CN117264031A - 一种B群脑膜炎奈瑟菌fHBP重组蛋白及其应用 - Google Patents
一种B群脑膜炎奈瑟菌fHBP重组蛋白及其应用 Download PDFInfo
- Publication number
- CN117264031A CN117264031A CN202311564091.7A CN202311564091A CN117264031A CN 117264031 A CN117264031 A CN 117264031A CN 202311564091 A CN202311564091 A CN 202311564091A CN 117264031 A CN117264031 A CN 117264031A
- Authority
- CN
- China
- Prior art keywords
- fhbp
- recombinant protein
- protein
- adjuvant
- group
- 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.)
- Granted
Links
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 title claims abstract description 29
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 title claims abstract description 29
- 108700026633 Neisseria meningitidis factor H-binding Proteins 0.000 title abstract description 6
- 229960005486 vaccine Drugs 0.000 claims abstract description 22
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 18
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 18
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 9
- 238000004519 manufacturing process Methods 0.000 claims abstract description 4
- 239000002671 adjuvant Substances 0.000 claims description 17
- 241000588650 Neisseria meningitidis Species 0.000 claims description 8
- 201000009906 Meningitis Diseases 0.000 claims description 7
- 239000000203 mixture Substances 0.000 claims description 7
- 239000002773 nucleotide Substances 0.000 claims description 7
- 125000003729 nucleotide group Chemical group 0.000 claims description 7
- 239000013598 vector Substances 0.000 claims description 7
- 229940124912 Neisseria meningitidis vaccine Drugs 0.000 claims description 3
- 102000004127 Cytokines Human genes 0.000 claims description 2
- 108090000695 Cytokines Proteins 0.000 claims description 2
- 108010040721 Flagellin Proteins 0.000 claims description 2
- 241000241413 Propolis Species 0.000 claims description 2
- AZDRQVAHHNSJOQ-UHFFFAOYSA-N alumane Chemical class [AlH3] AZDRQVAHHNSJOQ-UHFFFAOYSA-N 0.000 claims description 2
- 239000003638 chemical reducing agent Substances 0.000 claims description 2
- -1 cpGDNA Proteins 0.000 claims description 2
- 239000003814 drug Substances 0.000 claims description 2
- 239000003937 drug carrier Substances 0.000 claims description 2
- 230000003308 immunostimulating effect Effects 0.000 claims description 2
- 239000002502 liposome Substances 0.000 claims description 2
- 229940115272 polyinosinic:polycytidylic acid Drugs 0.000 claims description 2
- 229940069949 propolis Drugs 0.000 claims description 2
- 229930182490 saponin Natural products 0.000 claims description 2
- 150000007949 saponins Chemical class 0.000 claims description 2
- 235000017709 saponins Nutrition 0.000 claims description 2
- 229920001184 polypeptide Polymers 0.000 claims 1
- 102000004196 processed proteins & peptides Human genes 0.000 claims 1
- 108090000765 processed proteins & peptides Proteins 0.000 claims 1
- 239000000427 antigen Substances 0.000 abstract description 8
- 102000036639 antigens Human genes 0.000 abstract description 8
- 108091007433 antigens Proteins 0.000 abstract description 8
- 230000001900 immune effect Effects 0.000 abstract description 5
- 238000002156 mixing Methods 0.000 abstract description 4
- 238000001228 spectrum Methods 0.000 abstract description 2
- 101710186862 Factor H binding protein Proteins 0.000 description 54
- 239000000243 solution Substances 0.000 description 11
- 235000018102 proteins Nutrition 0.000 description 9
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 8
- 150000004676 glycans Chemical class 0.000 description 8
- 239000005017 polysaccharide Substances 0.000 description 8
- 229920001282 polysaccharide Polymers 0.000 description 8
- 238000000746 purification Methods 0.000 description 6
- 210000004027 cell Anatomy 0.000 description 5
- 239000000463 material Substances 0.000 description 5
- KZMAWJRXKGLWGS-UHFFFAOYSA-N 2-chloro-n-[4-(4-methoxyphenyl)-1,3-thiazol-2-yl]-n-(3-methoxypropyl)acetamide Chemical compound S1C(N(C(=O)CCl)CCCOC)=NC(C=2C=CC(OC)=CC=2)=C1 KZMAWJRXKGLWGS-UHFFFAOYSA-N 0.000 description 4
- 241000699670 Mus sp. Species 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 239000011780 sodium chloride Substances 0.000 description 4
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 4
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 238000010828 elution Methods 0.000 description 3
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 3
- 230000001939 inductive effect Effects 0.000 description 3
- 238000000034 method Methods 0.000 description 3
- 238000010369 molecular cloning Methods 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 210000002966 serum Anatomy 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- 238000002965 ELISA Methods 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- 108010042653 IgA receptor Proteins 0.000 description 2
- 241000588677 Neisseria meningitidis serogroup B Species 0.000 description 2
- PXHVJJICTQNCMI-UHFFFAOYSA-N Nickel Chemical compound [Ni] PXHVJJICTQNCMI-UHFFFAOYSA-N 0.000 description 2
- 102100034014 Prolyl 3-hydroxylase 3 Human genes 0.000 description 2
- 206010040047 Sepsis Diseases 0.000 description 2
- 239000012505 Superdex™ Substances 0.000 description 2
- WNROFYMDJYEPJX-UHFFFAOYSA-K aluminium hydroxide Chemical compound [OH-].[OH-].[OH-].[Al+3] WNROFYMDJYEPJX-UHFFFAOYSA-K 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 229940090821 bexsero Drugs 0.000 description 2
- 230000000903 blocking effect Effects 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 230000003053 immunization Effects 0.000 description 2
- 238000002649 immunization Methods 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 239000002808 molecular sieve Substances 0.000 description 2
- 239000013642 negative control Substances 0.000 description 2
- 239000000825 pharmaceutical preparation Substances 0.000 description 2
- 239000012898 sample dilution Substances 0.000 description 2
- 208000013223 septicemia Diseases 0.000 description 2
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 description 2
- 238000002604 ultrasonography Methods 0.000 description 2
- NHBKXEKEPDILRR-UHFFFAOYSA-N 2,3-bis(butanoylsulfanyl)propyl butanoate Chemical compound CCCC(=O)OCC(SC(=O)CCC)CSC(=O)CCC NHBKXEKEPDILRR-UHFFFAOYSA-N 0.000 description 1
- UAIUNKRWKOVEES-UHFFFAOYSA-N 3,3',5,5'-tetramethylbenzidine Chemical compound CC1=C(N)C(C)=CC(C=2C=C(C)C(N)=C(C)C=2)=C1 UAIUNKRWKOVEES-UHFFFAOYSA-N 0.000 description 1
- 206010001052 Acute respiratory distress syndrome Diseases 0.000 description 1
- 206010001361 Adrenal haemorrhage Diseases 0.000 description 1
- 208000023275 Autoimmune disease Diseases 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 108020004705 Codon Proteins 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 108010060123 Conjugate Vaccines Proteins 0.000 description 1
- 206010019280 Heart failures Diseases 0.000 description 1
- 206010061216 Infarction Diseases 0.000 description 1
- 102000004895 Lipoproteins Human genes 0.000 description 1
- 108090001030 Lipoproteins Proteins 0.000 description 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 1
- 208000009525 Myocarditis Diseases 0.000 description 1
- SQVRNKJHWKZAKO-PFQGKNLYSA-N N-acetyl-beta-neuraminic acid Chemical compound CC(=O)N[C@@H]1[C@@H](O)C[C@@](O)(C(O)=O)O[C@H]1[C@H](O)[C@H](O)CO SQVRNKJHWKZAKO-PFQGKNLYSA-N 0.000 description 1
- 206010030113 Oedema Diseases 0.000 description 1
- 208000013616 Respiratory Distress Syndrome Diseases 0.000 description 1
- 206010040070 Septic Shock Diseases 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- ILRRQNADMUWWFW-UHFFFAOYSA-K aluminium phosphate Chemical compound O1[Al]2OP1(=O)O2 ILRRQNADMUWWFW-UHFFFAOYSA-K 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 206010003246 arthritis Diseases 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- SQVRNKJHWKZAKO-UHFFFAOYSA-N beta-N-Acetyl-D-neuraminic acid Natural products CC(=O)NC1C(O)CC(O)(C(O)=O)OC1C(O)C(O)CO SQVRNKJHWKZAKO-UHFFFAOYSA-N 0.000 description 1
- 238000009739 binding Methods 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000005018 casein Substances 0.000 description 1
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 description 1
- 235000021240 caseins Nutrition 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 210000003169 central nervous system Anatomy 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 230000024203 complement activation Effects 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 208000035475 disorder Diseases 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 206010014801 endophthalmitis Diseases 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000013604 expression vector Substances 0.000 description 1
- 238000011010 flushing procedure Methods 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000002008 hemorrhagic effect Effects 0.000 description 1
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 1
- 239000005457 ice water Substances 0.000 description 1
- 230000006058 immune tolerance Effects 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 230000007574 infarction Effects 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 238000010255 intramuscular injection Methods 0.000 description 1
- 239000007927 intramuscular injection Substances 0.000 description 1
- 208000037909 invasive meningococcal disease Diseases 0.000 description 1
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 230000031700 light absorption Effects 0.000 description 1
- 201000002818 limb ischemia Diseases 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 101150095079 menB gene Proteins 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 229910052759 nickel Inorganic materials 0.000 description 1
- 230000004768 organ dysfunction Effects 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 208000008494 pericarditis Diseases 0.000 description 1
- 230000002093 peripheral effect Effects 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 230000020971 positive regulation of blood coagulation Effects 0.000 description 1
- 230000018338 positive regulation of fibrinolysis Effects 0.000 description 1
- 229940023143 protein vaccine Drugs 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 210000002345 respiratory system Anatomy 0.000 description 1
- 230000036303 septic shock Effects 0.000 description 1
- 208000017520 skin disease Diseases 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 229940035144 trumenba Drugs 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/22—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Neisseriaceae (F)
-
- 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
- A61K39/02—Bacterial antigens
- A61K39/095—Neisseria
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
-
- 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
- A61P31/04—Antibacterial agents
-
- 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
- C12N15/70—Vectors or expression systems specially adapted for E. coli
-
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Zoology (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Molecular Biology (AREA)
- Public Health (AREA)
- Biophysics (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- Veterinary Medicine (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Plant Pathology (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Physics & Mathematics (AREA)
- Epidemiology (AREA)
- Mycology (AREA)
- Communicable Diseases (AREA)
- Oncology (AREA)
- Immunology (AREA)
- Gastroenterology & Hepatology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Neurology (AREA)
- Neurosurgery (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Peptides Or Proteins (AREA)
Abstract
本发明提供一种B群脑膜炎奈瑟菌fHBP重组蛋白及其应用。该fHBP重组蛋白的氨基酸序列如SEQ ID NO:1所示。该fHBP重组蛋白诱导抗fHBP的免疫学效果优于本发明中fHBP三个变体等质量比混合所带来的免疫学效果。应用本发明的fHBP重组蛋白制备疫苗,只需要制备单个蛋白分子,生产工艺更为简单,却能产生优于多个蛋白混合疫苗的免疫效果,同时其抗原谱比现有疫苗更广、能够覆盖更多类型的变体菌株。
Description
技术领域
本发明涉及生物医药技术领域,特别涉及B群脑膜炎奈瑟菌fHBP重组蛋白及其应用。
背景技术
流行性脑脊髓膜炎(简称流脑)是一种由脑膜炎奈瑟菌 (Neisseriameningitidis)导致的以急性脑脊髓膜炎和败血症为主要症状的通过呼吸道传播的传染病。根据荚膜多糖的特征可将脑膜炎奈瑟菌分为 A、B、C、Y、W135等13个血清群。当前上市的四价脑膜炎球菌多糖或多糖蛋白结合疫苗可有效预防 A、C、Y和W135 致病血清群,减少侵袭性脑膜炎球菌疾病发生。
B群脑膜炎奈瑟菌(Neisseria meningitidis group B,MenB)俗称B群脑膜炎球菌。与 A、C、Y、W135血清群荚膜多糖不同,MenB荚膜多糖的主要成分与人中枢神经***抗原N–乙酰神经氨酸聚合物结构相似,可导致人体对MenB荚膜多糖产生免疫耐受,且有诱发自身免疫疾病的危险,因此MenB荚膜多糖不适合用作候选疫苗抗原。
目前对MenB疫苗的开发主要集中在非荚膜多糖免疫原,因子H结合蛋白(fHBP)是一种脂蛋白,它几乎在所有MenB菌株细胞膜上表达。fHBP的一个重要功能是与人补体调节因子H相结合,使脑膜炎奈瑟菌逃避宿主的杀菌作用,提高在血液中的存活能力。将fHBP作为MenB疫苗抗原,引发的抗fHBP抗体可以直接激活经典补体途径溶菌,也可以阻止fH与细菌表面结合。因此,fHBP是目前最有前景的一种疫苗抗原。
目前国外已上市的MenB疫苗主要有两款,Bexsero和Trumenba。Bexsero包含国外流行株的外膜囊泡以及其他多种脑膜炎球菌抗原的制剂。Trumenba含有吸附在磷酸铝上的两个脂化MenB fHBP抗原。上述2款MenB蛋白疫苗中fHBP蛋白序列主要针对fHBP 变体1(fHBP V1)和fHBP 变体3(fHBP V3),无法覆盖fHBP类型以fHBP 变体2(fHBP V2)为主的流行毒株。研发具有广泛保护性、更加安全有效、降低生产成本的 MenB 疫苗具有十分重要的意义。
发明内容
为了解决目前B群脑膜炎奈瑟菌疫苗技术和供应种类不足,本发明提供了一种B群脑膜炎奈瑟菌fHBP重组蛋白及其应用。
本发明提供如下具体的技术方案。
本发明提供一种fHBP重组蛋白,其包含如SEQ ID NO:1所示的氨基酸序列,所述的fHBP重组蛋白能同时诱导抗fHBP 变体1(fHBP V1)、fHBP 变体2(fHBP V2)和fHBP变体3(fHBP V3)的抗体。
在一些实施方案中,本发明的fHBP重组蛋白还包含纯化标签,如组氨酸标签。
在一些实施方案中,本发明提供一种fHBP重组蛋白,其氨基酸序列如SEQ ID NO:1所示。
本发明提供一种fHBP V1,其氨基酸序列为SEQ ID NO:2。
本发明提供一种fHBP V2,其氨基酸序列为SEQ ID NO:3。
本发明提供一种fHBP V3,其氨基酸序列为SEQ ID NO:4。
本发明提供编码上述fHBP重组蛋白的核苷酸,具体的核苷酸序列是本领域技术人员通过密码子表等常规手段容易获得的。
本发明提供包含编码上述fHBP重组蛋白的核苷酸的载体。
在一些实施方案中,本发明提供的载体包括克隆载体和表达载体。
本发明还提供包含上述核苷酸或载体的宿主细胞。
在一些实施方案中,表达上述载体的宿主细胞为CHO或E.coli。
本发明提供一种免疫组合物,其含有本发明所述的fHBP重组蛋白。
优选地,本发明的免疫组合物还包含药学上可接受的载体。
本发明提供一种B群脑膜炎奈瑟菌疫苗,其含有上述的免疫组合物和佐剂。
在一些实施方案中,本发明的疫苗含有的佐剂选自:铝盐类佐剂、弗氏完全佐剂、蜂胶佐剂、水油佐剂、细胞因子、CpGDNA、鞭毛蛋白、基因工程减毒素、免疫刺激复合物、脂质体、皂苷类、Poly(I:C)佐剂中的至少一种。
本发明提供上述B群脑膜炎奈瑟菌fHBP重组蛋白、免疫组合物或疫苗的药物制剂,所述药物用于预防或治疗B 群脑膜炎奈瑟菌相关疾病。
本发明中的B群脑膜炎奈瑟菌相关疾病包括但不限于:脑脊髓膜炎、败血症、脓毒性休克、关节炎、心肌炎、心包炎、眼内炎、脑膜炎、出血性皮肤病、激活血纤蛋白溶解和血液凝结,器官功能失调,例如肾、肺和心力衰竭,肾上腺出血和肌肉梗死,毛细血管渗漏,水肿,外周肢体缺血,呼吸窘迫综合征。
优选地,所述的B群脑膜炎奈瑟菌相关疾病为脑脊髓膜炎。
基于上述技术方案,本发明具有以下有益效果:
本发明的fHBP重组蛋白诱导抗fHBP的免疫学效果优于本发明中fHBP三个变体(fHBP V1、fHBP V2 和fHBP V3)等质量比混合所带来的免疫学效果。同时,本发明fHBP重组蛋白诱导抗fHBP的免疫学效果优于本领域已知的其他fHBP重组蛋白(在实施例中作为对照分子)分子等质量比混合所带来的免疫学效果。使用本发明的fHBP重组蛋白制备疫苗,只需要制备单个蛋白分子,生产工艺更为简单,却能产生优于多个蛋白混合疫苗的免疫效果,同时其抗原谱比现有疫苗更广、能够覆盖更多类型的变体菌株。
附图说明
图1为fHBP重组蛋白纯化后的SDS-PAGE鉴定结果,箭头指示的条带为断裂条带。
图2为fHBP重组蛋白和对照分子诱导抗fHBP抗体滴度图,其中A为诱导抗fHBP V1的抗体滴度图, B为诱导抗fHBP V2的抗体滴度图, C为诱导抗fHBP V3的抗体滴度图。
具体实施方式
以下结合实例对本发明的原理和特征进行描述,所举实例只用于解释本发明,并非用于限定本发明的范围。在进一步描述本发明具体实施方式之前,应理解,本发明的保护范围不局限于下述特定的具体实施方案;还应当理解,本发明实施例中使用的术语是为了描述特定的具体实施方案,而不是为了限制本发明的保护范围。下列实施例中未注明具体条件的试验方法,通常按照常规条件,或者按照各制造商所建议的条件。当实施例给出数值范围时,应理解,除非本发明另有说明,每个数值范围的两个端点以及两个端点之间任何一个数值均可选用。除非另外定义,本发明中使用的所有技术和科学术语与本技术领域技术人员通常理解的意义相同。除实施例中使用的具体方法、设备、材料外,根据本技术领域的技术人员对现有技术的掌握及本发明的记载,还可以使用与本发明实施例中所述的方法、设备、材料相似或等同的现有技术的任何方法、设备和材料来实现本发明。下列实施例中所用的实验材料,如无特殊说明,均为自常规试剂公司购买得到。
实施例1 fHBP重组蛋白的表达和纯化
1. 实验材料:
囊式滤器(Bricap C01:180cm2)购自Cobetter,膜包购自Millipore,HisTrapexcel购自Cytiva,分子筛(Superdex 200pg 10/300 GL)购自Cytiva。
2. 实验方法:
2.1、根据分子克隆的常规技术制备SEQ ID NO:1所示的fHBP重组蛋白。
对照分子1:通过分子克隆的常规手段制备SEQ ID NO:2所示的fHBP V1、SEQ IDNO:3所示的fHBP V2、SEQ ID NO:4所示的fHBP V3,由fHBP V1、fHBP V2和fHBP V3等质量比混合得到对照分子1。
对照分子2:由两种现有技术的fHBP重组蛋白A和B等质量比混合得到,其中蛋白A的氨基酸序列为SEQ ID NO:5,蛋白B的氨基酸序列为SEQ ID NO:6,上述的蛋白A和蛋白B,本领域技术人员均容易通过分子克隆的常规手段制备得到。
2.2、诱导表达条件:接种BL21(DE3)阳性单克隆菌种于800 mL LB(Amp+)培养基中, 37℃,220 rpm下培养4-5 h,待培养液OD600约0.6-0.8时,将菌液转移至18℃,加入IPTG 至终浓度为0.5 mM, 200 rpm下诱导蛋白表达16 h。
2.3、离心收菌:将培养完毕的菌液室温下7000 g离心收集菌体,丢弃培养液,用8
0 mL 150 mM NaCl,20 mM Tris 7.4溶液重悬菌体。
2.4、超声破碎:将重悬菌液置于冰水浴中进行超声破碎。变幅杆2号,50%功率,超声3 s,停7 s,超声总时长为12 min。
2.5、离心收集目的蛋白:13000 g,4℃,30 min,收集细胞破碎上清。
2.6、Histrap纯化目的蛋白:冲洗溶液为20mM Tris-HCl,150mM NaCl,pH 7.4;洗脱溶液为20mM Tris-HCl,150mM NaCl,500mM咪唑,pH 7.4。使用Histrap excel-5ml NI柱纯化;冲洗溶液平衡Histrap excel-5ml 10 CV后,上样;上样结束用冲洗溶液冲洗10CV层析柱;用 2% 洗脱溶液冲洗10CV,洗去杂蛋白;用2%-100% 洗脱溶液线性洗脱目的蛋白15CV;洗脱结束后SDS-PAGE检测蛋白纯度。
2.7、SEC纯化目的蛋白:收集镍柱纯化后的目的蛋白用浓缩管浓缩至1ml后进行分子筛(Superdex 200pg 10/300 GL)分离纯化,纯化buffer为20mM Tris-HCl, 150mM NaCl,pH 7.4,洗脱结束后SDS-PAGE检测蛋白纯度,SDS-PAGE鉴定纯化后的fHBP重组蛋白如图1所示。BCA法测定蛋白浓度,适当温度保存用于后续结合反应。
实施例2:动物免疫试验
(1) 实验材料
小鼠:5-6周龄的BALB/c品系的雌性小鼠(购自广东维通利华实验动物技术有限公司)
佐剂:氢氧化铝佐剂,其他试剂耗材均为商品化常规试剂耗材。
(2)实验步骤
如表1所示,在第0天和第14天分别免疫接种含有实施例1制备得到的fHBP重组蛋白的疫苗、包含对照分子1的疫苗和包含对照分子2的疫苗,接种方法为肌肉注射,接种剂量为9μg/剂/只,每剂疫苗并混合75μg的氢氧化铝佐剂。在第28天采血分离血清,用于抗体IgG的检测。
表1. 动物免疫方案
(3)结合抗体检测:
先用fHBP三种变体蛋白包被ELISA板,将包被好的ELISA板在Blocker Casein inPBS封闭液(购自ThermoFisher)中封闭1-4小时;将表1中由各实验组小鼠在免疫结束所采集的血清样本从1:300开始进行三倍梯度稀释至656100,将稀释液加入每个孔,阴性对照为样本稀释液,孵育2-3小时,用HRP偶联的羊抗鼠二抗孵育1小时后,用TMB底物进行显色,显色完成后用1 M盐酸终止反应,用酶标仪测定吸光值(OD),主波长450nm,参考波长620nm,样品OD值=OD450-OD620,终止后5分钟内完成测定。实验结果见图2。
小鼠免疫试验结果发现,实验组的疫苗诱导抗fHBP变体V1、V2和V3的抗体滴度均高于对照分子1和对照分子2。可见,本发明的fHBP重组蛋白制备得到的疫苗能够诱导小鼠产生更高滴度的血清抗体,具有更优异的免疫原性。
综上所述,上述各实施例仅为本发明的较佳实施例而已,并不用以限定本发明的保护范围,凡在本发明的精神和原则之内,所做的任何修改、等同替换、改进等,皆应包含在本发明的保护范围内。
Claims (8)
1.一种fHBP重组蛋白,其特征在于,所述的重组蛋白包含如SEQ ID NO:1所示的氨基酸序列。
2.编码如权利要求1所述的fHBP重组蛋白的核苷酸。
3.包含权利要求2所述核苷酸的载体。
4.包含权利要求2所述的核苷酸或权利要求3所述的载体的宿主细胞。
5.一种免疫组合物,其特征在于,包含权利要求1所述的fHBP重组蛋白和药学上可接受的载体。
6.一种B群脑膜炎奈瑟菌疫苗,其特征在于,其包含权利要求5所述的免疫组合物和佐剂。
7.根据权利要求6所述的疫苗,其特征在于,其中所述的佐剂选自:铝盐类佐剂、弗氏完全佐剂、蜂胶佐剂、水油佐剂、细胞因子、CpGDNA、鞭毛蛋白、基因工程减毒素、免疫刺激复合物、脂质体、皂苷类、Poly(I:C)佐剂中的至少一种。
8.权利要求1所述的fHBP重组蛋白、权利要求5所述的免疫组合物或权利要求6-7任一项所述的疫苗用于制备预防或治疗B群脑膜炎奈瑟菌引起的脑脊髓膜炎的药物的用途。
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202311462742 | 2023-11-03 | ||
CN2023114627421 | 2023-11-03 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN117264031A true CN117264031A (zh) | 2023-12-22 |
CN117264031B CN117264031B (zh) | 2024-01-30 |
Family
ID=89218281
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202311564091.7A Active CN117264031B (zh) | 2023-11-03 | 2023-11-22 | 一种B群脑膜炎奈瑟菌fHBP重组蛋白及其应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN117264031B (zh) |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102028941A (zh) * | 2011-02-11 | 2011-04-27 | 中国医学科学院医学生物学研究所 | 一种b群脑膜炎球菌重组蛋白嵌合疫苗及其制备方法 |
CN107823638A (zh) * | 2017-11-05 | 2018-03-23 | 北京智飞绿竹生物制药有限公司 | 一种b群脑膜炎球菌重组嵌合蛋白疫苗及其制备方法 |
CN110903400A (zh) * | 2019-11-08 | 2020-03-24 | 苏州微超生物科技有限公司 | B群脑膜炎球菌fHBP多变体融合蛋白及其制备方法与应用 |
US20210277069A1 (en) * | 2018-08-09 | 2021-09-09 | Glaxosmithkline Biologicals Sa | Modified meningococcal fhbp polypeptides |
-
2023
- 2023-11-22 CN CN202311564091.7A patent/CN117264031B/zh active Active
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102028941A (zh) * | 2011-02-11 | 2011-04-27 | 中国医学科学院医学生物学研究所 | 一种b群脑膜炎球菌重组蛋白嵌合疫苗及其制备方法 |
CN107823638A (zh) * | 2017-11-05 | 2018-03-23 | 北京智飞绿竹生物制药有限公司 | 一种b群脑膜炎球菌重组嵌合蛋白疫苗及其制备方法 |
US20210277069A1 (en) * | 2018-08-09 | 2021-09-09 | Glaxosmithkline Biologicals Sa | Modified meningococcal fhbp polypeptides |
CN110903400A (zh) * | 2019-11-08 | 2020-03-24 | 苏州微超生物科技有限公司 | B群脑膜炎球菌fHBP多变体融合蛋白及其制备方法与应用 |
Non-Patent Citations (1)
Title |
---|
CYNTHIA BURMAN等: "A review of the immunogenicity, safety and current recommendations for the meningococcal serogroup B vaccine, MenB-FHbp", J CLIN PHARM THER, vol. 45, no. 2, pages 270 - 281 * |
Also Published As
Publication number | Publication date |
---|---|
CN117264031B (zh) | 2024-01-30 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
KR100205462B1 (ko) | 라임 질병에 대한 왁진 | |
FI113009B (fi) | Menetelmä rokotteen valmistamiseksi Neisseria meningitidis-bakteeria vastaan sekä Neisseria meningitidis -kanta | |
FI106844B (fi) | Menetelmä Moraxella catarrhalisin antigeeneihin liittyvien koostumusten ja vasta-aineiden valmistamiseksi sekä DNA-segmentti, yhdistelmävektori ja yhdistelmäisäntäsolu | |
FI111336B (fi) | Menetelmä Staphylococcus epidermidis tyyppi I:n polysakkaridiantigeenin sisältävän rokotteen ja antigeeninvastaisen hyperimmunoglobuliinin valmistamiseksi | |
JP2002517218A (ja) | ワクチン | |
JPH09224680A (ja) | 肺炎球菌タンパクの構造遺伝子 | |
JPH08502417A (ja) | ヘモフィルス外膜タンパク質 | |
RU2194757C2 (ru) | ФРАГМЕНТ ДНК, КОДИРУЮЩИЙ ФРАГМЕНТ БЕЛКА РЕЦЕПТОРА ТРАНСФЕРРИНА ШТАММА Haemophilus (ВАРИАНТЫ), ПЛАЗМИДНЫЙ ВЕКТОР (ВАРИАНТЫ), РЕКОМБИНАНТНЫЙ БЕЛОК (ВАРИАНТЫ), ВЫДЕЛЕННЫЙ И ОЧИЩЕННЫЙ БЕЛОК (ВАРИАНТЫ), ИММУНОГЕННАЯ КОМПОЗИЦИЯ И СПОСОБ ПОЛУЧЕНИЯ ВЫДЕЛЕННОГО И ОЧИЩЕННОГО БЕЛКА | |
CN105884902B (zh) | 肺炎支原体蛋白抗原表位的融合蛋白及其制备、应用 | |
AU752794B2 (en) | Treatment and diagnosis of staphylococcal infections | |
JPS63503545A (ja) | ワクチン及び診断マーカーとして有用なネイセリア・ゴナリーア・レクチン及びこのレクチンの製法 | |
CN102757481A (zh) | 金黄色葡萄球菌肠毒素b的功能表位、与其特异性结合的单克隆抗体及其应用 | |
US9802988B2 (en) | Engineered type IV pilin of Clostridium difficile | |
CN107823638B (zh) | 一种b群脑膜炎球菌重组嵌合蛋白疫苗及其制备方法 | |
JP2012526059A (ja) | 腸球菌由来のポリペプチド、及びワクチン接種のためのその使用 | |
CN117264031B (zh) | 一种B群脑膜炎奈瑟菌fHBP重组蛋白及其应用 | |
CN111304224B (zh) | B群脑膜炎奈瑟氏菌重组菌毛蛋白Fim及其制备方法和应用 | |
CN110257405B (zh) | 牛支原体乙醇脱氢酶基因及其编码蛋白与应用 | |
JPH01225495A (ja) | 抗g−csf誘導体、nd28モノクローナル抗体 | |
CN116693632A (zh) | 非洲猪瘟病毒抗原表位肽、单克隆抗体及其用途 | |
CN114249824B (zh) | 杂交瘤细胞hEGF-3A8及其产生的单克隆抗体和应用 | |
CN107041950B (zh) | 一种多房棘球蚴多表位疫苗ltb-ae的设计、制备方法和应用 | |
JPH03115299A (ja) | フイブロネクチン結合タンパク質及びその製法 | |
CN111748042B (zh) | 一种含有内毒素的非洲猪瘟融合蛋白及其制备方法和应用 | |
CN110540966B (zh) | 人流感嗜血杆菌表面蛋白单克隆抗体及抗原捕获elisa试剂盒 |
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 |