CN117986388B - Recombinant protein, monkey pox vaccine and preparation method and application thereof - Google Patents
Recombinant protein, monkey pox vaccine and preparation method and application thereof Download PDFInfo
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- 238000002360 preparation method Methods 0.000 title description 7
- 241000700627 Monkeypox virus Species 0.000 claims abstract description 36
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- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/12—Viral antigens
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- C12N2710/24011—Poxviridae
- C12N2710/24111—Orthopoxvirus, e.g. vaccinia virus, variola
- C12N2710/24134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
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Abstract
The invention discloses a recombinant protein, which is the recombinant protein of the following a) or b): a) The amino acid sequence is a recombinant protein of SEQ ID No. 2; b) The recombinant protein which can code for the activity of the monkey pox antigen is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in SEQ ID No. 2. The invention screens proper recombinant virus subunit vaccine from EEV and IMV surface antigen, and the toxicity attack protection rate of immune group shows that the bigeminal fusion recombinant protein can generate 100% immunity protection rate. Is a high-efficiency and safe technical means, and solves the problems of unstable protection effect, easy secondary infection, strong toxicity and the like of the existing inactivated vaccine and the existing attenuated vaccine. The recombinant protein and the biological material can be used for preparing a monkey pox virus subunit vaccine and can be applied to the prevention of monkey pox.
Description
Technical Field
The invention relates to an in vitro diagnosis technology, in particular to a recombinant protein, a monkey pox vaccine, a preparation method and application thereof.
Background
Since month 5 of 2022, the number of Monkey Pox Virus (MPV) infection cases outside of the african primary region has dramatically increased. The World Health Organization (WHO) has announced that the global monkey poxvirus outbreak constitutes an international public health emergency. The monkey poxvirus and smallpox virus (smallpox) and vaccinia virus (VACV) belong to the genus orthopoxvirus of the family poxviridae and are double-stranded DNA viruses. Vaccination with an orthopoxvirus vaccine provides cross-immune protection against infection with other orthopoxviruses. Thus, two VACV-based vaccines with reduced activity (ACAM 2000 and JYNNEOS) have been urgently approved by the U.S. food and drug administration for the prevention of monkey pox. However, these vaccines have been shown to be associated with a number of serious adverse events, including myocarditis and pericarditis, particularly in certain individual populations, such as eczema patients and pregnant women. To date, they have only been approved for pre-exposure prophylaxis in monkey pox high exposure individuals, and not for general public use. Thus, in the context of reduced immunity of the world's smallpox, there is an urgent need for a safer, more effective new vaccine against the potentially increased monkey pox epidemic.
The monkey poxvirus has a large and complex proteome containing more than 200 proteins. There are two main modes of infection for monkey poxviruses, extracellular membrane viruses (EEVs) and Intracellular Mature Viruses (IMVs), and screening for appropriate recombinant viral subunit vaccines from surface antigens of EEVs and IMVs may be a highly efficient and safe technique.
CN116102663a discloses a monkey poxvirus B6R antigen, its preparation method and application.
CN116024265A discloses a method for preparing monkey poxvirus M1R antigen secretion protein and nucleic acid molecules used therefor.
CN116603060a discloses a monkey pox virus subunit vaccine, a preparation method and application thereof, and relates to the field of biotechnology. The anti-acne virus antigen comprises an antigen and an adjuvant, wherein the antigen components are monkey pox virus antigen proteins M1R, A, L, A, 35R and B6R, and His labels are added to the tail parts of the monkey pox virus antigen proteins M1R, A, 29, L, A and 35R or B6R.
CN116082521a discloses a multi-immunogen chimeric or mixed antigen against poxviruses (particularly monkey poxviruses), the chimeric or mixed antigen comprising three immunogens: (1) a monkey poxvirus a35R protein, (2) a monkey poxvirus M1R protein, and (3) a monkey poxvirus B6R protein, capable of eliciting an immune response against both infectious viral particles, intracellular mature viral particles (IMV) and extracellular enveloped viral particles (EEV).
CN116218879a discloses an mRNA vaccine against orthopoxvirus, which encodes and secretively expresses surface protein M1R, B6R, C19L, E8L, H3L derived from a monkey poxvirus 2022 epidemic strain.
CN116712536a discloses an anti-poxvirus vaccine comprising mRNA of the following antigens and/or fusion antigens: a fusion antigen fused to two or more monkey poxvirus antigens or parts of antigens selected from the group consisting of: a35R, M1R, B R and a29L.
The invention provides a bigeminal recombinant protein for preventing monkey pox, a construction method and application thereof, and aims to solve the problems that the protection effect of the existing inactivated vaccine and attenuated vaccine is unstable, secondary infection is easy to cause, virulence returns and the like. The technology can be used for preparing the subunit vaccine of the monkey pox virus and is applied to the prevention of the monkey pox.
Disclosure of Invention
In certain embodiments, the invention first provides a recombinant protein that is a recombinant protein of a) or b) as follows:
a) The amino acid sequence is a recombinant protein of SEQ ID No. 2;
b) The recombinant protein which can code for the activity of the monkey pox antigen is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in SEQ ID No. 2.
In certain embodiments, the present invention also provides a biological material associated with the recombinant protein described above, which is any one of the following B1) to B8):
b1 A nucleic acid molecule encoding the recombinant protein of claim 1;
b2 An expression cassette comprising the nucleic acid molecule of B1);
b3 A recombinant vector comprising the nucleic acid molecule of B1),
B4 A recombinant vector comprising the expression cassette of B2);
b5 A recombinant microorganism comprising the nucleic acid molecule of B1);
b6 A recombinant microorganism comprising the expression cassette of B2);
B7 A recombinant microorganism containing the recombinant vector of B3);
b8 A recombinant microorganism comprising the recombinant vector of B4);
in certain embodiments, the vectors include E.coli, yeast expression vectors, and other expression vectors;
In certain embodiments, the recombinant microorganism comprises an engineered bacterium.
In certain embodiments, the nucleic acid molecule is a nucleic acid molecule as set forth in 1) or 2) or 3) or 4) below:
1) The coding sequence is a DNA molecule or a cDNA molecule of SEQ ID No.1 in a sequence table;
2) The nucleic acid sequence is a DNA molecule of SEQ ID No.1 in a sequence table;
3) A cDNA molecule or a genomic DNA molecule having 75% or more identity to the nucleotide sequence defined in 1) or 2) and encoding the recombinant protein;
4) Hybridizing under stringent conditions with the nucleotide sequence defined in 1) or 2) and encoding a cDNA molecule or genomic DNA molecule of the recombinant protein described above.
In certain embodiments, the invention also provides any one of the following uses of the recombinant proteins described above:
c1 Preparing a product for preventing infection of monkey pox virus;
c2 Preventing infection by monkey poxvirus;
c3 Preparing a product for preventing or treating diseases caused by the monkey poxvirus;
C4 Preventing or treating diseases caused by the monkey poxvirus;
c5 Preparing a product for preventing or treating monkey pox caused by infection by a monkey pox virus;
c6 Preventing or treating monkey pox caused by infection with a monkey pox virus;
optionally, the use is for non-therapeutic purposes.
In certain embodiments, the invention also provides any one of the following applications of the above-described biological material:
c1 Preparing a product for preventing infection of monkey pox virus;
c2 Preventing infection by monkey poxvirus;
c3 Preparing a product for preventing or treating diseases caused by the monkey poxvirus;
C4 Preventing or treating diseases caused by the monkey poxvirus;
c5 Preparing a product for preventing or treating monkey pox caused by infection by a monkey pox virus;
c6 Preventing or treating monkey pox caused by infection with a monkey pox virus;
optionally, the use is for non-therapeutic purposes.
In certain embodiments, the invention also provides a method for producing a monkey pox vaccine comprising the step of expressing a recombinant protein using the biological material described above.
In certain embodiments, the invention also provides a monkey pox vaccine prepared by the above method.
In certain embodiments, the invention also provides the use of a monkey pox vaccine for the prevention of a monkey pox virus or for the treatment of monkey pox, the use being for non-therapeutic purposes.
Compared with the prior art, the invention has at least the following beneficial effects:
The invention screens proper recombinant virus subunit vaccine from EEV and IMV surface antigen, and the toxicity attack protection rate of immune group shows that the bigeminal fusion recombinant protein can generate 100% immunity protection rate. Is a high-efficiency and safe technical means, and solves the problems of unstable protection effect, easy secondary infection, strong toxicity and the like of the existing inactivated vaccine and the existing attenuated vaccine. The recombinant protein and the biological material can be used for preparing a monkey pox virus subunit vaccine and can be applied to the prevention of monkey pox.
Drawings
FIG. 1 purified polyacrylamide gel electrophoresis analysis of recombinant protein L1R-Plinker-EEV1-HIS 6. 1. Before IPTG induction; 2. ultrasonic disruption of bacterial precipitate after IPTG induction; 3. ultrasonically crushing a bacterial supernatant after IPTG induction; 4. elution with 50mM imidazole; 5. elution with 100mM imidazole; 6. elution with 200mM imidazole; 7. elution with 300mM imidazole; m: protein molecular weight indicates standard.
FIG. 2 antibody titres of the bivalent fusion recombinant protein L1R-Plinker-EEV1-HIS 6 after immunization of mice.
Detailed Description
In order to make the technical problems, technical solutions and advantages to be solved more apparent, the following detailed description will be given with reference to the accompanying drawings and specific embodiments.
Example 1
1. Obtaining monkey pox antigen gene sequences from NCBI database and codon optimizing same
One candidate antigen (IMV membrane protein L R and EEV type-I membrane glycoprotein, respectively) was selected from each of the monkey pox virus Intracellular Mature Virus (IMV) and extracellular membrane virus (EEV) surface antigens. Extracting IMV membrane protein L R and EEV type-I membrane glycoprotein gene data, removing a transmembrane region sequence, performing gene synthesis, connecting the two sequences by using a linker sequence, constructing a bivalent recombinant protein subunit vaccine gene expression sequence, and performing artificial synthesis. The nucleotide sequence of the coding gene of the duplex fusion recombinant protein is shown as SEQ ID NO. 1.
SEQ ID NO.1：
ATGGGTGCAGCCGCCTCCTTCCAGACAACCGTGAACACCCTGTACG
AGAGAATCTCTTCCAAGCTGGAGCAAGAGGCAAATGCGTCTGCAAAGA
CCAAATGCCACATCGAAATTGGTAACTTCTACATCAGACAGAACCATGGC
TGCAATATCACCGTGAAGAACATGTGTAGCGCGGATGCAGACGCCCAGC
TCGATGCTGTTCTGTCTGCTGCGACAGAGACATATAGCGGACTGACACCA
GAGCAGAAGGCATATGTGCCCGCCATGTTCACCGCAGCACTGAACATTC
AGACCTCTGTGAATACCGTGGTTCGTGATTTTGAGAACTACGTGAAGCA
GACCTGTAATTCCAGTGCCGTGGTTGATAACAAGCTGAAGATTCAGAATG
TTATCATCGATGAATGTTATGGCGCTCCTGGTAGTCCGACCAATCTGGAAT
TTATTAACACGGGCAGCAGTAAAGGCAATTGTGCCATCAAGGCTCTGATG
CAGCTGACTACCAAGGCAACCACCCAGATCGCGCCGCGTCAGGTGGCTG
GCACCGGCGTCCAGGCCGAAGCCGCAGCCAAAGAAGCAGCGGCCAAG
GAGGCCGCTGCTAAGGAGGCCGCCGCAAAAGCGCTGGAAGCAGAGGCC
GCGGCAAAAGAAGCTGCGGCAAAAGAAGCAGCAGCAAAAGAAGCAGC
CGCTAAGGCCATGAAGACCATTTCCGTGGTTACCCTGCTGTGTGTTCTGC
CTGCCGTTGTTTATAGTACCTGTACCGTACCGACCATGAACAACGCGAAA
CTGACCAGCACCGAGACCAGCTTCAACGACAAACAGAAAGTGACCTTT
ACCTGTGACTCCGGTTACCATTCTCTGGACCCGCCTGCTGTTTGCGAGAC
AGACAAGTGGAAATACGAAAACCCGTGTAAACGGATGTGCACTGTTTCG
GATTATGTTAGCGAACTGTATGACAAGCCTCTGTATGAAGTTAATTCCACC
ATGACCCTGAGCTGCAATGGTGAAACCAAATATTTTAGATGCGAAGAAA
AAAATGGCAATACTTCCTGGAACGACACCGTGACCTGTCCTCGCGCCGA
ATGCCAGCCTCTCCAGCTGGAACATGGTAGCTGTCAGCCGGTTAAAGAA
AAATATAGCTTTGGTGAGTACATGACCATCAACTGTGATGTCGGCTACGA
AGTGATTGGTGTGAGCTACATCTCATGTACAGCCAATAGCTGGAACGTCA
TCCCAAGTTGCCAGCAGAAGTGCGATATTCCGTCCCTAAGTAATGGTCTG
ATCAGCGGTAGCACCTTTTCCATTGGCGGAGTGATCCACCTGTCTTGTAA
AAGCGGCTTTACACTGACCGGTAGCCCTAGCAGCACCTGCATTGATGGC
AAGTGGAACCCCATTCTGCCGACCTGCGTTAGATCCAACGAGGAATTTG
ATCCTGTGGATGATGGTCCGGATGATGAGACCGACCTGTCTAAGCTGTCT
AAGGATGTCGTGCAGTATGAACAAGAGATCGAATCCCTGGAAGCAACCTACCAC；
The recombinant protein for preventing monkey pox is a bivalent fusion recombinant protein L1R-Plinker-EEV1, and the amino acid sequence of the recombinant protein is shown as SEQ ID NO. 2.
SEQ ID NO.2：
MGAAASFQTTVNTLYERISSKLEQEANASAKTKCHIEIGNFYIRQNHGCNITVKNMCSADADAQLDAVLSAATETYSGLTPEQKAYVPAMFTAALNIQTSVNTVVRDFENYVKQTCNSSAVVDNKLKIQNVIIDECYGAPGSPTNLEFINTGSSKGNCAIKALMQLTTKATTQIAPRQVAGTGVQAEAAAKEAAAKEAAAKEAAAKALEAEAAAKEAAAKEAAAKEAAAKAMKTISVVTLLCVLPAVVYSTCTVPTMNNAKLTSTETSFNDKQKVTFTCDSGYHSLDPPAVCETDKWKYENPCKRMCTVSDYVSELYDKPLYEVNSTMTLSCNGETKYFRCEEKNGNTSWNDTVTCPRAECQPLQLEHGSCQPVKEKYSFGEYMTINCDVGYEVIGVSYISCTANSWNVIPSCQQKCDIPSLSNGLISGSTFSIGGVIHLSCKSGFTLTGSPSSTCIDGKWNPILPTCVRSNEEFDPVDDGPDDETDLSKLSKDVVQYEQEIESLEATYH;
2. Constructing a codon-optimized bigeminal recombinant protein coding gene into a recombinant protein expression vector:
(1) Using a rigid protein linker: a (EAAAK) 4ALEA(EAAAK)4 A, the IMV membrane protein L R gene and the EEV type-I membrane glycoprotein gene after codon optimization are connected to obtain the expression gene sequence of the bigeminal fusion recombinant protein L1R-Plinker-EEV 1.
(2) The DNA was chemically synthesized and cloned into the prokaryotic expression vector pET28 a.
3. The invention relates to a bigeminal recombinant protein induction expression method for preventing monkey pox, which comprises the following steps:
Transferring the recombinant expression vector pET28a-L1R-Plinker-EEV1 into competent cells of E.coli Rosetta (DE 3) to obtain E.coli genetic engineering expression strain Ec-L1R-Plinker-EEV1 capable of expressing the multi-fusion recombinant protein L1R-Plinker-EEV1, and purifying by IPTG induced expression and Ni column affinity chromatography to obtain the recombinant protein L1R-Plinker-EEV1-HIS 6. The purified L1R-Plinker-EEV1-HIS 6 recombinant protein was analyzed by SDS-PAGE electrophoresis, as shown in FIG. 1.
The induction expression method of the recombinant protein specifically comprises the following steps:
(1) After the gene engineering expression bacteria Ec-L1R-Plinker-EEV1 are induced to express by IPTG, lysozyme is added, bacterial cells are crushed by ultrasound, and bacterial liquid after ultrasound is centrifuged for 30min at the temperature of 12000g and 4 ℃, and the supernatant is collected.
(2) Combining the supernatant after centrifugation with a nickel column; washing the column with eluting buffer to remove impurity protein; eluting the impurity protein with an elution buffer containing 50mM imidazole and an elution buffer containing 100mM imidazole respectively; the target protein was eluted with elution buffer containing 200mM and 300mM imidazole, and the protein eluate was collected.
(3) And (3) loading the collected protein eluent into a dialysis bag, immersing the dialysis bag into PBS for 3-4 times, changing the PBS for dialysis every 3 hours, and concentrating by using an ultrafiltration tube after the dialysis is finished to obtain the purified target protein L1R-Plinker-EEV1-HIS 6.
(4) The purified target protein concentration was measured using BCA protein concentration measurement kit.
3. Preparation and immunoprotection analysis experiment of monkey pox genetic engineering recombinant bivalent subunit vaccine
(1) Preparation of subunit vaccine
Diluting the purified bigeminal fusion recombinant protein L1R-Plinker-EEV1-HIS 6 to 100 mu-F/ml, and uniformly mixing with aluminum hydroxide gel according to a volume ratio of 1:1 to prepare the subunit vaccine.
(2) Subunit vaccine immunogenicity analysis experiment
SPF grade C57/BL6 mice of 4 weeks old are divided into 6 groups, 6 mice in each group, subunit vaccines prepared by immunizing the bivalent fusion recombinant protein respectively in the first 5 groups are immune groups, the 6 th group is PBS control group, and PBS with the same volume is injected. Immunization was emulsified with an equal volume of aluminum hydroxide gel, the immunization period was 7 days, the immunization schedule was 4 immunizations, and the immunization regimen was leg intramuscular inoculation. Serum samples of immunized mice were collected weekly for antibody titer change level detection starting one week after the first immunization of the mice by tail-end blood collection, and the serum was tested for antibody titer by indirect ELISA and analyzed for statistics and data by GRAPHPAD PRISM software. The results are shown in figure 2, and the result shows that the subunit vaccine prepared by the bigeminal fusion recombinant protein can stimulate the organism to produce antibodies, and the antibodies have long maintenance time, which indicates that the prepared bigeminal fusion recombinant protein has good immunogenicity and can stimulate the organism to produce the antibodies with strong binding capacity and long maintenance time.
FIG. 2 shows the determination of antibody titers of anti-L1R and anti-EEV1 in the serum of mice on days 0,7, 14, 21, 28, respectively, after immunization of mice with the bivalent fusion recombinant protein L1R-Plinker-EEV1-HIS 6. And statistical and data analysis was performed using GraphPadPrism 9 software. As a result, the subunit vaccine prepared by the bigeminal fusion recombinant protein can effectively stimulate the organism to produce protective antibodies. p <0.05 represents the statistical difference. In the upper graph, p <0.01; * Represents p <0.001; * P <0.0001.
(3) Subunit vaccine immunoprotection experiments
① Experimental mice grouping and immunization
Negative control (PBS group): and injecting PBS into the virus-fighting group.
Experimental group (vaccine group): and (3) injecting a bivalent subunit vaccine into the virus-attacking group.
② Toxicity attack protective experiment
After 4 immunizations, negative control (PBS group) and experimental group were given intramuscular injections of lethal doses of VACV virus (5 x 10 6 PFU/mouse). One week after the challenge, mice were observed daily for survival symptoms, mice weight and death recorded, the dead mice were examined by dissection, and lesions were observed. The results of the toxicity attack protection experiment are shown in table 1. The toxicity attack protection rate of the immune group indicates that the bigeminal fusion recombinant protein can generate 100% of the immune protection rate.
TABLE 1 toxicity counteracting protective test results
While the foregoing is directed to the preferred embodiments of the present invention, it will be appreciated by those skilled in the art that various modifications and adaptations can be made without departing from the principles of the present invention, and such modifications and adaptations are intended to be comprehended within the scope of the present invention.
Claims (9)
1. The recombinant protein is characterized in that the amino acid sequence of the recombinant protein is shown as SEQ ID No. 2.
2. The biomaterial associated with the recombinant protein of claim 1, which is any one of the following B1) to B8):
b1 A nucleic acid molecule encoding the recombinant protein of claim 1;
b2 An expression cassette comprising the nucleic acid molecule of B1);
b3 A recombinant vector comprising the nucleic acid molecule of B1),
B4 A recombinant vector comprising the expression cassette of B2);
b5 A recombinant microorganism comprising the nucleic acid molecule of B1);
b6 A recombinant microorganism comprising the expression cassette of B2);
B7 A recombinant microorganism containing the recombinant vector of B3);
B8 A recombinant microorganism containing the recombinant vector of B4).
3. The biomaterial according to claim 2, characterized in that: the vector comprises an escherichia coli or yeast expression vector.
4. The biomaterial according to claim 2, characterized in that: the recombinant microorganism comprises engineering bacteria.
5. The biomaterial according to claim 2, characterized in that: the nucleic acid molecule sequence is shown as SEQ ID No. 1.
6. The use of the recombinant protein of claim 1 for:
c1 Preparing a product for preventing monkey pox caused by infection of monkey pox virus.
7. The following uses of the biomaterial of claim 2:
c1 Preparing a product for preventing monkey pox caused by infection of monkey pox virus.
8. A method for producing a monkey pox vaccine comprising the step of expressing a recombinant protein using the biomaterial according to claim 2.
9. A monkey pox vaccine prepared by the method of claim 8.
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| CN116603060A (en) * | 2023-06-07 | 2023-08-18 | 深圳鹏泊生物科技有限公司 | Monkey poxvirus subunit vaccine and preparation method and application thereof |
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| CN116603060A (en) * | 2023-06-07 | 2023-08-18 | 深圳鹏泊生物科技有限公司 | Monkey poxvirus subunit vaccine and preparation method and application thereof |
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