WO2024046312A1 - 一种重组蛋白及其在制备呼吸道合胞病毒疫苗中的应用 - Google Patents

一种重组蛋白及其在制备呼吸道合胞病毒疫苗中的应用 Download PDF

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WO2024046312A1
WO2024046312A1 PCT/CN2023/115508 CN2023115508W WO2024046312A1 WO 2024046312 A1 WO2024046312 A1 WO 2024046312A1 CN 2023115508 W CN2023115508 W CN 2023115508W WO 2024046312 A1 WO2024046312 A1 WO 2024046312A1
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protein
cx3c
seq
recombinant
respiratory syncytial
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PCT/CN2023/115508
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English (en)
French (fr)
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王弋
刘昕
郭伟丽
赖强
李静怡
罗维方
郑飞
吴先戈
李志军
史战鹏
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广州源博医药科技有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/155—Paramyxoviridae, e.g. parainfluenza virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C07K14/08—RNA viruses
    • C07K14/115—Paramyxoviridae, e.g. parainfluenza virus
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C07K14/08—RNA viruses
    • C07K14/115—Paramyxoviridae, e.g. parainfluenza virus
    • C07K14/135—Respiratory syncytial virus
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention belongs to the field of biotechnology, and specifically relates to a recombinant protein and its application in preparing respiratory syncytial virus vaccine.
  • Respiratory syncytial virus belongs to the genus Pneumovirus of the family Paramyxoviridae and is one of the important pathogens causing respiratory infections in infants and young children, leading to hospitalization and even death.
  • the diameter of RSV virus is 120 to 300nm, single-stranded negative-strand RNA, and its genome size is 15222bp.
  • RSV contains 11 genes, encoding 10 proteins, including 3 envelope proteins (F, G, SH), 2 matrix proteins (M, M2), 3 nucleocapsid proteins (N, P, L) and 2 There are two non-structural proteins (NS1, NS2).
  • the virus structure is shown in Figure 1.
  • RSV respiratory syncytial virus
  • FI-RSV formalin-inactivated vaccine
  • FI-RSV makes infants and young children susceptible to wild strain RSV, possibly because FI-RSV does not induce enough RSV neutralizing antibodies, does not induce local immunity, and does not trigger cytotoxic T lymphocytes in the vaccinee ( CTL) response, once infected with wild-type RSV (wtRSV) virus, it will directly produce cytopathic effects in the lower respiratory tract.
  • the main problems existing in the current development of RSV vaccines are as follows: 1 The infant's immature immune system and high levels of maternally transmitted antibodies in the body may suppress the immune response, making it possible that the baby may not be able to produce a sufficient immune response to the vaccine; 2 The RSV vaccine There are problems with protection, making it more difficult to develop a vaccine.
  • a variety of vaccine candidates are currently being developed for different immune targets, including live attenuated vaccines, subunit vaccines, vector vaccines and nanoparticle vaccines. Among them, subunit vaccines and live attenuated vaccines are the most promising and have been used in clinical trials. . Because subunit vaccines are more suitable for older and high-risk children, there are also maternal immunizations.
  • the first object of the present invention is to provide a recombinant protein.
  • a second object of the present invention is to provide biological materials related to the recombinant protein of the first aspect.
  • a third aspect of the present invention aims to provide a method for preparing the recombinant protein of the first aspect.
  • the purpose of the fourth aspect of the present invention is to provide the use of the recombinant protein of the first aspect of the present invention and/or the biological material of the second aspect and/or the preparation method of the third aspect in preparing products.
  • the fifth aspect of the present invention aims to provide a diagnostic antigen.
  • a sixth aspect of the present invention aims to provide a vaccine.
  • the object of the seventh aspect of the present invention is to provide a complete kit.
  • An eighth aspect of the present invention aims to provide a method for preventing respiratory syncytial virus infection or respiratory syncytial virus disease.
  • a first aspect of the present invention provides a recombinant protein, comprising: respiratory syncytial virus SH protein (SH) and respiratory syncytial virus G protein (G(CX3C)) containing a CX3C motif.
  • SH respiratory syncytial virus SH protein
  • G(CX3C) respiratory syncytial virus G protein
  • the recombinant protein further includes: a carrier protein (Carrier); the carrier protein is used to present the SH protein of the respiratory syncytial virus and the G protein of the respiratory syncytial virus containing the CX3C motif.
  • Carrier carrier protein
  • the carrier protein can be any carrier known to those skilled in the art that is suitable for presenting antigens, or any other molecular construct can be used, as long as the antigen can be effectively presented to the immune system.
  • the carrier protein includes at least one of thrombospondin, cholera toxin, hemocyanin, bovine serum albumin, leukotoxin (lktA), and human serum albumin; further preferably, the carrier protein includes leukocytes. toxin.
  • the SH protein (SH) is any one of a1) to a3):
  • SH protein that has the same function as SEQ ID NO.5 by substituting and/or deleting and/or adding one or more amino acids to SEQ ID NO.5;
  • the leukotoxin (lktA) is any one of b1) to b3):
  • a leukotoxin that has the same function as SEQ ID NO.6 by substituting and/or deleting and/or adding one or more amino acids to SEQ ID NO.6;
  • a leukotoxin that has 99%, 98%, 97%, 96%, 95%, 94%, 93% or 92% homology with SEQ ID NO.6 and has the same function as SEQ ID NO.6.
  • the G protein of the respiratory syncytial virus containing the CX3C motif is any one of c1) to c3):
  • a G protein that has 99%, 98%, 97%, 96%, 95%, 94%, 93% or 92% homology with SEQ ID NO.7 and has the same function as SEQ ID NO.7.
  • the recombinant protein contains: (SH) n -Carrier-(G(CX3C)) m ; wherein, m and n are independently selected from 1, 2, 3, 4, 5, 6, 7, and 8.
  • the SHs are connected through a connecting peptide.
  • the G(CX3C) are connected through a connecting peptide.
  • the connecting peptide is a flexible connecting peptide.
  • the amino acid in the connecting peptide is selected from one or more of Gly, Ser, Pro, Ala and Glu.
  • the connecting peptide is selected from (GGGGS) k , (GGGS) k , (GGS) k , (GS) k or (G) k , where k is selected from 1, 2, 3, 4, 5 or 6.
  • the connecting peptide is GSGS (SEQ ID NO. 8).
  • the SH is connected to the carrier protein through FP.
  • the recombinant protein includes: (SH) 6 -Carrier-G(CX3C) 3 , (SH) 8 -Carrier-(G(CX3C)) 1 , (SH) 6 -Carrier-(G(CX3C)) 6. At least one of (SH) 3 -Carrier-(G(CX3C)) 6 .
  • the recombinant protein includes: (SH) 6 -Carrier-G(CX3C) 3 , (SH) 6 -Carrier-(G(CX3C)) 6 , (SH) 3 -Carrier-(G(CX3C)) At least one of 6 .
  • the recombinant protein is any one of d1) to d3):
  • a second aspect of the present invention provides biological materials related to the recombinant protein of the first aspect.
  • the biological material is any one of e1) to e12):
  • e2) an expression cassette containing the nucleic acid molecule described in e1);
  • e3 A recombinant vector containing the nucleic acid molecule described in e1);
  • e10 A transgenic cell line containing the expression cassette described in e2);
  • the transgenic animal cell line does not contain reproductive material.
  • sequence of the nucleic acid molecule encoding the recombinant protein of the first aspect is any one of f1) to f3):
  • a third aspect of the present invention provides a method for preparing the recombinant protein of the first aspect, which is expressed by culturing the recombinant microorganism and/or transgenic cell line of the second aspect of the present invention.
  • the recombinant microorganism is a protein whose expressed amino acid sequence is SEQ ID No. 3 obtained by introducing pET-28a-(RSV-SH) 6 -lktA-(G(CX3C)) 3 into E. coli BL21(DE3) Recombinant microorganism, the pET-28a-(RSV-SH) 6 -lktA-(G(CX3C)) 3 is represented by replacing the sequence between the NcoI and XhoI sites of the vector pET-28a with SEQ ID No. 4 The recombinant vector obtained from the DNA fragment.
  • the expression is induced expression.
  • the induced expression is induced by IPTG.
  • a fourth aspect of the present invention provides the use of the recombinant protein of the first aspect of the present invention and/or the biological material of the second aspect and/or the preparation method of the third aspect in preparing products;
  • the product is any one from g1) to g4):
  • the vaccine is used to prevent and/or treat respiratory syncytial virus infection and/or diseases caused by respiratory syncytial virus.
  • the diagnostic antigen is used for diagnosing respiratory syncytial virus and/or diseases caused by respiratory syncytial virus.
  • the disease caused by respiratory syncytial virus includes at least one of pneumonia, bronchiolitis and otitis media.
  • a fifth aspect of the present invention provides a diagnostic antigen, comprising: the recombinant protein of the first aspect of the present invention.
  • the diagnostic antigen is used for diagnosing respiratory syncytial virus and/or diseases caused by respiratory syncytial virus.
  • the disease caused by respiratory syncytial virus includes at least one of pneumonia, bronchiolitis and otitis media.
  • a sixth aspect of the present invention provides a vaccine, comprising: the recombinant protein of the first aspect of the present invention and/or the biological material of the second aspect of the present invention.
  • the vaccine is used to prevent and/or treat respiratory syncytial virus infection and/or diseases caused by respiratory syncytial virus.
  • the disease caused by respiratory syncytial virus includes at least one of pneumonia, bronchiolitis and otitis media.
  • the vaccine comprises the recombinant protein of the first aspect of the present invention and an adjuvant.
  • Adjuvants suitable for the vaccine of the present invention include adjuvants that can enhance the antibody response against B cell epitopes in respiratory syncytial virus, and can enhance Adjuvants for cell-mediated responses against T cell epitopes in respiratory syncytial virus, these adjuvants are well known in the art.
  • the adjuvant is an aluminum adjuvant, an oil emulsion adjuvant such as oil-in-water, water-in-oil, and two-way emulsion, or an adjuvant derived from microorganisms such as peptidoglycan (PG), gram-negative bacterial outer membrane lipopolysaccharide (Lipopolysccharide, LPS), mycobacteria and their components (such as muramyl dipeptide), GpG oligonucleotide (GpG ODN), cholera toxin (CT), particulate antigen delivery systems such as liposomes, At least one of polymeric microspheres, inert nanospheres, immunostimulating complexes (ISCOM), cytokines, polysaccharides such as inulin (MPI), and natural sources such as propolis (propolis) and saponin (Sapoin) kind.
  • PG peptidoglycan
  • LPS gram-negative bacterial outer membrane lipopolys
  • the adjuvant is at least one of aluminum hydroxide adjuvant (Alum), Freund's complete adjuvant (FCA), MF59 adjuvant, and immune stimulating complex (ISCOMS) adjuvant; further, it is an immune stimulating complex adjuvant. (ISCOMS) adjuvant.
  • Al aluminum hydroxide adjuvant
  • FCA Freund's complete adjuvant
  • ISCOMS immune stimulating complex adjuvant
  • Vaccines are typically formulated for parenteral administration. Typical immunization is vaccination by the nasal route, but the invention also contemplates oral and subcutaneous (SC), intramuscular (IM), intravenous (IV), intraperitoneal (IP) or intradermal (ID) injection. .
  • SC subcutaneous
  • IM intramuscular
  • IV intravenous
  • IP intraperitoneal
  • ID intradermal
  • the vaccine is administered in a manner compatible with the dosage formulation and in an amount such as a therapeutically effective amount and an immunogenically effective amount.
  • the amount administered depends on the subject being treated, the ability of the subject's immune system to synthesize antibodies, and the expected maintenance level of protection.
  • the exact amount of active ingredient to be administered depends on the physician's judgment and will vary from individual to individual.
  • the appropriate schedule for initial administration and booster vaccination may vary, but typically a further injection or other administration will be given at some interval (weeks or months) after the initial administration.
  • a seventh aspect of the present invention provides a complete kit, including the vaccine of the sixth aspect of the present invention and a container for vaccination.
  • the container is a medical syringe.
  • An eighth aspect of the present invention provides a method for preventing respiratory syncytial virus infection or preventing respiratory syncytial virus disease, by administering an effective amount of the recombinant protein of the first aspect of the present invention and/or the second aspect to a subject of biological materials and/or vaccines in the sixth aspect.
  • the subject is a mammal; further a primate, further a human.
  • the number of administrations is one or more times; further, it is 1 to 3 times.
  • the mode of administration is intramuscular injection.
  • an effective amount means an amount effective in achieving the intended purpose.
  • an effective amount to prevent a disease refers to an amount that can effectively prevent, prevent or delay the occurrence of a disease (eg, respiratory syncytial virus infection).
  • the invention provides a recombinant protein, which includes: respiratory syncytial virus SH protein (SH) and respiratory syncytial virus G protein (G(CX3C)) containing a CX3C motif, which improves low molecular weight antigens SH and G(CX3C).
  • SH respiratory syncytial virus SH protein
  • G(CX3C) respiratory syncytial virus G protein
  • the recombinant protein has good immunogenicity and high neutralizing antibody titer, and the recombinant protein targets two immune sites and has a good protective effect, and the effect is better than the SH protein containing respiratory syncytial virus
  • the recombinant protein of (SH) and the recombinant protein containing the G protein of respiratory syncytial virus (G(CX3C)) containing the CX3C motif can be used to prepare vaccines, respiratory syncytial virus antibodies, anti-respiratory syncytial virus serum and diagnostic antigens and other products.
  • the present invention uses polylinked SH protein to further increase the molecular weight of the recombinant protein, improve its immunogenicity, neutralizing antibody titer and protective effect.
  • adjuvants into the vaccine provided by the present invention can quickly stimulate strong humoral immunity and cellular immunity. It can also be used as a mucosal adjuvant to induce mucosal immune response, produce sIgA, and only form complexes with antigens with many hydrophobic groups. body.
  • Figure 1 is a schematic diagram of the structure of the RSV virus.
  • Figure 2 is an agarose gel electrophoresis detection picture of the (RSV-SH) 6 -lktA-(G(CX3C)) 3 gene PCR amplification product.
  • Figure 3 is a diagram showing the enzyme digestion identification results of the recombinant plasmid (RSV-SH) 6 -lktA-(G(CX3C)) 3 -pET-28a.
  • Figure 4 is a graph showing the results of SDS-PAGE identification of the effect of induction on the expression of (RSV-SH) 6 -lktA-(G(CX3C)) 3 -pET-28a.
  • Figure 5 is a diagram showing the results of Western bolt identification (RSV-SH) 6 -lktA-(G(CX3C)) 3 .
  • Figure 6 is a picture of the purification results of SDS-PAGE detection (RSV-SH) 6 -lktA-(G(CX3C)) 3 .
  • Figure 7 is a Coomassie brilliant blue standard curve graph.
  • Figure 8 is a graph showing the detection results of mouse serum SH protein-specific antibody IgG one day before the second immunization and the third immunization.
  • Figure 9 is a graph showing the detection results of mouse serum SH protein-specific antibody IgG1 one day before the second immunization and the third immunization.
  • Figure 10 is a graph showing the detection results of mouse serum SH protein-specific antibody IgG2a one day before the second immunization and the third immunization.
  • Figure 11 is a graph showing the detection results of mouse serum CX3C protein-specific antibody IgG one day before the second immunization and the third immunization.
  • Figure 12 is a graph showing the detection results of mouse serum CX3C protein-specific antibody IgG1 one day before the second immunization and the third immunization.
  • Figure 13 is a graph showing the detection results of mouse serum CX3C protein-specific antibody IgG2a one day before the second immunization and the third immunization.
  • Figure 14 is a graph showing the detection results of mouse serum SH protein-specific antibody IgG1/IgG2a one day before the second immunization and the third immunization.
  • Figure 15 is a graph showing the detection results of mouse serum CX3C protein-specific antibody IgG1/IgG2a one day before the second immunization and the third immunization.
  • Figure 16 is a graph showing the results of neutralizing antibody titer detection in mice after challenge.
  • Figure 17 is a graph showing the results of virus titer detection in mouse lungs after challenge.
  • reagents, methods and equipment used in the following examples are conventional reagents, methods and equipment in this technical field.
  • the PCR amplification product of the target gene (RSV-SH) 6 -lktA-(G(CX3C)) 3 was detected by agarose gel electrophoresis. The results are shown in Figure 2. A strip containing approximately 2450 bp of both sides of the sequence was amplified. The band is consistent with the size (2178bp) of the target gene (RSV-SH) 6 -lktA-(G(CX3C)) 3 , indicating that the target gene was amplified successfully.
  • Ligation system 6 ⁇ L of target gene fragment; 2 ⁇ L of pET-28a; 1 ⁇ L of T4 ligase; 2 ⁇ L of 10 ⁇ T4 DNA Ligase Buffer; 9 ⁇ L of ddH 2 O; total volume 20 ⁇ L.
  • Connection conditions Connect on a PCR machine at 16°C for 3 hours.
  • Enzyme digestion system 2 ⁇ L of recombinant plasmid (200ng); 0.5 ⁇ L of NcoI; 0.5 ⁇ L of XhoI; 1 ⁇ L of 10 ⁇ Cutsmart buffer; 6 ⁇ L of ddH 2 O; total volume 10 ⁇ L.
  • Enzyme digestion conditions 37°C constant temperature water bath for 2 hours.
  • the recombinant plasmid (RSV-SH) 6 -lktA-(G(CX3C)) 3 -pET-28a obtained above was subjected to restriction enzyme digestion and identification.
  • the restriction sites used were NcoI and Xhol.
  • the restriction product was about 2200 bp, as shown in Figure 3 (M is DNA marker (5K), 1 is pET-28a-(RSV-SH) 6 -lktA-(G(CX3C)) 3 recombinant plasmid, 2 is pET-28a empty, 3 is (RSV-SH) 6 -lktA-(G(CX3C)) 3 /pET-28a(NcoI+XhoI)), it can be seen that the sizes are consistent with the expected size, verifying the recombinant plasmid (RSV-SH) 6 -lktA-(G(CX3C)) 3 -pET -28a is correct.
  • M is DNA marker (5K)
  • 1 is pET-28a-(RSV-SH) 6 -lktA-(G(CX3C)) 3 recombinant plasmid
  • 2 is pET-28a empty
  • 3 is (RSV-
  • OD 600 Measure the OD 600 after culturing in a shaker at 37°C for about 3 hours.
  • the OD 600 is between 0.6 and 0.8, which is the appropriate density of induced cells;
  • Electrophoresis program 2 110V, 90mA, until the indicator bromophenol blue reaches the lower edge of the gel, end the electrophoresis, and peel off the gel;
  • the target protein was detected by Western Blotting method.
  • the results are shown in Figure 5 (M: protein marker (100KD), 1: pET-28a-(RSV-SH) 6 -lktA-(G(CX3C)) 3 before induction, 2: pET -28a-(RSV-SH) 6 -lktA-(G(CX3C)) 3 after induction), using mouse anti-His-IgG as the primary antibody and goat anti-mouse IgG-HRP as the secondary antibody, the results after induction
  • the protein expressed in the liquid can be combined with the mouse anti-His-IgG primary antibody.
  • Centrifuge the induced bacteria resuspend the bacterial pellet at a ratio of 60mL bacterial solution/L lysate (containing 50mM glycine, 0.1M NaCl, pH 8.0), and use 25% power to ultrasonically crush the bacteria.
  • a ratio of 60mL bacterial solution/L lysate containing 50mM glycine, 0.1M NaCl, pH 8.0
  • use 25% power to ultrasonically crush the bacteria.
  • Place the bottle containing the bacterial suspension in an ice-water bath take a 30 ⁇ L sample of the broken liquid after sonication, which is the complete lysis liquid. Then take 30 ⁇ L of the broken fluid, centrifuge at 12,000 rpm for 10 min, and take the supernatant into another tube. This sample is the lysis supernatant.
  • the centrifuged pellet is resuspended in 30 ⁇ L of water. This sample is the lysis pellet.
  • the remaining broken liquid was centrifuged at 12,000
  • His-Ni affinity chromatography column was first manually washed with 10 volumes of filtered ddH 2 O, and then equilibrated with 5 volumes of pH9.08M urea;
  • SDS-PAGE was used to detect (RSV-SH) 6 -lktA-(G(CX3C)) 3 protein purification results, as shown in Figure 6 (M: Protein Marker (100kd), 1: Flow-through fluid Ft (inclusion body lysate), 2-5: 30mM imidazole eluent, 6-9: 100mM imidazole eluent), peaks appear at 30mM and 100mM imidazole eluent respectively. Samples were taken for SDS-PAGE detection. It can be seen that there are differences between Marker70 and 100KD. Obvious target band.
  • SEQ ID NO.3 The single underline is the amino acid sequence of the leukotoxin, SEQ ID NO.6; the double underline is the amino acid sequence of the G protein of respiratory syncytial virus containing the CX3C motif, SEQ ID NO.7; the wave
  • the line is the amino acid sequence of SH protein, SEQ ID NO.5); its corresponding nucleotide sequence is:
  • bovine serum albumin (BSA) as a reference, measure the UV absorbance value of Coomassie Brilliant Blue G-250 at 595nm, and draw a standard curve (as shown in Figure 7) for protein quantification of each target protein and the control group.
  • BSA bovine serum albumin
  • FCA Freund's adjuvant
  • ISOCOMs ISOCOMs
  • MF59 MF59
  • aluminum adjuvant to prepare a vaccine of 50ug/mL
  • FCA Freund's adjuvant
  • FI-RSV inactivated vaccine
  • the mice were divided into groups (aluminum adjuvant group, FCA group, MF59 group, ISCOMs group, FI-RSV group and PBS group).
  • the mice were Balb/c mice, 6 to 8 weeks old, 5 in each group. Three immunizations were carried out on days 0, 14, and 28, with 0.1 mL of each dose injected intramuscularly.
  • FI-RSV and PBS were injected in the same way.
  • Mouse serum collection Collect 50 to 100 ⁇ L of blood through the tail vein one day before the second and third immunizations, place it in a 1.5 mL sterilized EP tube, place it at 37°C for 2 hours, transfer to 4°C and let it stand overnight. , serum can be separated the next day, 10000rpm, 10min, collect the supernatant and store at -20°C.
  • mice IgG, IgG1, and IgG2a secondary antibodies (Goat Anti-Mouse IgG (H+L) HRP, supplier Affinity Biosciences, Cat. No. S0002) diluted with the antibody diluent required for various experiments and incubate for 2 hours, discard and use Wash with TBST three times;
  • the IgG1/IgG2a ratio in the FI-RSV group was between 10 and 300 times, indicating that the immune response induced by it tends to the TH2 pathway.
  • the above results show that the fusion protein can induce specific antibodies against SH and G protein CX3C, and will not cause the problem of uneven immune response.
  • mice cover the mice with a glass cup, put ether inside, fascinate the mouse until it becomes unsteady and slide it under the cup, then take it out, pinch the back of the mouse and use a pipette to apply it to the nostril of the mouse.
  • the results are shown in Figure 17.
  • the ISCOMs group had the best protective effect on the mice, with a significant difference from the PBS group (P ⁇ 0.001), followed by In the FCA group and the MF59 group (P ⁇ 0.01), the aluminum adjuvant group had a relatively worse protective effect than the first three groups, but it was also different from the PBS group (P ⁇ 0.05).
  • mice Take 5 ⁇ g of (RSV-SH) 6 -lktA-(G(CX3C)) 3 , SH-lktA (amino acid sequence is 1 to 639 of SEQ ID NO. 3), lktA-G(cx3c)( in step 3 respectively.
  • the amino acid sequence is 162 to 725 of SEQ ID NO. 3) was mixed with ISOCOM adjuvant (the protein concentration was 50ug/mL) and the mice were immunized.
  • the mice were grouped according to Table 1.
  • the mice were Balb/c mice, 6 to 8 weeks old, 5 in each group. Immunize by intramuscular injection three times on days 1, 14, and 28.
  • step 3 blood is collected for IgG and antibody neutralization titer detection (the method is the same as step 3); RSV challenge is performed two weeks after three immunizations. Four days after intoxication, take lung tissue and perform virus titration to determine its virus protection ability (the method is the same as step 4).
  • the results show that SH-lktA fusion protein alone does not have neutralizing activity after immunization, while (RSV-SH) 6 -lktA-(G(CX3C)) 3 and lktA-G(cx3c) both have certain neutralizing activity.
  • the amino acid sequence of RSV-SH is SEQ ID NO.5
  • the amino acid sequence of lktA is SEQ ID NO.6
  • the amino acid sequence of G(CX3C) is SEQ ID NO. 7.
  • RSV-SH is connected through GSGS, G(CX3C is connected through GSGS, and RSV-SH and lktA are connected through FP; after mixing the above recombinant protein with ISOCOM adjuvant (the concentration of the protein is 50ug/mL ) to immunize mice.
  • the mice are Balb/c mice, aged 6 to 8 weeks, with 5 mice in each group. Immunize by intramuscular injection three times on 1, 14, and 28 days. , after the third immunization, blood was collected for IgG and antibody neutralization titer detection (the method is the same as step 3).

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Abstract

本发明属于生物技术领域，具体涉及一种重组蛋白及其应用。该重组蛋白，包含：呼吸道合胞病毒的SH蛋白(SH)和包含CX3C模序的呼吸道合胞病毒的G蛋白(G(CX3C))，提高了低分子量抗原SH和G(CX3C)蛋白免疫原性，使重组蛋白免疫原性好、中和抗体滴度高，并且该重组蛋白针对两个免疫位点，具有较好的保护作用，可用于制备疫苗、呼吸道合胞病毒抗体、抗呼吸道合胞病毒血清和诊断抗原等产品。

Description

一种重组蛋白及其在制备呼吸道合胞病毒疫苗中的应用 技术领域
本发明属于生物技术领域，具体涉及一种重组蛋白及其在制备呼吸道合胞病毒疫苗中的应用。
背景技术
呼吸道合胞病毒(respiratory syncytial virus，RSV)属于副黏病毒科肺病毒属，是引起婴幼儿呼吸道感染的重要病原体之一，导致患者住院治疗甚至死亡。RSV病毒的直径为120至300nm，单股负链RNA，其基因组大小为15222bp。RSV含有11个基因，编码10个蛋白分别为3种囊膜蛋白(F、G、SH)，2种基质蛋白(M、M2)，3种核衣壳蛋白(N、P、L)和2种非结构蛋白(NS1、NS2)，病毒结构示意如图1所示。
目前，RSV(respiratory syncytial virus)是婴幼儿病毒性肺炎和毛细支气管炎的主要病原体之一，对老人和免疫缺陷成人也有危害，且与支气管哮喘的发生发展密切相关。RSV感染临床表现以低热、下呼吸道感染病变多见，婴幼儿多呈阵咳、憋喘症状，甚至死亡。只有采用特异性抗体进行被动免疫治疗，重症者治疗困难，所以RSV疫苗的研制非常迫切。在过去的40多年里RSV的研究有一定的发展，虽然有疫苗进行了临床实验，但仍然没有疫苗上市，RSV疫苗已被WHO列为全球优先发展疫苗之一。
20世纪60年代研制的福尔马林灭活疫苗(FI-RSV)给婴幼儿肌肉注射免疫后，不仅不能预防RSV感染反而在感染后使病情加重，甚至有的引起死亡，死者的肺组织内有嗜酸性粒细胞浸润，主要与Th1/Th2平衡紊乱有关。用FI-RSV免疫会触发Th2型应答，局部产生大量趋化因子和细胞因子，引起淋巴细胞和嗜酸粒细胞浸润，导致炎症和支气管狭窄，而免疫复合物的沉积会进一步加重患儿的病情。另外，研究表明，FI-RSV使婴幼儿对野生株RSV易感，可能是因为FI-RSV没有诱生足够的RSV中和抗体，未诱导局部免疫，没有触发接种者的细胞毒性T淋巴细胞(CTL)应答，一旦感染野生型RSV(wtRSV)病毒就会直接在下呼吸道产生细胞病变。
目前研制RSV疫苗中存在的问题主要有以下几点：①婴儿的免疫系统不成熟和体内的高水平母传抗体可能会抑制免疫应答，使之可能无法对疫苗产生足够的免疫答；②RSV疫苗的保护性存在问题，增加了研制疫苗的难度。目前正在研制多种针对不同免疫对象的候选疫苗，包括减毒活疫苗、亚单位疫苗、载体疫苗和纳米颗粒疫苗等，其中亚单位疫苗和减毒活疫苗最有前途，已被用于临床试验。由于亚单位疫苗相对于大龄和高危的儿童，还有母体免疫更适合。
发明内容
为了克服现有技术所存在的不足，本发明的第一方面的目的，在于提供一种重组蛋白。
本发明的第二方面的目的，在于提供与第一方面的重组蛋白相关的生物材料。
本发明的第三方面的目的，在于提供第一方面的重组蛋白的制备方法。
本发明的第四方面的目的，在于提供本发明的第一方面的重组蛋白和/或第二方面的生物材料和/或第三方面的制备方法在制备产品中的应用。
本发明的第五方面的目的，在于提供一种诊断抗原。
本发明的第六方面的目的，在于提供一种疫苗。
本发明的第七方面的目的，在于提供成套试剂盒。
本发明的第八方面的目的，在于提供一种预防呼吸道合胞病毒感染或预防呼吸道合胞病毒的疾病的方法。
为了实现上述目的，本发明所采取的技术方案是：
本发明的第一个方面，提供一种重组蛋白，包含：呼吸道合胞病毒的SH蛋白(SH)和包含CX3C模序的呼吸道合胞病毒的G蛋白(G(CX3C))。
优选地，所述重组蛋白还包含：载体蛋白(Carrier)；所述载体蛋白用于呈递所述呼吸道合胞病毒的SH蛋白和所述包含CX3C模序的呼吸道合胞病毒的G蛋白。
优选地，所述载体蛋白可以是本领域技术人员公知的适用于呈递抗原的任意载体，也可以使用任意其他分子构建体，只要能有效地提呈抗原到免疫系统。
优选地，所述载体蛋白包含血小板反应蛋白、霍乱毒素、血蓝蛋白、牛血清白蛋白、白细胞毒素(lktA)、人血清白蛋白中的至少一种；进一步优选地，所述载体蛋白包含白细胞毒素。
优选地，所述SH蛋白(SH)为a1)至a3)中任一种：
a1)氨基酸序列如SEQ ID NO.5所示的SH蛋白；
a2)将SEQ ID NO.5经过一个或几个氨基酸的取代和/或缺失和/或添加且与SEQ ID NO.5具有相同功能的SH蛋白；
a3)与SEQ ID NO.5具有99％、98％、97％、96％、95％、94％、93％或92％的同源性且与SEQ ID NO.5具有相同功能的SH蛋白。
优选地，所述白细胞毒素(lktA)为b1)至b3)中任一种：
b1)氨基酸序列如SEQ ID NO.6所示的白细胞毒素；
b2)将SEQ ID NO.6经过一个或几个氨基酸的取代和/或缺失和/或添加且与SEQ ID NO.6具有相同功能的白细胞毒素；
b3)与SEQ ID NO.6具有99％、98％、97％、96％、95％、94％、93％或92％的同源性且与SEQ ID NO.6具有相同功能的白细胞毒素。
优选地，所述包含CX3C模序的呼吸道合胞病毒的G蛋白为c1)至c3)中任一种：
c1)氨基酸序列如SEQ ID NO.7所示的G蛋白；
c2)将SEQ ID NO.7经过一个或几个氨基酸的取代和/或缺失和/或添加且与SEQ ID NO.7具有相同功能的G蛋白；
c3)与SEQ ID NO.7具有99％、98％、97％、96％、95％、94％、93％或92％的同源性且与SEQ ID NO.7具有相同功能的G蛋白。
优选地，所述重组蛋白包含：(SH)n-Carrier-(G(CX3C))m；其中，m、n独立选自于1、2、3、4、5、6、7、8。
优选地，所述n大于等于2时，所述SH之间通过连接肽连接。
优选地，所述m大于等于2时，所述G(CX3C)之间通过连接肽连接。
优选地，所述连接肽为柔性连接肽。
优选地，所述连接肽中的氨基酸选自Gly、Ser、Pro、Ala以及Glu中的一种或多种。
优选地，所述连接肽选自(GGGGS)k、(GGGS)k、(GGS)k、(GS)k或(G)k，其中k选自1，2，3，4，5或6。
优选地，所述连接肽为GSGS(SEQ ID NO.8)。
优选地，所述SH与载体蛋白通过FP连接。
优选地，所述重组蛋白包含：(SH)6-Carrier-G(CX3C)3、(SH)8-Carrier-(G(CX3C))1、(SH)6-Carrier-(G(CX3C))6、(SH)3-Carrier-(G(CX3C))6中的至少一种。
优选地，所述重组蛋白包含：(SH)6-Carrier-G(CX3C)3、(SH)6-Carrier-(G(CX3C))6、(SH)3-Carrier-(G(CX3C))6中的至少一种。
优选地，所述重组蛋白为d1)至d3)中任一种：
d1)氨基酸序列如SEQ ID NO.3所示的蛋白；
d2)将SEQ ID NO.3经过一个或几个氨基酸的取代和/或缺失和/或添加且与SEQ ID NO.3具有相同功能的蛋白；
d3)与SEQ ID NO.3具有99％、98％、97％、96％、95％、94％、93％或92％的同源性且与SEQ ID NO.3具有相同功能的蛋白。
本发明的第二个方面，提供与第一个方面的重组蛋白相关的生物材料。
优选地，所述生物材料为e1)至e12)中的任一种：
e1)编码第一方面的重组蛋白的核酸分子；
e2)含有e1)所述核酸分子的表达盒；
e3)含有e1)所述核酸分子的重组载体；
e4)含有e2)所述表达盒的重组载体；
e5)含有e1)所述核酸分子的重组微生物；
e6)含有e2)所述表达盒的重组微生物；
e7)含有e3)所述重组载体的重组微生物；
e8)含有e4)所述重组载体的重组微生物；
e9)含有e1)所述核酸分子的转基因细胞系；
e10)含有e2)所述表达盒的转基因细胞系；
e11)含有e3)所述重组载体的转基因细胞系；
e12)含有e4)所述重组载体的转基因细胞系。
优选地，所述转基因动物细胞系不包含繁殖材料。
优选地，所述编码第一方面的重组蛋白的核酸分子的序列为f1)至f3)中任一种：
f1)如SEQ ID NO.4所示；
f2)将SEQ ID NO.4经过一个或几个核苷酸的取代和/或缺失和/或添加且与SEQ ID NO.4具有相同功能的核甘酸序列；
f3)与SEQ ID NO.4具有99％、98％、97％、96％、95％、94％、93％或92％的同源性且与SEQ ID NO.4具有相同功能的核苷酸序列。
本发明的第三个方面，提供第一方面的重组蛋白的制备方法，通过培养本发明第二个方面的重组微生物和/或转基因细胞系表达所述重组蛋白。
优选地，所述重组微生物为将pET-28a-(RSV-SH)6-lktA-(G(CX3C))3导入大肠杆菌BL21(DE3)得到的表达氨基酸序列是SEQ ID No.3的蛋白的重组微生物，所述pET-28a-(RSV-SH)6-lktA-(G(CX3C))3为将载体pET-28a的NcoI和XhoI位点之间的序列替换为SEQ ID No.4所示的DNA片段得到的重组载体。
优选地，所述表达为诱导表达。
优选地，所述诱导表达通过IPTG诱导。
本发明的第四个方面，提供本发明第一个方面的重组蛋白和/或第二个方面的生物材料和/或第三个方面的制备方法在制备产品中的应用；
所述产品为g1)至g4)中任一种：
g1)疫苗；
g2)呼吸道合胞病毒抗体；
g3)抗呼吸道合胞病毒血清；
g4)诊断抗原。
优选地，所述疫苗用于预防和/或治疗呼吸道合胞病毒感染和/或呼吸道合胞病毒引起的疾病。
优选地，所述诊断抗原用于诊断呼吸道合胞病毒和/或呼吸道合胞病毒引起的疾病。
优选地，所述呼吸道合胞病毒引起的疾病包含肺炎、毛细支气管炎和中耳炎中的至少一种。
本发明的第五个方面，提供一种诊断抗原，包含：本发明第一个方面的重组蛋白。
优选地，所述诊断抗原用于诊断呼吸道合胞病毒和/或呼吸道合胞病毒引起的疾病。
优选地，所述呼吸道合胞病毒引起的疾病包含肺炎、毛细支气管炎和中耳炎中的至少一种。
本发明的第六方面，提供一种疫苗，包含：本发明第一个方面的重组蛋白和/或本发明第二个方面的生物材料。
优选地，所述疫苗用于预防和/或治疗呼吸道合胞病毒感染和/或呼吸道合胞病毒引起的疾病。
优选地，所述呼吸道合胞病毒引起的疾病包含肺炎、毛细支气管炎和中耳炎中的至少一种。
优选地，所述疫苗包含本发明第一个方面的重组蛋白和佐剂，适用于本发明疫苗的佐剂包括可增强针对呼吸道合胞病毒中B细胞表位的抗体反应的佐剂，以及可增强细胞介导的针对呼吸道合胞病毒中T细胞表位的反应的佐剂，这些佐剂是本领域所熟知的。
优选地，所述佐剂为铝佐剂，油乳佐剂如水包油、油包水、双向型乳液，微生物来源类佐剂如肽聚糖(PG)、革兰氏阴性菌外膜脂多糖(Lipopolysccharide,LPS)、分枝杆菌及其组分(如胞壁酰二肽)、GpG寡核苷酸(GpG ODN)、霍乱毒素(Cholera toxin,CT)，微粒抗原递送体系如脂质体、聚合微球体、惰性纳米微球、免疫刺激复合物(Immunostimulating comples,ISCOM)、细胞因子，多糖类如菊粉(MPI)，天然来源类如蜂胶(propolis)、皂苷(Sapoin)中的至少一种。
优选地，所述佐剂为氢氧化铝佐剂(Alum)、弗氏完全佐剂(FCA)、MF59佐剂、免疫刺激复合物(ISCOMS)佐剂中的至少一种；进一步为免疫刺激复合物(ISCOMS)佐剂。
疫苗典型地被配制用于肠胃外施用。典型的免疫接种是通过鼻腔途径的疫苗接种，但本发明还考虑了口腔和皮下(SC)、肌内(IM)、静脉内(IV)、腹膜内(IP)或真皮内(ID)注射实现。
上述疫苗是以与剂量配方相容的方式，以及诸如治疗有效量和免疫原性有效量的用量被施用的。施用量取决于接受治疗的对象、该对象的免疫系统合成抗体的能力，以及预期的保 护程度。需施用的活性成分的准确数量取决于医师的判断，个体不同，用量也不同。最初施用和加强接种的合适方案也可变化，但典型地在首次施用后的一定间隔时间(数周或数月)后再进行1次注射或以其它方式施用。
本发明的第七个方面，提供成套试剂盒，包含本发明第六个方面的疫苗及用于所述疫苗接种的容器。
优选地，所述容器为医用注射器。
本发明的第八个方面，提供预防呼吸道合胞病毒感染或预防呼吸道合胞病毒的疾病的方法，向受试者施用有效量的本发明第一个方面的重组蛋白和/或第二个方面的生物材料和/或第六个方面的疫苗。
优选地，所述受试者为哺乳动物；进一步为灵长类动物，更进一步为人。
优选地，所述施用的次数为一次或多次；进一步为1至3次。
优选地，所述施用的方式为肌内注射。
“有效量”是指能够有效实现预期目的的量。例如，预防疾病(例如呼吸道合胞病毒感染)有效量是指，能够有效预防、阻止或延迟疾病(例如呼吸道合胞病毒感染)的发生的量。
本发明的有益效果是：
本发明提供一种重组蛋白，包含：呼吸道合胞病毒的SH蛋白(SH)和包含CX3C模序的呼吸道合胞病毒的G蛋白(G(CX3C))，提高了低分子量抗原SH和G(CX3C)蛋白免疫原性，使重组蛋白免疫原性好、中和抗体滴度高，并且该重组蛋白针对两个免疫位点，具有较好的保护作用，效果优于包含呼吸道合胞病毒的SH蛋白(SH)的重组蛋白以及含包含CX3C模序的呼吸道合胞病毒的G蛋白(G(CX3C))的重组蛋白，可用于制备疫苗、呼吸道合胞病毒抗体、抗呼吸道合胞病毒血清和诊断抗原等产品。
进一步地，本发明采用多联SH蛋白进一步提高了重组蛋白的分子量，提高其免疫原性，中和抗体滴度及保护作用。
进一步地，本发明提供地疫苗中引入佐剂，可以快速激发强烈的体液免疫和细胞免疫，也可作为粘膜佐剂，诱导黏膜免疫应答，产生sIgA，且只与疏水基团多的抗原形成复合体。
附图说明
图1是RSV病毒结构示意图。
图2是(RSV-SH)6-lktA-(G(CX3C))3基因PCR扩增产物的琼脂糖凝胶电泳检测图。
图3是重组质粒(RSV-SH)6-lktA-(G(CX3C))3-pET-28a的酶切鉴定结果图。
图4是SDS-PAGE鉴定诱导对(RSV-SH)6-lktA-(G(CX3C))3-pET-28a表达影响的结果图。
图5是Western bolt鉴定(RSV-SH)6-lktA-(G(CX3C))3的结果图。
图6是SDS-PAGE检测(RSV-SH)6-lktA-(G(CX3C))3纯化结果图。
图7是考马斯亮蓝标准曲线图。
图8是第二次免疫、第三次免疫前一天小鼠血清SH蛋白特异性抗体IgG的检测结果图。
图9是第二次免疫、第三次免疫前一天小鼠血清SH蛋白特异性抗体IgG1的检测结果图。
图10是第二次免疫、第三次免疫前一天小鼠血清SH蛋白特异性抗体IgG2a的检测结果图。
图11是第二次免疫、第三次免疫前一天小鼠血清CX3C蛋白特异性抗体IgG的检测结果图。
图12是第二次免疫、第三次免疫前一天小鼠血清CX3C蛋白特异性抗体IgG1的检测结果图。
图13是第二次免疫、第三次免疫前一天小鼠血清CX3C蛋白特异性抗体IgG2a的检测结果图。
图14是第二次免疫、第三次免疫前一天小鼠血清SH蛋白特异性抗体IgG1/IgG2a的检测结果图。
图15是第二次免疫、第三次免疫前一天小鼠血清CX3C蛋白特异性抗体IgG1/IgG2a的检测结果图。
图16是攻毒后小鼠中和抗体滴度检测结果图。
图17是攻毒以后小鼠肺部病毒滴度检测结果图。
具体实施方式
以下结合具体的实施例及附图对本发明的内容作进一步详细的说明。
应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。
除非特别说明，下列实施例中采用的试剂、方法和设备为本技术领域常规试剂、方法和设备。
除非特别说明，以下实施例所用试剂和材料均为市购。
除非特别说明，以下实施例采用的免疫学、生物化学、化学、分子生物学、微生物学、细胞生物学、基因组学和重组DNA等是本领域的常规技能。参见萨姆布鲁克(Sambrook)、弗里奇(Fritsch)和马尼亚蒂斯(Maniatis)，《分子克隆：实验室手册》(MOLECULAR CLONING:A LABORATORY MANUAL)，第2次编辑(1989)；《当代分子生物学实验手册》(CURRENT PROTOCOLS IN MOLECULAR BIOLOGY)(F.M.奥苏贝尔(F.M.Ausubel)等人编辑，(1987))；《酶学方法》(METHODS IN ENZYMOLOGY)系列(学术出版公司)：《PCR2:实用方法》(PCR2:A PRACTICAL APPROACH)(M.J.麦克弗森(M.J.MacPherson)、B.D.黑姆斯(B.D.Hames)和G.R.泰勒(G.R.Taylor)编辑(1995))、哈洛(Harlow)和拉内(Lane)编辑(1988)《抗体：实验室手册》(ANTIBODIES,A LABORATORY MANUAL)，以及《动物细胞培养》(ANIMAL CELL CULTURE)(R.I.弗雷谢尼(R.I.Freshney)编辑(1987))。
实施例1
1.表达载体(RSV-SH)6-lktA-(G(CX3C))3pET-28a的构建
A.抗原蛋白SH的基因引物
正向引物SH-NcoI-F(SEQ ID NO.1)：ATACCATGGTGAACAAATTGTGCGAGTATAACGTG；
反向引物G(CX3C)-His-XhoI-R(SEQ ID NO.2)TCAGTGGTGGTGGTGGTGGTGCTCGAGGGTATTGACGCGAGCGC。
B.PCR扩增目的基因(RSV-SH)6-lktA-(G(CX3C))3体系
模板(RSV-SH)6-lktA-(G(CX3C))3-PMD18-T质粒(将SEQ ID NO.4所示的(RSV-SH)6-lktA-(G(CX3C))3插入PMD18-T的NcoI、XhoI之间)：1μL(10ng)；引物SH-NcoI-F：1μL(10μM)；引物G(CX3C)-His-XhoI-R：1μL(10μM)；2×PrimeSTAR：25μL；ddH2O：22μL；总 量50μL。
C.PCR扩增目的基因(RSV-SH)6-lktA-(G(CX3C))3程序
98℃10min；98℃10s，55℃15s，72℃30s，30个循环；72℃4min；4℃10min。
将目的基因(RSV-SH)6-lktA-(G(CX3C))3的PCR扩增产物进行琼脂糖胶凝电泳检测，结果如图2所示，扩增出包含两侧序列约2450bp的条带，与目的基因(RSV-SH)6-lktA-(G(CX3C))3大小(2178bp)相符，说明目的基因扩增成功。
D.PCR基因片段胶回收
a)制备1％的琼脂糖凝胶，将PCR产物与Loading Buffer在1％的琼脂糖凝胶点样孔加样，并用100V电压电泳，根据Loading Buffer的位置决定电泳时间；
b)在紫外灯下将凝胶电泳所得的目的条带切下，转移到1.5mL的EP管中；
c)加入2倍体积的溶胶液，在65℃放置20min直到胶块完全溶解；
d)将溶胶混合液加入胶回收的柱子中后12000rpm离心1min，将离心的液体重新倒回柱子中离心过柱，小心弃去离心液；
e)加入700μL漂洗液12000rpm离心1min，弃去离心液，重新加入700μL漂洗液12000rpm离心1min，弃去离心液；
f)将柱子12000rpm离心2min，然后将离心甩出来的溶液弃去，将柱子放到新的EP管中37℃放置5min；
g)在柱子中间加入30μL ddH2O，放置2min，12000rpm离心1min，将离心下来的液体重新加到柱子中间放置2min，12000rpm离心1min；
h)将提取出来的目的基因片段取3μL进行凝胶电泳定量，剩下的胶回收片段存储在-20℃冰箱以备后面实验使用。
E.目的基因(RSV-SH)6-lktA-(G(CX3C))3和pET-28a双酶切(NcoI和XhoI)
a)目的基因酶切体系
目的基因胶回收片段10μL；NcoI 0.5μL；XhoI 0.5μL；10×Cutsmart buffer 2μL；ddH2O 7μL；总量20μL。
b)pET-28a酶切体系
pET-28a 2μL；NcoI 0.5μL；XhoI 0.5μL；10×Cutsmart buffer 2μL；ddH2O 5μL；总量10μL。
c)连接
把双酶切和凝胶电泳纯化后的目的基因片段和pET-28a按3：1的质量比例连接。
连接体系：目的基因片段6μL；pET-28a 2μL；T4 ligase 1μL；10×T4 DNA Ligase Buffer 2μL；ddH2O 9μL；总量20μL。
连接条件：在PCR仪上16℃连接3小时。
F.转化
a)将提前制备好的感受态细胞DH5α放在冰水浴中预冷，将连接产物在超净台中加入到预冷的感受态细胞中，冰水浴30min；
b)在42℃金属浴中热激2min，再放入冰水浴中冰浴2min；
c)将冰浴过的感受态混合物加入l mL LB培养基，在37℃震摇30min；
d)12000rpm离心30s，在超净台去除850μL上清，将沉淀与无抗LB培养基混匀，加在Kan+固体LB培养基平板上进行涂板；
e)37℃放置18h使单菌落长到足够大。
G.酶切鉴定
将转化长出的单菌落挑起，接种到LB培养基中并加入0.1％的卡那霉素，37℃摇菌12h后提质粒，进行酶切鉴定。
酶切体系：重组质粒2μL(200ng)；NcoI 0.5μL；XhoI 0.5μL；10×Cutsmart buffer 1μL；ddH2O 6μL；总量10μL。
酶切条件：37℃恒温水浴2小时。
将上述得到的重组质粒(RSV-SH)6-lktA-(G(CX3C))3-pET-28a进行酶切鉴定，所用酶切位点为NcoI和Xhol，酶切产物约2200bp，由图3(M为DNA marker(5K)，1为pET-28a-(RSV-SH)6-lktA-(G(CX3C))3重组质粒，2为pET-28a空载，3为(RSV-SH)6-lktA-(G(CX3C))3/pET-28a(NcoI+XhoI))可知，均与预期大小一致，验证了重组质粒(RSV-SH)6-lktA-(G(CX3C))3-pET-28a是正确的。
2.目的蛋白(RSV-SH)6-lktA-(G(CX3C))3的表达与纯化
A.制备表达目的蛋白的菌并诱导表达
a)将步骤1制备的(RSV-SH)6-lktA-(G(CX3C))3-pET-28a(DH5α)菌种接种到LB培养基中，37℃摇菌12h后进行转接，37℃摇菌12h后提质粒；
b)将质粒转化BL21(DE3)感受态，涂板后37℃放置18h；
c)挑单菌落接种到LB培养基(加入0.1％Kan+)中，37℃摇菌12h，然后按1：100转接到新鲜LB培养基中；
d)37℃摇床中培养约3小时后测定OD600，OD600为0.6至0.8之间，为合适的诱导菌体密度；
e)向d)中达到合适的诱导菌体密度的菌液中添加终浓度为1mM的IPTG，于37℃，220rpm振荡培养4小时，诱导目的蛋白表达；
f)取上述经过诱导表达的菌液40μL，加入10μL 5×loading buffer，沸水中煮10min；取未经IPTG诱导的菌液同样处理(即诱导前的菌液)，作为阴性对照样品。
B.SDS-PAGE
用浓缩胶浓度为5％，分离胶浓度为10％的SDS-PAGE胶检测目的蛋白的表达，根据诱导前和诱导后全菌蛋白条带的差异判断是否诱导出目的蛋白；
a)在SDS-PAGE胶上加样10μL，电泳程序1：85V，60mA，30min；
b)电泳程序2：110V，90mA，至指示剂溴酚蓝跑电泳到凝胶的下边缘，结束电泳，剥出凝胶；
c)用100mL考马斯亮蓝染色液进行1.5h染色；
b)脱色，将染色液倒出来并用清水漂洗几遍，加入适量脱色液脱色，脱到染色液染成蓝色以后要换脱色液。
SDS-PAGE结果如图4(M：蛋白marker(100KD)，1：pET-28a空载，2：pET-28a-(RSV-SH)6-lktA-(G(CX3C))3诱导前，3：pET-28a-(RSV-SH)6-lktA-(G(CX3C))3诱导后)所示，可见诱导后样品的70至100KDa处有目标条带，与目标蛋白大小一致，而空载和诱导前则无目标条带，说明(RSV-SH)6-lktA-(G(CX3C))3在大肠杆菌中经诱导后得到表达。
C.Western blotting鉴定蛋白
a)将全菌煮样(参照A中f)步骤)并进行SDS-PAGE电泳，并进行转膜；
b)取出转膜，将膜放入平皿中用10mL封闭液进行封闭，在4℃冰箱封闭过夜；
c)用封闭液或TBST稀释一抗，将一抗加入到平皿中，室温摇床震摇lh；
d)每次用20mL TTBS洗膜，每次在摇床上室温震摇lh，洗膜4次；
e)加入二抗，室温摇床震摇孵育1h；孵育结束后如上述洗膜4次；
f)加入底物反应液DAB显色，30s后用ddH2O清洗，终止反应；
通过Western Blotting方法来检测目的蛋白，结果如图5(M：蛋白marker(100KD)，1：pET-28a-(RSV-SH)6-lktA-(G(CX3C))3诱导前，2：pET-28a-(RSV-SH)6-lktA-(G(CX3C))3诱导后)所示，以鼠抗His-IgG为一抗，用羊抗鼠IgG-HRP作为二抗，结果诱导后菌液表达的蛋白可以和鼠抗His-IgG一抗结合，在阴性对照组(诱导前的菌液)的相同位置无阳性反应，并且诱导后菌液表达的蛋白大小与SDS-PAGE上的条带大小相同，说明之前表达的蛋白条带就是所述目的蛋白(RSV-SH)6-lktA-(G(CX3C))3。
D.蛋白的纯化
a)(RSV-SH)6-lktA-(G(CX3C))3包涵体的制备
将可以诱导出目的蛋白的菌种按1％的比例接种到LB培养基中，并同时加入0.1％Kan+，37℃摇菌4.5h，取少量菌液用酶标仪在600nm的波长测OD值，当OD600值达到0.6至0.8的时候加入终浓度为1mM的IPTG进行诱导表达，加入IPTG以后继续震摇诱导4h；
将诱导完的菌离心，按60mL菌液/L裂解液(包含50mM甘氨酸、0.1M NaCl、pH 8.0)的比例将菌沉淀重悬起来，用25％的功率将菌超声破碎，破碎的过程中装菌悬液的瓶子置于冰水浴；将超声完的破碎液取30μL留样，该样品为裂解全液。然后取30μL破碎液，12000rpm离心10min，上清取到另外一管，该样品为裂解上清，离心沉淀用30μL体积水重悬起来，该样品为裂解沉淀。剩余破碎液12000rpm离心完成固液分离，得到包涵体。
将留样加入等体积的2×Loading Buffer(30μL)，在开水中煮沸15min，取15μL进行凝胶电泳定量，根据目的蛋白在上清和沉淀中存在的情况断定蛋白以包涵体的形式存在。
b)(RSV-SH)6-lktA-(G(CX3C))3蛋白的纯化
将制备好的包涵体用pH9.0 8M尿素和用移液器吹悬溶解，得到包涵体裂解液，然后12000rpm离心10min；
将His-Ni亲和层析柱先用10体积的过滤过的ddH2O手工过柱清洗，再用5体积pH9.08M尿素进行平衡；
将包涵体裂解液的离心上清用1mL的注射器缓慢加样到柱子上，密封放置30min；
用5体积的蛋白裂解液(pH9.0 8M尿素)洗脱柱子；
用4体积30mM咪哇的洗脱缓冲液洗脱柱子，并收集洗脱液；
用5体积100mM咪哇的洗脱缓冲液洗脱柱子，并收集洗脱液；
用10体积过滤过的ddH2O洗柱子，然后用5体积的20％乙醇洗柱子，最后将柱子盖好，置于冰箱中。
采用SDS-PAGE检测(RSV-SH)6-lktA-(G(CX3C))3蛋白纯化结果，如图6(M：蛋白Marker(100kd)，1：流穿液Ft(包涵体裂解液)，2-5:30mM咪唑洗脱液，6-9：100mM咪唑洗脱液)所示，分别在30mM和100mM咪唑洗脱处出峰，取样进行SDS-PAGE检测，可见在Marker70和100KD之间有明显目标条带。
即(RSV-SH)6-lktA-(G(CX3C))3，其氨基酸序列为：
(SEQ ID NO.3：其中，单下划线为白细胞毒素的氨基酸序列，SEQ ID NO.6；双下划线为包含CX3C模序的呼吸道合胞病毒的G蛋白的氨基酸序列，SEQ ID NO.7；波浪线为SH蛋白的氨基酸序列，SEQ ID NO.5)；其对应的核苷酸序列为：

c)蛋白的浓缩
采用蛋白浓缩离心柱浓缩蛋白，采用40℃，3500g进行离心，浓缩20倍以后离心去掉析出的蛋白，将浓缩后的蛋白进行SDS-PAGE进行初步蛋白定量，然后使用Bradford用酶标仪进行精确定量。
3.重组蛋白(RSV-SH)6-lktA-(G(CX3C))3免疫原性评价
用牛血清白蛋白(BSA)做参照，测定考马斯亮蓝G-250在595nm处的紫外吸光值，绘制标准曲线(如图7)，用于对各目的蛋白和对照组进行蛋白定量，上述实验得到的目的蛋白的浓度为5.3mg/mL。
B.不同免疫佐剂免疫效果研究
小鼠分组
取目的蛋白分别用弗氏佐剂(FCA)、ISOCOMs、MF59、铝佐剂进行配制成50ug/mL的疫苗；同时取106TCID50/0.1mL甲醛灭活的RSV病毒，用铝佐剂进行配置成灭活疫苗(FI-RSV，5×106TCID50/mL)。将小鼠进行分组(铝佐剂组、FCA组、MF59组、ISCOMs组、FI-RSV组和PBS组)，小鼠为Balb/c小鼠，鼠龄6至8周，每组5只。按0，14，28天进行三次免疫，每种采用肌肉注射的方式免疫0.1mL，FI-RSV和PBS按照同样的方式进行注射。
血清样品收集
小鼠血清收集：第二次免疫、第三次免疫前一天通过尾静脉进行采血50至100μL，置于1.5mL灭菌处理后的EP管中，37℃放置2h，转入4℃静置过夜，次日可见血清分离，10000rpm，10min，收集上清，-20℃保存。
C.小鼠血清中SH和G(CX3C)蛋白特异性抗体IgG的检测
a)分别使用SH和CX3C多肽作为抗原与包被液混匀，每孔保持抗原量5μg，50μL/孔，设立空白组(仅含包被液)，4℃包被过夜；
b)第二天弃去包被液用TBST清洗3遍，加入封闭液(TBST+20％FBS+5％蔗糖+0.05％硫柳汞)，100μL/孔，封闭2h；
c)弃去封闭液用TPBS清洗，加入封闭液稀释的各个组的小鼠血清一抗孵育2h，弃去并用TBST清洗3遍；
e)加入各种实验所需要抗体稀释液稀释的小鼠IgG、IgG1、IgG2a的二抗(Goat Anti-Mouse IgG(H+L)HRP，供应商Affinity Biosciences，货号S0002)孵育2h，弃去并用TBST清洗三遍；
f)加入100μL底物缓冲液37℃反应10min，见有明显的颜色反应加入终止液对反应进行终止；
g)用酶标仪在492nm波长读取OD值，并进行计算。
小鼠第二、第三次免疫SH蛋白和CX3C蛋白后特异性抗体IgG、IgG1、IgG2a的检测结果分别如图8、图9、图10、图11、图12、图13所示，由图可见，经过三次免疫以后，通过检测三次免疫血清，结果证明第二次免疫后、第三次免疫后，铝佐剂组、FCA组、MF59组和ISCOMs组的SH蛋白和CX3C蛋白特异性IgG抗体滴度显著高于PBS组(P<0.05)，并且第三次免疫后，铝佐剂组、FCA组、MF59组和ISCOMs组的SH蛋白和CX3C蛋白特异性IgG抗体滴度高于FI-RSV组，其中ISCOMs组滴度最高；另外，SH蛋白和CX3C蛋白特异性抗体的各组别中所有的IgG1/IgG2a比值2至7倍之间(图14、图15)，说明免疫极化趋向平衡；而FI-RSV组IgG1/IgG2a比值10至300倍之间，说明其诱导的免疫反应趋向于TH2途径。以上结果表明，该融合蛋白具有诱导针对SH和G蛋白CX3C的特异抗体，并不会出现免疫反应不均衡的问题。
D.病毒中和抗体滴度的检测
(1)第一天，铺2.5×104个/孔Hep-2细胞于96孔细胞培养板中。
(2)第二天，各组的三次免疫后血清于56℃水浴孵育30min。
(3)用DMEM培养基稀释血清，稀释起始比例为1：2，2倍倍比稀释。
(4)向血清稀释液中加入纯化后的RSV(60PFU/100μL)，混合后放置于37℃孵育1小时，得到混合液。
(5)将混合液100μL加入到铺有单层Hep-2细胞的96孔板中，37℃孵育1小时。同时设置正常细胞对照组(不含病毒)和只加病毒的病毒对照组(不含血清)。
(6)病毒培养4天后，弃培养基，待病毒组相对于正常细胞对照组的细胞病变达到90％以上，弃掉细胞板中的液体，加入0.5‰染液(500mg结晶紫，用200mL无水乙醇溶解后加水稀释至1000mL)，100μL/孔，染色30min。
(7)弃去染色液，流水冲洗至水流澄清，吸干水分，加脱色液(500mL无水乙醇，1mL冰乙酸，加水稀释至1000mL)100μL/孔，室温放置5min，混匀。
(8)以630nm为参比波长，于波长570nm处用酶标仪测定吸光值，并通过半数抑制浓度，计算血清中和滴度。
结果分析：由图16分析可知，ISCOMs组的中和抗体滴度最高，同甲醛灭活的FI-RSV中和滴度差别不大，证明其对病毒具有较好中和作用；其余3组同样检测出较高的中和抗体滴度，证明四个疫苗组均有明显的中和RSV病毒的效果。
4.攻毒保护试验
将第三次免疫一周后的小鼠用玻璃杯罩起来，在里面放乙醚，将小鼠迷到站不稳滑到杯子底下即取出，捏着小鼠背用移液器往小鼠鼻孔上滴加100μL 106pfu/mL RSV病毒液，让小 鼠鼻孔冒泡将病毒液吸入呼吸道。将小鼠至于通风的地方换气，直到小鼠清醒可以爬动为止，将其放回鼠笼。
A.攻毒以后小鼠肺部病毒滴度
小鼠攻毒3天后，检测小鼠肺部病毒滴度，结果如图17所示，ISCOMs组对小鼠的攻毒保护作用最好，与PBS组存在明显差异(P<0.001)，其次是FCA组和MF59组(P<0.01)，铝佐剂组攻毒保护作用相对较前三组差，但也与PBS组存在差异(P<0.05)。
5.不同重组蛋白的免疫效果研究
分别取5μg的步骤3的(RSV-SH)6-lktA-(G(CX3C))3、SH-lktA(氨基酸序列为SEQ ID NO.3的1至639位)、lktA-G(cx3c)(氨基酸序列为SEQ ID NO.3的162至725位)同ISOCOM佐剂进行混合后(蛋白的浓度为50ug/mL)免疫小鼠。将小鼠按照表1进行分组，小鼠为Balb/c小鼠，鼠龄6至8周，每组5只。按1、14、28天时间进行三次肌肉注射的方式免疫，免疫第三次后采血进行IgG和抗体中和滴度检测(方法同步骤3)；免疫三次后过两周进行RSV攻毒，攻毒四天后取肺部组织，进行病毒滴定，确定其病毒保护能力(方法同步骤4)。结果(见表1)表明，单独SH-lktA融合蛋白免疫后不具有中和活性，而(RSV-SH)6-lktA-(G(CX3C))3和lktA-G(cx3c)都具有一定的中和活性；另外(RSV-SH)6-lktA-(G(CX3C))3攻毒后肺部未检出病毒，而其他两个SH-lktA和lktA-G(cx3c)肺部有少量的病毒检出，但也远远低于未免疫组。说明(RSV-SH)6-lktA-(G(CX3C))3攻毒保护效果明显优于SH-lktA和lktA-G(cx3c)。因此，本发明提供的蛋白(RSV-SH)6-lktA-(G(CX3C))3同时能够诱导SH和G(cx3c)的特异性抗体，并诱发更好的保护效果。
表1
参照步骤1、2制备得到以下重组蛋白：(RSV-SH)8-lktA-(G(CX3C))1、(RSV-SH)6-lktA-(G(CX3C))6、(RSV-SH)3-lktA-(G(CX3C))6：其中，RSV-SH的氨基酸序列为SEQ ID NO.5，lktA的氨基酸序列为SEQ ID NO.6，G(CX3C)的氨基酸序列为SEQ ID NO.7，RSV-SH之间通过GSGS相连，G(CX3C之间通过GSGS相连，RSV-SH和lktA之间通过FP连接；将上述重组蛋白同ISOCOM佐剂进行混合后(蛋白的浓度为50ug/mL)免疫小鼠。将小鼠按照表2进行分组，小鼠为Balb/c小鼠，鼠龄6至8周，每组5只。按1、14、28天时间进行三次肌肉注射的方式免疫，免疫第三次后采血进行IgG和抗体中和滴度检测(方法同步骤3)。结果(见表2)表明：(RSV-SH)8-lktA-(G(CX3C))1、(RSV-SH)6-lktA-(G(CX3C))6、(RSV-SH)3-lktA-(G(CX3C))6同时能够诱导SH和G(cx3c)的特异性抗体，并诱发更好的保护效 果。
表2
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。

Claims (10)

  1. 一种重组蛋白，包含：呼吸道合胞病毒的SH蛋白和包含CX3C模序的呼吸道合胞病毒的G蛋白。
  2. 根据权利要求1所述的重组蛋白，其特征在于：
    所述重组蛋白还包含载体蛋白；
    优选地，所述重组蛋白包含：(SH)n-Carrier-(G(CX3C))m；其中，m、n独立选自于1、2、3、4、5、6、7、8；SH为呼吸道合胞病毒的SH蛋白；Carrier为载体蛋白；G(CX3C)为包含CX3C模序的呼吸道合胞病毒的G蛋白；
    优选地，所述载体蛋白包含血小板反应蛋白、霍乱毒素、血蓝蛋白、牛血清白蛋白、白细胞毒素、人血清白蛋白中的至少一种。
  3. 根据权利要求2所述的重组蛋白，其特征在于：
    所述SH蛋白为a1)至a3)中任一种：
    a1)氨基酸序列如SEQ ID NO.5所示的SH蛋白；
    a2)将SEQ ID NO.5经过一个或几个氨基酸的取代和/或缺失和/或添加且与SEQ ID NO.5具有相同功能的SH蛋白；
    a3)与SEQ ID NO.5具有99％、98％、97％、96％、95％、94％、93％或92％的同源性且与SEQ ID NO.5具有相同功能的SH蛋白；
    优选地，所述白细胞毒素为b1)至b3)中任一种：
    b1)氨基酸序列如SEQ ID NO.6所示的白细胞毒素；
    b2)将SEQ ID NO.6经过一个或几个氨基酸的取代和/或缺失和/或添加且与SEQ ID NO.6具有相同功能的白细胞毒素；
    b3)与SEQ ID NO.6具有99％、98％、97％、96％、95％、94％、93％或92％的同源性且与SEQ ID NO.6具有相同功能的白细胞毒素；
    优选地，所述包含CX3C模序的呼吸道合胞病毒的G蛋白为c1)至c3)中任一种：
    c1)氨基酸序列如SEQ ID NO.7所示的G蛋白；
    c2)将SEQ ID NO.7经过一个或几个氨基酸的取代和/或缺失和/或添加且与SEQ ID NO.7具有相同功能的G蛋白；
    c3)与SEQ ID NO.7具有99％、98％、97％、96％、95％、94％、93％或92％的同源性且与SEQ ID NO.7具有相同功能的G蛋白；
    优选地，所述重组蛋白包含：(SH)6-Carrier-G(CX3C)3、(SH)8-Carrier-(G(CX3C))1、(SH)6-Carrier-(G(CX3C))6、(SH)3-Carrier-(G(CX3C))6中的至少一种。
  4. 与权利要求1至3任一项所述的重组蛋白相关的生物材料；
    所述生物材料为e1)至e12)中的任一种：
    e1)编码权利要求1至3任一项所述的重组蛋白的核酸分子；
    e2)含有e1)所述核酸分子的表达盒；
    e3)含有e1)所述核酸分子的重组载体；
    e4)含有e2)所述表达盒的重组载体；
    e5)含有e1)所述核酸分子的重组微生物；
    e6)含有e2)所述表达盒的重组微生物；
    e7)含有e3)所述重组载体的重组微生物；
    e8)含有e4)所述重组载体的重组微生物；
    e9)含有e1)所述核酸分子的转基因细胞系；
    e10)含有e2)所述表达盒的转基因细胞系；
    e11)含有e3)所述重组载体的转基因细胞系；
    e12)含有e4)所述重组载体的转基因细胞系。
  5. 权利要求1至3任一项所述的重组蛋白的制备方法，通过培养权利要求4中所述的重组微生物和/或转基因细胞系表达所述重组蛋白。
  6. (1)至(3)中任一项在制备产品中的应用；
    所述产品为g1)至g4)中任一种：
    g1)疫苗；
    g2)呼吸道合胞病毒抗体；
    g3)抗呼吸道合胞病毒血清；
    g4)诊断抗原；
    (1)权利要求1至3任一项所述的重组蛋白；
    (2)权利要求4所述的生物材料；
    (3)权利要求5所述的制备方法；
    优选地，所述疫苗用于预防和/或治疗呼吸道合胞病毒感染和/或呼吸道合胞病毒引起的疾病；
    优选地，所述诊断抗原用于诊断呼吸道合胞病毒和/或呼吸道合胞病毒引起的疾病。
  7. 一种诊断抗原，包含：权利要求1至3任一项所述的重组蛋白。
  8. 一种疫苗，包含：权利要求1至3任一项所述的重组蛋白和/或权利要求4所述的生物 材料。
  9. 根据权利要求8所述的疫苗，其特征在于：
    所述疫苗包含权利要求1至3任一项所述的重组蛋白和佐剂。
  10. 成套试剂盒，包含权利要求8或9所述的疫苗及用于所述疫苗接种的容器。
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