CN117222740A - mRNA vaccines encoding PcrV and/or OprF-I proteins - Google Patents

Abstract

The application provides an mRNA vaccine encoding pcrV and/or OprF-I proteins, the mRNA molecule encoding 1) pcrV protein; 2) At least one of the OprF proteins and OprI proteins. The vaccine prepared from mRNA designed by the application has excellent preventing and/or treating effects on diseases caused by pseudomonas aeruginosa.

Description

mRNA vaccines encoding pcrV and/or OprF-I proteins

Technical Field

The application relates to the biomedical field, in particular to an mRNA vaccine for encoding pcrV and/or OprF-I proteins.

Background

Pseudomonas aeruginosa (Pseudomonas aeruginosa, PA) is a gram-negative, non-fermentative and aerobic opportunistic pathogen with diverse phenotypes that are widely present in the natural and human living environment and commonly infects patients with impaired immune defense mechanisms, such as Cystic Fibrosis (CF), sepsis and oncology patients. Pseudomonas aeruginosa is generally nonpathogenic, but under specific conditions, chronic inflammation which can cause secondary infection or mixed infection is one of important pathogenic bacteria of nosocomial infection, and is also one of pathogenic bacteria with highest incidence rate such as ICU ward, burn, war wound and the like. PA infection can occur in any tissues and parts of the human body, and can also cause systemic infection such as endocarditis, pneumonia, septicemia and the like, and the mortality rate of the systemic infection is more than 20 percent.

Currently, due to the abuse of antibiotics and the like, the drug resistance problem of PA is increasingly serious, the pan-drug resistant pseudomonas aeruginosa (PDR-PA) and the multi-drug resistant pseudomonas aeruginosa (MDR-PA) are generated, and the separation rate of the drug resistant PA is increased year by year. Because of antibiotic resistance and slow development of new antibiotics, it is an important strategy to develop safe and effective vaccines, with the urgent need to find new "non-antibiotic therapies". Since the 60 s of the 20 th century, at least 60 vaccines against pseudomonas aeruginosa have been developed, and vaccine targets include Lipopolysaccharide (LPS), extracellular polysaccharide (extracellular polysaccharide, EPS), flagella, outer membrane proteins (outer membrane protein, OMP), bacterial toxins, outer membrane vesicles (outer membrane vesicle, OMV), and the like. According to different technical routes, the vaccine can be divided into component vaccines, subunit vaccines, attenuated live vaccines, whole-cell inactivated vaccines and carrier vaccines. To date, four vaccines have entered phase iii clinical trials, namely seven-valent lipopolysaccharide vaccine developed by the company of pyrogine, type a and type B bivalent flagella vaccine developed by the company of immno, octavalent lipopolysaccharide-endotoxin a conjugate vaccine developed by the company of switzerland Serum and Vaccine Institute, and vaccine IC43 developed by the company of Valneva Austria GmbH and using pseudomonas aeruginosa outer membrane protein OprF190-342 and OprI21-83 fusion protein as antigens, but all of these four vaccines have failed, and no pseudomonas aeruginosa vaccine is currently marketed in batches.

mRNA (Messenger RNA) is an intermediate Messenger that transmits genetic information in DNA to proteins. mRNA vaccine treatment refers to in vitro synthesis of mRNA, introduction of mRNA into a body, translation of mRNA into cells to express antigen protein, stimulation of an in vivo immune system by antigen, activation of humoral immunity and cellular immunity, and rapid immune response and self-protection when the body is contacted with alloantigen (cell/virus) again.

In recent years, related technologies in the field of RNA molecules have been developed in breakthrough, mRNA vaccines have achieved a certain research result on various infectious diseases such as influenza virus, ebola virus, zika virus and the like, mRNA vaccines transmit mRNA to cells, and protein is produced by expression, so that an organism is immune protected. The mRNA vaccine has the following main outstanding advantages compared with the traditional vaccine (including inactivated vaccine, attenuated vaccine, subunit vaccine and virus vector vaccine): (1) mRNA vaccines can induce strong humoral immunity in host cells, activate cellular immunity including cytotoxic T cells and activate innate immunity through specific recognition of pattern recognition receptors; (2) The mRNA vaccine has strong designability, simple and convenient preparation flow, rapid standardized production and short research and development period; (3) The mRNA vaccine research and development cost is high in controllability and relatively low in cost; (4) mRNA vaccine has high safety, can undergo natural degradation process after entering the body, can control side effects more accurately, and does not have risks of integration, induced gene mutation and exogenous viral infection. In addition, with the development of mRNA structure engineering, such as modification of 5' end cap structure and nucleotide, optimization of mRNA sequence, the defects of poor stability, low translation efficiency and easy degradation of mRNA are greatly improved, and the application of mRNA therapy is promoted. In recent years, mRNA has been widely used in cell programming and vaccine research and has shown great potential for use. Meanwhile, mRNA vaccine is also applied to the research of various infectious disease vaccines, such as CMV vaccine of Moderna company in the United states, zika vaccine and the like, which enter clinical research. At present, no safe and effective pseudomonas aeruginosa vaccine exists.

Disclosure of Invention

According to a first aspect, in an embodiment, there is provided an mRNA molecule encoding at least one of PcrV protein, oprF-I protein;

from the N-terminal to the C-terminal, the PcrV protein contains the following amino acid sequence:

MEVRNLNAARELFLDELLAASAAPASAEQEELLALLRSERIVLAHAGQPLSEAQVLKALAWLLAANPSAPPGQGLEVLREVLQARRQPGAQWDLREFLVSAYFSLHGRLDEDVIGVYKDVLQTQDGKRKALLDELKALTAELKVYSVIQSQINAALSAKQGIRIDAGGIDLVDPTLYGYAVGDPRWKDSPEYALLSNLDTFSGKLSIKDFLSGSPKQSGELKGLSDEYPFEKDNNPVGNFATTVSDRSRPLNDKVNEKTTLLNDTSSRYNSAVEALNRFIQKYDSVLRDILSAI(SEQ ID No.7)；

from the N-terminus to the C-terminus, the OprF protein contains the following amino acid sequence:

MNAFAAPAPEPVADVCSDSDNDGVCDNVDKCPDTPANVTVDANGCPAVAEVVRVQLDVKFDFDKSKVKENSYADIKNLADFMKQYPSTSTTVEGHTDSVGTDAYNQKLSERRANAVRDVLVNEYGVEGGRVNAVGYGESRPVADNATAEGRAINRRVE(SEQ ID No.8)；

from the N-terminus to the C-terminus, the OprI protein contains the following amino acid sequence:

HSKETEARLTATEDAAARAQARADEAYRKADEALGAAQKAQQTADEANERALRMLEKASRK(SEQ ID No.9)。

according to a second aspect, in an embodiment, there is provided a lipid nanoparticle loaded with the mRNA molecule of the first aspect.

According to a third aspect, in an embodiment, there is provided a protein comprising an amino acid sequence encoded by the mRNA of the first aspect.

According to a fourth aspect, in one embodiment, there is provided a DNA molecule encoding the mRNA molecule of the first aspect.

According to a fifth aspect, in an embodiment, a recombinant plasmid is provided comprising the DNA molecule of the fourth aspect.

According to a sixth aspect, in an embodiment, there is provided a vaccine comprising the mRNA molecule of the first aspect, the lipid nanoparticle of the second aspect, the protein of the third aspect, the DNA molecule of the fourth aspect or the recombinant plasmid of the fifth aspect.

According to a seventh aspect, in one embodiment, there is provided an antibody which is produced by induction and isolation of the vaccine of the sixth aspect.

According to an eighth aspect, in an embodiment there is provided the use of an mRNA molecule according to the first aspect for the manufacture of a medicament for the treatment and/or prophylaxis of a disease.

mRNA vaccines encoding pcrV and/or OprF-I proteins according to the above embodiments. The vaccine prepared from mRNA designed by the application has excellent preventing and/or treating effects on diseases caused by pseudomonas aeruginosa.

Drawings

FIG. 1 is a schematic diagram of the constituent elements of an mRNA vaccine;

FIG. 2.1 is a map of plasmid pVAX1-pcrV in example 1;

FIG. 2.2 is a map of plasmid pVAX1-OprF-I in example 1;

FIG. 3 is a chemical formula of Cap1 Cap of example 1;

FIG. 4 is a schematic representation of the transcription initiation of T7RNA polymerase in example 1;

FIGS. 5.1 and 5.2 are graphs of capillary electrophoresis analyses of IVT transcribed fragments of example 1;

FIG. 6 is a structural formula of a compound starting material for preparing mRNA-LNP in example 1;

FIG. 7 is a graph of the DLS test results of mRNA-pcrv-LNP of example 1;

FIG. 8 is a graph of the DLS test results for mRNA-OprF/I-LNP in example 1;

FIG. 9 is a graph showing the linear relationship between mRNA concentration and fluorescence intensity in example 1;

FIG. 10 is a graph of the results of the mRNA-LNP cytotoxicity test in example 1;

FIG. 11 is a photograph showing the result of coloration of the cell expression of WB-identified mRNA in example 1;

FIG. 12 is a graph showing the results of purification of the expression of the pcrV and OprF-I proteins of example 1;

FIG. 13 is a schematic diagram of the immunization and blood collection procedure of the mouse in example 1;

FIG. 14.1 is a graph of total IgG antibody titres detected one week after second immunization of mice with mRNA-pcrv-LNP (5, 25 μg) of example 1;

FIG. 14.2 is a graph of total IgG antibody titers detected one week after second immunization of mice with mRNA-OprF-I-LNP (5, 25 μg) in example 1;

FIG. 14.3 is a graph showing IgM antibody titer detection results 7 days after immunization of the vaccine of example 1;

FIG. 14.4 is a graph showing the results of the titer assays of the IgG subtypes (IgG 1 and IgG2 a) induced by mRNA-pcrv-LNP, mRNA-OprF-I-LNP in example 1;

FIG. 15 is a schematic diagram of the construction flow of the burn model of the mice in example 1;

FIG. 16 is a graph showing the results of the toxicity test in example 1.

Detailed Description

The application will be described in further detail below with reference to the drawings by means of specific embodiments. In the following embodiments, numerous specific details are set forth in order to provide a better understanding of the present application. However, one skilled in the art will readily recognize that some of the features may be omitted in various situations, or replaced by other materials, methods. In some instances, related operations of the present application have not been shown or described in the specification in order to avoid obscuring the core portions of the present application, and may be unnecessary to persons skilled in the art from a detailed description of the related operations, which may be presented in the description and general knowledge of one skilled in the art.

Furthermore, the described features, operations, or characteristics of the description may be combined in any suitable manner in various embodiments. Also, various steps or acts in the method descriptions may be interchanged or modified in a manner apparent to those of ordinary skill in the art. Thus, the various orders in the description and drawings are for clarity of description of only certain embodiments, and are not meant to be required orders unless otherwise indicated.

The numbering of the components itself, e.g. "first", "second", etc., is used herein merely to distinguish between the described objects and does not have any sequential or technical meaning.

As used herein, an open reading frame (open reading frame, ORF) is the normal nucleotide sequence of a structural gene, and the reading frame from the start codon to the stop codon can encode a complete polypeptide chain, with no stop codon present that interrupts translation.

Herein, unless otherwise specified, the amino acid sequences are all from the N-terminus (i.e., the terminus with an overhanging amino group) to the C-terminus (i.e., the terminus with an overhanging carboxyl group). The N-terminal refers to an amino group (-NH) with a protruding amino acid sequence ₂ ) The C-terminal refers to the terminal having a protruding carboxyl group (-COOH) on the amino acid sequence.

Herein, unless otherwise specified, the nucleotide sequences are all from the 5 'end to the 3' end.

The type III secretion system of Pseudomonas aeruginosa is a large syringe-like complex composed of more than 20 proteins, and can directly inject some virulence factors and other effector proteins into host cells, causing damage to the host cells and playing an important role in bacterial infectious diseases. PcrV is one of the important components of the type III secretion system of pseudomonas aeruginosa, which is capable of forming homomultimers, constituting "tubes" for transporting virulence factors and effector proteins by the secretion system, "key components of the type III secretion system of pseudomonas aeruginosa. Given the important role of PcrV in the pathogenesis of pseudomonas aeruginosa, this protein is an important target for the therapeutic antibodies against pseudomonas aeruginosa infection. The polyclonal antibody and the monoclonal antibody of the anti-PcrV prove that the anti-PcrV can inhibit the secretion of a III type secretion system and protect organisms from being invaded by pseudomonas aeruginosa. Similarly, pcrV is also an important candidate vaccine molecule, but due to its ability to form homomultimers, etc., the expression production of soluble recombinant proteins is limited. In one embodiment, the present application provides nucleic acid vaccines encoding PcrV, particularly mRNA vaccines, that address this problem well.

The outer membrane protein of pseudomonas aeruginosa is inlaid on the outer membrane of PA lipopolysaccharide and phospholipid layers, and is porin and other structural functional components on the surface of bacteria. The OprF and OprI selected in the present application are 2 important outer membrane proteins that are highly conserved in PA surface exposure and antigen. OprF is a PA bacterial surface nonspecific channel pore that is rich in beta-helical structures. Through regulation of quorum sensing networks, are involved in the virulence of PA. OprF can affect the formation of outer membrane vesicles by modulating Pseudomonas quinolone signaling (POS) levels. The cell-free expression system is adopted to express the OprF porin, and the functional characterization can be stably combined in the lipid bilayer membrane, so that the analysis of the structure and the function of the OprF is facilitated. OprF can be used as a safe protective immunogen in all pathogenic and environmental strains of PA and is an important candidate for the development of PA vaccines. OprI is a lipoprotein in the inner monolayer of the PA cell outer membrane, enriched in a-helix structures. OprI is a good carrier molecule for vaccination, acting with TLR2/4 of Antigen Presenting Cells (APCs) and epithelial cells, the terminal lipid tail can elicit an immune response, its adhesion to tracheal and intestinal epithelium aids in the development of mucosal protein vaccines. OprI is a novel target of cationic antibacterial peptide (cationic antimicrobial peptides, AMP), participates in the sensitivity of PA to hRNase7 and alpha helix cationic AMP, plays a role by inhibiting the antibacterial activity and increasing the permeability of bacterial cell membranes, can be used for screening medicines with drug resistance Pa infection, and is hopeful for developing novel vaccines.

According to a first aspect, in an embodiment, there is provided an mRNA molecule encoding at least one of the following proteins: 1) PcrV protein; 2) OprF proteins and OprI proteins;

from the N-terminal to the C-terminal, the PcrV protein contains the following amino acid sequence:

MEVRNLNAARELFLDELLAASAAPASAEQEELLALLRSERIVLAHAGQPLSEAQVLKALAWLLAANPSAPPGQGLEVLREVLQARRQPGAQWDLREFLVSAYFSLHGRLDEDVIGVYKDVLQTQDGKRKALLDELKALTAELKVYSVIQSQINAALSAKQGIRIDAGGIDLVDPTLYGYAVGDPRWKDSPEYALLSNLDTFSGKLSIKDFLSGSPKQSGELKGLSDEYPFEKDNNPVGNFATTVSDRSRPLNDKVNEKTTLLNDTSSRYNSAVEALNRFIQKYDSVLRDILSAI(SEQ ID No.7)；

from the N-terminus to the C-terminus, the OprF protein contains the following amino acid sequence:

MNAFAAPAPEPVADVCSDSDNDGVCDNVDKCPDTPANVTVDANGCPAVAEVVRVQLDVKFDFDKSKVKENSYADIKNLADFMKQYPSTSTTVEGHTDSVGTDAYNQKLSERRANAVRDVLVNEYGVEGGRVNAVGYGESRPVADNATAEGRAINRRVE(SEQ ID No.8)；

from the N-terminus to the C-terminus, the OprI protein contains the following amino acid sequence:

HSKETEARLTATEDAAARAQARADEAYRKADEALGAAQKAQQTADEANERALRMLEKASRK(SEQ ID No.9)。

in one embodiment, the PcrV selected in the present application is conserved, a highly desirable and important vaccine candidate antigen.

In one embodiment, the amino acid sequence shown in SEQ ID No.8 is located in the same amino acid sequence as the amino acid sequence shown in SEQ ID No. 9. Namely, the amino acid sequence shown in SEQ ID No.8 and the amino acid sequence shown in SEQ ID No.9 are positioned on the same amino acid sequence in a tandem manner.

In one embodiment, the amino acid sequence shown in SEQ ID No.8 and the amino acid sequence shown in SEQ ID No.9 can be expressed independently by independent nucleotide sequences and expression vectors, or can be expressed simultaneously in the same expression vector by nucleotide sequences in tandem. Preferably simultaneously.

In one embodiment, the amino acid sequence shown as SEQ ID No.8 is concatenated with the amino acid sequence shown as SEQ ID No.9 by a first linker sequence.

In one embodiment, the C-terminus of the amino acid sequence shown in SEQ ID No.8 is tandem to the N-terminus of the first linker sequence and the N-terminus of the amino acid sequence shown in SEQ ID No.9 is tandem to the C-terminus of the first linker sequence. In one embodiment, the amino acid sequence shown in SEQ ID No.8 has a signal peptide sequence in tandem at the N-terminus.

In another embodiment, the C-terminus of the amino acid sequence shown in SEQ ID No.9 is tandem to the N-terminus of the first linker sequence and the N-terminus of the amino acid sequence shown in SEQ ID No.8 is tandem to the C-terminus of the first linker sequence. In one embodiment, the amino acid sequence shown in SEQ ID No.9 has a signal peptide sequence in tandem at the N-terminus.

In one embodiment, the C-terminal of the amino acid sequence shown in SEQ ID No.7 is serially connected with a second linker sequence and a tailing amino acid sequence in sequence.

In one embodiment, the amino acid sequence shown in SEQ ID No.7 has a signal peptide sequence in tandem at the N-terminus.

In one embodiment, after the amino acid sequences shown in SEQ ID No.8 and SEQ ID No.9 are connected in series, a second linker sequence and a tailing amino acid sequence are sequentially connected in series at the C end of the amino acid sequence of the series.

In one embodiment, the first linker sequence comprises glycine (G), serine (S).

In one embodiment, the second linker sequence comprises glycine, serine.

In one embodiment, the first linker sequence includes, but is not limited to, the following amino acid sequences: GSGSGSGSGS.

In one embodiment, the second linker sequence includes, but is not limited to, the following amino acid sequences: GGGS.

In one embodiment, the tailing amino acid sequence contains at least one histidine (H).

In one embodiment, the tailing amino acid sequence contains 6 to 12 histidines.

In one embodiment, the mRNA encoding PcrV protein contains the nucleotide sequence:

in one embodiment, the mRNA encoding the OprF protein contains the nucleotide sequence:

in one embodiment, the mRNA encoding the OprI protein contains the nucleotide sequence:

in one embodiment, the mRNA molecule comprises, in order from the 5 'end to the 3' end: a 5' cap structure, a 5' UTR (Untranslated Region, non-coding region) sequence, a nucleotide sequence encoding a signal peptide, a nucleotide sequence encoding a PcrV protein and/or an OprF protein and an OprI protein, a 3' UTR sequence, a polyadenylation sequence (PolyA).

In one embodiment, the 5' end Cap sub-structure includes, but is not limited to, any of Cap0, cap1, cap2, and any other structure.

In one embodiment, the 5'utr, 3' utr sequences are independently derived from at least one of a natural protein, an artificial protein.

In one embodiment, the native protein includes, but is not limited to, any of α -globulin, β -globulin, heat shock protein HSP 70.

In one embodiment, the mRNA molecule is unmodified or modified.

In one embodiment, the modification comprises: at least one of pseudouridine triphosphate, N1-methyl pseudouridine triphosphate modification.

In one embodiment, the PcrV protein comprises the amino acid sequence depicted in SEQ ID No. 1.

In one embodiment, the mRNA molecule encoding the PcrV protein comprises the nucleotide sequence shown in SEQ ID No. 10.

In one embodiment, the OprF protein and the OprI protein (i.e., the OprF-I fusion protein) comprise the amino acid sequence shown in SEQ ID No. 6.

In one embodiment, the mRNA encoding the OprF protein and the OprI protein comprises the nucleotide sequence shown in SEQ ID No. 11.

According to a second aspect, in an embodiment, lipid nanoparticles (Lipid Nanoparticle, LNP) loaded with the mRNA molecules of the first aspect are provided. Lipid nanoparticles are a type of nanoparticle formed using lipids.

In one embodiment, the lipid nanoparticle comprises at least one of a cationic lipid, a neutral helper phospholipid, cholesterol, a polyethylene glycol modified lipid. The ionized lipid is a modified lipid that ionizes in an acidic environment when the LNP is endocytosed by the cell, thereby allowing the LNP to escape from the endosome. Ionizable cationic lipids are the most critical lipids, which are the determining factors for mRNA delivery and transfection efficiency. The neutral auxiliary phospholipid is generally saturated phospholipid, can improve the phase transition temperature of the cationic liposome, supports the formation of lamellar lipid double-layer structure and stabilizes the structural arrangement of the lamellar lipid double-layer structure; cholesterol has stronger membrane fusion property, and promotes the intracellular uptake and cytoplasmic entry of mRNA; PEG (polyethylene glycol) phosphatide is positioned on the surface of the lipid nanoparticle, so that the hydrophilicity of the lipid nanoparticle is improved, the PEG (polyethylene glycol) phosphatide is prevented from being rapidly cleared by an immune system, particle aggregation is prevented, and the stability is improved.

In one embodiment, the cationic lipid includes, but is not limited to, methyl 4- (N, N-dimethylamino) butyrate (diimine) methyl ester (DLin-MC 3-DMA, CAS number 1224606-06-7).

In one embodiment, the neutral helper phospholipids include, but are not limited to, distearoyl phosphatidylcholine.

In one embodiment, the polyethylene glycol modified lipids include, but are not limited to, at least one of 1, 2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol 2000, 2- [ (polyethylene glycol) -2000] -N, N-tetracosacetamide, 1, 2-distearoyl-rac-glycerol-3-methoxypolyethylene glycol 2000 (DSG-PEG 2000), or N- (carbonyl-methoxypolyethylene glycol 2000) -1, 2-distearoyl-sn-glycerol-3-phosphatidylethanolamine sodium salt, preferably at least one of 1, 2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol 2000 (structural formula see mPEG2000-DMG in FIG. 6, CAS number 160743-62-4), 2- [ (polyethylene glycol) -2000] -N, N-tetracosacetamide.

In one embodiment, the cationic lipid is on a molar basis: neutral helper phospholipid: cholesterol: polyethylene glycol modified lipid = 50:38.5:10:1.5.

according to a third aspect, in an embodiment, there is provided a protein comprising an amino acid sequence encoded by the mRNA of the first aspect.

According to a fourth aspect, in one embodiment, there is provided a DNA molecule encoding the mRNA molecule of the first aspect.

According to a fifth aspect, in an embodiment, a recombinant plasmid is provided comprising the DNA molecule of the fourth aspect.

According to a sixth aspect, in an embodiment, there is provided a vaccine comprising the mRNA molecule of the first aspect, the lipid nanoparticle of the second aspect, the fusion protein of the third aspect, the DNA molecule of the fourth aspect or the recombinant plasmid of the fifth aspect.

According to a seventh aspect, in one embodiment, there is provided an antibody which is produced by induction and isolation of the vaccine of the sixth aspect.

In one embodiment, the antibodies include, but are not limited to, at least one of IgG1, igG2 a.

According to an eighth aspect, in an embodiment there is provided the use of an mRNA molecule according to the first aspect for the manufacture of a medicament for the treatment and/or prophylaxis of a disease.

In one embodiment, the disease comprises a bacterial-induced disease.

In one embodiment, the bacteria include, but are not limited to, pseudomonas.

In one embodiment, the bacteria include, but are not limited to, pseudomonas aeruginosa.

In one embodiment, the application provides an mRNA vaccine for preventing and treating Pseudomonas aeruginosa infection, which encodes the protein PcrV important for the three secretion system of Pseudomonas aeruginosa and OprF-I fusion protein in outer membrane protein, and is delivered in Lipid Nanoparticles (LNP).

In one embodiment, the application designs and synthesizes mRNA with encoding the fusion protein of the PcrV protein and the OprF-I, wherein the Cap of the mRNA is Cap1 structure, the Cap is obtained by the mRNA co-transcription capping method, and the natural guanine nucleotide in the mRNA is replaced by N1-methyl pseudouridine. Three groups of immunized Balb/c mice, namely, a blank, 10 mug and 30 mug, are arranged after the PCrV-mRNA and the OprF-I-mRNA are wrapped by LNP, so that the immune response of the vaccine is evaluated, and after the mice are immunized twice, a toxicity attack test is carried out on burn mice by adopting pseudomonas aeruginosa PAO1 strain to evaluate the protection effect of the vaccine. Experimental results show that mRNA can be transcribed with high quality, and high-level protein expression can be realized at the cellular level; LNP particle size of mRNA coatedUniform, high encapsulation efficiency, and high titer of antigen-specific antibodies can be induced by immunizing mice; toxicity test shows that the pair of PcrV-mRNA vaccine is 50xLD ₅₀ The protective effect (i.e. survival rate of mice) of the challenge dose is up to 100%. The OprF-I-mRNA vaccine has a protective effect of 50 to 66.7 percent.

In one embodiment, as shown in FIG. 1, which is a schematic diagram of the constituent elements of an mRNA vaccine, the mRNA drug and vaccine constituent elements comprise, in order from 5 'to 3': a 5' end cap structure, a KOZAK sequence, a 5' non-coding region (5 ' utr), an open reading frame (Open Reading Frame, ORF), a 3' non-coding region (3 ' utr), and a poly adenine tail (PolyA). Whether mRNA can express exogenous genes with high efficiency is closely related to the composition and nucleotide modification of each element, so that to obtain an mRNA vaccine capable of expressing exogenous target genes with high efficiency, multiple aspects need to be considered and continuous screening and optimization are needed.

Example 1

The specific design flow of this embodiment is as follows:

1. construction of template Gene

The coding sequence was designed based on the amino acid sequences described above, with reference to the PcrV protein sequence and the OprF, oprI (Uniport: PA 1706) protein sequence, and with secretion signal peptide and 6 xhis tag added at the N-and C-terminus. The following sequence 1 is the amino acid sequence encoded by the ORF of mRNA. 5'UTR and KOZAK sequences (SEQ ID NO: 2) were added to the 5' end of the ORF, and 3'UTR and poly A (PolyA) sequences were added to the 3' end. The gene fragment is synthesized by Jin Wei intelligent company and cloned into pVAX1 vector to obtain template plasmid pVAX1-pcrV. FIG. 2.1 shows the plasmid pVAX1-pcrV, and FIG. 2.2 shows the plasmid pVAX 1-OprF-I.

In this example, the signal peptide sequence is the signal peptide sequence of TPA protein (sequence 3); the sequence of the 5' UTR is shown as sequence 4; the 3' UTR is shown in sequence 5; polyA has a length of 80 to 150.

The amino acid sequence encoded by the mRNA-pcrV ORF (sequence 1):

MEVRNLNAARELFLDELLAASAAPASAEQEELLALLRSERIVLAHAGQPLSEAQVLKALAWLLAANPSAPPGQGLEVLREVLQARRQPGAQWDLREFLVSAYFSLHGRLDEDVIGVYKDVLQTQDGKRKALLDELKALTAELKVYSVIQSQINAALSAKQGIRIDAGGIDLVDPTLYGYAVGDPRWKDSPEYALLSNLDTFSGKLSIKDFLSGSPKQSGELKGLSDEYPFEKDNNPVGNFATTVSDRSRPLNDKVNEKTTLLNDTSSRYNSAVEALNRFIQKYDSVLRDILSAIGGGSHHHHHH*(SEQ ID No.1)；

wherein the sequence indicated by the bold underline is the signal peptide sequence.

Kozak sequence (sequence 2):

GCCACCAUGG(SEQ ID No.2)

the "AUG" at the end of the Kozak sequence is the start codon of the ORF region.

The secretion signal peptide Tissue Plasminogen Activator (TPA) sequence (sequence 3) is as follows:

MDAMKRGLCCVLLLCGAVFVSP(SEQ ID No.3)

the 5' UTR sequence (sequence 4) is as follows:

the 3' UTR sequence (SEQ ID NO: 5) is as follows:

the amino acid sequence encoded by the ORF region of mRNA-OprF-I (SEQ ID NO: 6) is as follows:

MNAFAAPAPEPVADVCSDSDNDGVCDNVDKCPDTPANVTVDANGCPAVAEVVRVQLDVKFDFDKSKVKENSYADIKNLADFMKQYPSTSTTVEGHTDSVGTDAYNQKLSERRANAVRDVLVNEYGVEGGRVNAVGYGESRPVADNATAEGRAINRRVE/>HSKETEARLTATEDAAARAQARADEAYRKADEALGAAQKAQQTADEANERALRMLEKASRKGGGSHHHHHH*(SEQ ID No.6)；

in sequence 6, the bolded portion is a Linker sequence (Linker). The sequence indicated by the bold underline is the signal peptide sequence.

The mRNA sequence of PcrV is as follows:

wherein, each area is described as follows:

5' UTR: italics; kozak sequence:GCCACC ORF: thickening; 3' UTR: double underlining; polyA: and drawing a wavy line downwards. mRNA sequence encoding signal peptide: and (3) thick underlining. mRNA sequence encoding histidine tag: and underlined in phantom.

The mRNA sequence of OprF-I is as follows:

wherein, each area is described as follows:

5' UTR: italics; kozak sequence:GCCACC ORF: thickening the black body; 3' UTR: double underlining; polyA: drawing down wavy lines; (GS) 5 junction sequence: dot underscores. mRNA encoding a signal peptide: and (3) thick underlining. mRNA sequence encoding histidine tag: and underlined in phantom.

2. Plasmid template amplification and linearization

The pVAX1-pcrV plasmid and pVAX1-OprF-I plasmid were used to transform DH 5. Alpha. Competent cells, which were plated on Kan+ (kanamycin) -resistant solid LB plates, cultured at 37℃for 12-15 hours, single colonies were picked up and added to Kan+ (kanamycin) -resistant liquid LB medium, cultured at 37℃at 250rpm for 12-15 hours, and the cells were collected by centrifugation, and amplified plasmids were extracted using a commercial plasmid extraction kit. The XhoI close to the PolyA tail is selected as the linearization enzyme cutting site of the amplified plasmid, the linearization degree is detected by DNA agarose gel electrophoresis, the linearized plasmid DNA is recovered by a PCR product recovery kit, and the DNA concentration and the DNA quality are obtained by measuring the OD260 and the OD260/OD280 values by a Nanodrop ultramicro nucleic acid instrument.

3. mRNA in vitro transcription and purification

In vitro synthesis of RNA (IVU) mRNA complementary to one strand of template DNA was synthesized using DNA (linearized plasmid or PCR product) containing the sequence of U7 promoter (UAAUACGACUCACUAUAGGG) or SP6 promoter (AUUUAGGUGACACUAUAG) as template, using NUP as substrate under the action of U7 or SP6 RNA polymerase, and enhancing the stability of mRNA by adding cap structure at the 5 'end and ployA tail at the 3' end.

Capping of mRNA may be performed by co-transcription capping or post-transcription capping, and tailing may be performed by template transcription tailing or post-transcription enzymatic tailing. In this example, capping and tailing methods were co-transcribed capping and template tailing, respectively, with a 5' cap structure, in this example m7 gppppn (fig. 3). In co-transcriptional capping, cap analogs are added directly to the IVU, which directly produce the corresponding 5 '-capped mRNA by RNA polymerase with relaxed substrate specificity incorporated at the 5' -end. Since the cap analogue lacks free 5' -triphosphate, no internal incorporation of the cap analogue occurs during IVU. In this embodiment, the Urilinker company is usedReagenU AG (3 ' OMe) is a cap structure (FIG. 3), and as shown in FIG. 4, the transcription initiation scheme of U7RNA polymerase is shown, and under the action of U7 polymerase, transcription is initiated with 5' AG 3 '.

The specific transcription reaction is set according to the proportion of each component in the table 1, and the steps are as follows in sequence: (1) adding RNase free wax and NUPs; (2) Adding inThe reagent U AG (3' OMe) was mixed well and centrifuged to collect the liquid; (3) Adding 10X UranscripUion Buffer, mixing, and centrifugingCollecting liquid; (4) adding a linearized DNA template; (5) Adding Murine RNase InhibiUor, yeasU Inorganic PyrophosphaUa se and U7RNA Polymerase, mixing, and centrifuging to collect the liquid; (6) reacting at 37 ℃ for 2-3 hours. After the reaction, DNAaseI was added to remove the template DNA, and the transcript was purified by Monarch RNA cleanup kiU (NEB). The concentration of mRNA was calculated from the read value of the Nano drop ultraviolet spectrophotometer, and the purity of mRNA was measured by capillary electrophoresis 2100 Bioanalyzer. The results shown in FIGS. 5.1 and 5.2 show that the size of the in vitro transcribed mRNA molecule is as expected and that the integrity and purity are high.

TABLE 1 proportion of each component in the in vitro transcription reaction of mRNA

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4. mRNA-LNP preparation

The structural formula of the compound raw material used in this step is shown in fig. 6, and the mRNA-loaded lipid nanoparticle contains four components, namely, ionizable cationic phospholipids (ionizable lipids), neutral auxiliary phospholipids, cholesterol (CholesUerol), and polyethylene glycol-modified phospholipids (pegaued). The preparation process of the specific LNP comprises the following steps: ionizable lipids (DLin-MC 3-DMA, structural formula: sm-102, CAS number: 1224606-06-7 in FIG. 6), cholesterol (CholesUerol), helper lipids (DSPC, distearoyl phosphatidylcholine), 1, 2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol 2000 (structural formula: mPEG2000-DMG, CAS number: 160743-62-4 in FIG. 6) were used in an amount of 50:38.5:10:1.5 in absolute ethanol, the total concentration is 10mg/mL, and an organic phase is formed; mRNA encoding antigen protein was dissolved in sodium citrate buffer (50 mM, pH 4) at a total concentration of 0.1mg/mL to form an aqueous phase; the organic phase and the aqueous phase were combined according to 1:3, uniformly mixing the mixture at a speed of 12mL/min by using a microfluidic device to obtain LNP-mRNA mixed solution; LNP-mRNA mixture was sterilized with PBS (10 mM, pH 7.2) The mixture was diluted 40-fold and transferred to pre-sterilizationUlUra-15 centrifugal filter (cut-off = 100 KDa). To achieve buffer exchange and product concentration, centrifugation was completed in 15-30 min at 4000 Xg and repeated three times after addition of fresh PBS, and concentration gave mRNA-LNP at a concentration of 2 mg/mL. The final product was stored at 4 ℃ until use.

5. mRNA-LNP particle size and uniformity determination

Particle size and uniformity of LNP were measured using Dynamic Light Scattering (DLS). The specific operation is as follows: concentrated samples were again diluted in sterile PBS at a volume ratio of 1:100 and repeated three times using HORIBA-SZ100 equipment at dispersion angles of 25 ° and 90 ° to give LNP particle size distribution and PDI values (Polymer dispersiUy index, polymer dispersion index). Particle size results are given as a ratio of particle size to strength, while predicting the stability of the LNP dispersion system.

The DLS test results of the mRNA-pcrv-LNP shown in FIG. 7, which showed that the LNP had an average particle size of 91.1nm and a PDI index of 0.237, showed that the LNP was uniformly distributed in particle size, and the peak intensity of 91% indicated that the nanoparticles were aggregated in a very small amount on the surface.

The DLS test results of the mRNA-OprF-I-LNP shown in FIG. 8, which showed that the average particle size of LNP was 110.9nm and the PDI index was 0.214, indicate that the prepared LNP had a uniform particle size distribution, a peak intensity of 95.1%, indicating that a very small amount of nanoparticles were aggregated on the surface.

6. mRNA-LNP encapsulation efficiency assay

mRNA encapsulation efficiency Using QuanU-iU ^TM RiboGreen ^TM The RNA kit is used for measuring, and the measuring principle is as follows: quanU-iU ^TM The RNA reagent is an ultrasensitive fluorescent nucleic acid stain capable of detecting 1-200 ng of nucleic acid in solution, and the nucleic acid stain cannot penetrate LNP, so that only free nucleic acid not entrapped by LNP can be bound. Uriuon-100 asAs a surfactant, a demulsifier is often used, and LNP-mRNA obtained by treatment with 1% Uriuon-100 can release the entrapped nucleic acid to give the total nucleic acid amount. The drug loading rate is obtained by calculating the difference of the nucleic acid amount before and after demulsification and then dividing the drug loading rate by the total nucleic acid amount, and the encapsulation rate is obtained by the following formula:

encapsulation efficiency (%) = (post-demulsification basis-pre-demulsification basis)/post-demulsification basis.

The specific operation method is as follows: rRNA standard solutions of different concentrations (i.e., 1000, 500, 250, 125, 62.5, 31.25, 15.625, and 0 ng/mL) were prepared in UE (uri-EDUA) buffer. The mRNA LNP to be tested was dissolved in UEbuffer to prepare a sample of about 250ng/mL mRNA. Similar samples were also prepared in UE buffer supplemented with Uriuon-X100 surfactant (0.5%). mu.L of each sample (including standard solution and mRNA LNP sample) was added to the microwells of the 96-well plate. Subsequently, 100 μl 1 was added: 200 dilution of Ribogreen reagent. After 5 minutes of incubation in darkness and room temperature, fluorescence intensities were recorded using cyuoion 3 (biolek, winooski, VU, USA) with excitation and emission applied at 485 and 528nm, respectively. Fluorescence obtained from mRNA LNP samples dispersed in UE and UE/uri uon-X100 was theoretically attributed to free (unencapsulated) and total mRNA, respectively. Nevertheless, the Ribogreen reagent penetrated the LNP slightly even in the absence of Uriuon-X100. Thus, the fluorescence obtained from the filtered sample dispersed in the UE is subtracted from the fluorescence emitted by a similar unfiltered sample in the UE. A standard curve drawn using a standard solution is used to convert the fluorescence intensity into a concentration, and finally, the encapsulation efficiency is calculated according to a formula. The two mRNAs were encapsulated separately and the encapsulation efficiency was calculated using the same standard curve. The standard curve shown in FIG. 9 shows the linear relationship between mRNA concentration and fluorescence intensity (R ² =0.9993), the encapsulation efficiency was calculated using a standard curve, obtaining an encapsulation efficiency of about 95%.

7. mRNA-PCrV and mRNA-OprF-I cell transfection

Cell transfection of mRNA-PcrV and mRNA-OprF-I was delivered by both LNP and lip 3000. The transfection method of mRNA-LNP is to directly add LNP coated with mRNA-PcrV and mRNA-OprF-I respectively into 293U cells grown to about 70% with mRNA concentration of about 0.5 μg/cm ² . The lip3000 transfection mode is carried out according to the instruction, 293U cells which can stably grow are selected from six-well plates for plating, and transfection is carried out when the cells grow to 70-80%. According to the specification, firstly preparing a solution A and a solution B; and (3) solution A: 200. Mu.L of OpUi-MEM diluted 4. Mu.g mRNA; diluting solution B with 200. Mu.L of OpUi-MEM to obtain 10. Mu.L of lipo3000 (i.e. lipofeUamin 3000), mixing solution A and solution B, standing for 5min, adding solution B into solution A, mixing, and standing for 20min at room temperature. At 20min of the resting of the A, B mixture, the six-well plate was washed, the culture medium in the dish was aspirated, and the plate was washed once with PBS or serum-free medium. After the A, B mixture was left to stand, a transfection reagent was added to each well for 48 hours.

8. mRNA-LNPs cytotoxicity assays

To investigate the possible cytotoxicity of mRNA-LNPs, HEK 293U cells were used for adherent culture and different doses of mRNA were added to observe the cytotoxicity of mRNA-LNP. Briefly, PBS, blank LNP, and mRNA-loaded LNPLNP-pcrV and LNP-OprF-I were added 4-6 hours after seeding the cells in 96-well plates. After 72 hours of addition, 10. Mu.LCCK 8 was added to each well and incubated for 0.5 to 4 hours. The CKK8 reagent contains WSU-8, and the chemical name of the WSU-8 is as follows: 2- (2-methoxy-4-nitrophenyl) -3- (4-nitrophenyl) -5- (2, 4-disulfonic acid benzene) -2H-tetrazole monosodium salt. It is reduced by dehydrogenase in cells to yellow Formazan product (Formazan dye) with high water solubility under the action of electron carrier 1-methoxy-5-methylphenazinium dimethyl sulfate (1-MeUhoxy PMS), the number of Formazan compounds generated is proportional to the number of living cells, and the absorbance (OD) at 450nm is measured by an enzyme-labeled instrument to quantify the number of cells per well. The results of fig. 10 show that the blank LNP is slightly cytotoxic and that the mRNA-loaded LNP is substantially similar to the blank LNP.

9. Cell expression of WB-identified mRNA

Cell culture supernatants and cell lysates obtained from transfection of cells with PcrV-mRNA, pcrV-mRNA-LNP and mRNA-OprF/I, mRNA-OprF-I-LNP were assayed for the desired expression of the protein of interest by WB. Adding 6×loading buffer into cell culture supernatant and cell lysate, and performing polyacrylamide gel electrophoresis in boiling water bath for 5 min; gel electrotransfer, transferring proteins to PVDF; blocking with 1% BSA at room temperature for 60min, and washing with PBSU for 3 times; incubation with ani-His-HRP antibody for 2h at room temperature, washing 3 times with ubsu, and then developing and scanning with enhanced chemiluminescent kit (ECL).

FIG. 11 is a photograph showing the result of color development, which shows that PcrV-mRNA and mRNA-OprF-I can express the target protein at a high level after transfection of cells and can be secreted outside the cells with high efficiency, regardless of whether lipofeUamin3000 or LNP is used as a delivery means.

10. Expression purification of PcrV and OprF-I proteins

Cloning of the PcrV and OprF-I protein encoding genes into the pEU21a expression vector (InviUgen) with a C-terminal 6 XHis tag and transformation of E.coli BL ₂₁ (DE ₃ ) pLysS competent cells. Single colonies carrying the protein of interest plasmid expressed recombinant PCrV and OprF-I proteins carrying 6 consecutive histidine residues under IPUG induction at 37 ℃. Centrifugally collecting thalli, crushing the thalli by using a high-pressure homogenizer, and centrifuging at high speed to obtain supernatant, wherein the supernatant is firstly subjected to rough purification by using a NUA-Ni affinity chromatographic column, and then subjected to fine purification by using a size exclusion chromatography.

FIG. 12 shows SDS-PAGE of proteins, and it can be seen that both the PcrV and OprF-I proteins have been prepared in high purity and high yield.

11. Immunization and blood sampling of mice

The immunization and blood collection flow of the mice is schematically shown in FIG. 13, wherein the PcrV-mRNA-LNP, mRNA-OprF-I-LNP and blank LNP were used for immunizing balb/c mice by intramuscular injection, and the immunization dose was set at 5 and 25 μg in two doses, and the immunization volume was 100 μl. A second boost was performed 3 weeks after the first immunization, with the same dose, volume and immunization pattern as the first. Blood was collected from the orbital venous plexus at weeks 1, 3 and 5 of primary immunization, and serum was isolated for testing.

12. Determination of mouse specific antibody titers by enzyme-linked immunosorbent assay (ELISA)

For serum separated before and after immunization, the specific antibody titer is determined by adopting an indirect ELISA method, and the specific operation steps are as follows: coating a blank ELISA plate with PCrV and OprF expressed by escherichia coli, and coating liquid at 4 ℃ overnight; PBSU is washed 3 times, 1% BSA is added into each hole for sealing for 2 hours at 37 ℃, PBSU is washed 3 times, 100 mu L of mouse serum which is diluted in a gradient is added into each hole, the mixture is reacted for 60 minutes at 37 ℃, and the mixture is gently shaken; PBSU is washed 3 times, 100 mu L of HRP-labeled rabbit anti-mouse IgG or IgG1, igG2A (1:1000 dilution) is added, the mixture is allowed to act at 37 ℃ for 60min, and the mixture is gently shaken; PBSU is washed for 3 times, added with 100 mu L of HRP chromogenic substrate UMB and reacted in the dark at room temperature for 15 min; 100. Mu.L of 2% sulfuric acid stop solution was added; OD450 detects absorbance. FIGS. 14.1 to 14.4 show ELISA results. Wherein FIGS. 14.1 and 14.2 are total IgG antibody titers detected one week after second immunization of mice with mRNA-pcrv-LNP (5, 25 μg) and mRNA-OprF-I-LNP (5, 25 μg), respectively, each dose group was repeated 2 times. In the figure, the abscissa shows dilution factors of mouse immune serum, and the ordinate shows readings of OD450, wherein each column in FIG. 14.1 shows, from left to right, mRNA-pcrv-LNP-5 μg-1, mRNA-pcrv-LNP-2, mRNA-pcrv-LNP-25 μg-1, mRNA-pcrv-LNP-25 μg-2, and blank (blank) groups, and each column in FIG. 14.2 shows, from left to right, mRNA-OprF-I-LNP-5 μg-1, mRNA-OprF-I-LNP-2, mRNA-OprF-I-LNP-25 μg-1, and mRNA-OprF-I-LNP-25 μg-2, respectively. FIG. 14.3 shows IgM antibody detection results 7 days after vaccine immunization. The subtype of the antibody was further analyzed on the basis of detection of the total IgG titer of the antibody, and the results in FIG. 14.4 show that mRNA-pcrv-LNP induced IgG1 and IgG2a antibody levels were significantly higher than mRNA-OprF-I-LNP induced IgG1 and IgG2a antibody levels. In summary, experiments show that mRNA vaccine prepared in this example can induce effective humoral immunity in mice.

13. Establishment of burn model for mice

FIG. 15 shows a schematic flow chart of the construction of a burn model in mice, BALB/c mice were immunized twice and one month apart. All mice (immunized and non-immunized groups, body weight 20-30 g) were first anesthetized with 250 μl of 2.5% averuin, then depilated with depilatory cream on the right side of the mice waist and body back, finally a metal block of 22mm diameter, 100mm length, 165g weight was heated to 104 ℃ and a burn of 8s was applied to the shaved area of the animals to produce a grade 3 burn. Immediately thereafter, mice received peritoneal injections of 500 μl of 0.9% saline and 40 μl meloxicam (1 mg/kg), once every 24h, to prevent them from being stimulated and feeling painful.

14. Determination of half-death (LD 50) in Pseudomonas aeruginosa infected mice

The half-lethal dose refers to the minimum bacterial count that can cause 50% of the death of experimental animals under certain conditions, and the determination method is specifically as follows: bacterial concentration was first measured by measuring the readings of OD600 using a nanodrop micro-spectrophotometer, then Pseudomonas aeruginosa strains (PAO 1) of different concentrations (10≡2, 10≡3, 10≡4, 5×10≡4, 5×10≡5, 10≡5, 10≡8) were subcutaneously injected into the burn centres of all mice, the death of the mice was analysed by statistical analysis, and finally the results were analysed using SPSS software and LD50, 2×LD50, 5×LD50 and 10×LD50 doses were determined, the test repeated 3 times.

15. Evaluation of mRNA vaccine protection Effect by mouse challenge

In this embodiment, the protection effect of the vaccine is evaluated by a toxicity test of pseudomonas aeruginosa, and the specific implementation method is as follows: on day 14 after the second injection of mRNA vaccine, pseudomonas aeruginosa strain (PAO 1) was subcutaneously injected at 50×ld50 dose in the center of burns of mice (vaccine immunized group and control group), and wound, death of the mice were noted by observation. For dead mice, the mice were dissected for each organ and the amount of bacteria was measured. The results of the challenge experiment in FIG. 16 show that none of the 5. Mu.g and 25. Mu.g mice immunized with the PcrV-mRNA vaccine in advance died, i.e., the survival rate was 100%, compared to all mice in the LNP blank immunization control group, showing that the vaccine has a 100% protective effect. The survival rate of 5 mug group mice of the mRNA-OprF-I vaccine can reach 50 percent, and the survival rate of 25 mug group mice of the vaccine can reach 66.67 percent.

The foregoing description of the application has been presented for purposes of illustration and description, and is not intended to be limiting. Several simple deductions, modifications or substitutions may also be made by a person skilled in the art to which the application pertains, based on the idea of the application.

SEQUENCE LISTING

<110> university of south science and technology

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agcaguaccc uagcacaagc accaccgugg agggccacac cgacagcgug ggcaccgacg 540

ccuacaauca gaagcugagc gagagaagag ccaacgccgu gagagacgug cuggugaacg 600

aguacggcgu ggagggcggc agagugaacg ccgugggcua cggcgagagc agacccgugg 660

cugacaacgc caccgccgag ggcagagcca ucaacagaag aguggagggc agcgggagcg 720

gcagcggcag cggcagccac uccaaggaga ccgaggcuag acugaccgcc acagaggacg 780

ccgccgcuag agcccaagcu agagcugacg aggccuacag aaaggccgac gaggcccugg 840

gcgccgcuca gaaggcucag cagaccgccg acgaggcuaa ugagagagcc cugagaaugc 900

uggagaaggc cucccggaag ggcgggggca gccaccauca ccaucaccac ugagcggccg 960

cugagcggcc gcugauaaua ggcuggagcc ucgguggcca ugcuucuugc cccuugggcc 1020

uccccccagc cccuccuccc cuuccugcac ccguaccccc guggucuuug aauaaagucu 1080

gagugggcgg caaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1140

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1200

aaaaaaaaaa a 1211

<210> 12

<211> 882

<212> RNA

<213> artificial sequence

<400> 12

auggagguga gaaaccugaa cgccgcccgg gagcuguucc uggacgagcu ccuggccgcu 60

uccgccgccc ccgccuccgc ugagcaagag gaacugcugg cucugcugag aagcgagaga 120

aucguccugg cccacgccgg ccaaccccug uccgaggccc aaguccugaa agcucuggcc 180

uggcugcugg cugccaaucc uagcgccccu cccggccaag gccuggaggu gcugagagag 240

gugcugcaag cuagaagaca gcccggcgcu cagugggacc ugagagaguu ccuggugagc 300

gccuacuuca gccugcacgg cagacuggac gaggacguga ucggcgugua caaggacgug 360

cugcagaccc aagacggcaa gagaaaggcc cugcuggacg agcucaaggc ccucaccgcc 420

gagcugaagg uguacagcgu gauucagagc caaaucaacg ccgcccugag cgccaagcaa 480

ggcaucagaa ucgacgccgg cggcaucgac cugguggacc ccacccugua cggcuacgcc 540

gugggcgacc cuagauggaa ggacagcccc gaguacgccc ugcugagcaa ccuggacacc 600

uucagcggca agcugagcau caaggacuuc cugagcggca gccccaagca gagcggcgag 660

cugaagggcc ugagcgacga guaccccuuc gagaaggaca acaaccccgu gggcaacuuc 720

gccaccaccg ugagcgacag aagcagaccc cugaacgaca aggugaacga gaagaccacc 780

cugcugaacg acacaagcag cagauacaac agcgccgugg aggcccugaa cagauucauu 840

cagaaguacg acagcgugcu gagagacauc cugagcgcca uc 882

<210> 13

<211> 474

<212> RNA

<213> artificial sequence

<400> 13

augaacgcuu ucgcugcucc cgcccccgag cccguggccg auguguguag cgacagcgac 60

aacgacggcg ugugcgacaa cguggacaaa uguccugaca ccccugcuaa cgugaccgug 120

gacgccaacg gcugcccugc cguggccgag guggugagag ugcagcugga cgugaaguuc 180

gacuucgaca agagcaaggu gaaggagaac agcuacgccg acaucaagaa ccuggccgac 240

uucaugaagc aguacccuag cacaagcacc accguggagg gccacaccga cagcgugggc 300

accgacgccu acaaucagaa gcugagcgag agaagagcca acgccgugag agacgugcug 360

gugaacgagu acggcgugga gggcggcaga gugaacgccg ugggcuacgg cgagagcaga 420

cccguggcug acaacgccac cgccgagggc agagccauca acagaagagu ggag 474

<210> 14

<211> 183

<212> RNA

<213> artificial sequence

<400> 14

cacuccaagg agaccgaggc uagacugacc gccacagagg acgccgccgc uagagcccaa 60

gcuagagcug acgaggccua cagaaaggcc gacgaggccc ugggcgccgc ucagaaggcu 120

cagcagaccg ccgacgaggc uaaugagaga gcccugagaa ugcuggagaa ggccucccgg 180

aag 183

Claims (10)

1. An mRNA molecule encoding at least one of the following proteins: 1) PcrV protein; 2) OprF proteins and OprI proteins;

from the N-terminal to the C-terminal, the PcrV protein contains the following amino acid sequence:

MEVRNLNAARELFLDELLAASAAPASAEQEELLALLRSERIVLAHAGQPLSEAQVLKALAWLLAANPSAPPGQGLEVLREVLQARRQPGAQWDLREFLVSAYFSLHGRLDEDVIGVYKDVLQTQDGKRKALLDELKALTAELKVYSVIQSQINAALSAKQGIRIDAGGIDLVDPTLYGYAVGDPRWKDSPEYALLSNLDTFSGKLSIKDFLSGSPKQSGELKGLSDEYPFEKDNNPVGNFATTVSDRSRPLNDKVNEKTTLLNDTSSRYNSAVEALNRFIQKYDSVLRDILSAI(SEQ ID No.7)；

from the N-terminus to the C-terminus, the OprF protein contains the following amino acid sequence:

MNAFAAPAPEPVADVCSDSDNDGVCDNVDKCPDTPANVTVDANGCPAVAEVVRVQLDVKFDFDKSKVKENSYADIKNLADFMKQYPSTSTTVEGHTDSVGTDAYNQKLSERRANAVRDVLVNEYGVEGGRVNAVGYGESRPVADNATAEGRAINRRVE(SEQ ID No.8)；

from the N-terminus to the C-terminus, the OprI protein contains the following amino acid sequence:

HSKETEARLTATEDAAARAQARADEAYRKADEALGAAQKAQQTADEANERALRMLEKASRK(SEQ ID No.9)。

2. the mRNA molecule of claim 1, wherein the amino acid sequence set forth in SEQ ID No.8 is located in the same amino acid sequence as the amino acid sequence set forth in SEQ ID No. 9;

preferably, the amino acid sequence shown in SEQ ID No.8 is in tandem with the amino acid sequence shown in SEQ ID No.9 via a first linker sequence;

preferably, the C-terminal of the amino acid sequence shown in SEQ ID No.8 is connected in series to the N-terminal of the first linker sequence, and the N-terminal of the amino acid sequence shown in SEQ ID No.9 is connected in series to the C-terminal of the first linker sequence;

preferably, the C-terminal of the amino acid sequence shown in SEQ ID No.9 is connected in series to the N-terminal of the first linker sequence, and the N-terminal of the amino acid sequence shown in SEQ ID No.8 is connected in series to the C-terminal of the first linker sequence;

preferably, the C end of the amino acid sequence shown in SEQ ID No.7 is sequentially connected with a second joint sequence and a tailing amino acid sequence in series;

preferably, after the amino acid sequences shown in SEQ ID No.8 and SEQ ID No.9 are connected in series, a second joint sequence and a tailing amino acid sequence are sequentially connected in series at the C end of the amino acid sequence of the series sequence;

preferably, the first linker sequence comprises glycine, serine;

preferably, the second linker sequence comprises glycine, serine;

preferably, the first linker sequence comprises the amino acid sequence: GSGSGSGSGS;

preferably, the second linker sequence comprises the amino acid sequence: GGGS;

preferably, the tailing amino acid sequence contains at least one histidine;

preferably, the tailing amino acid sequence contains 6-12 histidines;

preferably, the mRNA encoding the PcrV protein contains the nucleotide sequence:

AUGGAGGUGAGAAACCUGAACGCCGCCCGGGAGCUGUUCCUGGACGAGCUCCUGGCCGCUUCCGCCGCCCCCGCCUCCGCUGAGCAAGAGGAACUGCUGGCUCUGCUGAGAAGCGAGAGAAUCGUCCUGGCCCACGCCGGCCAACCCCUGUCCGAGGCCCAAGUCCUGAAAGCUCUGGCCUGGCUGCUGGCUGCCAAUCCUAGCGCCCCUCCCGGCCAAGGCCUGGAGGUGCUGAGAGAGGUGCUGCAAGCUAGAAGACAGCCCGGCGCUCAGUGGGACCUGAGAGAGUUCCUGGUGAGCGCCUACUUCAGCCUGCACGGCAGACUGGACGAGGACGUGAUCGGCGUGUACAAGGACGUGCUGCAGACCCAAGACGGCAAGAGAAAGGCCCUGCUGGACGAGCUCAAGGCCCUCACCGCCGAGCUGAAGGUGUACAGCGUGAUUCAGAGCCAAAUCAACGCCGCCCUGAGCGCCAAGCAAGGCAUCAGAAUCGACGCCGGCGGCAUCGACCUGGUGGACCCCACCCUGUACGGCUACGCCGUGGGCGACCCUAGAUGGAAGGACAGCCCCGAGUACGCCCUGCUGAGCAACCUGGACACCUUCAGCGGCAAGCUGAGCAUCAAGGACUUCCUGAGCGGCAGCCCCAAGCAGAGCGGCGAGCUGAAGGGCCUGAGCGACGAGUACCCCUUCGAGAAGGACAACAACCCCGUGGGCAACUUCGCCACCACCGUGAGCGACAGAAGCAGACCCCUGAACGACAAGGUGAACGAGAAGACCACCCUGCUGAACGACACAAGCAGCAGAUACAACAGCGCCGUGGAGGCCCUGAACAGAUUCAUUCAGAAGUACGACAGCGUGCUGAGAGACAUCCUGAGCGCCAUC(SEQ ID No.12)；

preferably, the mRNA encoding the OprF protein contains the nucleotide sequence:

AUGAACGCUUUCGCUGCUCCCGCCCCCGAGCCCGUGGCCGAUGUGUGUAGCGACAGCGACAACGACGGCGUGUGCGACAACGUGGACAAAUGUCCUGACACCCCUGCUAACGUGACCGUGGACGCCAACGGCUGCCCUGCCGUGGCCGAGGUGGUGAGAGUGCAGCUGGACGUGAAGUUCGACUUCGACAAGAGCAAGGUGAAGGAGAACAGCUACGCCGACAUCAAGAACCUGGCCGACUUCAUGAAGCAGUACCCUAGCACAAGCACCACCGUGGAGGGCCACACCGACAGCGUGGGCACCGACGCCUACAAUCAGAAGCUGAGCGAGAGAAGAGCCAACGCCGUGAGAGACGUGCUGGUGAACGAGUACGGCGUGGAGGGCGGCAGAGUGAACGCCGUGGGCUACGGCGAGAGCAGACCCGUGGCUGACAACGCCACCGCCGAGGGCAGAGCCAUCAACAGAAGAGUGGAG(SEQ ID No.13)；

preferably, the mRNA encoding the OprI protein contains the nucleotide sequence:

CACUCCAAGGAGACCGAGGCUAGACUGACCGCCACAGAGGACGCCGCCGCUAGAGCCCAAGCUAGAGCUGACGAGGCCUACAGAAAGGCCGACGAGGCCCUGGGCGCCGCUCAGAAGGCUCAGCAGACCGCCGACGAGGCUAAUGAGAGAGCCCUGAGAAUGCUGGAGAAGGCCUCCCGGAAG(SEQ ID No.14)；

preferably, the mRNA molecule comprises, in order from the 5 'end to the 3' end: a 5' cap structure, a 5' UTR sequence, a nucleotide sequence encoding a signal peptide, a nucleotide sequence encoding a pcrV protein and/or an OprF, oprI protein, a 3' UTR sequence, a polyadenylation sequence;

preferably, the 5' end Cap substructure comprises Cap0, cap1, cap2;

preferably, the 5'utr, 3' utr sequences are independently derived from at least one of natural proteins, synthetic proteins;

preferably, the natural protein comprises any one of alpha-globulin, beta-globulin and heat shock protein HSP 70;

preferably, a Kozak sequence is also contained between the 5' utr sequence and the nucleotide sequence encoding the signal peptide;

preferably, the mRNA molecule is unmodified or modified;

preferably, the modification comprises: pseudouridine triphosphate modification, N1-methyl pseudouridine triphosphate modification;

preferably, the PcrV protein comprises the amino acid sequence shown in SEQ ID No. 1;

preferably, the mRNA molecule encoding the PcrV protein comprises the nucleotide sequence shown in SEQ ID No. 10;

preferably, the OprF protein and OprI protein comprise the amino acid sequence shown in SEQ ID No. 6;

preferably, the mRNA encoding the OprF protein and the OprI protein comprises the nucleotide sequence shown as SEQ ID No. 11.

3. Lipid nanoparticle loaded with an mRNA molecule according to any one of claims 1-2.

4. A protein comprising an amino acid sequence encoded by the mRNA of any one of claims 1-2.

5. A DNA molecule encoding the mRNA molecule of any one of claims 1-2.

6. A recombinant plasmid comprising the DNA molecule of claim 5.

7. A vaccine comprising the mRNA molecule of any one of claims 1-2, the lipid nanoparticle of claim 3, the protein of claim 4, the DNA molecule of claim 5, or the recombinant plasmid of claim 6.

8. An antibody, wherein said antibody is produced by induction and isolation of the vaccine of claim 7.

9. Use of an mRNA molecule according to any one of claims 1 to 2, or a protein according to claim 4, or a DNA molecule according to claim 5, or a recombinant plasmid according to claim 6, for the preparation of a medicament for the treatment and/or prophylaxis of a disease.

10. The use according to claim 9, wherein the disease comprises a bacterial-induced disease;

preferably, the bacteria comprise the order pseudomonas;

preferably, the bacteria comprise pseudomonas aeruginosa.