodiments, the present invention first provides a recombinant protein, and the amino acid sequence of the recombinant protein is as shown in SEQ ID No.1 or SEQ ID No.2.

[0009] In some embodiments, the present invention also provides a biological material related to the above recombinant protein, which is any one of the following B1) to B8):

[0010] B1) A nucleic acid molecule encoding the above recombinant protein;

[0011] B2) An expression cassette containing the nucleic acid molecule described in B1);

[0012] B3) A recombinant vector containing the nucleic acid molecule described in B1),

[0013] B4) A recombinant vector containing the expression cassette described in B2);

[0014] B5) A recombinant microorganism containing the nucleic acid molecule described in B1);

[0015] B6) A recombinant microorganism containing the expression cassette described in B2);

[0016] B7) A recombinant microorganism containing the recombinant vector described in B3);

[0017] B8) A recombinant microorganism containing the recombinant vector described in B4);

[0018] In some embodiments, the vector includes Escherichia coli, yeast expression vectors, and other expression vectors;

[0019] In some embodiments, the recombinant microorganism includes an engineered bacterium.

[0020] In some embodiments, the nucleic acid molecule is a DNA molecule or cDNA molecule capable of encoding SEQ ID No.1 or SEQ ID No.2.

[0021] In some embodiments, the present invention also provides any one of the following applications of the above recombinant protein:

[0022] C1) Preparing a product for preventing monkeypox virus infection;

[0023] C2) Preparing a product for preventing or treating diseases caused by monkeypox virus;

[0024] C3) Preparing a product for preventing or treating monkeypox caused by monkeypox virus infection.

[0025] In some embodiments, the present invention also provides any one of the following applications of the above-mentioned biomaterials:

[0026] C1) Preparing a product for preventing monkeypox virus infection;

[0027] C2) Preparing a product for preventing or treating diseases caused by monkeypox virus;

[0028] C3) Preparing a product for preventing or treating monkeypox caused by monkeypox virus infection.

[0029] In some embodiments, the present invention also provides a method for producing a monkeypox vaccine, which comprises the step of expressing a recombinant protein by using the above-mentioned biomaterial.

[0030] In some embodiments, the present invention also provides a monkeypox vaccine, which is prepared by the above-mentioned method.

[0031] Compared with the prior art, the present invention has at least the following beneficial effects:

[0032] The present invention screens suitable recombinant virus subunit vaccines from the surface antigens of EEV and IMV. The challenge protection rate of the immunized group shows that the dual-fusion recombinant protein and the triple-fusion recombinant protein can produce a 100% immune protection rate. It is an efficient and safe technical means, which solves the problems existing in the current inactivated vaccines and attenuated vaccines, such as unstable protection effect, easy secondary infection, and reversion of virulence. The recombinant proteins and biomaterials of the present invention can be used to prepare monkeypox virus subunit vaccines for preventing monkeypox. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 Electrophoresis diagrams of the triple-fusion protein and the dual-fusion protein;

[0034] Figure 2 Serum titer diagrams of the triple-fusion protein vaccine group and the dual-fusion protein vaccine;

[0035] Figure 3 Activation percentage diagrams of CD4+ T cells in the triple-fusion protein vaccine group and the dual-fusion protein vaccine group. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0036] To make the technical problems, technical solutions and advantages to be solved by the present invention clearer, the following will be described in detail with reference to the accompanying drawings and specific embodiments.

[0037] Example 1. Construction of Fusion Protein

[0038] 1. Obtain the monkeypox antigen gene sequence from the NCBI database and optimize its codons

[0039] (1) Construct a dual-fusion protein by selecting the A29L protein and M1R protein of the monkeypox virus. A29L is a glycoprotein on the surface of the monkeypox virus and plays an important role in the binding and invasion of the virus to cells; M1R is a core protein and plays a key role in the assembly and maturation of the virus. Both are important antigens on the surface of the monkeypox virus.

[0040] To construct a triple-fusion protein, additionally select the A35R protein. The A35R protein is of great significance in the immune regulation of monkeypox virus infection.

[0041] (2) Design the structure of the fusion protein

[0042] For the dual-fusion protein (A29L-M1R), introduce a linker sequence (Linker) between A29L and M1R. Here, GGGGS (glycine-glycine-glycine-serine-glycine) is selected as the Linker. Its sequence is: A29L-GGGGS-M1R.

[0043] The amino acid sequence of the dual-fusion protein is SEQ ID NO:1: KAAKNPETKREAMVKAYGDDNEETLKQRLTQLEKKITNITKKFEQIEKCCKRNDEVLFRLENHAETLRAAMISLAKKIDVQTGRHPYE GGGGS CDIEIGNFYIRQNHGCNITVKNMCSADADAQLDAVLSAATETYSGLTPEQKAYVPAMFTAALNITTSVNTVVRDFENYVKQTCNSQAVVDNKLKIQSVIIDECYGAPGSPTNLEFINTGSSKGNCAIKALMQLTTKATTQIAPRQVAGTGVQFYMIVIGVIILAALFSYYAKRMLFTSTNDKIKLILANKENVHWTTYMDTFFRTSPMIIATTDIQN;

[0044] (Note that the underlined part is the linker sequence);

[0045] For the triple-fusion protein (A29L-M1R-A35R), the same or similar Linker is used for connection. For example: A29L-GGGGS-M1R-GGGGS-A35R.

[0046] The amino acid sequence of the triple fusion protein is SEQ ID NO:2: KAAKNPETKREAMVKAYGDDNEETLKQRLTQLEKKITNITKKFEQIEKCCKRNDEVLFRLENHAETLRAAMISLAKKIDVQTGRHPYE GGGGS CDIEIGNFYIRQNHGCNITVKNMCSADADAQLDAVLSAATETYSGLTPEQKAYVPAMFTAALNITTSVNTVVRDFENYVKQTCNSQAVVDNKLKIQSVIIDECYGAPGSPTNLEFINTGSSKGNCAIKALMQLTTKATTQIAPRQVAGTGVQFYMIVIGVIILAALFSYYAKRMLFTSTNDKIKLILANKENVHWTTYMDTFFRTSPMIIATTDIQN; GGGGS TYDHKESCNGLSYQGSCYILHSDYASFEDAKANCAAESSTLPNKS DVLTTWLIDYVEDTWGSDGNPITKTTSDYQDSQVSQEVRKYFCT (Note: The underlined part is the linker sequence);

[0047] The specific A29L protein sequence (SEQ ID NO:3):

[0048] KAAKNPETKREAMVKAYGDDNEETLKQRLTQLEKKITNITKKFEQIEK CCKRNDEVLFRLENHAETLRAAMISLAKKIDVQTGRHPYE;

[0049] The specific M1R protein sequence (SEQ ID NO:4):

[0050] CDIEIGNFYIRQNHGCNITVKNMCSADADAQLDAVLSAATETYSGLTPEQKA

[0051] YVPAMFTAALNITTSVNTVVRDFENYVKQTCNSQAVVDNKLKIQSVIIDEC

[0052] YGAPGSPTNLEFINTGSSKGNCAIKALMQLTTKATTQIAPRQVAGTGVQFY

[0053] MIVIGVIILAALFSYYAKRMLFTSTNDKIKLILANKENVHWTTYMDTFFRTSPMIIATTDIQN;

[0054] Specific A35R protein sequence (SEQ ID NO:5):

[0055] TYDHKESCNGLSYQGSCYILHSDYASFEDAKANCAAESSTLPNKSDVLTTW LIDYVEDTWGSDGNPITKTTSDYQDSQVSQEVRKYFCT;

[0056] Example 2. Preparation method of fusion protein

[0057] 1. Gene cloning

[0058] According to the selected amino acid sequences of A29L, M1R and A35R, synthesize the gene nucleotide fragments of A29L, M1R or A35R, and ligate them with an expression vector pET-28a containing a suitable promoter (CMV promoter), ribosome entry site (IRES), HIS tag and terminator. Use restriction enzymes to cut out the corresponding sites of the vector and the target gene, and then carry out a ligation reaction through T4 ligase. For 2-in-1 or 3-in-1 fusion proteins, ensure that the ligation order and Linker sequence are accurate.

[0059] 2. Transformation of expression vector into Escherichia coli

[0060] Transform the ligated expression vectors of pET-28a-A29L-GGGGS-M1R and pET-28a-A29L-GGGGS-M1R-GGGGS-A35R into Escherichia coli DH5α competent cells. Spread the bacterial solution evenly on an LB plate containing ampicillin and culture overnight at 37°C.

[0061] Pick monoclonal colonies and inoculate them into an LB liquid medium containing the same antibiotic, and culture them with shaking at 37°C and 200 rpm. When the OD600 of the bacterial solution reaches 0.4 - 0.6, induce the expression of the fusion protein with IPTG (final concentration of 0.5 mM - 1 mM). The induction time is 4 - 8 hours.

[0062] 3. Protein expression and purification

[0063] After the induction ended, the bacterial liquid was collected and centrifuged (10000 rpm, 15 minutes, 4°C) to collect the bacterial cells. The bacterial cells were resuspended in lysis buffer (50 mM Tris-HCl, pH 8.0, 100 mM NaCl, 0.5% Triton X-100) and sonicated. The sonicated bacterial liquid was centrifuged at high speed (15000 rpm, 30 minutes, 4°C), and the supernatant was taken.

[0064] The supernatant was preliminarily purified by nickel column (His-Tag) affinity chromatography. Gradient elution was performed using elution buffer (50 mM Tris-HCl, pH 8.0, 300 mM NaCl) containing different concentrations of imidazole (20 mM, 50 mM, 250 mM, 500 mM imidazole eluent), and the elution peak containing the fusion protein was collected.

[0065] The preliminarily purified fusion protein was further purified by ion exchange chromatography (Q-Sepharose) to obtain a high-purity fusion protein, and the results were as Figure 1 shown.

[0066] Figure 1 The results showed that the molecular weight of A29L-GGGGS-M1R (2-link fusion protein) was 34.41 kDa; the molecular weight of A29L-GGGGS-M1R-GGGGS-A35R (3-link fusion protein) was 44.70 kDa.

[0067] Example 3: Preparation and testing effect of the fusion protein vaccine

[0068] 1. Vaccine preparation

[0069] The purified fusion protein (2-link or 3-link fusion protein) was mixed with an appropriate adjuvant (aluminum hydroxide adjuvant) at a certain ratio (protein:adjuvant = 1:1, protein concentration was 1 mg / ml) to prepare the monkeypox recombinant protein vaccine.

[0070] 2. Immune effect test

[0071] Animal model selection: BALB / c mice were used for the experiment. The mice were randomly divided into four groups: 2-link fusion protein vaccine group, 3-link fusion protein vaccine group, A29L control group (injected with an equal amount of A29L protein), and blank control group (injected with an equal amount of normal saline).

[0072] Immunization protocol: The mice in the vaccine groups were intramuscularly injected with an immunization dose of 10 μg / mouse, and immunized three times at week 0, week 2, and week 4. The control group and the blank control group were injected with the corresponding reagents according to the same protocol.

[0073] Serum antibody detection: One week after each immunization (i.e., the 1st week, the 3rd week, and the 5th week), orbital blood of mice was collected and serum was separated. Serum containing antibodies against A29L was detected by enzyme-linked immunosorbent assay (ELISA), see Figure 2 .

[0074] Figure 2 The results showed that the serum titers of antibodies against A29L in the triple fusion protein vaccine group and the double fusion protein vaccine group were significantly higher than those in the A29L control group and the blank control group.

[0075] Cellular immunity detection: Three days after the last immunization (the 4th week), the spleens of mice were removed and spleen cell suspensions were prepared. The activation of CD4+ T cells in spleen cells was detected by flow cytometry, see Figure 3 .

[0076] Figure 3 The results showed that the activation percentages of CD4+ T cells in the triple fusion protein vaccine group and the double fusion protein vaccine group were significantly higher than those in the A29L control group.

[0077] Protection effect test: Immunized mice were challenged by intranasal or intramuscular inoculation with monkeypox virus, and negative control (PBS group) and experimental groups were intramuscularly inoculated with a lethal dose of VACV virus (5×10 6 PFU / mouse). One week after virus challenge, the survival symptoms of mice were observed daily, the body weights of mice were recorded, and the death conditions were recorded. The dead mice were dissected and pathological changes were observed. The results showed that mice in the triple fusion protein vaccine group and the double fusion protein vaccine group had no symptoms of disease after challenge, their body weights were stable, and there was no death. The protection rates were both 100%, while mice in the negative control (PBS group) showed obvious symptoms of disease, their body weights decreased sharply, and the mortality rate was 80%.

[0078] In summary, the double or triple monkeypox fusion protein vaccine constructed by the present invention has good immunogenicity and protection effect, and is expected to become an effective vaccine for preventing monkeypox.

[0079] The above is the preferred embodiment of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the principle described in the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A recombinant protein, characterized in that, The amino acid sequence of the recombinant protein is as shown in SEQ ID No.1 or SEQ ID No.

2.

2. The biological material related to the recombinant protein according to claim 1 is any one of the following B1) to B8): B1) A nucleic acid molecule encoding the recombinant protein according to claim 1; B2) An expression cassette containing the nucleic acid molecule described in B1); B3) A recombinant vector containing the nucleic acid molecule described in B1), B4) A recombinant vector containing the expression cassette described in B2); B5) A recombinant microorganism containing the nucleic acid molecule described in B1); B6) A recombinant microorganism containing the expression cassette described in B2); B7) A recombinant microorganism containing the recombinant vector described in B3); B8) A recombinant microorganism containing the recombinant vector described in B4).

3. The biomaterial according to claim 2, wherein: The vector includes Escherichia coli, yeast expression vectors, and other expression vectors.

4. The biomaterial according to claim 2, wherein: The recombinant microorganism includes engineered bacteria.

5. The biomaterial according to claim 2, characterized in that: The nucleic acid molecule is a DNA molecule or cDNA molecule capable of encoding SEQ ID No.1 or SEQ ID No.

2.

6. Any of the following applications of the recombinant protein according to claim 1: C1) Preparing a product for preventing monkeypox virus infection; C2) Preparing a product for preventing or treating diseases caused by monkeypox virus; C3) Preparing a product for preventing or treating monkeypox caused by monkeypox virus infection.

7. Any of the following applications of the biological material according to claim 2: C1) Preparing a product for preventing monkeypox virus infection; C2) Preparing a product for preventing or treating diseases caused by monkeypox virus; C3) Preparing a product for preventing or treating monkeypox caused by monkeypox virus infection.

8. A production method of a monkeypox vaccine, characterized in that, It includes the step of expressing the recombinant protein using the biological material described in claim 2.

9. A monkeypox vaccine, characterized in that, The vaccine is prepared by the method described in claim 8.

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