Preparation method and application of recombinant HPIV3-F protein trimer vaccine

Abstract

The invention relates to a fusion protein, the fusion protein comprises an F2 region and an F1 region of an F protein of HPIV3 and a foldon protein, after the F protein of HPIV3 is mutated, the stability and expression efficiency can be improved, the F protein of HPIV3 has a neutralizing effect with an antibody and can be used for preparing a vaccine, and the vaccine can stimulate an organism to generate a neutralizing antibody aiming at HPIV and has an immune protection effect.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to a fusion protein including HPIV3-F protein, a preparation method and application thereof. Background Art

[0002] Human parainfluenza virus (HPIV) mainly causes acute respiratory tract infection in children under 3 years old, and its clinical incidence rate is second only to respiratory syncytial virus infection. More than half of the cases are caused by HPIV type 3 (HPIV3), which poses a great threat to the life and health of newborns and infants. At present, there is a lack of effective therapeutic drugs and preventive vaccines for HPIV-3 infection. Most of the designs for human parainfluenza virus type 3 vaccines are based on live attenuated vaccines. The first step for HPIV-3 to infect host cells is to bind the HN protein on the virus particle to the sialic acid receptor of the host cell membrane protein, and then the F protein mediates the fusion of the virus envelope with the host cell membrane, prompting the virus to enter the host cell. The F protein must be cleaved by the host cell hydrolase into two subunits F1 and F2 connected by disulfide bonds before the F protein has fusion activity. It is necessary to develop a vaccine that can be stably expressed and has good immune protection. Summary of the invention

[0003] In order to overcome the above defects, the present invention provides a fusion protein including HPIV3-F protein, a preparation method and application thereof. Specifically,

[0004] In a first aspect, the present invention provides a fusion protein comprising the F protein of HPIV3 (HPIV3-F protein) and foldon protein, wherein the F protein comprises an F2 region and an F1 region, and the F1 region comprises at least an HRB (HeptadRepeat B) region.

[0005] Preferably, the HRB region includes positions 436-453 of SEQ ID No.1.

[0006] Preferably, the F2 region and the F1 region are connected via a cleavage region.

[0007] Preferably, the fusion protein further comprises a signal peptide.

[0008] Preferably, the F protein of the fusion protein is directly or indirectly connected to the foldon protein.

[0009] More preferably, the indirect connection comprises connection via a connecting peptide. In a specific embodiment, the connecting peptide comprises GS, GGGS (SEQ ID No.9), GSGGGGSG (SEQ ID No.10), GGS, GGGGS (SEQ ID No.11), GSGS (SEQ ID No.12), GGSGS (SEQ ID No.13), GSGGS (SEQ ID No.14), GGSGS (SEQ ID No.15), GGGSS (SEQ ID No.16) or multiple repeats thereof, etc.

[0010] Preferably, the foldon protein includes a cleavage site, which is recognized or cleaved by a specific protease; more preferably, the protease is selected from but not limited to one or a combination of two of the following proteases: non-human enterokinase, tobacco etch virus protease, protease derived from Bacillus subtilis, protease derived from Bacillus amyloliquefaciens, protease derived from rhinovirus, papain, homologs of insect papain or homologs of crustacean papain.

[0011] In a specific embodiment, the protease is derived from the 3C protease of rhinovirus (HRV-3C). Preferably, the fusion protein also includes a tag protein, which refers to a polypeptide or protein fused and expressed with the target protein using DNA in vitro recombination technology to facilitate the expression, detection, tracing and / or purification of the target protein. More preferably, the tag may be a Flag tag, a His tag, an MBP tag, an HA tag, a myc tag, a GST tag and / or a SUMO tag, etc. In a specific embodiment, the tag protein includes 8×His and 6×His.

[0012] More preferably, the fusion protein includes the F2 region of the F protein of HPIV3, the cleavage region, the F1 region of the F protein of HPIV3, the connecting peptide, and the foldon protein in sequence from the N-terminus to the C-terminus; further preferably, the fusion protein also includes a signal peptide at the N-terminus, and / or an enzyme cleavage site and a tag protein after the C-terminus of the foldon protein. More preferably, the enzyme cleavage site is connected to the upstream and / or downstream functional region at its N and / or C-terminus through a single amino acid, and in a specific embodiment, the single amino acid is G.

[0013] More preferably, the amino acid sequence of the F2 region of the F protein of the HPIV3 comprises:

[0014] (A1), 26-112 of SEQ ID No. 1 or 6,

[0015] (A2) a protein having the same function obtained by replacing and / or deleting and / or adding one or more amino acid residues of any one of (A1);

[0016] (A3) A protein having 80% or more identity with any one of (A1) to (A2) and having the same function.

[0017] More preferably, the amino acid sequence of the F1 region of the F protein of the HPIV3 comprises:

[0018] (B1), 149-491 of SEQ ID No. 1 or 6;

[0019] (B2) a protein having the same function obtained by replacing and / or deleting and / or adding one or more amino acid residues of any one of (B1);

[0020] (B3) A protein having 80% or more identity with any one of (B1) to (B2) and having the same function.

[0021] More preferably, the foldon protein comprises:

[0022] (C1), SEQ ID No. 1 or 6, 496-522,

[0023] (C2) a protein having the same function obtained by replacing and / or deleting and / or adding one or more amino acid residues of any of (C1);

[0024] (C3) A protein having 80% or more identity with any one of (C1) to (C2) and having the same function.

[0025] More preferably, the signal peptide comprises:

[0026] (D1), SEQ ID No.1 or 6, 1-25,

[0027] (D2) a protein having the same function obtained by replacing and / or deleting and / or adding one or more amino acid residues of any one of (D1);

[0028] (D3) A protein having 80% or more identity with any one of (D1) to (D2) and having the same function.

[0029] More preferably, the amino acid sequence of the HRV-3C enzyme cleavage site includes:

[0030] (E1), 524-531 of SEQ ID No. 1 or 6;

[0031] (E2) A polypeptide having the same function obtained by replacing and / or deleting and / or adding one or more amino acid residues of any one of (E1);

[0032] (E3) A polypeptide having 80% or more identity with any one of (E1) to (E2) and having the same function.

[0033] More preferably, the fusion protein comprises:

[0034] (F1), SEQ ID No. 1 or 6;

[0035] (F2) A polypeptide having the same function obtained by replacing and / or deleting and / or adding one or more amino acid residues of any one of (F1);

[0036] (F3) A polypeptide having 80% or more identity with any one of (F1) to (F2) and having the same function.

[0037] Herein, identity refers to amino acid sequence identity. The identity of amino acid sequences can be determined using homology search sites on the Internet, such as the BLAST webpage on the NCBI homepage website. For example, in Advanced BLAST2.1, by using blastp as a program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as a Matrix, setting Gap existence cost, Per residue gap cost and Lambda ratio to 11, 1 and 0.85 (default values) respectively and searching to calculate the identity of amino acid sequences, the value of identity (%) can then be obtained.

[0038] Herein, the 80% or greater identity may be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

[0039] Preferably, the fusion protein can form a trimer.

[0040] The second aspect of the present invention provides a nucleotide molecule, which encodes any one of the above-mentioned fusion proteins.

[0041] Preferably, the nucleic acid molecule comprises DNA and / or RNA, such as recombinant DNA, or mRNA.

[0042] More preferably, the nucleotide molecule comprises:

[0043] (G1), SEQ ID No.7 or 8,

[0044] (G2), complementary, degenerate or transcribed sequence of (G1),

[0045] (G3) A DNA molecule or mRNA that has 75% or more identity with the DNA molecule or mRNA defined in (G1) or (G2) and encodes the corresponding protein in the fusion protein.

[0046] Those skilled in the art can easily mutate the nucleotide sequence encoding the above fusion protein of the present invention by using known methods, such as directed evolution and point mutation. Those artificially modified nucleotides having 75% or higher identity with the nucleotide sequence encoding the above fusion protein are all derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention as long as they encode the above fusion protein and have the same function.

[0047] The identity refers to the sequence similarity with the nucleic acid sequence being compared. "Identity" includes nucleotide sequences having 75% or higher, 80% or higher, or 85% or higher, or 90% or higher, or 95% or higher identity with the nucleotide sequence shown in the present invention. Identity can be evaluated with the naked eye or computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences.

[0048] The aforementioned 75% or more identity may be 80%, 85%, 90% or 95% or more identity.

[0049] The third aspect of the present invention provides a vector, wherein the vector comprises any one of the above-mentioned nucleotide molecules.

[0050] Preferably, the vector further includes a regulatory factor, such as a promoter for initiating transcription of the above polypeptide or fusion protein encoding gene sequence, and may also include a terminator for terminating transcription of the above polypeptide or fusion protein encoding gene sequence. Further, the vector may also include an enhancer sequence.

[0051] The vector described herein refers to a vector that can carry exogenous DNA, mRNA or target gene into host cells for amplification and expression. The vector can be a cloning vector or an expression vector, including but not limited to: plasmid, phage (such as lambda phage or M13 filamentous phage, etc.), cosmid (i.e., cosmid), Ti plasmid, viral vector (such as retrovirus (including lentivirus), adenovirus, adeno-associated virus, etc.). In one or more embodiments of the present invention, the vector is a pUC57 vector and / or a PKS001 (pKS001) vector.

[0052] In a fourth aspect, the present invention provides a host cell, wherein the host cell comprises any one of the above-mentioned nucleotide molecules or vectors.

[0053] The host cell (also referred to as recipient cell) described herein can be a plant cell or an animal cell. The host cell can be understood to refer not only to a specific recipient cell, but also to the progeny of such a cell, and due to natural, accidental or intentional mutations and / or changes, the progeny may not necessarily be completely identical to the original parent cell, but is still included in the scope of the host cell. Suitable host cells are known in the art, wherein: the plant cell may be Arabidopsis thaliana, tobacco (Nicotiana tabacum), corn (Zea mays), rice (Oryza sativa), wheat (Triticum aestivum) and the like, but is not limited thereto; the animal cell may be a mammalian cell (e.g., Chinese hamster ovary cell (CHO cell), African green monkey kidney cell (Vero cell), baby hamster kidney cell (BHK cell), mouse breast cancer cell (C127 cell), human kidney epithelial cell line or its derivative strain, such as HEK293, 293T / 17, etc., human HeLa cell, fibroblast, bone marrow cell line, T cell or NK cell, etc.), avian cell (e.g., chicken or duck cell), amphibian cell (e.g., African clawed frog (Xenopus laevis) cell or giant salamander (Andrias davidianus) cells), fish cells (such as grass carp, carp, rainbow trout or catfish cells), insect cells (such as Sf21 cells or Sf-9 cells), etc. but not limited thereto. In one or more embodiments of the present invention, the host cell is CHO-K1 cell, 293T cell.

[0054] Preferably, the host cell may also be a microorganism, and the microorganism described herein may be a bacterium, a fungus, an actinomycete, a protozoa, an algae or a virus. Wherein, the bacterium may be from Escherichia sp., Erwinia sp., Agrobacterium sp., Flavobacterium sp., Alcaligenes sp., Pseudomonas sp., Bacillus sp., etc., but not limited thereto, for example, the bacterium may be Escherichia coli, Bacillus subtilis or Bacillus pumilus. In one or more embodiments of the present invention, the microorganism is a TOP10 competent cell.

[0055] In a fifth aspect, the present invention provides a method for preparing the above-mentioned fusion protein, the preparation method comprising introducing the above-mentioned nucleotide molecule or vector encoding the fusion protein into a host cell, and culturing the host cell.

[0056] Preferably, the preparation method comprises screening a monoclonal cell line with high and stable expression level.

[0057] More preferably, the screening method includes conventional screening methods in the art, such as adding screening reagents, comparing the biological activity of clone pools by ELISA, and the like.

[0058] In a sixth aspect, the present invention provides an application of any of the above fusion proteins, nucleotide molecules, vectors or host cells, wherein the application includes any of the following:

[0059] (1) Use in the preparation of products for preventing and / or treating diseases caused by HPIV infection;

[0060] (2) Use in the preparation of products for inducing immune responses to HPIV virus antigens;

[0061] (3) Use in the prevention and / or treatment of diseases caused by HPIV virus infection;

[0062] (4) Application in inducing immune response to HPIV virus antigens.

[0063] The product described herein may be a reagent or a drug. The reagent may be a diagnostic reagent, and the drug may be a therapeutic or preventive drug. More preferably, the drug is a preventive drug, such as a vaccine.

[0064] Preferably, the preparation further comprises screening, for example, using the above fusion protein as a target to screen preventive or therapeutic candidate drugs or diagnostic agents.

[0065] The products described in (1) and (2) may be vaccines or antibodies against HPIV viruses, and the antibodies include full-length antibodies or antigen-binding fragments (such as Fab fragments, Fv fragments, Fab′ fragments, F(ab′)2 fragments, single-chain antibodies (ScFv), nanobodies (single-domain antibodies), bispecific antibodies or minimum recognition units (MRU), etc. but not limited thereto).

[0066] Furthermore, the HPIV antibody may be a neutralizing antibody that specifically binds to the F protein of HPIV. The neutralizing antibody may be a high-titer neutralizing antibody against multiple epidemic strains of HPIV.

[0067] In the above applications, the diseases caused by the HPIV virus infection may include respiratory system infection, digestive system infection, cardiovascular system infection, and / or nervous system infection, etc.

[0068] Preferably, the respiratory system infection may include respiratory tract infection and / or lung infection.

[0069] Preferably, the digestive system infection may include intestinal diseases, anorexia, nausea, vomiting, abdominal pain and / or diarrhea.

[0070] More preferably, the respiratory tract infection may include severe acute respiratory syndrome, hypoxemic respiratory failure, sepsis, septic shock, nasopharyngitis, rhinitis, pharyngitis, tracheitis and / or bronchitis.

[0071] More preferably, the lung infection may include pneumonia and / or lung injury.

[0072] Preferably, the product for preventing and / or treating diseases caused by HPIV virus infection comprises a vaccine.

[0073] In an eighth aspect, the present invention provides a drug, which comprises any one of the above-mentioned fusion proteins, nucleotide molecules, vectors, host cells or antibodies.

[0074] Preferably, the drug is a vaccine.

[0075] More preferably, the vaccine further comprises an adjuvant and / or a vaccine delivery system.

[0076] More preferably, the adjuvant may be a substance that can stimulate the body to produce a stronger humoral and / or cellular immune response to the antigen inoculated with it. The adjuvant described herein may be known to those skilled in the art, including but not limited to: plant adjuvants (such as alkylamines, phenolic components, quinine, saponin, sesquiterpenes, proteins, polypeptides, polysaccharides, glycolipids, phytohemagglutinins, etc.), bacterial adjuvants (such as cholera toxin, Escherichia coli heat-labile toxin, bacterial lipopolysaccharides, etc.), aluminum adjuvants and other inorganic adjuvants (such as calcium adjuvants), cytokines and nucleic acid adjuvants (such as monocyte colony stimulating factor, leukocyte factor IL-1, IL-2, IL-4, IL-5, IL-6, IFN-γ, CpG motifs, nucleic acid vectors, etc.), emulsion adjuvants (such as Freund's adjuvant). The adjuvant may be a pharmaceutically acceptable adjuvant.

[0077] It is well known to those skilled in the art that in order to enhance the immunogenicity of antigenic proteins, in addition to adding compounds with immunopotentiating effects as adjuvants, the gene combination can be adjusted to allow them to be expressed in a granular structure; or they can be aggregated in vitro and encapsulated in liposomes or capsule microspheres.

[0078] Preferably, the vaccine further comprises a vaccine delivery system.

[0079] The vaccine delivery system can be a type of substance that can carry antigenic substances to the body's immune system, store and exert its antigenic effect therein for a long time. The vaccine delivery system described herein can be an aluminum salt gel adjuvant vaccine delivery system, an emulsion adjuvant vaccine delivery system, a liposome adjuvant vaccine delivery system or a nano adjuvant vaccine delivery system.

[0080] Furthermore, the medicine or vaccine also includes one or more pharmaceutically acceptable carriers.

[0081] The pharmaceutically acceptable carrier may be a diluent, an excipient, a filler, a binder, a wetting agent, a disintegrant, an absorption promoter, an adsorption carrier, a surfactant or a lubricant but is not limited thereto.

[0082] The vaccine for preventing infection of the present invention can be an intramuscular liquid injection, an intravenous liquid injection, an intranasal liquid injection, an intradermal liquid injection or a subcutaneous liquid injection.

[0083] In a ninth aspect, the present invention also provides a method for producing an immune response, which may include administering any of the above vaccines to a subject.

[0084] In the above method, after the vaccine is administered to a subject, an immune response against HPIV virus can be induced in the subject. The immune response can be a cellular immune response, or a humoral immune response, or a cellular immune response and a humoral immune response.

[0085] The cellular immune response may include a B cell immune response and a T cell immune response.

[0086] The subject described herein can be a human or a non-human animal.

[0087] Furthermore, the non-human animal may be a non-human mammal.

[0088] The non-human mammal may be any one of mice, rats, guinea pigs, hamsters, pigs, dogs, sheep, monkeys, rabbits, cats, cows, and horses, but is not limited thereto.

[0089] The subjects described herein include, but are not limited to, healthy subjects, symptomatic infected subjects, asymptomatic infected subjects, or recovered subjects (subjects who have recovered after infection).

[0090] The administration described herein includes, but is not limited to, intramuscular injection, subcutaneous injection, intradermal injection, intravenous injection, arterial injection, intraperitoneal injection, microneedle injection, mucosal administration, oral administration, oral and nasal spraying, or aerosol inhalation.

[0091] Preferably, the above-mentioned HPIV virus is HPIV3 virus.

[0092] In summary, compared with the prior art, the present invention has the following beneficial effects:

[0093] This study successfully expressed and purified the parainfluenza antigen molecule preF protein by designing and using a eukaryotic expression system. This protein molecule can form a trimer structure with uniform particle size and stable structure. It can be used in vaccine research and development, and provides a reference for the research and application of vaccines designed in other trimer forms. BRIEF DESCRIPTION OF THE DRAWINGS

[0094] Figure 1 : Trimer design scheme.

[0095] Figure 2 : Amino acid sequence comparison of multiple candidate antigen molecules.

[0096] Figure 3 :Construction strategy for cloning eukaryotic expression vector of parainfluenza preF trimer antigen protein.

[0097] Figure 4 :Purification of HPIV3-F trimer antigen molecule.

[0098] Figure 5 :Molecular screening analysis of target antigen protein.

[0099] Figure 6 : SDS-PAGE analysis of eukaryotic expressed proteins.

[0100] Figure 7 : Maldi-ToF analysis.

[0101] Figure 8 : Trimeric protein immune serum neutralization test. DETAILED DESCRIPTION

[0102] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0103] The experimental methods in the following examples are conventional methods unless otherwise specified, and are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. The materials and reagents used in the examples are listed below, and the others can be obtained from commercial sources unless otherwise specified.

[0104] Example 1 Construction of eukaryotic expression system plasmid

[0105] 1. Sequence structure

[0106] An HPIV3 fusion protein comprising a mutated HPIV3 F protein and a foldon sequence was designed.

[0107] Sequence design principles: From N-terminus to C-terminus, it includes: signal peptide, tightly connected HPIV3-F protein sequence, connected flexible linker peptide, then connected foldon sequence, then connected enzyme cleavage site, such as HRV-3C cleavage site, and finally connected tag protein (see Figure 1 ).

[0108] SEQ ID No.1: Amino acid sequence of HPIV3 fusion protein:

[0109]

[0110] Among them, positions 1-25: signal peptide region; positions 26-112: F2 region of HPIV3F protein; positions 113-148: cleavage region; positions 149-491: F1 region of HPIV3F protein; positions 492-496: flexible linker region; positions 497-522: foldon sequence; positions 524-531: HRV 3C restriction site; positions 533-540: His tag.

[0111] Simultaneously design multiple antigens, trimer combinations, foldon sequences, and linker sequences or their mutations:

[0112] SEQ ID No. 2 completely removes the F2-F1 cleavage site;

[0113] SEQ ID No.3 adds a GSG linker to the F2-F1 cleavage region;

[0114] The F2 protein is completely removed from the sequence of SEQ ID No. 4;

[0115] SEQ ID No. 5 removes the HRB linker region of the F protein;

[0116] SEQ ID No. 6 is a tagged trimer vaccine sequence designed based on the sequences of other epidemic strains.

[0117] For details on sequence alignment, see Figure 2 .

[0118] SEQ ID No.2 completely removes the F2-F1 cleavage site

[0119]

[0120] SEQ ID No.3F2-F1 cutting region adds GSG linker

[0121]

[0122] SEQ ID No. 4 completely removes the F2 protein;

[0123]

[0124] SEQ ID No.5 removes the HRB linker region of the F protein

[0125]

[0126]

[0127] SEQ ID No.6 is a tagged trimer vaccine sequence designed based on other epidemic strain sequences

[0128]

[0129] 2. Construction Method

[0130] The PKS001 vector plasmid (purchased from Zhongshan Kangtian Shenghe Biotechnology Co., Ltd.) and the plasmid containing the nucleotide sequence encoding the HPIV3 preF protein (synthesized by Nanjing GenScript Biotechnology Co., Ltd.) were double-digested with HindⅢ and NotⅠ restriction endonucleases. The vector and fragments were connected with T4 ligase and transformed into Escherichia coli strain Top10. Positive clones were screened and plasmids were extracted. After sequencing, the correct plasmids were extracted in large quantities. A high-quality plasmid containing the nucleotide sequence encoding the HPIV3 preF antigen was obtained (see Figure 3 ).

[0131] Among them, the nucleotide sequence encoding SEQ ID NO.1 is shown as SEQ ID NO.7, and the nucleotide sequence encoding SEQ ID NO.6 is shown as SEQ ID NO.8.

[0132] SEQ ID NO.7

[0133]

[0134] SEQ ID NO.8

[0135]

[0136]

[0137] The nucleotide sequences encoding SEQ ID NO. 2-5 are not listed, and those skilled in the art can obtain them by making adaptive adjustments based on the amino acid sequences of SEQ ID NO. 2-5 and referring to SEQ ID NO. 7 and 8.

[0138] Example 2 Transformation of 293T cells, cell culture, protein purification and expression analysis

[0139] The high-quality plasmid with the nucleotide sequence encoding the HPIV preF antigen successfully constructed in Example 1 was used to transform 293T cells, and the "suspension-sensitive" transfection reagent-DNA complex was prepared according to the following method: 300 μg of plasmid DNA was diluted with serum-free medium to a final volume of 5 mL; 600 μL of "suspension-sensitive" transfection reagent (Polymer, M5 HiPerSuspension Cell Special Transfection Reagent MF388) was diluted with serum-free medium to a final volume of 5 mL; gently mix and incubate at room temperature for 5 minutes; after incubation for 5 minutes, the diluted plasmid DNA was added to the diluted "suspension-sensitive" transfection reagent and gently mixed. Incubate at room temperature for 20 minutes to allow the DNA-"suspension-sensitive" transfection reagent complex to form. After complete incubation, 10 mL of the DNA "suspension-sensitive" transfection reagent complex was added to 290 mL of growth medium containing 293 suspension cells, so that the final cell density was approximately 1×10 6 cell / mL.

[0140] The cells were cultured in a shaker at 37°C, 5% CO2, and 150 rpm. Protein purification and expression analysis were performed after 6 days.

[0141] RESULTS AND CONCLUSION: Protein culture medium was obtained using 293T transiently transfected cells.

[0142] Purification and expression analysis can be seen in the following steps:

[0143] 1. Purification of His-tagged target protein

[0144] Methods: The plasmids corresponding to SEQ ID No.1-6 were transiently transfected into 293T cells. The cell culture fluid cultured for 6 days was centrifuged at 8000r / min for 20 minutes, and the supernatant was collected and filtered using a 0.45μm filter membrane (Jin Teng, catalog number: JTSF 025013 / 014) to obtain about 300mL of supernatant cell fluid, which was purified using an AKTA protein purifier.

[0145] The specific steps are as follows: The purification column is filled with 5ml of filler (Ni-TED Purose 6Fast Flow, Jiaxing Qianchun Biotechnology Co., Ltd., A42302-06). After connecting the purification column to AKTA, debug the purification system, use the balance solution (20mM PB+0.5M NaCl, PH7.4) to balance the column, and the flow rate is set to 3mL / min. After balancing, the flow rate is set to 2mL / min for loading. After all samples pass through the purification column, use the balance solution at a flow rate of 3mL / min to rinse until the absorption value is stable. Then use the washing solution (20mM PB+0.5M NaCl+30mM imidazole, PH7.4) to rinse the column, and the flow rate is set to 3mL / min. Then use the eluent (20mM PB+0.5M NaCl+0.5M imidazole, PH7.4) to elute the target protein, and the flow rate is set to 3mL / min.

[0146] Results and conclusion: The expression of SEQ ID No.1-6 was performed, and the expression levels were found to be quite different, among which the expression levels of SEQ ID No.1 and 6 were similar. SEQ ID No.2-3 could not obtain the protein that could be purified, and SEQ ID 4 changed the F protein structure to obtain the target size protein. SEQ ID 5 was a mutant that removed the HRB linker, and the protein could be purified, and the protein expression level was similar to SEQ ID No.1 and 6.

[0147] Taking SEQ ID No.1 and SEQ ID No.6 as examples, the purified His-tagged protein was initially obtained. The concentration was 5 mg / ml and the volume was about 10 ml (see Figure 4 ).

[0148] 2. Molecular sieve

[0149] Method: The collected elution peaks were concentrated and then separated on a Geldex 200PG high-resolution agarose gel chromatography column (YEASEN, catalog number: HB220526) molecular sieve column. The specific steps are as follows: After connecting the chromatography column to AKTA, debug the purification system, use the balance solution (20mM PB+0.15M NaCl) to balance the column, and set the flow rate to 1mL / min. The sample to be separated enters the chromatography column through the sample loop, and the column is continuously rinsed with the balance solution to collect samples with different absorption peaks. After the absorption peak is concentrated, subsequent experiments are carried out.

[0150] Results Figure 5 Taking SEQ ID No.1 as an example, two specific peaks can be obtained after the purified protein passes through the molecular sieve. The first peak is the polymer form of the antigen molecule, and the second peak is the trimer of the antigen molecule; the concentration of peak 2 is about 4 mg / ml, about 5 ml.

[0151] 3. SDS-PAGE

[0152] Methods: Add 20 μL of 5× protein (SEQ ID No.1) loading buffer to 80 μL of fusion protein solution, treat at 95℃ for 10 min and centrifuge. Take 30 μL of supernatant for SDS-PAGE analysis, and observe protein expression, purification and separation after staining. The protein gel concentration is 10%, and the buffer is 1×Tris-MOPS-SDS Running buffer (MACKLIN, NONE6600).

[0153] Results Figure 6 :

[0154] In the left figure, lane 1 is the protein marker, lane 2 is the non-reduced sample of peak 1, lane 3 is the reduced sample of peak 1, lane 4 is the non-reduced sample of peak 2, lane 5 is the reduced sample of peak 2, and lane 6 is the sample before the molecular sieve experiment.

[0155] The right figure is a comparison of the purification of SEQ ID No.1 (Seq1) and SEQ ID No.6 (Seq6) proteins, lane 1 is the protein marker, lane 3 is the Seq1 protein sample, and lane 4 is the Seq6 protein sample.

[0156] Conclusion: A high-purity protein band can be observed at 55kDa in SDS-PAGE gel, which is consistent with the predicted size of the antigen molecule protein monomer. The protein purity is estimated to be above 95%.

[0157] 4. Intact Molecular Weight Analysis

[0158] Methods: The concentrated molecular sieve sample peak 2 sample was sent to Shanghai Microspectro Detection Technology Group Co., Ltd. for MALDI-TOF intact molecular weight analysis.

[0159] Results Figure 7 : The test results showed that the actual molecular weight of the target area protein was 188.7 kDa: which was basically consistent with the estimated trimer size, proving that the peak was a trimer.

[0160] Example 3 Activity detection of target protein

[0161] Method: The total protein of SEQ ID No. 1, 4, 5, and 6 purified in Example 2 and the trimer protein obtained by molecular sieve were verified by ELISA experiment, and PBS was used as blank control. The specific steps are as follows: 200 ng protein was coated with coating solution for each sample well at 4°C overnight. The primary antibody used was PIV3 neutralizing antibody (6WRP, see the literature: Boonyaratanakornkit, J., Singh, S., Weidle, C., Rodarte, J., Bakthavatsalam, R., Perkins, J., Stewart-Jones, GBE, Kwong, PD, McGuire, AT, Pancera, M., and Taylor, JJ (2021). Protective antibodies against human parainfluenza virus type 3 infection. mAbs 13.10.1080 / 19420862.2021.1912884.

[0162] ), and the secondary antibody used was Peroxidase-Conjugated Goat anti-Human IgG (H+L), 1:5000. After color development, the plate was read and the data was stored.

[0163] The results showed that the titers of the total protein and trimer protein of SEQ ID No.1 and SEQ ID No.6 were all above 1000, indicating that the purified target protein had HPIV3 viral protein binding activity. SEQ ID No.4 changed the F protein structure and had no binding activity. SEQ ID 5 was a mutant with the HRB linker removed, and its binding activity was significantly reduced, with a titer of only 100.

[0164] Example 4 Evaluation of the effect of mouse immune antigen molecules

[0165] Method: The trimer protein obtained by the purified molecular sieve of SEQ ID No. 1 in Example 2 was used to immunize mice, and 20 C57BL / 6 female mice aged 6-8 weeks were selected and randomly divided into 3 groups. The first and second immunizations were separated by 3 weeks, and serum was collected two weeks after the second immunization for ELISA titer detection. The immunization scheme is shown in Table 1, and the ELISA immunization effect is shown in Table 2.

[0166] Table 1. Antigen molecule immunization scheme

[0167] Group Immunogen Adjuvant Immune volume Immunization Immunization times Number of animals 1 PBS - 0.1ml im 2 5 2 Target antigen 10 μg 0.2mgAL(OH)3+CPG 0.1ml im 2 5 3 Target antigen 10 μg 0.2mgAL(OH)3 0.1ml im 2 5

[0168] Table 2. Antigen molecule immune effect

[0169] Group 21 days after free 14 days after the second free ELISA titer Mean ELISA titer Mean <![CDATA[10 μg target antigen + 0.2 mg AL(OH)3]]> 1600 7680 10μg target antigen + 0.2mg AL(OH)3 + CPG 3040 20480 PBS ND ND

[0170] The above results show that the target antigen can effectively induce the body to produce antibodies, which is significantly different from the control group, and the double adjuvant group is also significantly different from the single adjuvant group.

[0171] Example 5 Serum neutralization effect detection

[0172] method:

[0173] Use 1*PBS to wash the Vero-E6 cells grown in a T75 flask twice, add 0.25% trypsin, place at 37°C for 2 minutes, resuspend the cells and centrifuge at 4000rpm for 5 minutes. Resuspend the cells with culture medium and count them, evenly spread the Vero-E6 cells in a 96-well cell culture plate, and let them stand for 1 hour to adhere to the wall. Place in a cell culture incubator at 37°C and 5% CO2 and culture for 24 hours. After taking out the HPIV3 virus from -80 degrees, melt it in a 37-degree water bath, dilute it to 40 times, and place it on ice. Use 1*PBS to dilute the mouse serum obtained in Example 4 in multiples, and evenly spread the virus on the 96-well plate with diluted mouse serum with a spray gun, and incubate it in a 37-degree incubator for 1 hour. Then inoculate the cells with the mixture of serum and virus and culture it for 1.5 hours. After that, the serum and toxin mixture was discarded, and 200μl of the covering layer (DMEM+2% serum+1% double antibody+1.5% methylcellulose) was added, and the plate was placed in a 37-degree incubator for 25 hours. Invert and gently remove, and after shaking off the covering layer, 100μl of tissue fixative was added to each well and incubated at room temperature for 30 minutes. After discarding the tissue fixative, in order to improve the permeability of the cells, 100μl of 1% TRITON X-100 dissolved in PBS was added to each well, and incubated at room temperature for 30 minutes. After discarding the permeation solution, 10% BSA in PBS was added to block and incubate for 1 hour. After discarding the blocking solution, 100μl of primary antibody was added to each well, 6WRP (1:250), and incubated overnight at 4°C. After washing the plate 3 times with 1*PBST, 100μl of secondary antibody was added to each well, HRP-labeled goat anti-human (1:4000), and incubated at 37°C for 45 minutes. After washing the plate 5 times with 1*PBST, add 50μl of TrueBlue colorimetric solution to each well and develop the color for 5 minutes until a good color is produced. Remove the dye and store at room temperature away from light. Blue spots represent infected and pathological cells, which are photographed and analyzed using a CTL enzyme-linked immunospot analyzer (see Figure 8 ). 8 replicates, in Figure 8 A, B, H, etc. represent replicates, and no reagents were added to columns 1 and 12.

[0174] Table 3. Neutralization experiment design and data

[0175] List Serum dilution multiple average value SD 2 No serum added 597 45 3 65610 583 41 4 21870 561 29 5 7290 474 67 6 2430 244 53 7 810 90 31 8 270 29 10 9 90 8 3 10 30 3 1 11 10 2 1

[0176] The results showed that the mouse serum produced by the trimer antigen molecule had a neutralizing immune effect, and its effect was negatively correlated with the dilution.

[0177] The trimer vaccine sequence SEQ ID No.6 designed based on the sequences of other epidemic strains can achieve similar effects to SEQ ID No.1, and its protein expression level, purification effect, trimer mouse immunization effect and serum neutralization effect are all similar.

[0178] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0179] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

[0180] In addition, various embodiments of the present invention may be arbitrarily combined, and as long as they do not violate the concept of the present invention, they should also be regarded as the contents disclosed by the present invention.

Claims

1. A fusion protein, characterized in that The fusion protein includes the F protein of HPIV3 (HPIV3-F protein) and foldon protein, the F protein includes the F2 region and the F1 region, and the F1 region includes at least the HRB region.

2. The fusion protein according to claim 1, characterized in that The F2 region and the F1 region are connected via a cleavage region; Preferably, the fusion protein further comprises a signal peptide; Preferably, the F protein of the fusion protein is directly or indirectly linked to the foldon protein; Preferably, the foldon protein includes a restriction site, and / or, The fusion protein also includes a tag protein.

3. The fusion protein according to claim 1 or 2, characterized in that The fusion protein includes, from the N-terminus to the C-terminus, the F2 region of the F protein of HPIV3, the cleavage region, the F1 region of the F protein of HPIV3, the connecting peptide, and the foldon protein; Preferably, the fusion protein further comprises a signal peptide at the N-terminus, and / or further comprises a restriction site and a tag protein after the C-terminus of the foldon protein; More preferably, the amino acid sequence of the F2 region of the F protein of the HPIV3 comprises: (A1), 26-112 of SEQ ID No. 1 or 6, (A2) a protein having the same function obtained by replacing and / or deleting and / or adding one or more amino acid residues of any one of (A1); (A3) a protein having more than 80% identity with any one of (A1)-(A2) and having the same function; The amino acid sequence of the F1 region of the F protein of the HPIV3 includes: (B1), 149-491 of SEQ ID No. 1 or 6; (B2) a protein having the same function obtained by replacing and / or deleting and / or adding one or more amino acid residues of any one of (B1); (B3) a protein having more than 80% identity with any of (B1)-(B2) and having the same function; The foldon protein comprises: (C1), SEQ ID No. 1 or 6, 496-522, (C2) a protein having the same function obtained by replacing and / or deleting and / or adding one or more amino acid residues of any of (C1); (C3) a protein having more than 80% identity with any of (C1)-(C2) and having the same function; The signal peptide comprises: (D1), SEQ ID No.1 or 6, 1-25, (D2) a protein having the same function obtained by replacing and / or deleting and / or adding one or more amino acid residues of any one of (D1); (D3) a protein having more than 80% identity with any of (D1)-(D2) and having the same function; and / or, Preferably, the restriction site is the HRV-3C restriction site. More preferably, the amino acid sequence of the HRV-3C restriction site includes: (E1), 524-531 of SEQ ID No. 1 or 6; (E2) A polypeptide having the same function obtained by replacing and / or deleting and / or adding one or more amino acid residues of any one of (E1); (E3), a polypeptide having more than 80% identity with any one of (E1)-(E2) and having the same function; Further preferably, the fusion protein comprises: (F1), SEQ ID No. 1 or 6; (F2) A polypeptide having the same function obtained by replacing and / or deleting and / or adding one or more amino acid residues of any one of (F1); (F3) A polypeptide having 80% or more identity with any one of (F1) to (F2) and having the same function.

4. A nucleotide molecule, characterized in that The nucleotide molecule encodes the fusion protein according to any one of claims 1-3.

5. The nucleotide molecule according to claim 4, characterized in that The nucleotide molecule includes DNA and / or RNA. Preferably, the nucleotide molecule includes: (G1), SEQ ID No.7 or 8, (G2), complementary, degenerate or transcribed sequence of (G1), (G3) A DNA molecule or mRNA that has 75% or more identity with the DNA molecule or mRNA defined in (G1) or (G2) and encodes the corresponding protein in the fusion protein.

6. A carrier, characterized in that The vector comprises the nucleotide molecule according to any one of claims 4-5.

7. A host cell, characterized in that The host cell comprises the nucleotide molecule according to any one of claims 4-5 or the vector according to claim 6.

8. A method for preparing the fusion protein according to any one of claims 1 to 3, characterized in that: The preparation method comprises introducing the nucleotide molecule according to any one of claims 4-5 or the vector according to claim 6 into a host cell, and culturing the host cell.

9. A use of the fusion protein according to any one of claims 1 to 3, the nucleotide molecule according to any one of claims 4 to 5, the vector according to claim 6 or the host cell according to claim 7, the use comprising any one of the following: (1) Use in the preparation of products for preventing and / or treating diseases caused by HPIV infection; (2) Use in the preparation of products for inducing immune responses to HPIV antigens; (3) Use in the prevention and / or treatment of diseases caused by HPIV infection; (4) Application in inducing immune response to HPIV antigens.

10. A drug, characterized in that The drug comprises the fusion protein of any one of claims 1-3, the nucleotide molecule of any one of claims 4-5, the vector of claim 6 or the host cell of claim 7. Preferably, the drug is a vaccine. More preferably, the vaccine further comprises an adjuvant and / or a vaccine delivery system.

Images