CN110054668B - Respiratory syncytial virus pre-fusion F protein and application thereof - Google Patents

Respiratory syncytial virus pre-fusion F protein and application thereof Download PDF

Info

Publication number
CN110054668B
CN110054668B CN201910337141.5A CN201910337141A CN110054668B CN 110054668 B CN110054668 B CN 110054668B CN 201910337141 A CN201910337141 A CN 201910337141A CN 110054668 B CN110054668 B CN 110054668B
Authority
CN
China
Prior art keywords
ser
asn
leu
thr
protein
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN201910337141.5A
Other languages
Chinese (zh)
Other versions
CN110054668A (en
Inventor
何金生
马尧
付远辉
彭向雷
郑妍鹏
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Beijing Jiaotong University
Original Assignee
Beijing Jiaotong University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Beijing Jiaotong University filed Critical Beijing Jiaotong University
Priority to CN201910337141.5A priority Critical patent/CN110054668B/en
Publication of CN110054668A publication Critical patent/CN110054668A/en
Application granted granted Critical
Publication of CN110054668B publication Critical patent/CN110054668B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K39/12Viral antigens
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12Antivirals
    • A61P31/14Antivirals for RNA viruses
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K16/00Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1027Paramyxoviridae, e.g. respiratory syncytial virus
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983Viruses
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K2039/51Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525Virus
    • A61K2039/5258Virus-like particles
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K2039/51Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53DNA (RNA) vaccination
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011Details
    • C12N2760/18011Paramyxoviridae
    • C12N2760/18511Pneumovirus, e.g. human respiratory syncytial virus
    • C12N2760/18522New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011Details
    • C12N2760/18011Paramyxoviridae
    • C12N2760/18511Pneumovirus, e.g. human respiratory syncytial virus
    • C12N2760/18534Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08RNA viruses
    • G01N2333/115Paramyxoviridae, e.g. parainfluenza virus
    • G01N2333/135Respiratory syncytial virus

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Virology (AREA)
  • Molecular Biology (AREA)
  • Immunology (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Hematology (AREA)
  • Biochemistry (AREA)
  • Biomedical Technology (AREA)
  • Urology & Nephrology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Microbiology (AREA)
  • Veterinary Medicine (AREA)
  • Physics & Mathematics (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Pathology (AREA)
  • General Physics & Mathematics (AREA)
  • Genetics & Genomics (AREA)
  • Biotechnology (AREA)
  • Cell Biology (AREA)
  • Biophysics (AREA)
  • Analytical Chemistry (AREA)
  • Food Science & Technology (AREA)
  • Epidemiology (AREA)
  • Pulmonology (AREA)
  • Mycology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Communicable Diseases (AREA)
  • Oncology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Peptides Or Proteins (AREA)

Abstract

The invention provides a respiratory syncytial virus pre-fusion F protein and application thereof, belonging to the technical field of respiratory syncytial virus vaccines. The F protein before fusion is a wild type F protein, wherein after amino acids at 106 th, 108 th and 109 th positions are mutated from Arg to Asn, amino acid at 104 th position is mutated from Asn to Cys, amino acid at 155 th position is mutated from Ser to Cys, or amino acid at 58 th position is mutated from Thr to Cys, and amino acid at 190 th position is mutated from Ser to Cys. The invention mutates the first furin cleavage site of wild type F protein, so that the structure of Pre-F is more stable, the expression level is not reduced, and the fusion Pre-epitope expressed by M1-F in 293T cell is obviously improved; the second mutation of the amino acid site of the F protein leads the F protein to form a disulfide bond, has more stable epitope before fusion compared with the wild type, and is suitable for other virus strains of human RSV; the vaccine is applicable to various vaccine forms taking RSV F protein as antigen, such as: nucleic acid vaccines, protein vaccines, vector vaccines and recombinant virus particle vaccines.

Description

Respiratory syncytial virus pre-fusion F protein and application thereof
Technical Field
The invention relates to the technical field of respiratory syncytial virus vaccines, in particular to a respiratory syncytial virus pre-fusion F protein and application thereof.
Background
Human Respiratory Syncytial Virus (RSV) is an enveloped, non-segmented, single-stranded RNA virus belonging to the Pneumoviridae (pneumovirus family) that is capable of causing severe lower respiratory tract infections in infants and elderly. The number of serious lower respiratory tract infection caused by RSV in children under 5 years old is 3 million and the number of death is more than 6 million all year round. The population of all-cause deaths from RSV infection accounts for 6.7% of all-cause deaths in infants from 1 month of age to 1 week of age. 100% of infants are infected with RSV virus at least 1 time within 3 years of age, with peak infection occurring at 2 to 4 months of age. Children under 5 years of age are at risk of severe disease from RSV infection, and the virus is repeatedly infected. Although RSV has been widely recognized since 1956, no vaccine effective against RSV infection has been developed to date.
Fusion glycoprotein (F) of RSV (respiratory syncytial virus) is envelope protein and has adhesion function and fusion function. At the same time, the F protein is used as a major candidate antigen for RSV vaccine development due to its ability to produce highly effective neutralizing antibodies, and its high degree of conservation across different RSV strains.
The F protein belongs to the type I integral membrane protein, and consists of 574 amino acids of inactive precursors (F0). Three of F0 formed trimers, and when transported through the Golgi apparatus, the host furin cleaved between amino acids 109 and 110 and between amino acids 136 and 137 of F0. After cleavage, the middle 27 amino acids short peptide (P27) was released, while the remaining two F2 and F1 were linked by disulfide bonds (Cys 69-Cys 212 and Cys 37-Cys 439) to form the mature F protein structure. At the N-terminal of the F1 protein, there is a highly hydrophobic Fusion Peptide (FP), which is located in the hydrophobic cavity of the protein and protected from the external hydrophilic environment. When the F protein is present on the surface of a virus or a cell, the structure is not stable, but is in a high-energy metastable Pre-F structure. Subsequently, the N-terminal of the F1 protein undergoes a series of drastic structural changes, and the process induces the occurrence of membrane fusion, thereby achieving the aim of viral infection of cells. At the same time, this process also results in the conversion of the F protein from a high-energy metastable Pre-F structure to a stable Post-fusion F protein structure (Post-F).
McLellan et al first studied the structure of the Pre-F protein. They pass throughDisulfide bonds were formed by mutations at amino acids 155 and 290 (S155C and S290C) of the F protein, mutations at amino acids 190 and 207 (S190F and V207L) maintained structural stability, and the transmembrane and intracellular regions of the F protein were replaced at the C-terminus of F1 with a T4 bacteriophage fibrin trimerization domain, thereby purifying a stable prefusion F protein trimer, designated DS-Cav 1. Current studies show that pre-fusion F contains two pre-fusion specific epitopes (II)
Figure BDA0002039507140000021
And Site V). The neutralizing antibodies generated by the two epitopes have remarkably enhanced neutralizing activity compared with four epitopes (Site I, Site II, Site III and Site IV) contained in F before fusion and F after fusion. Therefore, the key to the study of RSV vaccines using F protein as the main protective antigen is how to maintain the stability of the F protein fusion pro-structure to enhance the production of highly neutralizing antibodies.
Disclosure of Invention
The invention aims to provide a respiratory syncytial virus pre-fusion F protein mutant with remarkably improved pre-fusion epitope and thermal stability, so as to solve the technical problems in the background technology.
In order to achieve the purpose, the invention adopts the following technical scheme:
in one aspect, the invention provides a respiratory syncytial virus pre-fusion F protein, which is the wild-type F protein and further comprises at least one second mutation after the first mutation of the amino acids 106, 108 and 109, wherein the second mutation is selected from the group consisting of:
a) the 104 th amino acid is mutated from Asn to Cys, the 155 th amino acid is mutated from Ser to Cys,
b) the 58 th amino acid is mutated from Thr to Cys, and the 190 th amino acid is mutated from Ser to Cys.
Furthermore, the first mutation is to change from Arg to Asn at amino acids 106, 108 and 109.
In one aspect, the present invention provides a method for preparing the respiratory syncytial virus pre-fusion F protein, which comprises the following steps:
step 1: carrying out first mutation on a first furin cleavage site of the wild type F protein, wherein the amino acid sequence after the first mutation is shown as SEQ ID NO: 2 is shown in the specification; the amino acid sequence of the wild type F protein is shown as SEQ ID NO: 1 is shown in the specification;
step 2: subjecting the polypeptide mutated in step 1 to at least one second mutation selected from the group consisting of:
a) the amino acid at the 104 th site is mutated from Asn to Cys, the amino acid at the 155 th site is mutated from Ser to Cys, and the amino acid sequence is shown as SEQ ID NO: as shown in figure 3, the first and second,
b) the 58 th amino acid is mutated from Thr to Cys, the 190 th amino acid is mutated from Ser to Cys, and the amino acid sequence is shown as SEQ ID NO: 4, respectively.
In one aspect, the invention also provides a gene encoding a polypeptide after a first mutation as described above.
The nucleotide sequence of the gene is shown as SEQ ID NO: 5, respectively.
In one aspect, the invention also provides the application of the respiratory syncytial virus pre-fusion F protein in the preparation of a medicament for preventing respiratory syncytial virus infection.
In one aspect, the invention also provides the use of the respiratory syncytial virus pre-fusion F protein in the preparation of respiratory syncytial virus antibodies.
In one aspect, the invention also provides the application of the F protein before the respiratory syncytial virus fusion in the preparation of diagnostic reagents for the respiratory syncytial virus.
In one aspect, the invention also provides the application of the F protein before the respiratory syncytial virus fusion in the preparation of nucleic acid vaccines, protein vaccines, virus-like particle vaccines and vector vaccines of the respiratory syncytial virus.
The invention has the beneficial effects that: the first furin cleavage site of the mutant F protein enables the structure of Pre-F to be more stable, compared with wild type F protein, the expression amount is not reduced, and the fusion Pre-epitope expressed by M1-F in 293T cell is obviously higher than F; the second mutation of the F protein amino acid site enables the F protein amino acid site to form a disulfide bond so as to stabilize the epitope before fusion, compared with the wild type, the F protein amino acid site has more stable epitope before fusion and better protection in animal experiments, and the mutation of the furin site and the mutation of the disulfide bond are suitable for other virus strains of human RSV; the vaccine is applicable to various vaccine forms taking RSV F protein as antigen, such as: nucleic acid vaccines, protein vaccines, virus-like particle vaccines, and vector vaccines.
Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts.
FIG. 1 is an expression profile of a mutated F protein after furin cleavage site according to an embodiment of the present invention.
FIG. 2 is an expression profile of the epitopes of neutralizing antibodies after mutant plasmid transfection of mutated F protein after furin cleavage sites, according to an embodiment of the present invention.
FIG. 3 is a map showing the expression level of a recombinant plasmid after mutation based on M1-F according to an embodiment of the present invention.
FIG. 4 is a diagram showing the epitope of neutralizing antibody expressed by each recombinant plasmid after mutation based on M1-F according to the embodiment of the present invention.
FIG. 5 is a schematic diagram of the thermal stability analysis of M1-F mutant at 55 ℃ according to the example of the present invention.
FIG. 6 is a diagram showing the expression level of M1-F mutant and the thermal stability analysis at 37 ℃, 4 ℃ and-20 ℃ in the example of the present invention.
FIG. 7 is a schematic diagram of the humoral immune response, neutralizing antibodies and protective immune results induced by the M1-F encoding mutant plasmid in mice according to the present invention.
FIG. 8 is a graph showing the scoring of inflammation in the lungs after H & E staining in accordance with an embodiment of the present invention.
Detailed Description
Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to the same or similar elements or modules having the same or similar functionality throughout. The embodiments described below with reference to the accompanying drawings are illustrative only for the purpose of explaining the present invention, and are not to be construed as limiting the present invention.
It will be understood by those skilled in the art that, unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the prior art and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.
For the convenience of understanding of the embodiments of the present invention, the following description will be further explained by taking specific embodiments as examples with reference to the drawings, and the embodiments are not to be construed as limiting the embodiments of the present invention.
It will be understood by those of ordinary skill in the art that the figures are merely schematic representations of one embodiment and that the elements or devices in the figures are not necessarily required to practice the present invention.
Examples
Test 1: and (3) constructing a recombinant plasmid capable of expressing the F protein with the mutated furin cleavage site.
Based on the optimized RSV Long strain F protein gene, the furin cutting sites of the RSV Long strain F protein gene are mutated, the amino acid sequence and the DNA sequence of each mutant are shown in a table 1, wherein the bold positions of fonts are the furin cutting sites. F is genome-optimized RSV F protein, M1-F is a mutant with a mutated first furin cleavage site, M2-F is a mutant with a mutated second furin cleavage site, and U-F is an uncleaved mutant, i.e., a mutant with two sites mutated simultaneously.
Table 1:
Figure BDA0002039507140000051
Figure BDA0002039507140000061
in addition, this experiment constructed mutant DS-Cav1 in a prefusion structure for use as a control, but the C-terminus of F1 retained the transmembrane region (TM region) and intracellular region (CT region) structures of the original F1. In the experiment, M1-F, M2-F and U-F plasmids constructed by an overlap extension PCR method are added with 2 mu l of Dpn I enzyme after PCR to degrade a template sequence. After mixing, the mixture was incubated at 37 ℃ for 30 minutes. Then, the site-directed mutant plasmid is transformed, and gene sequencing is carried out to identify whether recombination is successful. Construction of DS-Cav1 was accomplished using a site-directed mutagenesis kit. The successfully sequenced recombinant plasmid is subjected to plasmid big extraction, a gel imaging system is used for identifying double enzyme digestion results, the correctly identified plasmid is detected by OD260 and 280 through a spectrophotometer, the DNA concentration and purity of the plasmid are determined, and the plasmid is stored at the temperature of minus 20 ℃.
We construct the recombinant plasmid by two-step site-directed mutagenesis method two, after PCR reaction, add 2 μ l Dpn I enzyme, degrade the template sequence. After mixing, the mixture is placed at 37 ℃ and incubated for more than 5 minutes. Then, the site-directed mutant plasmid is transformed, and gene sequencing is carried out to identify whether the mutation is successful. The successfully sequenced recombinant plasmid is subjected to plasmid large extraction and agarose gel electrophoresis to identify double digestion results, then the DNA concentration is determined, and the recombinant plasmid is stored at the temperature of minus 20 ℃.
Test 2: the expression level of the recombinant plasmid is detected by a Western Blot method, which comprises the following steps:
(1) on the first day, 6X 105/well 293T cells were seeded in 6-well plates.
(2) The next day, plasmid transfection was performed when the cell confluence reached around 80%. 100 mul of serum-free DMEM medium is put into a 1.5ml centrifuge tube, 2 mug of plasmid is added, and the mixture is vortexed and shaken to be mixed evenly. Then 100 mul serum-free DMEM medium is put into a 1.5ml centrifuge tube, 4 mul Lipofectamine2000 is added, and vortex shaking is carried out to mix evenly. Standing for 10 minutes, mixing the plasmid solution and the liposome solution in the two centrifuge tubes, vortexing, shaking and mixing uniformly, and standing for 20 minutes. Then, the solution was added to one well of a 6-well plate, gently shaken, and then cultured in a cell incubator.
(3) On day four, the cell culture was discarded and PBS was added to gently wash the cells.
(4) Cells were lysed by adding 300. mu.l of cell lysate.
(5) After lysis, the cells were collected, centrifuged at 12000rpm at 4 ℃ for 20 minutes, and the supernatant was collected.
(6) Reducing SDS-PAGE electrophoresis samples, adding beta mercaptoethanol and Loading buffer. And only Loading buffer is added in the non-reducing SDS-PAGE electrophoresis.
(7) A12% SDS-PAGE gel was prepared for electrophoresis of the samples. Firstly, preparing separation glue, adding the separation glue into a glue groove, and sealing the glue with distilled water to keep the glue surface horizontal. After standing at room temperature for 30 minutes, the separation gel had solidified. Sucking out upper layer distilled water, adding separation gel, rapidly inserting into a comb for gel running, standing at room temperature for 30 min, and storing at 4 deg.C for use.
(8) After the electrophoresis is finished, the protein is transferred to an NC membrane by a wet transfer method.
(9) NC membranes were blocked for 2 hours in 5% skim milk in TBS.
(10) After TBS washing, NC membranes were incubated in skim milk containing human Motavizumab antibody and murine GAPDH antibody (1:2000 dilution) and incubated at 37 ℃ for 1 hour.
(11) NC membranes were washed 5 times with TBST.
(12) NC membranes were incubated in skim milk containing goat anti-human IRDye 800 and goat anti-mouse IRDye 700(1:2000 dilution) for 1 hour at 37 ℃.
(13) The NC membrane was washed 5 times with TBST, and the Western Blot results were detected in a two-color infrared fluorescence imaging system and photographed.
As shown in FIG. 1, Western Blot was used to analyze the expression level of the F protein and its mutants expressed by 293T cells. The detection antibody is Motavizumab. The left lane in the figure is Marker, and the bands 1-14 in the sample are internal reference GAPDH. F is fusion protein, M1-F is a mutant mutating a first furin cleavage site, M2-F is a mutant mutating a second furin cleavage site, U-F is a mutant mutating two furin cleavage sites simultaneously, and DS-Cav1 is F protein before fusion with unchanged C-terminal.
Test 3: and (3) detecting the content of each epitope of the recombinant plasmid expression protein in situ by using an immunofluorescence method.
(1) 293T cells were seeded at 6X 105 cells/well in 6-well plates, and after 24 hours the cell confluence reached about 80%, and the cell culture medium was replaced with fresh one.
(2) And (4) plasmid transfection. The method is the same as 3.2.2.1. The amount of plasmid was 2. mu.g/well and the amount of Lipofectamine2000 was 4. mu.l/well.
(3) 48 hours after transfection, the cell culture medium was discarded and PBS was added to gently wash the cells.
(4) 5% skim milk blocked the cells for 2 hours.
(5) After washing with PBS, the cells were incubated in skim milk containing antibodies recognizing different epitopes (Motavizumab, D25, MPE8 and AM14) at 37 ℃ for 1 hour.
(6) Cells were washed 3 times with PBST.
(7) Cells were incubated in skim milk containing goat anti-human Alexa Fluor488(1:2000 dilution) for 1 hour at 37 ℃.
(8) Cells were washed 3 times with PBST.
(9) Cell nucleus dye DAPI was added for 30 min.
(10) Cells were washed 3 times with PBST and photographed under a fluorescence microscope and analyzed for fluorescence intensity using an ImageJ launcher. In the final analysis of the results, the epitope content of group F was set to 1, and the other group contents were expressed as multiples of the relative F content.
As shown in figure 2, after the F protein and the mutant plasmid thereof transfect 293T cells, the expression of the high-efficiency neutralizing antibody epitope in the F protein and the mutant thereof is detected in situ by using an immunofluorescence method. Motavizumab (A), D25(B), MPE8(C) and AM14(D) antibodies were used to recognize Site II,
Figure BDA0002039507140000081
III and V epitopes.
As shown in FIG. 3, M1-F-1 and M1-F-5 are two sets of mutations in the second step (N104C and S155C, T58C and S190C). The recombinant plasmid is expressed in 293T cells, and the expression amount is analyzed by Western Blot after SDS-PAGE.
As shown in FIG. 4, immunofluorescence demonstrated that expression of antibody epitopes was efficiently neutralized after transfection of cells with M1-F, DS-Cav1, M1-F-1 and M1-F-5. Motavizumab (A), D25(B), MPE8(C) and AM14(D) were used to recognize epitope Site II,
Figure BDA0002039507140000091
III and IV.
Test 4: the M1-F protein mutant expressed by the recombinant plasmid is tested for stability at 55 ℃.
(1) 293T cells were seeded at 3X 105/well in 12-well plates.
(2) Plasmid transfection, 50 μ l serum-free DMEM medium is placed in a 1.5ml centrifuge tube, 1 μ g plasmid is added, and vortex shaking is carried out to mix evenly. And then 50 mul of serum-free DMEM medium is put into a 1.5ml centrifuge tube, 2 mul of Lipofectamine2000 is added, and the mixture is vortexed and uniformly mixed. Standing for 10 minutes, mixing the plasmid solution and the liposome solution in the two centrifuge tubes, vortexing, shaking and mixing uniformly, and standing for 20 minutes. Then, the solution was added to one well of a 6-well plate, gently shaken, and then cultured in a cell incubator.
(3) Two days after transfection, the cell culture medium was discarded and PBS was added to gently wash the cells.
(4) Add 300. mu.l PBS to resuspend the cells.
(5) And (4) subpackaging the uniformly mixed cells.
(6) The dispensed cells were incubated in a water bath at 55 ℃ for 0 min, 0.5 min, 1 min, 3 min, 5 min and 10 min, respectively, and stored on ice after the incubation was completed.
(7) After mixing each sample, 2. mu.l was put on NC membrane and each sample was repeated three times.
(8) After the sample was sufficiently absorbed on the membrane, the NC membrane was placed in 5% skim milk in TBS configuration and blocked for 1 hour at room temperature.
(9) After TBS washing, the cells were added to skim milk containing antibodies recognizing different epitopes (Motavizumab, D25, MPE8 and AM14), and incubated at room temperature for 1 hour.
(10) NC membranes were washed 3 times with TBST.
(11) NC membranes were incubated in skim milk containing horseradish peroxidase-labeled goat anti-human IgG (1:2000 dilution) for 1 hour at room temperature.
(12) NC membranes were washed 3 times with TBST.
(13) The washed NC membrane was developed with ECL Western Blotting Substrate.
(14) After development, the image was taken by using a gel imaging system, and the development intensity was analyzed by ImageJ launcher.
(15) Finally, the stability of the mutants was expressed as a percentage of the epitope before fusion relative to the mean gray value. The calculation formula is as follows: the percentage of the relative mean gray value of the sample is PX/P0, PX is the ratio of the mean gray value of the epitope of the antibody at a certain time point to the mean gray value of the Motavizumab at the time point, and P0 is the ratio of the mean gray value of the epitope of the antibody in the non-stimulated group of the mutant to the mean gray value of the Motavizumab in the non-stimulated group of the mutant.
Test 5: and (3) comprehensively detecting the expression quantity of each recombinant plasmid and the heat stability of the mutant protein at different temperatures.
(1) 293T cells were seeded in 12-well plates and the cell confluence reached around 80% after 24 hours.
(2) And (4) plasmid transfection. The amount of plasmid was 1. mu.g/well and the amount of Lipofectamine2000 was 2. mu.l/well.
(3) 48 hours after transfection, the cell culture medium was discarded and PBS was added to gently wash the cells.
(4) An additional 300. mu.l of PBS was added to resuspend the cells.
(5) And (4) subpackaging the uniformly mixed cells.
(6) The dispensed cells were incubated at 37 deg.C, 4 deg.C and-20 deg.C for 0, 1, 2, 3 and 4 days, respectively. (different initial times of seeding cells, and finally collecting cells incubated for different lengths of time on the same day).
(7) After incubation was complete, 2. mu.l of each sample was mixed and placed on NC membrane, and each sample was repeated three times.
(8) After the sample was sufficiently absorbed on the membrane, the NC membrane was placed in 5% skim milk in PBS and blocked at room temperature for 1 hour.
(9) After washing with PBS, skim milk containing antibodies recognizing different epitopes (Motavizumab, D25, MPE8 and AM14) was added thereto and incubated at room temperature for 1 hour.
(10) NC membranes were washed 3 times with TBST.
(11) NC membranes were incubated in skim milk containing horseradish peroxidase-labeled goat anti-human IgG (1:2000 dilution) for 1 hour at room temperature.
(12) NC membranes were washed 3 times with TBST.
(13) The washed NC membrane was developed with ECL Western Blotting Substrate.
(14) After development, the image was taken by using a gel imaging system, and the development intensity was analyzed by ImageJ launcher.
(15) Finally, the residual content of the epitope before fusion of the mutant is expressed as relative average gray scale value of the epitope before fusion. The calculation formula is as follows: the relative average gray value of the sample is VX RX, VX is the gray average value of the epitope of a certain antibody at a certain time point, and RX is the ratio of the gray average value of the Motavizumab in the unstimulated group of the mutant to the gray average value of the Motavizumab at the time point.
As shown in FIG. 5, Dot Blot analysis of the prefusion epitopes ((II))
Figure BDA0002039507140000111
And Site V) and the preponderant epitope (Site III) before fusion were expressed on the cell surface two days after transfection. Specific pre-fusion F monoclonal antibodies D25 and AM14 and a pre-fusion F dominant epitope antibody MPE8 were used to recognize protein epitopes adsorbed on NC membranes.
As shown in FIG. 6, the F protein expressed on the cell surface was heat-inactivated at 37 ℃, 4 ℃ and-20 ℃ for 1 to 4 days, respectively. The expression level and stability of the protein were analyzed by Dot Blot.
Test 6: immunization with the mutant plasmid with enhanced coding stability and RSV challenge experiments.
The experimental animals were kept and the experimental procedures were carried out according to the procedures established by the institutional animal care and management committee (IACUC) of the university of qinghua. The experimental animals are SPF BALB/c female mice with the age of 6-8 weeks and the weight of about 20 g. The mice were purchased from Beijing Wittiulihua laboratory animal technology GmbH and bred in the laboratory animal center of Qinghua university.
(1) Purchased mice were randomly grouped into 5 mice each. The amount of DNA immunized per mouse was 30. mu.g, diluted in 0.1ml of PBS. The immunization method is intramuscular injection, the injection point is bilateral thigh quadriceps muscle of the mouse, and electroporation stimulation is carried out at the injection point after the injection to enhance the transfection efficiency. The control group was 0.1ml of PBS without DNA.
(2) Three weeks after the primary immunization (day 21), a second booster immunization was performed, followed by three additional weeks apart (day 42), three total immunizations.
(3) The FI-RSV lung pathology control group was immunized at 35 days with an inactivated RSV 1X 106 PFU/mouse diluted in 0.1ml PBS with 4mg/ml aluminum adjuvant. The immunization mode is intramuscular injection.
(4) The final immunization is carried out 14 days later for virus challenge (FI-RSV is 21 days after the initial immunization), the challenge-read dose is 1 multiplied by 106 PFU/mouse, the volume is 30 mul, and the virus challenge mode is nasal drip.
Test 7: and (5) detecting serum antibodies.
(1) Purified RSV virus was diluted in pre-chilled PBS, coated in 96-well plates at 1 × 105 PFU/well RSV and coated overnight at 4 ℃.
(2) Discard the coating solution and add PBS to wash once.
(3) The washing solution was discarded, and PBS blocking solution containing 5% fetal bovine serum was added thereto, followed by standing at 37 ℃ for 1 hour.
(4) The blocking solution was discarded and washed 3 times with PBST wash.
(5) A gradient dilution of mouse serum in blocking solution was added (first dilution 1:100, 2-fold down). Incubate at 37 ℃ for 1 hour.
(6) PBST wash was washed 5 times.
(7) A1: 2000 dilution of goat anti-mouse IgG antibody labeled with horseradish peroxidase was added (for subtype analysis, goat anti-mouse IgG1 or IgG2a antibody labeled with horseradish peroxidase was added). Incubate at 37 ℃ for 1 hour.
(8) PBST wash was washed 5 times.
(9) Adding ELISA developing solution to develop for 15 minutes at room temperature in a dark place.
(10) 2mol/L of H is added2SO4The color development is stopped, and the absorbance under the condition of 450nm is detected by an enzyme-labeling instrument.
(11) The positive results were determined as follows: the color value of the sample is more than 0.2 greater than the corresponding dilution multiple contrast color value, and the color value of the sample is more than 2 times of the corresponding dilution multiple contrast color value, so that the color result of the dilution multiple sample is determined to be positive.
Test 8: and (5) detecting a neutralizing antibody.
(1) After immunization the sera were incubated in a 56 ℃ water bath for 30 min.
(2) The antibody was diluted with DMEM medium containing 2% FBS at an initial dilution ratio of 1:2, 2-fold downward dilution.
(3) Purified rRSV-EGFP (1000 PFU/100. mu.l) was added, mixed and incubated at 37 ℃ for 1 hour.
(4) Mu.l of the mixture was added to a 96-well plate plated with a monolayer of HEp-2 cells and cultured at 37 ℃ for 48 hours.
(6) After 2 days of virus culture, the fluorescence intensity of the green fluorescent protein in each well is detected in an EnSpire 2300 Multilabel Reader, and the experimental data is positive when the fluorescence intensity of the experimental group is less than half of that of the non-added mouse serogroup. The positive value with the greatest dilution of antibody in each group was the final neutralizing antibody titer for that group.
Test 9: pulmonary virus titers were measured following challenge of mice with RSV.
(1) The challenged mice were sacrificed 4 days later and lung tissue was taken and washed in PBS.
(2) The lung tissue was weighed and 1ml of PBS containing 1% FBS was added to 0.1g of lung tissue.
(3) Lung tissue was ground and then centrifuged at 10000g for 1 minute, and 100. mu.l of the supernatant was collected.
(4) And adding 700 mu l of Trizol solution into the supernatant, and repeatedly blowing and uniformly mixing by using a pipette gun.
(5) Then, 200. mu.l of chloroform was added thereto, vortexed for 15 seconds, and allowed to stand at 4 ℃ for 10 minutes.
(6) The mixture after standing was centrifuged at 12000g at 4 ℃ for 15 minutes.
(7) The tube was gently removed and the layering occurred after centrifugation. The uppermost colorless aqueous layer was separated.
(8) About 500. mu.l of isopropanol was added to the aqueous layer of about 500. mu.l, and the mixture was mixed well and allowed to stand at 4 ℃ for 10 minutes.
(9) The mixture was centrifuged at 12000g at 4 ℃ for 10 minutes.
(10) Carefully remove the supernatant to avoid touching the pellet and add 500. mu.l of 75% ethanol solution.
(11) Gently mixed, the precipitate was thoroughly contacted with a 75% ethanol solution and allowed to stand at 4 ℃ for 10 minutes.
(12) After centrifugation at 12000g and 4 ℃ for 10 minutes, the supernatant was discarded.
(13) The clean bench was allowed to stand to evaporate the residual organic solvent, and 30. mu.l of RNase-free deionized water was added to resuspend the RNA.
(14) The concentration of the well-solubilized RNA was measured.
(15) The extracted RNA was Reverse transcribed into cDNA using Reverse Transcription kit Reverse Transcription System according to the instructions. Mu.g of RNA was mixed with 1. mu.l of random primer and 5. mu.l of RNase-free water was added.
(16) After mixing, incubation was carried out at 70 ℃ for 5 minutes and immediately placed on ice.
(17) Preparing a reaction system: mu.l of GoScript5 × Reaction Buffer, 1. mu.l of PCR Nucleotide Mix, 4. mu.l of MgCl2, 0.5. mu.l of Recombinant RNase viral Inhibitor, 1. mu.l of reverse transcriptase, and 15. mu.l of RNase-free water.
(18) Mu.l of the mixture of RNA and primers previously placed on ice was added to 15. mu.l of the reaction system and mixed well.
(19) Incubation was carried out at 25 ℃ for 5 minutes (annealing), at 42 ℃ for 1 hour (extension) and at 70 ℃ for 15 minutes (inactivation of reverse transcriptase). Subsequently, the reverse-transcribed cDNA was stored at-20 ℃.
(20) And (3) detecting the copy number of the cDNA after reverse transcription by using a fluorescent quantitative PCR instrument and an RT-qPCR method.
(21) And (3) designing a primer of real-time quantitative PCR (RT-qPCR) by taking the N gene preserved in the RSV Long strain as a template.
An upstream primer: RSA-15 '-AGATCAACTTCTGTCATCCAGCAA-3'
A downstream primer: RSA-25 '-GCACATCATAATTAGGAGTATCAAT-3'
(22) The standard substance for RT-qPCR quantification is a recombinant plasmid pMD18-T-N containing RSV Long strain N gene and taking pMD18-T as a vector (TaKaRa).
(23) Real-time quantitative PCR amplification is performed in a two-step process.
(24) Pre-denaturation at 95 ℃ for 15 minutes, PCR reaction at 95 ℃ for 15 seconds and 60 ℃ for 1 minute for 40 cycles, fluorescence data were collected and quantitative analysis was performed.
Test 10: and (3) analyzing the lung tissue damage of the mice after immunization.
In order to detect whether the vaccine has the effect of enhancing the lung tissue diseases of the mice, the lung tissue damage degree of the mice after challenge is analyzed by an immunohistochemical method, and the method comprises the following steps:
(1) lung tissues of mice 4 days after challenge were collected, washed with PBS, and then soaked in 10% formalin solution for more than 24 hours.
(2) The soaked tissue is embedded in paraffin, and paraffin section processing is carried out, wherein the section thickness is 5 mu m.
(3) The treated paraffin sections were subjected to hematoxylin-eosin staining (H & E).
(4) Stained tissues were photographed under a microscope and more than 10 fields were selected for each tissue.
(5) The photographs were scored for peribronchiolitis, perivascular inflammation and interstitial inflammation, respectively. The judgment standard is as follows: the score range of each index is 0-4 points, 0 point is no pathological change, and 4 points are severe pathological change. FI-RSV was used as a positive control.
As shown in FIG. 7, (A) IgG serum antibodies after 2 and 3 immunizations. Sera were collected 2 weeks after immunization. (B) Serum neutralizing antibody titers. The values are expressed as the reciprocal of the highest dilution of neutralizing antibody, i.e., the highest dilution of antibody with half the reduction in fluorescence intensity relative to the control. Serum was collected 14 days after the last immunization. The red line is the lowest detected Line (LOD). (C) RT-qPCR detects the copy number of RSV contained per microgram RNA in lung homogenates.
As shown in fig. 8, the H & E stained lung inflammation score was used in a semi-quantitative mode from 0-4 (0 means no lung pathology, 4 means extremely severe lung pathology). The evaluation was perivascular inflammation (A), peribronchiolar inflammation (B) and interstitial inflammation (C).
In conclusion, after the F protein before the respiratory syncytial virus fusion mutates the first furin cleavage site, the structure of Pre-F is more stable, compared with the wild type F protein, the expression amount is not reduced, and the Pre-fusion epitope expressed by M1-F in 293T cells is obviously higher than F; the second mutation of the F protein amino acid site enables the F protein amino acid site to form a disulfide bond so as to stabilize the epitope before fusion, compared with the wild type, the F protein amino acid site has more stable epitope before fusion and better protection in animal experiments, and the mutation of the furin site and the mutation of the disulfide bond are suitable for other virus strains of human RSV; the vaccine is applicable to various vaccine forms taking RSV F protein as antigen, such as: DNA vaccines, protein vaccines, adenovirus vaccines and recombinant virion vaccines.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are included in the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.
Sequence listing
<110> Beijing university of transportation
<120> respiratory syncytial virus pre-fusion F protein mutant and construction method thereof
<130> 2019
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 574
<212> PRT
<213> respiratory syncytial virus (respiratory syncytial virus)
<400> 1
Met Glu Leu Pro Ile Leu Lys Ala Asn Ala Ile Thr Thr Ile Leu Ala
1 5 10 15
Ala Val Thr Phe Cys Phe Ala Ser Ser Gln Asn Ile Thr Glu Glu Phe
20 25 30
Tyr Gln Ser Thr Cys Ser Ala Val Ser Lys Gly Tyr Leu Ser Ala Leu
35 40 45
Arg Thr Gly Trp Tyr Thr Ser Val Ile Thr Ile Glu Leu Ser Asn Ile
50 55 60
Lys Glu Asn Lys Cys Asn Gly Thr Asp Ala Lys Val Lys Leu Met Lys
65 70 75 80
Gln Glu Leu Asp Lys Tyr Lys Asn Ala Val Thr Glu Leu Gln Leu Leu
85 90 95
Met Gln Ser Thr Pro Ala Ala Asn Asn Arg Ala Arg Arg Glu Leu Pro
100 105 110
Arg Phe Met Asn Tyr Thr Leu Asn Asn Thr Lys Lys Thr Asn Val Thr
115 120 125
Leu Ser Lys Lys Arg Lys Arg Arg Phe Leu Gly Phe Leu Leu Gly Val
130 135 140
Gly Ser Ala Ile Ala Ser Gly Ile Ala Val Ser Lys Val Leu His Leu
145 150 155 160
Glu Gly Glu Val Asn Lys Ile Lys Ser Ala Leu Leu Ser Thr Asn Lys
165 170 175
Ala Val Val Ser Leu Ser Asn Gly Val Ser Val Leu Thr Ser Lys Val
180 185 190
Leu Asp Leu Lys Asn Tyr Ile Asp Lys Gln Leu Leu Pro Ile Val Asn
195 200 205
Lys Gln Ser Cys Arg Ile Ser Asn Ile Glu Thr Val Ile Glu Phe Gln
210 215 220
Gln Lys Asn Asn Arg Leu Leu Glu Ile Thr Arg Glu Phe Ser Val Asn
225 230 235 240
Val Gly Val Thr Thr Pro Val Ser Thr Tyr Met Leu Thr Asn Ser Glu
245 250 255
Leu Leu Ser Leu Ile Asn Asp Met Pro Ile Thr Asn Asp Gln Lys Lys
260 265 270
Leu Met Ser Asn Asn Val Gln Ile Val Arg Gln Gln Ser Tyr Ser Ile
275 280 285
Met Ser Ile Ile Lys Glu Glu Val Leu Ala Tyr Val Val Gln Leu Pro
290 295 300
Leu Tyr Gly Val Ile Asp Thr Pro Cys Trp Lys Leu His Thr Ser Pro
305 310 315 320
Leu Cys Thr Thr Asn Thr Lys Glu Gly Ser Asn Ile Cys Leu Thr Arg
325 330 335
Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala Gly Ser Val Ser Phe Phe
340 345 350
Pro Gln Ala Glu Thr Cys Lys Val Gln Ser Asn Arg Val Phe Cys Asp
355 360 365
Thr Met Asn Ser Leu Thr Leu Pro Ser Glu Val Asn Leu Cys Asn Val
370 375 380
Asp Ile Phe Asn Pro Lys Tyr Asp Cys Lys Ile Met Thr Ser Lys Thr
385 390 395 400
Asp Val Ser Ser Ser Val Ile Thr Ser Leu Gly Ala Ile Val Ser Cys
405 410 415
Tyr Gly Lys Thr Lys Cys Thr Ala Ser Asn Lys Asn Arg Gly Ile Ile
420 425 430
Lys Thr Phe Ser Asn Gly Cys Asp Tyr Val Ser Asn Lys Gly Val Asp
435 440 445
Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr Val Asn Lys Gln Glu Gly
450 455 460
Lys Ser Leu Tyr Val Lys Gly Glu Pro Ile Ile Asn Phe Tyr Asp Pro
465 470 475 480
Leu Val Phe Pro Ser Asp Glu Phe Asp Ala Ser Ile Ser Gln Val Asn
485 490 495
Glu Lys Ile Asn Gln Ser Leu Ala Phe Ile Arg Lys Ser Asp Glu Leu
500 505 510
Leu His Asn Val Asn Ala Gly Lys Ser Thr Thr Asn Ile Met Ile Thr
515 520 525
Thr Ile Ile Ile Val Ile Ile Val Ile Leu Leu Ser Leu Ile Ala Val
530 535 540
Gly Leu Leu Leu Tyr Cys Lys Ala Arg Ser Thr Pro Val Thr Leu Ser
545 550 555 560
Lys Asp Gln Leu Ser Gly Ile Asn Asn Ile Ala Phe Ser Asn
565 570
<210> 2
<211> 574
<212> PRT
<213> respiratory syncytial virus (respiratory syncytial virus)
<400> 2
Met Glu Leu Pro Ile Leu Lys Ala Asn Ala Ile Thr Thr Ile Leu Ala
1 5 10 15
Ala Val Thr Phe Cys Phe Ala Ser Ser Gln Asn Ile Thr Glu Glu Phe
20 25 30
Tyr Gln Ser Thr Cys Ser Ala Val Ser Lys Gly Tyr Leu Ser Ala Leu
35 40 45
Arg Thr Gly Trp Tyr Thr Ser Val Ile Thr Ile Glu Leu Ser Asn Ile
50 55 60
Lys Glu Asn Lys Cys Asn Gly Thr Asp Ala Lys Val Lys Leu Met Lys
65 70 75 80
Gln Glu Leu Asp Lys Tyr Lys Asn Ala Val Thr Glu Leu Gln Leu Leu
85 90 95
Met Gln Ser Thr Pro Ala Ala Asn Asn Asn Ala Asn Asn Glu Leu Pro
100 105 110
Arg Phe Met Asn Tyr Thr Leu Asn Asn Thr Lys Lys Thr Asn Val Thr
115 120 125
Leu Ser Lys Lys Arg Lys Arg Arg Phe Leu Gly Phe Leu Leu Gly Val
130 135 140
Gly Ser Ala Ile Ala Ser Gly Ile Ala Val Ser Lys Val Leu His Leu
145 150 155 160
Glu Gly Glu Val Asn Lys Ile Lys Ser Ala Leu Leu Ser Thr Asn Lys
165 170 175
Ala Val Val Ser Leu Ser Asn Gly Val Ser Val Leu Thr Ser Lys Val
180 185 190
Leu Asp Leu Lys Asn Tyr Ile Asp Lys Gln Leu Leu Pro Ile Val Asn
195 200 205
Lys Gln Ser Cys Arg Ile Ser Asn Ile Glu Thr Val Ile Glu Phe Gln
210 215 220
Gln Lys Asn Asn Arg Leu Leu Glu Ile Thr Arg Glu Phe Ser Val Asn
225 230 235 240
Val Gly Val Thr Thr Pro Val Ser Thr Tyr Met Leu Thr Asn Ser Glu
245 250 255
Leu Leu Ser Leu Ile Asn Asp Met Pro Ile Thr Asn Asp Gln Lys Lys
260 265 270
Leu Met Ser Asn Asn Val Gln Ile Val Arg Gln Gln Ser Tyr Ser Ile
275 280 285
Met Ser Ile Ile Lys Glu Glu Val Leu Ala Tyr Val Val Gln Leu Pro
290 295 300
Leu Tyr Gly Val Ile Asp Thr Pro Cys Trp Lys Leu His Thr Ser Pro
305 310 315 320
Leu Cys Thr Thr Asn Thr Lys Glu Gly Ser Asn Ile Cys Leu Thr Arg
325 330 335
Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala Gly Ser Val Ser Phe Phe
340 345 350
Pro Gln Ala Glu Thr Cys Lys Val Gln Ser Asn Arg Val Phe Cys Asp
355 360 365
Thr Met Asn Ser Leu Thr Leu Pro Ser Glu Val Asn Leu Cys Asn Val
370 375 380
Asp Ile Phe Asn Pro Lys Tyr Asp Cys Lys Ile Met Thr Ser Lys Thr
385 390 395 400
Asp Val Ser Ser Ser Val Ile Thr Ser Leu Gly Ala Ile Val Ser Cys
405 410 415
Tyr Gly Lys Thr Lys Cys Thr Ala Ser Asn Lys Asn Arg Gly Ile Ile
420 425 430
Lys Thr Phe Ser Asn Gly Cys Asp Tyr Val Ser Asn Lys Gly Val Asp
435 440 445
Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr Val Asn Lys Gln Glu Gly
450 455 460
Lys Ser Leu Tyr Val Lys Gly Glu Pro Ile Ile Asn Phe Tyr Asp Pro
465 470 475 480
Leu Val Phe Pro Ser Asp Glu Phe Asp Ala Ser Ile Ser Gln Val Asn
485 490 495
Glu Lys Ile Asn Gln Ser Leu Ala Phe Ile Arg Lys Ser Asp Glu Leu
500 505 510
Leu His Asn Val Asn Ala Gly Lys Ser Thr Thr Asn Ile Met Ile Thr
515 520 525
Thr Ile Ile Ile Val Ile Ile Val Ile Leu Leu Ser Leu Ile Ala Val
530 535 540
Gly Leu Leu Leu Tyr Cys Lys Ala Arg Ser Thr Pro Val Thr Leu Ser
545 550 555 560
Lys Asp Gln Leu Ser Gly Ile Asn Asn Ile Ala Phe Ser Asn
565 570
<210> 3
<211> 574
<212> PRT
<213> respiratory syncytial virus (respiratory syncytial virus)
<400> 3
Met Glu Leu Pro Ile Leu Lys Ala Asn Ala Ile Thr Thr Ile Leu Ala
1 5 10 15
Ala Val Thr Phe Cys Phe Ala Ser Ser Gln Asn Ile Thr Glu Glu Phe
20 25 30
Tyr Gln Ser Thr Cys Ser Ala Val Ser Lys Gly Tyr Leu Ser Ala Leu
35 40 45
Arg Thr Gly Trp Tyr Thr Ser Val Ile Thr Ile Glu Leu Ser Asn Ile
50 55 60
Lys Glu Asn Lys Cys Asn Gly Thr Asp Ala Lys Val Lys Leu Met Lys
65 70 75 80
Gln Glu Leu Asp Lys Tyr Lys Asn Ala Val Thr Glu Leu Gln Leu Leu
85 90 95
Met Gln Ser Thr Pro Ala Ala Cys Asn Asn Ala Asn Asn Glu Leu Pro
100 105 110
Arg Phe Met Asn Tyr Thr Leu Asn Asn Thr Lys Lys Thr Asn Val Thr
115 120 125
Leu Ser Lys Lys Arg Lys Arg Arg Phe Leu Gly Phe Leu Leu Gly Val
130 135 140
Gly Ser Ala Ile Ala Ser Gly Ile Ala Val Cys Lys Val Leu His Leu
145 150 155 160
Glu Gly Glu Val Asn Lys Ile Lys Ser Ala Leu Leu Ser Thr Asn Lys
165 170 175
Ala Val Val Ser Leu Ser Asn Gly Val Ser Val Leu Thr Ser Lys Val
180 185 190
Leu Asp Leu Lys Asn Tyr Ile Asp Lys Gln Leu Leu Pro Ile Val Asn
195 200 205
Lys Gln Ser Cys Arg Ile Ser Asn Ile Glu Thr Val Ile Glu Phe Gln
210 215 220
Gln Lys Asn Asn Arg Leu Leu Glu Ile Thr Arg Glu Phe Ser Val Asn
225 230 235 240
Val Gly Val Thr Thr Pro Val Ser Thr Tyr Met Leu Thr Asn Ser Glu
245 250 255
Leu Leu Ser Leu Ile Asn Asp Met Pro Ile Thr Asn Asp Gln Lys Lys
260 265 270
Leu Met Ser Asn Asn Val Gln Ile Val Arg Gln Gln Ser Tyr Ser Ile
275 280 285
Met Ser Ile Ile Lys Glu Glu Val Leu Ala Tyr Val Val Gln Leu Pro
290 295 300
Leu Tyr Gly Val Ile Asp Thr Pro Cys Trp Lys Leu His Thr Ser Pro
305 310 315 320
Leu Cys Thr Thr Asn Thr Lys Glu Gly Ser Asn Ile Cys Leu Thr Arg
325 330 335
Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala Gly Ser Val Ser Phe Phe
340 345 350
Pro Gln Ala Glu Thr Cys Lys Val Gln Ser Asn Arg Val Phe Cys Asp
355 360 365
Thr Met Asn Ser Leu Thr Leu Pro Ser Glu Val Asn Leu Cys Asn Val
370 375 380
Asp Ile Phe Asn Pro Lys Tyr Asp Cys Lys Ile Met Thr Ser Lys Thr
385 390 395 400
Asp Val Ser Ser Ser Val Ile Thr Ser Leu Gly Ala Ile Val Ser Cys
405 410 415
Tyr Gly Lys Thr Lys Cys Thr Ala Ser Asn Lys Asn Arg Gly Ile Ile
420 425 430
Lys Thr Phe Ser Asn Gly Cys Asp Tyr Val Ser Asn Lys Gly Val Asp
435 440 445
Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr Val Asn Lys Gln Glu Gly
450 455 460
Lys Ser Leu Tyr Val Lys Gly Glu Pro Ile Ile Asn Phe Tyr Asp Pro
465 470 475 480
Leu Val Phe Pro Ser Asp Glu Phe Asp Ala Ser Ile Ser Gln Val Asn
485 490 495
Glu Lys Ile Asn Gln Ser Leu Ala Phe Ile Arg Lys Ser Asp Glu Leu
500 505 510
Leu His Asn Val Asn Ala Gly Lys Ser Thr Thr Asn Ile Met Ile Thr
515 520 525
Thr Ile Ile Ile Val Ile Ile Val Ile Leu Leu Ser Leu Ile Ala Val
530 535 540
Gly Leu Leu Leu Tyr Cys Lys Ala Arg Ser Thr Pro Val Thr Leu Ser
545 550 555 560
Lys Asp Gln Leu Ser Gly Ile Asn Asn Ile Ala Phe Ser Asn
565 570
<210> 4
<211> 574
<212> PRT
<213> respiratory syncytial virus (respiratory syncytial virus)
<400> 4
Met Glu Leu Pro Ile Leu Lys Ala Asn Ala Ile Thr Thr Ile Leu Ala
1 5 10 15
Ala Val Thr Phe Cys Phe Ala Ser Ser Gln Asn Ile Thr Glu Glu Phe
20 25 30
Tyr Gln Ser Thr Cys Ser Ala Val Ser Lys Gly Tyr Leu Ser Ala Leu
35 40 45
Arg Thr Gly Trp Tyr Thr Ser Val Ile Cys Ile Glu Leu Ser Asn Ile
50 55 60
Lys Glu Asn Lys Cys Asn Gly Thr Asp Ala Lys Val Lys Leu Met Lys
65 70 75 80
Gln Glu Leu Asp Lys Tyr Lys Asn Ala Val Thr Glu Leu Gln Leu Leu
85 90 95
Met Gln Ser Thr Pro Ala Ala Asn Asn Asn Ala Asn Asn Glu Leu Pro
100 105 110
Arg Phe Met Asn Tyr Thr Leu Asn Asn Thr Lys Lys Thr Asn Val Thr
115 120 125
Leu Ser Lys Lys Arg Lys Arg Arg Phe Leu Gly Phe Leu Leu Gly Val
130 135 140
Gly Ser Ala Ile Ala Ser Gly Ile Ala Val Ser Lys Val Leu His Leu
145 150 155 160
Glu Gly Glu Val Asn Lys Ile Lys Ser Ala Leu Leu Ser Thr Asn Lys
165 170 175
Ala Val Val Ser Leu Ser Asn Gly Val Ser Val Leu Thr Cys Lys Val
180 185 190
Leu Asp Leu Lys Asn Tyr Ile Asp Lys Gln Leu Leu Pro Ile Val Asn
195 200 205
Lys Gln Ser Cys Arg Ile Ser Asn Ile Glu Thr Val Ile Glu Phe Gln
210 215 220
Gln Lys Asn Asn Arg Leu Leu Glu Ile Thr Arg Glu Phe Ser Val Asn
225 230 235 240
Val Gly Val Thr Thr Pro Val Ser Thr Tyr Met Leu Thr Asn Ser Glu
245 250 255
Leu Leu Ser Leu Ile Asn Asp Met Pro Ile Thr Asn Asp Gln Lys Lys
260 265 270
Leu Met Ser Asn Asn Val Gln Ile Val Arg Gln Gln Ser Tyr Ser Ile
275 280 285
Met Ser Ile Ile Lys Glu Glu Val Leu Ala Tyr Val Val Gln Leu Pro
290 295 300
Leu Tyr Gly Val Ile Asp Thr Pro Cys Trp Lys Leu His Thr Ser Pro
305 310 315 320
Leu Cys Thr Thr Asn Thr Lys Glu Gly Ser Asn Ile Cys Leu Thr Arg
325 330 335
Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala Gly Ser Val Ser Phe Phe
340 345 350
Pro Gln Ala Glu Thr Cys Lys Val Gln Ser Asn Arg Val Phe Cys Asp
355 360 365
Thr Met Asn Ser Leu Thr Leu Pro Ser Glu Val Asn Leu Cys Asn Val
370 375 380
Asp Ile Phe Asn Pro Lys Tyr Asp Cys Lys Ile Met Thr Ser Lys Thr
385 390 395 400
Asp Val Ser Ser Ser Val Ile Thr Ser Leu Gly Ala Ile Val Ser Cys
405 410 415
Tyr Gly Lys Thr Lys Cys Thr Ala Ser Asn Lys Asn Arg Gly Ile Ile
420 425 430
Lys Thr Phe Ser Asn Gly Cys Asp Tyr Val Ser Asn Lys Gly Val Asp
435 440 445
Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr Val Asn Lys Gln Glu Gly
450 455 460
Lys Ser Leu Tyr Val Lys Gly Glu Pro Ile Ile Asn Phe Tyr Asp Pro
465 470 475 480
Leu Val Phe Pro Ser Asp Glu Phe Asp Ala Ser Ile Ser Gln Val Asn
485 490 495
Glu Lys Ile Asn Gln Ser Leu Ala Phe Ile Arg Lys Ser Asp Glu Leu
500 505 510
Leu His Asn Val Asn Ala Gly Lys Ser Thr Thr Asn Ile Met Ile Thr
515 520 525
Thr Ile Ile Ile Val Ile Ile Val Ile Leu Leu Ser Leu Ile Ala Val
530 535 540
Gly Leu Leu Leu Tyr Cys Lys Ala Arg Ser Thr Pro Val Thr Leu Ser
545 550 555 560
Lys Asp Gln Leu Ser Gly Ile Asn Asn Ile Ala Phe Ser Asn
565 570
<210> 5
<211> 1725
<212> DNA
<213> respiratory syncytial virus (respiratory syncytial virus)
<400> 5
atggagctgc ctatcctgaa ggccaacgcc atcaccacaa ttctggccgc cgtgaccttc 60
tgttttgcca gcagccagaa catcaccgag gagttctacc agagcacctg tagcgccgtg 120
agcaagggct atctgagcgc cctgagaacc ggctggtaca ccagcgtgat caccatcgag 180
ctgagcaaca tcaaggagaa caagtgcaac ggcaccgacg ccaaggtgaa gctgatgaag 240
caggagctgg acaagtacaa gaacgccgtg accgaactgc agctgctgat gcagtctacc 300
cctgccgcca acaacaacgc caacaacgag ctgccccggt tcatgaacta caccctgaac 360
aacaccaaga aaaccaacgt gaccctgagc aagaagcgga agcggagatt cctgggcttt 420
ctgctgggag tgggctctgc catcgcctct ggcatcgccg tgtctaaggt gctgcacctg 480
gagggagagg tgaacaagat caagagcgcc ctgctgagca ccaataaggc cgtggtgagc 540
ctgagcaatg gcgtgagcgt gctgacaagc aaggtgctgg acctcaagaa ctacatcgac 600
aagcagctgc tgcccatcgt gaacaagcag agctgccgga tcagcaacat cgagaccgtg 660
atcgagttcc agcagaagaa caaccggctg ctggagatca ccagggagtt cagcgtgaat 720
gtgggcgtga ccacccctgt gagcacctac atgctgacca acagcgagct gctgagcctg 780
atcaacgaca tgcccatcac caacgaccag aagaagctga tgtccaacaa cgtgcagatc 840
gtgcggcagc agagctacag catcatgtcc atcatcaagg aggaggtgct ggcttacgtg 900
gtgcagctgc ctctgtacgg cgtgatcgac accccttgct ggaagctgca caccagccct 960
ctgtgcacca ccaataccaa ggagggcagc aacatctgcc tgaccaggac cgatagaggc 1020
tggtactgcg acaatgccgg cagcgtgagc ttctttccac aggccgagac ctgtaaggtg 1080
cagagcaacc gggtgttctg cgacaccatg aacagcctga ccctgccttc tgaggtgaac 1140
ctgtgcaacg tggacatctt caaccccaag tacgactgca agatcatgac cagcaagacc 1200
gacgtgagca gcagcgtgat tacaagcctg ggcgccatcg tgagctgtta cggcaagacc 1260
aagtgcaccg ccagcaacaa gaaccgcggc atcatcaaga ccttcagcaa cggctgcgac 1320
tacgtgagca acaagggcgt ggatacagtg agcgtgggca acaccctgta ctacgtcaac 1380
aagcaggagg gcaagagcct gtacgtgaag ggcgagccca tcatcaactt ctacgacccc 1440
ctggtgttcc ctagcgacga gttcgatgcc agcatcagcc aggtgaacga gaagatcaac 1500
cagagcctgg ccttcatcag gaagagcgac gagctgctgc acaatgtgaa cgccggcaag 1560
agcaccacca acatcatgat caccaccatc atcatcgtga tcatcgtcat cctgctgtcc 1620
ctgattgctg tgggcctgct gctgtactgt aaggccagaa gcacccccgt gaccctgtct 1680
aaggatcagc tgagcggcat caacaacatc gccttctcca actga 1725

Claims (6)

1. A respiratory syncytial virus pre-fusion F protein, characterized by: the pre-fusion F protein is obtained by performing a first mutation on amino acids 106, 108 and 109 of a wild-type F protein and then performing a second mutation, wherein the second mutation is selected from the following group:
a) the 104 th amino acid is mutated from Asn to Cys, the 155 th amino acid is mutated from Ser to Cys,
b) the 58 th amino acid is mutated from Thr to Cys, and the 190 th amino acid is mutated from Ser to Cys;
the first mutation is that the amino acids at the 106 th site, the 108 th site and the 109 th site are mutated from Arg to Asn;
the amino acid sequence of the wild type F protein is shown as SEQ ID NO: 1 is shown.
2. The method of claim 1, comprising the steps of:
step 1: carrying out first mutation on a first furin cleavage site of the wild type F protein, wherein the amino acid sequence after the first mutation is shown as SEQ ID NO: 2 is shown in the specification;
step 2: subjecting the polypeptide mutated in step 1 to a second mutation selected from the group consisting of:
a) the 104 th amino acid is mutated from Asn to Cys, the 155 th amino acid is mutated from Ser to Cys,
b) the 58 th amino acid is mutated from Thr to Cys, and the 190 th amino acid is mutated from Ser to Cys.
3. Use of the respiratory syncytial virus pre-fusion F protein of any one of claims 1-2 in the manufacture of a medicament for the prevention of respiratory syncytial virus infection.
4. Use of the respiratory syncytial virus pre-fusion F protein of any one of claims 1-2 in the preparation of a respiratory syncytial virus antibody.
5. Use of the respiratory syncytial virus pre-fusion F protein of any one of claims 1-2 in the preparation of a diagnostic reagent for respiratory syncytial virus.
6. Use of the respiratory syncytial virus pre-fusion F protein of any one of claims 1-2 in the preparation of a nucleic acid vaccine, a protein vaccine, a virus-like particle vaccine, or a vector vaccine for respiratory syncytial virus.
CN201910337141.5A 2019-04-25 2019-04-25 Respiratory syncytial virus pre-fusion F protein and application thereof Active CN110054668B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201910337141.5A CN110054668B (en) 2019-04-25 2019-04-25 Respiratory syncytial virus pre-fusion F protein and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201910337141.5A CN110054668B (en) 2019-04-25 2019-04-25 Respiratory syncytial virus pre-fusion F protein and application thereof

Publications (2)

Publication Number Publication Date
CN110054668A CN110054668A (en) 2019-07-26
CN110054668B true CN110054668B (en) 2021-09-10

Family

ID=67320742

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201910337141.5A Active CN110054668B (en) 2019-04-25 2019-04-25 Respiratory syncytial virus pre-fusion F protein and application thereof

Country Status (1)

Country Link
CN (1) CN110054668B (en)

Families Citing this family (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110878128A (en) * 2019-11-04 2020-03-13 中国疾病预防控制中心病毒病预防控制所 Human respiratory syncytial virus mucosal vaccine and preparation method and application thereof
CN113881742A (en) * 2021-04-28 2022-01-04 武汉爱博泰克生物科技有限公司 Preparation method and application of new crown spike protein in post-fusion state
CN113563480B (en) * 2021-07-12 2023-04-28 成都维瑾柏鳌生物医药科技有限公司 CLD protein mutant and application thereof
WO2023103440A1 (en) * 2021-12-06 2023-06-15 厦门大学 Antibody for recognizing rsv pre-f protein and use thereof
CN116003536A (en) * 2022-09-23 2023-04-25 暨南大学 F protein mutant before fusion of respiratory syncytial virus and application thereof
CN116284266B (en) * 2022-11-21 2024-01-19 怡道生物科技(苏州)有限公司 Mutant respiratory syncytial virus pre-fusion F protein and application thereof
CN117886902A (en) * 2023-12-21 2024-04-16 易康生物(苏州)有限公司 Improved respiratory syncytial virus fusion F protein mutant and application thereof
CN117586358A (en) * 2024-01-19 2024-02-23 北京安百胜生物科技有限公司 Respiratory Syncytial Virus (RSV) polypeptides having immunogenicity

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9060975B2 (en) * 2013-03-14 2015-06-23 Mucosis Bv Heat-stable respiratory syncytial virus F protein oligomers and their use in immunological compositions
US20160228536A1 (en) * 2013-09-18 2016-08-11 Medimmune, Llc Recombinant respiratory syncytial virus (rsv) and vaccines
WO2017040387A2 (en) * 2015-08-31 2017-03-09 Technovax, Inc. Human respiratory syncytial virus (hrsv) virus-like particles (vlps) based vaccine
PE20181354A1 (en) * 2015-12-23 2018-08-22 Pfizer RSV PROTEIN F MUTANTS
WO2019032480A1 (en) * 2017-08-07 2019-02-14 Avatar Medical, Llc Conformationally stabilized rsv pre-fusion f proteins

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
Proteolytic Activation of Respiratory Syncytial Virus Fusion Protein;Gert Zimmer等;《Journal of Biological Chemistry》;20010824;第276卷(第34期);第31642-31650页 *
Synthetic construct fusion protein (F) gene, complete cds;GenBank;《NCBI》;20071017;EF566942.1 *

Also Published As

Publication number Publication date
CN110054668A (en) 2019-07-26

Similar Documents

Publication Publication Date Title
CN110054668B (en) Respiratory syncytial virus pre-fusion F protein and application thereof
CN113185613B (en) Novel coronavirus S protein and subunit vaccine thereof
WO2022262142A1 (en) Recombinant sars-cov-2 rbd tripolymer protein vaccine capable of generating broad-spectrum cross-neutralization activity, preparation method therefor, and application thereof
CN105669838B (en) Neutralizing epitopes from varicella-zoster virus gE protein and antibodies thereto
CN107660150A (en) The associated proteins of IL 18 (IL 18BP) and antibody are in inflammatory disease
CN103379916B (en) Respiratory syncytial virus vaccines
CA2834376A1 (en) Neutralizing antibodies to nipah and hendra virus
ES2843543T3 (en) Hendra and Nipah virus F-glycoprotein antibodies
CN110272473B (en) Influenza A universal virus-like particle and preparation method and application thereof
CN114989308B (en) Novel coronavirus chimeric nucleic acid vaccine and use thereof
CN113354717A (en) Novel coronavirus SARS-CoV-2 broad-spectrum polypeptide antigen and its specific neutralizing antibody and application
Perez et al. A single dose of an MVA vaccine expressing a prefusion-stabilized SARS-CoV-2 spike protein neutralizes variants of concern and protects mice from a lethal SARS-CoV-2 infection
CN113416245A (en) Neutralizing antibody capable of combining SARS-CoV-2 virus RBD protein and application thereof
CN115925822A (en) Recombinant protein and application thereof in preparation of respiratory syncytial virus vaccine
WO2022179535A1 (en) Anti-sars-cov-2 nucleocapsid protein monoclonal antibody, and preparation method therefor and use thereof
WO2022068846A1 (en) Novel coronavirus mrna vaccine, preparation method therefor and use thereof
WO2024002129A1 (en) Novel coronavirus trimer chimeric vaccine and use thereof
KR20150135231A (en) Human Antibody Specific To Human Metapneumovirus, or Antigen-Binding Fragment Thereof
WO2020128496A1 (en) Peste des petits ruminant virus (pprv) with chimeric n protein and corresponding vaccine
JP6903351B2 (en) Recombinant RSV antigen
CN115678906B (en) Optimized novel coronavirus chimeric nucleic acid vaccine and uses thereof
WO2021238854A1 (en) Monoclonal antibody against sars-cov-2 spike protein, preparation method therefor, and application thereof
CN114478717A (en) Recombinant novel coronavirus protein vaccine, preparation method and application thereof
CN111732667B (en) Peste des petits ruminants virus genetic engineering subunit vaccine
WO2023143176A1 (en) Broad-spectrum antibody of sars-cov-2 virus and use thereof

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant