Recombinant HPIV3-F protein nanoparticle vaccine as well as preparation method and application thereof

Abstract

The invention relates to a fusion protein, the fusion protein comprises a truncated F protein region of HPIV3 and ferritin or Ls protein, a fusion expression molecule of the F protein of HPIV3 and ferritin or Ls has a neutralization effect with an antibody and can be used for preparing a vaccine and obtaining nanoparticles with uniform particle size, and the vaccine can stimulate a body to generate a neutralizing antibody aiming at HPIV3 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 vaccine comprising HPIV3-F protein, a preparation method and an application thereof. Background Art

[0002] Human parainfluenza virus (HPIV3) mainly causes acute respiratory tract infections in children under 3 years old, with a clinical incidence second only to respiratory syncytial virus infection. More than half of the cases are caused by HPIV type 3 (HPIV3), posing a great threat to the life and health of newborns and infants. Currently, there is a lack of effective therapeutic drugs and preventive vaccines for HPIV-3 infection, and the design of vaccines for human parainfluenza virus type 3 is mostly based on live attenuated vaccines.

[0003] 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 viral envelope and the host cell membrane, allowing the virus to enter the host cell. The F protein must be cleaved by the host cell hydrolase into subunits F1 and F2 connected by two disulfide bonds before the F protein has fusion activity. At the same time, it is difficult to achieve effective immune protection using only the F protein as a vaccine antigen. It is necessary to develop a vaccine that can be stably expressed and has good immune protection. Summary of the invention

[0004] In view of the shortcomings of the prior art, the present invention aims to propose fusion protein nanoparticles including HPIV3-F protein, which solves the problems of fusion activity and immunogenicity of F protein through ferritin or dioxotetrahydropterin synthase polypeptide.

[0005] To achieve the above objectives, on the one hand, the present invention provides a fusion protein, which includes the F protein of HPIV3 (HPIV3-F protein) and a ferritin polypeptide or a dioxotetrahydropterin synthase polypeptide, the F protein includes an F2 region and an F1 region, and the F protein forms nanoparticles with the ferritin polypeptide or the dioxotetrahydropterin synthase polypeptide.

[0006] In some embodiments, the fusion protein comprises the following sequence from N-terminus to C-terminus: the F2 region, the F1 region and the ferritin polypeptide or the dioxotetrahydropterin synthase polypeptide, which are directly connected to each other by peptide bonds or by peptide segments.

[0007] In some embodiments, in the fusion protein, the F2 region and the F1 region are connected via a cleavage region;

[0008] Preferably, the fusion protein further comprises a signal peptide;

[0009] Preferably, the F protein of the fusion protein is directly or indirectly linked to the ferritin polypeptide or the dioxotetrahydropterin synthase polypeptide;

[0010] Preferably, the ferritin or the dioxotetrahydropterin synthase comprises a restriction site, and / or,

[0011] The fusion protein also includes a tag protein.

[0012] In some embodiments, the fusion protein includes, from N-terminus to C-terminus, a signal peptide, the F2 region of the F protein of HPIV3, a cleavage region, the F1 region of the F protein of HPIV3, a connecting peptide and a ferritin polypeptide or the dioxotetrahydropterin synthase polypeptide; preferably, the fusion protein also includes a restriction site and a tag protein after the C-terminus of the ferritin polypeptide or the dioxotetrahydropterin synthase polypeptide.

[0013] The tag protein refers to a polypeptide or protein that is 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 can 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 or 6×His.

[0014] In some embodiments, the fusion protein,

[0015] The amino acid sequence of the F2 region is selected from SEQ ID NO.1 or 2, or a protein having the same function obtained by replacing and / or deleting and / or adding one or less than ten amino acid residues of the amino acid sequence shown in SEQ ID NO.1 or 2;

[0016] The amino acid sequence of the F1 region is selected from SEQ ID NO. 3 or 4, or a protein having the same function obtained by replacing and / or deleting and / or adding one or ten amino acid residues of the amino acid sequence shown in SEQ ID NO. 3 or 4;

[0017] The cleavage region amino acid sequence is selected from SEQ ID NO.5 or 6;

[0018] The amino acid sequence of the ferritin polypeptide is shown in SEQ ID NO.9; and / or

[0019] The amino acid sequence of the dioxotetrahydropterin synthase polypeptide is shown in SEQ ID NO.10.

[0020] In some specific embodiments, the substitution and / or deletion and / or addition of less than ten amino acid residues is selected from the substitution and / or deletion and / or addition of less than two, three, four, five, six, seven, eight, nine, or ten amino acid residues.

[0021] In a second aspect, the present invention provides a nucleic acid molecule encoding the fusion protein as described above.

[0022] In a third aspect, the present invention provides a vector comprising the nucleic acid molecule as described above.

[0023] 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.

[0024] 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.

[0025] In a fourth aspect, the present invention provides a host cell, comprising the nucleic acid molecule or the vector as described above.

[0026] 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.

[0027] 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.

[0028] In a fifth aspect, the present invention provides a method for preparing the fusion protein as described above, the preparation method comprising introducing the nucleic acid molecule as described above or the vector as described above into a host cell, and culturing the host cell.

[0029] In a sixth aspect, the present invention provides a use of the fusion protein, the nucleic acid molecule, the vector or the host cell as described above, wherein the use comprises any of the following:

[0030] (1) Use in the preparation of products for preventing and / or treating diseases caused by HPIV infection;

[0031] (2) Use in the preparation of products for inducing immune responses to HPIV antigens;

[0032] (3) Use in the prevention and / or treatment of diseases caused by HPIV infection;

[0033] (4) Application in inducing immune response to HPIV antigens.

[0034] 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.

[0035] 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.

[0036] 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).

[0037] 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.

[0038] 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.

[0039] Preferably, the respiratory system infection may include respiratory tract infection and / or lung infection.

[0040] Preferably, the digestive system infection may include intestinal diseases, anorexia, nausea, vomiting, abdominal pain and / or diarrhea.

[0041] 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.

[0042] More preferably, the lung infection may include pneumonia and / or lung injury.

[0043] Preferably, the product for preventing and / or treating diseases caused by HPIV virus infection comprises a vaccine.

[0044] Any of the above, more preferably, the HPIV virus is HPIV3 virus.

[0045] In a seventh aspect, the present invention provides a drug, which comprises any one of the above-mentioned fusion proteins, nucleotide molecules, vectors, host cells or antibodies.

[0046] Preferably, the drug is a vaccine.

[0047] Preferably, the vaccine further comprises an adjuvant and / or a vaccine delivery system.

[0048] 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.

[0049] 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.

[0050] Preferably, the vaccine further comprises a vaccine delivery system.

[0051] 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.

[0052] Furthermore, the medicine or vaccine also includes one or more pharmaceutically acceptable carriers.

[0053] 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.

[0054] 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.

[0055] In an eighth 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.

[0056] 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.

[0057] The cellular immune response may include a B cell immune response and a T cell immune response.

[0058] The subject described herein can be a human or a non-human animal.

[0059] Furthermore, the non-human animal may be a non-human mammal.

[0060] 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.

[0061] 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).

[0062] 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.

[0063] In summary, compared with the prior art, the present invention has the following beneficial effects:

[0064] This study successfully expressed and purified nanoparticles formed by the fusion protein of parainfluenza antigen molecule preF protein and ferritin or LS polypeptide by designing and using a eukaryotic expression system. The nanoparticles have uniform particle size, stable structure, and immune protection, and can be used in vaccine research and development. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] Figure 1 : Design scheme of recombinant nanoparticle vaccine.

[0066] Figure 2 : Sequence alignment of recombinant particle vaccine design.

[0067] Figure 3 : Construction strategy of preF nanoparticles expressing eukaryotic HPIV3.

[0068] Figure 4 :Molecular screening analysis of target antigen molecules.

[0069] Figure 5 :Purification of HPIV3-F nanoparticle antigen molecules.

[0070] Figure 6 : Electron microscopy analysis of nanoparticles.

[0071] Figure 7 : Examination of the neutralization effect of nanoparticles. DETAILED DESCRIPTION

[0072] 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.

[0073] 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.

[0074] Example 1 Design of Nanoparticle Antigen Molecules for Expression of Parainfluenza F Protein in Eukaryotic System

[0075] F2 fragment 1 (SEQ ID NO. 1):

[0076]

[0077] F2 fragment 2 (SEQ ID NO. 2):

[0078]

[0079] F1 fragment 1 (SEQ ID NO. 3):

[0080]

[0081] F1 fragment 2 (SEQ ID NO. 4):

[0082]

[0083] F fragment cleavage region 1 (SEQ ID NO.5):

[0084] RTERFFGGVIGTIALGVATSAQITAAVALVEAKQAK

[0085] F fragment cleavage region 2 (SEQ ID NO.6):

[0086] RTERFFGGVIGTIALGVATSAQITAAVALVEAKQAR

[0087] Design principle of ferritin nanoparticle sequence: signal peptide (MLRGPGPGLLLAVLCLGTAVRCTEA (SEQ ID NO.7)) is tightly fused with HPIV3-F protein sequence, connected with flexible linker (GSGGGGSG (SEQ ID NO.8)) in the middle, and then tightly fused with ferritin sequence (SEQ ID NO.9)

[0088]

[0089] (See Figure 1 ).

[0090] Design principles of dioxotetrahydropterin synthase polymer nanoparticle sequence: the HPIV3-F protein sequence is tightly fused after the signal peptide (MLRGPGPGLLLAVLCLGTAVRCTEA, SEQ ID NO.7), a flexible linker (GSGGGGSG, SEQ ID NO.8) is connected in the middle, and then the Ls peptide sequence (SEQ ID NO.10) is tightly fused (QIYEGKLTAEGLRFGIVASRFNHALVDRLVEGAIDCIVRHGGSGGITLVRVPGSWEIPVAAG ELARKEDIDAVIAIGVLIRGATPHFDYIASEVSKGLADLSLELRKPITFGVITADTLEQAIERAG TKHGNKGWEAALSAIEMANLFKSLR) (see Figure 1 ).

[0091] 1. Sequence Structure

[0092] Based on the above mutation principle, fusion proteins including HPIV3 preF of various serotypes, ferritin fused nanoparticles or lumazine synthase (Ls) nanoparticle sequences were designed. For sequence alignment, see Figure 2 , see the following Seq1-Seq4 for details:

[0093] Seq1 (SEQ ID NO.11):

[0094]

[0095] Seq2 (SEQ ID NO.12):

[0096]

[0097] Seq3 (SEQ ID NO.13):

[0098]

[0099]

[0100] Seq4 (SEQ ID NO.14):

[0101]

[0102] The sequence alignment of Seq1-4 can be found in Figure 2 .

[0103] The structural differences and design principles are shown in Table 1:

[0104] Table 1: Nanoparticle design principles

[0105] Serial number design type Seq1 HPIV3 preF-ferritin Nanoparticles Seq2 HPIV3 preF-ferritin Nanoparticles Seq3 HPIV3 preF-Ls1 Nanoparticles Seq4 HPIV3 preF-Ls2 Nanoparticles

[0106] 2. Construction Method

[0107] The PKS001 vector plasmid (purchased from Zhongshan Kangtian Shenghe Biotechnology Co., Ltd.) and the plasmid containing the nucleotide molecule encoding the HPIV3 F protein (synthesized by Nanjing GenScript Biotechnology Co., Ltd.) were double-digested with HindⅢ and NotⅠ restriction endonucleases, and the vector and fragments were ligated with T4 ligase and transformed into Escherichia coli strain Top10 (see Figure 3 ), screen positive clones and extract plasmids. After sequencing, extract the correct plasmids in large quantities to obtain high-quality plasmids with nucleotide molecules encoding Seq1-4 antigens.

[0108] Example 2 Transformation, cell culture, protein purification and expression analysis of 293T cells

[0109] The high-quality plasmid Seq1-4 containing the nucleotide molecule encoding the HPIV3 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 HiPer Suspension 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. Cultured in a shaking incubator at 37°C, 5% CO2, 150 rpm. Protein purification and expression analysis were performed after 6 days.

[0110] Purification and expression analysis can be seen in the following steps:

[0111] 1. Purification of target protein in nanoparticles

[0112] Methods: After the 6-day seq1-seq4 cell culture fluid was centrifuged at 8000r / min for 20 minutes, 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. The specific steps are as follows: After connecting the purification column to AKTA, debug the purification system, use the balance solution (20mM PB, pH7.4) to balance the column, and the column filler is (Galanthus Nivalis Lectin (GNL), Agarose bound, Vector Laboratories, AL-1243-5). At this time, the flow rate is set to 3mL / min. After balancing, the flow rate is set to 2mL / min for loading. After all samples have passed 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 balance solution (20mM PB, pH7.4) to rinse the column, and the flow rate is set to 3mL / min. Then, the target protein was eluted with an eluent (1M α-methyl-D-mannoside), the flow rate was set to 1 mL / min, and 10 ml of protein solution was collected. After obtaining the primary pure protein, it was concentrated with a 30 kDa protein concentrator to obtain the primary pure solution of the target protein.

[0113] Results and conclusion: The purified nanoparticle protein was initially obtained. The concentration of the initial pure solution of different molecular proteins was different, ranging from 0.3-2 mg / ml, and the volume was about 1 ml.

[0114] 2. Molecular sieve

[0115] Method: The collected concentrated protein primary pure liquid was separated using a Superose 6Increase 10 / 300GL high-resolution gel chromatography column (Cytiva, catalog number: 10340913) molecular sieve column. The specific steps are as follows: After connecting the chromatography column to AKTA, debug the purification system, use the balance solution (1*PBS) 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.

[0116] Results Figure 4 : Taking Seq1 and Seq3 molecules as an example, after purification, the protein has a specific absorption peak at 11-13ml after passing through the molecular sieve. After collecting the sample, the protein is concentrated using a 30kDa protein concentrator.

[0117] 3. SDS-PAGE

[0118] Methods: Add 20 μL of 5× protein loading buffer to 80 μL of fusion protein solution or cell culture medium, 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 non-reduced sample is 80 μL of fusion protein solution, add 20 μl of 5× native protein loading buffer, centrifuge, and take the supernatant for detection. The protein gel concentration is 10%, and the buffer is 1×Tris-MOPS-SDS Runningbuffer (MACKLIN, NONE6600).

[0119] Results Figure 5 , where lane 1 is the protein marker, and lanes 2-4 are the purified seq1, seq2, and seq3 proteins, respectively. A high-purity protein band can be observed at 75 kDa in the SDS-PAGE gel, which is consistent with the predicted size of the antigen molecule protein, and the protein purity is estimated to be above 90%.

[0120] 4. EM Observation of Nanoparticle Protein

[0121] Method: Nanoparticles were observed using EM. The results are shown in Figure 6 For example, by using electron microscopy to photograph the seq1 protein, we can observe Figure 6In the left image, each dark particle in this field of view is the target protein. The right image is an image obtained by 2D-class clustering analysis of several nanoparticle protein images. It can be seen that the core diameter of the target nanoparticle protein is about 10-15nm, and the total diameter of the antigen and nanoparticles together is about 30nm. It is in line with expectations, proving that the purified protein structure is a nanoparticle, and the prepared fusion protein is very uniform, forming a stable nanoparticle. .

[0122] Example 3 Antigen Activity Detection

[0123] Methods: 6WRP can specifically detect HPIV3 pre-F protein. The primary antibody used was PIV3 neutralizing antibody (6WRP, see reference: 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.

[0124] 10.1080 / 19420862.2021.1912884.), the secondary antibody used was Peroxidase-Conjugated Goatanti-Human IgG (H+L), 1:5000. After color development, the plate was read and the data was stored. The purified seq1-seq4 proteins were coated with 1X PBS and placed overnight at 4°C, 200ng / well. After the coated plate was taken out from 4°C, the plate was washed 3 times, each wash volume was 300μl / well. If there was residual wash solution in the well after washing, it should be patted dry on absorbent paper. After washing the plate, 6WRP was added as the primary antibody at a ratio of 1:2500, 100μl / well, and 100μl of sample diluent was added as the blank control (Blk), duplicate wells were set, and incubated at 37°C for 60min. Add secondary antibody: discard the sample, wash the plate 3 times, each time with a volume of 300μl / well, if there is any residual solution in the well after washing, pat it dry on absorbent paper; add diluted secondary antibody, 100μl / well, cover with film, and incubate at 37℃ for 60min. Wash the 96-well plate 3 times, each time with a volume of 300μl / well, if there is any residual solution in the well after washing, pat it dry on absorbent paper, add single-component TMB colorimetric solution 1 (prepared in advance from Take out, balance to room temperature), 100 μl / well, color development at 25°C in the dark for 15 min. Put the ELISA plate into the ELISA reader, and measure the absorbance at a wavelength of 450 nm. Finally, determine the EC50 value for analysis.

[0125] The results are shown in Table 2:

[0126] Table 2: ELISA results of fusion protein and antibody

[0127] Fusion Protein ELISA Seq1 2.831 Seq2 2.525 Seq3 2.765 Seq4 1.945 Blk 0.034

[0128] This indicates that proteins purified by ferritin, Ls, and His tags all have antigen-binding activity.

[0129] Example 4 Evaluation of the effect of mouse immune antigen molecules

[0130] Method: The corresponding nanoparticle protein purified in Example 2 was used to immunize mice, and 30 C57BL / 6 female mice aged 6-8 weeks were selected and randomly divided into 5 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 3.

[0131] Table 3: Antigen molecule immunization scheme

[0132]

[0133]

[0134] The ELISA test was performed on the sera of immunized mice using the endpoint dilution method. The purified trimer protein was coated and 200 ng of protein was added to each well. The serum was diluted from 100 times with 1*PBS solution. The primary antibody used was 6WRP neutralizing antibody and the secondary antibody used was mouse secondary antibody. The ELISA titer was calculated.

[0135] Table 4 Results of serum immunity of mice after the second immunization

[0136]

[0137]

[0138] Example 5 Serum neutralization effect detection

[0139] method:

[0140] Vero-E6 cells grown in a T75 flask were washed twice with 1*PBS, 0.25% trypsin was added, and the cells were resuspended and centrifuged at 4000rpm for 5min after being placed at 37°C for 2min. The cells were resuspended with culture medium and counted, and the Vero-E6 cells were evenly spread in a 96-well cell culture plate and allowed to stand for 1 hour to adhere to the wall. They were placed in a cell culture incubator at 37°C and 5% CO2 and cultured for 24 hours. After the HPIV3 virus seed was taken out from -80 degrees, it was melted in a 37-degree water bath, diluted to 40 times, and placed on ice. The mouse serum obtained in Example 5 was diluted with 1*PBS in multiple ratios, and the virus was evenly spread on the 96-well plate with diluted mouse serum using a spray gun, and then the mixture of serum and virus was inoculated into the cells and incubated in a 37-degree incubator for 1.5 hours. After that, the mixture of serum and virus was discarded, and a covering layer (DMEM+2% serum+1% double antibody+1.5% methylcellulose) of 200μl was added, and it was placed in a 37-degree incubator and cultured for 25 hours. Invert and gently remove, shake off the covering layer, add 100μl of tissue fixative to each well and incubate at room temperature for 30 minutes. After discarding the tissue fixative, in order to improve cell permeability, add 1% TRITON X-100 dissolved in PBS, 100μl to each well, and incubate at room temperature for 30 minutes. After discarding the permeabilization solution, add 10% BSA in PBS to block and incubate for 1 hour. After discarding the blocking solution, add 100μl of primary antibody to each well, 6WRP (1:250), and incubate at 4℃ overnight. After washing the plate 3 times with 1*PBST, add 100μl of secondary antibody to each well, HRP-labeled goat anti-human (1:4000), and incubate at 37℃ for 45 minutes. After washing the plate 5 times with 1*PBST, add 50μl of TrueBlue colorimetric solution to each well and develop for 5 minutes until a good color is produced. Remove the stain and store at room temperature away from light. The blue spots represent cells that have been infected and have pathological changes, and they were photographed and analyzed using a CTL ELISA analyzer. The results are shown in Table 5.

[0141] Table 5. Antigen molecule immunospot results

[0142] Protein molecules CTL average mock 0 PBS 423 Antigen Seq1 42 AntigenSeq2 35 Antigen Seq3 78 Antigen Seq4 66

[0143] Results: The number of blue spots represents the severity of the disease. From the data, it can be observed that after the mouse immune serum without adding antigen molecules reacted with the virus neutralization, the number of spots obtained was about 423, which is the disease control value. The mice immunized with antigen molecules seq1-4 will greatly reduce the number of spots, reducing it to 10%-40%, indicating that the mouse serum produced by the ferritin nanoparticles and Ls nanoparticles of immune fusion F protein has the effect of neutralizing virus immunity. Secondly, it can be observed that the four antigen molecules Seq1, Seq2, Seq3, and Seq4 designed by the nanoparticles have similar effects. After immunizing mice with the same dose of 5μg, the antigen molecules of the nanoparticle structure have a good neutralization effect. In addition, the neutralization effects of Seq1 and Seq2 designed based on ferritin nanoparticles are slightly better than Seq3 and Seq4 designed based on Ls nanoparticles.

[0144] 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.

[0145] 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.

[0146] 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 a ferritin polypeptide or a dioxotetrahydropterin synthase polypeptide, the F protein includes an F2 region and an F1 region, and the F protein and the ferritin polypeptide or the dioxotetrahydropterin synthase polypeptide form nanoparticles; Preferably, the order of the fusion protein from N-terminus to C-terminus is: the F2 region, the F1 region and the ferritin polypeptide or the dioxotetrahydropterin synthase polypeptide, which are directly connected to each other by peptide bonds or by peptide segments.

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 ferritin polypeptide or the dioxotetrahydropterin synthase polypeptide; Preferably, the ferritin or the dioxotetrahydropterin synthase comprises 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, a signal peptide, an F2 region of the F protein of HPIV3, a cleavage region, an F1 region of the F protein of HPIV3, a connecting peptide, and a ferritin polypeptide or the dioxotetrahydropterin synthase polypeptide; Preferably, the fusion protein further comprises a restriction site and a tag protein after the C-terminus of the ferritin polypeptide or the dioxotetrahydropterin synthase polypeptide.

4. The fusion protein according to any one of claims 1 to 3, characterized in that The amino acid sequence of the F2 region is selected from SEQ ID NO.1 or 2, or a protein having the same function obtained by replacing and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID NO.1 or 2; The amino acid sequence of the F1 region is selected from SEQ ID NO. 3 or 4, or a protein having the same function obtained by replacing and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID NO. 3 or 4; The cleavage region amino acid sequence is selected from SEQ ID NO.5 or 6; The amino acid sequence of the ferritin polypeptide is shown in SEQ ID NO.9; and / or The amino acid sequence of the dioxotetrahydropterin synthase polypeptide is shown in SEQ ID NO.

10.

5. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the fusion protein according to any one of claims 1 to 4.

6. A carrier, characterized in that The vector comprises the nucleic acid molecule of claim 5.

7. A host cell, characterized in that The host cell comprises the nucleic acid molecule of claim 5 or the vector of claim 6.

8. A method for preparing the fusion protein according to any one of claims 1 to 4, characterized in that: The preparation method comprises introducing the nucleic acid molecule according to claim 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 4, the nucleic acid molecule according to claim 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 according to any one of claims 1 to 4, the nucleic acid molecule according to claim 5, the vector according to claim 6 or the host cell according to claim 7. Preferably, the drug is a vaccine, and more preferably, the vaccine further comprises an adjuvant.

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