CN116355827A - 一种治疗结核病的重组卡介苗药物及其应用 - Google Patents
一种治疗结核病的重组卡介苗药物及其应用 Download PDFInfo
- Publication number
- CN116355827A CN116355827A CN202310401070.7A CN202310401070A CN116355827A CN 116355827 A CN116355827 A CN 116355827A CN 202310401070 A CN202310401070 A CN 202310401070A CN 116355827 A CN116355827 A CN 116355827A
- Authority
- CN
- China
- Prior art keywords
- strain
- bcg
- recombinant
- tuberculosis
- recombinant bcg
- 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.)
- Pending
Links
- 201000008827 tuberculosis Diseases 0.000 title claims abstract description 48
- 239000003814 drug Substances 0.000 title claims abstract description 25
- 229960000190 bacillus calmette–guérin vaccine Drugs 0.000 title claims abstract description 19
- 241001467552 Mycobacterium bovis BCG Species 0.000 title claims description 3
- 230000001225 therapeutic effect Effects 0.000 claims abstract description 24
- 229940079593 drug Drugs 0.000 claims abstract description 9
- 108090000623 proteins and genes Proteins 0.000 claims description 35
- 238000011282 treatment Methods 0.000 claims description 10
- 238000000034 method Methods 0.000 claims description 7
- 230000006801 homologous recombination Effects 0.000 claims description 5
- 238000002744 homologous recombination Methods 0.000 claims description 5
- 239000002671 adjuvant Substances 0.000 claims description 4
- 230000001404 mediated effect Effects 0.000 claims description 3
- 239000004480 active ingredient Substances 0.000 claims description 2
- 238000004519 manufacturing process Methods 0.000 claims description 2
- 125000003275 alpha amino acid group Chemical group 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 11
- 238000011161 development Methods 0.000 abstract description 3
- 230000004957 immunoregulator effect Effects 0.000 abstract description 2
- 230000001024 immunotherapeutic effect Effects 0.000 abstract 2
- 241000699670 Mus sp. Species 0.000 description 37
- 210000001519 tissue Anatomy 0.000 description 26
- 210000004072 lung Anatomy 0.000 description 25
- 238000011081 inoculation Methods 0.000 description 13
- 208000015181 infectious disease Diseases 0.000 description 11
- 241000894006 Bacteria Species 0.000 description 9
- 239000013612 plasmid Substances 0.000 description 8
- 241000699666 Mus <mouse, genus> Species 0.000 description 6
- 238000010186 staining Methods 0.000 description 6
- 210000004027 cell Anatomy 0.000 description 5
- 229940124622 immune-modulator drug Drugs 0.000 description 5
- 229960005486 vaccine Drugs 0.000 description 5
- YQYJSBFKSSDGFO-UHFFFAOYSA-N Epihygromycin Natural products OC1C(O)C(C(=O)C)OC1OC(C(=C1)O)=CC=C1C=C(C)C(=O)NC1C(O)C(O)C2OCOC2C1O YQYJSBFKSSDGFO-UHFFFAOYSA-N 0.000 description 4
- 101100466198 Mus musculus Ptpra gene Proteins 0.000 description 4
- 241000187479 Mycobacterium tuberculosis Species 0.000 description 4
- 238000010276 construction Methods 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 230000036039 immunity Effects 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- 238000012163 sequencing technique Methods 0.000 description 4
- 238000002255 vaccination Methods 0.000 description 4
- 229920001817 Agar Polymers 0.000 description 3
- 208000032420 Latent Infection Diseases 0.000 description 3
- 241000187480 Mycobacterium smegmatis Species 0.000 description 3
- 239000008272 agar Substances 0.000 description 3
- 230000001580 bacterial effect Effects 0.000 description 3
- 208000037771 disease arising from reactivation of latent virus Diseases 0.000 description 3
- 239000012636 effector Substances 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 239000012634 fragment Substances 0.000 description 3
- 239000002955 immunomodulating agent Substances 0.000 description 3
- 230000008595 infiltration Effects 0.000 description 3
- 238000001764 infiltration Methods 0.000 description 3
- 210000004969 inflammatory cell Anatomy 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 230000001575 pathological effect Effects 0.000 description 3
- 230000002265 prevention Effects 0.000 description 3
- 230000000069 prophylactic effect Effects 0.000 description 3
- 229940021993 prophylactic vaccine Drugs 0.000 description 3
- 230000001681 protective effect Effects 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 238000011740 C57BL/6 mouse Methods 0.000 description 2
- 241001646725 Mycobacterium tuberculosis H37Rv Species 0.000 description 2
- 108700035964 Mycobacterium tuberculosis HsaD Proteins 0.000 description 2
- 241000187644 Mycobacterium vaccae Species 0.000 description 2
- 229930040373 Paraformaldehyde Natural products 0.000 description 2
- 102000004160 Phosphoric Monoester Hydrolases Human genes 0.000 description 2
- 208000022971 Tuberculous meningitis Diseases 0.000 description 2
- 208000036981 active tuberculosis Diseases 0.000 description 2
- 239000000443 aerosol Substances 0.000 description 2
- 150000001413 amino acids Chemical group 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 230000005745 host immune response Effects 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 231100000518 lethal Toxicity 0.000 description 2
- 230000001665 lethal effect Effects 0.000 description 2
- 208000001223 meningeal tuberculosis Diseases 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 239000013642 negative control Substances 0.000 description 2
- 239000012188 paraffin wax Substances 0.000 description 2
- 229920002866 paraformaldehyde Polymers 0.000 description 2
- 238000003752 polymerase chain reaction Methods 0.000 description 2
- 239000013641 positive control Substances 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- 238000011144 upstream manufacturing Methods 0.000 description 2
- IJJWOSAXNHWBPR-HUBLWGQQSA-N 5-[(3as,4s,6ar)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]-n-(6-hydrazinyl-6-oxohexyl)pentanamide Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)NCCCCCC(=O)NN)SC[C@@H]21 IJJWOSAXNHWBPR-HUBLWGQQSA-N 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 206010013453 Disseminated tuberculosis Diseases 0.000 description 1
- 108010042407 Endonucleases Proteins 0.000 description 1
- 102000004533 Endonucleases Human genes 0.000 description 1
- 101000854700 Homo sapiens Vacuolar protein sorting-associated protein 33B Proteins 0.000 description 1
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 1
- 206010027259 Meningitis tuberculous Diseases 0.000 description 1
- 201000006836 Miliary Tuberculosis Diseases 0.000 description 1
- 240000007019 Oxalis corniculata Species 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 108700019535 Phosphoprotein Phosphatases Proteins 0.000 description 1
- 108090000608 Phosphoric Monoester Hydrolases Proteins 0.000 description 1
- 102000001253 Protein Kinase Human genes 0.000 description 1
- 102000044159 Ubiquitin Human genes 0.000 description 1
- 108090000848 Ubiquitin Proteins 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 238000009098 adjuvant therapy Methods 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000002924 anti-infective effect Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000030609 dephosphorylation Effects 0.000 description 1
- 238000006209 dephosphorylation reaction Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 239000002552 dosage form Substances 0.000 description 1
- 229940072185 drug for treatment of tuberculosis Drugs 0.000 description 1
- 238000012224 gene deletion Methods 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 102000049033 human VPS33B Human genes 0.000 description 1
- 238000002649 immunization Methods 0.000 description 1
- 230000003053 immunization Effects 0.000 description 1
- 230000007365 immunoregulation Effects 0.000 description 1
- 238000009169 immunotherapy Methods 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000002458 infectious effect Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 230000015788 innate immune response Effects 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 238000010197 meta-analysis Methods 0.000 description 1
- 210000004877 mucosa Anatomy 0.000 description 1
- 210000004400 mucous membrane Anatomy 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 238000010827 pathological analysis Methods 0.000 description 1
- 230000020658 phagosome acidification Effects 0.000 description 1
- 230000003449 preventive effect Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000002250 progressing effect Effects 0.000 description 1
- 238000011321 prophylaxis Methods 0.000 description 1
- 108060006633 protein kinase Proteins 0.000 description 1
- 101150020232 ptpA gene Proteins 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 230000007480 spreading Effects 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 229960002109 tuberculosis vaccine Drugs 0.000 description 1
- 239000000814 tuberculostatic agent Substances 0.000 description 1
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 230000001018 virulence Effects 0.000 description 1
Images
Classifications
-
- 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
-
- 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
- A61K39/04—Mycobacterium, e.g. Mycobacterium tuberculosis
-
- 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
- A61P31/06—Antibacterial agents for tuberculosis
-
- 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
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
-
- 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
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C12N15/90—Stable introduction of foreign DNA into chromosome
- C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y301/00—Hydrolases acting on ester bonds (3.1)
- C12Y301/03—Phosphoric monoester hydrolases (3.1.3)
- C12Y301/03048—Protein-tyrosine-phosphatase (3.1.3.48)
-
- 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
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
- A61K2039/522—Bacterial cells; Fungal cells; Protozoal cells avirulent or attenuated
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/32—Mycobacterium
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Pulmonology (AREA)
- Animal Behavior & Ethology (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Communicable Diseases (AREA)
- Mycology (AREA)
- Veterinary Medicine (AREA)
- Pharmacology & Pharmacy (AREA)
- Public Health (AREA)
- Physics & Mathematics (AREA)
- Epidemiology (AREA)
- Immunology (AREA)
- Oncology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
本发明属于医药技术领域，具体公开一种治疗结核病的重组卡介苗免疫治疗药物及其应用。该重组卡介苗是在传统卡介苗菌株基础上敲除基因Mb2258的重组卡介苗，其不仅可以预防结核病的发生和发展，还能够作为治疗性免疫调节药物缓解结核病的进展，其预防和治疗结核病的效果均优于野生卡介苗株，因此是一种具有良好潜力的新型结核病免疫治疗药物。
Description
技术领域
本发明属于医药卫生领域，具体涉及一种重组卡介苗菌株BCG△Mb2258以及该重组菌株在结核病预防以及治疗方面的应用。
背景技术
结核病(Tuberculosis，TB)是由结核分枝杆菌(Mycobacterium tuberculosis，Mtb)感染引起的重要慢性致死性传染病。世界卫生组织发布的《2022全球结核病报告》显示：2021年全球新发TB患者约1060万，较2020年增加了4.5％；2021年TB致死人数约160万，超过2020年和2019年的死亡人数，又回到2017年的水平，结核病防控出现停滞甚至逆转，形势不容乐观。
目前唯一获得临床使用许可用于预防结核病的疫苗是卡介苗(BovisbacilleCalmette-Guérin，BCG)，它对新生儿和学龄前儿童的结核性脑膜炎具有50-80％左右的保护作用，但对肺结核的保护效果个体差异很大，且对成年人肺结核无明显的保护作用(Trunz BB,Fine P&Dye C(2006),Effect of BCG vaccination on childhoodtuberculous meningitis and miliary tuberculosis worldwide:a meta-analysis andassessment of cost-effectiveness.Lancet 367:1173–1180；Helen A Fletcher(2016),Sleeping Beauty and the Story of the Bacille Calmette-Guérin Vaccine.mBio30；7(4):e01370-16)，因此新型结核病疫苗开发迫在眉睫。另一方面，这些疫苗一般只能起到提前预防作用，而不具有治疗作用。另外除了活动性结核患者以外，社会上还有很大一部分人群属于结核潜伏感染者，即虽感染了Mtb但并未表现明显症状，此类人群的比例很大，急需治疗性药物以减缓其发病。例如，智飞生物开发的注射用母牛分枝杆菌(商品名：微卡)就是除了用于辅助治疗结核病，还可用于预防结核潜伏感染人群进展为结核病，因此具有更广的临床应用价值。
Mtb基因组共编码十余种真核样蛋白激酶和磷酸酯酶，它们通过直接靶向宿主蛋白进而抑制宿主免疫应答并促进Mtb存活。其中，PtpA在BCG中由Mb2258基因编码，属于低分子量酪氨酸磷酸酯酶。研究表明PtpA是直接靶向宿主的关键Mtb效应蛋白，可直接抑制宿主细胞吞噬体酸化以及抑制固有免疫信号通路活化等(Wang J et al.,(2015),Mycobacterium tuberculosis suppresses innate immunity by coopting the hostubiquitin system.Nature Immunology16(3):237-45；Bach H et al.,(2008),Mycobacterium tuberculosis virulence is mediated by PtpA dephosphorylation ofhuman vacuolar protein sorting 33B.Cell Host Microbe15；3(5):316-22)。本发明在此基础上进行了研究，其中发现有意想不到的结果。
发明内容
本发明构建了敲除Mb2258基因的重组BCG菌株，以解除传统BCG中PtpA效应蛋白对宿主固有免疫的抑制作用，进而更有效地提升机体的免疫水平。通过Mtb-小鼠感染模型，发现BCG重组菌株BCG△Mb2258可以预防结核病的发生，这与该基因的功能预期结果一致。但在研究中，意外发现其还能够作为治疗性免疫调节药物缓解结核病进展，其治疗结核病的效果优于野生卡介苗菌株。而这与通常情况下重组BCG菌株仅能作为疫苗预防结核病的发生的疫苗完全不同，由此完成本发明。
本发明在野生型BCG菌株的基础上敲除Mb2258基因从而构建重组BCG菌株，Mtb-小鼠感染实验表明该重组卡介苗菌株不仅在预防而且在治疗结核病方面的效果优于传统野生型卡介苗菌株。
本发明的第一方面是提供了一种治疗用途的BCG重组菌株(BCG△Mb2258)，该BCG重组菌株中的基因Mb2258被完全敲除。基因Mb2258的氨基酸序列为SEQ ID No.：1。
进一步地，在敲除过程中，采用的Mb2258基因同源臂序列为SEQ ID No.：2–5。
本发明的第二方面是提供上述治疗用途的BCG重组菌的构建方法，通过噬菌粒同源重组技术进行无痕敲除野生型BCG菌株中的基因Mb2258获得该BCG重组菌。
本发明第三方面提供一种治疗结核病的重组卡介苗药物，其含有根据所述的治疗用途的重组卡介苗菌株作为有效成分。
具体地，所述药物包含所述治疗用途的重组卡介苗菌株的培养物或其组分。进一步地，所述药物还包括药学上可接受的佐剂。
本发明第四方面尤其是提供所述的治疗用途的重组卡介苗菌株在制备治疗结核病的药物中的应用。
具体地，所述药物包含所述治疗用途的重组卡介苗菌株及其培养物或组分。
更具体地，所述药物还包括药学上可接受的佐剂，以适用的剂型和给药方式的药物形式等。
本发明在研究去除传统BCG中PtpA效应蛋白(即敲除Mb2258基因)对宿主固有免疫的抑制作用中，意外发现其具有很好的治疗结核病的用途，并经实验证实，在治疗性免疫调节药物的开发中具有应用价值。
附图说明
图1PCR鉴定重组BCG菌株BCG△Mb2258中Mb2258基因的敲除结果；
图2小鼠水平检测重组BCG菌株BCG△Mb2258作为TB预防性疫苗的保护力评价流程图；
图3不同卡介苗菌株作为TB预防性疫苗接种小鼠的肺组织Mtb荷菌量比较；
图4不同卡介苗菌株作为TB预防性疫苗接种小鼠的肺组织病理染色结果；
图5小鼠水平检测重组BCG菌株BCG△Mb2258作为TB治疗性免疫调节药物的有效性评价流程图；
图6不同卡介苗菌株作为TB治疗性免疫调节药物接种小鼠的肺组织Mtb荷菌量比较；
图7不同卡介苗菌株作为TB治疗性免疫调节药物接种小鼠的肺组织病理染色结果。
具体实施方式
下面通过具体实施例和附图对本发明进行详细和具体的介绍，以便更好的理解本发明，但是下述实施例并不限制本发明。
实施例中方法如无特殊说明的采用常规方法，使用的试剂如无特殊说明的使用市售试剂或按常规方法配制的试剂。
其中，实施例中用的序列信息如下：
SEQ ID No.：1Mb2258基因编码氨基酸序列
MSDPLHVTFVCTGNICRSPMAEKMFAQQLRHRGLGDAVRVTSAGTGNWHVGSCADERAA
GVLRAHGYPTDHRAAQVGTEHLAADLLVALDRNHARLLRQLGVEAARVRMLRSFDPRSG
THALDVEDPYYGDHSDFEEVFAVIESALPGLHDWVDERLARNGPSSEQ ID No.：2Mb2258基因左侧同源臂上游引物序列
TTTTTTTTCCATAAATTGGTACACGCCATGGTCAATGCC
SEQ ID No.：3Mb2258基因左侧同源臂下游引物序列
TTTTTTTTCCATTTCTTGGTCAGACACCTAGCGCCTC
SEQ ID No.：4Mb2258基因右侧同源臂上游引物序列
TTTTTTTTCCATAGATTGGATGCCCCGCCTAGCGTTCCTG
SEQ ID No.：5Mb2258基因右侧同源臂下游引物序列
TTTTTTTTCCATCTTTTGGGTCGAGGCGGCGTTTGCTGG
SEQ ID No.：6Mb2258基因左侧同源臂测序引物序列
AACAGCTTGTTCGACGGGAT
SEQ ID No.：7Mb2258基因右侧同源臂测序引物序列
ACGTTTTGGCATTCTTGCCC
实施例1重组BCG菌株BCG△Mb2258的构建及鉴定
本实施在野生型BCG(日本株)基础上利用同源重组技术构建了Mb2258基因缺失重组BCG菌株(BCG△Mb2258)，主要流程如下：
1.1构建p0004S-△Mb2258质粒
以BCG全基因组DNA为模板，分别设计两对针对Mb2258基因的左、右臂引物，即Mb2258-LS TTTTTTTTCCATAAATTGGTACACGCCATGGTCAATGCC(SEQ ID No.：2)、Mb2258-LA TTTTTTTTCCATTTCTTGGTCAGACACCTAGCGCCTC(SEQ ID No.：3)和Mb2258-RS TTTTTTTTCCATAGATTGGATGCCCCGCCTAGCGTTCCTG(SEQ ID No.：4)、Mb2258-RA TTTTTTTTCCATCTTTTGGGTCGAGGCGGCGTTTGCTGG(SEQ ID No.：5)。通过聚合酶链式反应(Polymerase Chain Reaction，PCR)扩增Mb2258基因左右两侧同源臂序列。反应条件：95℃3min；95℃15s；58℃15s；72℃1min，30个循环；72℃5min。采用Van91I(cat#FD0714，Thermo Scientific)酶切Mb2258基因左、右同源臂和p0004S质粒，连接酶切后的片段，转化DH5α感受态细胞，涂布于含有150μg/mL潮霉素(cat#10843555001，Roche)的LB固体平板上。挑取单克隆菌落并提取质粒，Van91I酶切验证确定阳性克隆。
1.2构建phAE159-△Mb2258穿梭质粒
用PacI(cat#FD2204，Thermo Scientific)内切酶分别酶切p0004S-△Mb2258质粒和phAE159质粒，连接酶切后的片段。通过噬菌体体外包装试剂盒(cat#MP5120，Epicentre)转化HB101感受态细胞，涂布于含有150μg/mL潮霉素的LB固体平板上。挑取单克隆菌落并提取质粒，PacI酶切验证确定阳性克隆。
1.3噬菌体扩增
取5μg phAE159-△Mb2258质粒电转入耻垢分枝杆菌mc2155感受态细胞中，加入700μL7H9培养基于37℃复苏4h，将复苏的菌液与0.7％琼脂混匀，铺于7H10平板上，30℃培养箱避光培养2–3d。
挑取4–5个噬菌斑到EP管中，加入200μL MP缓冲液，4℃过夜。取50μL上清液加至300μL新鲜培养的耻垢分枝杆菌mc2155菌液中，再与0.7％琼脂混匀，铺于7H10平板上。待噬菌斑扩大到相连后，加入适量MP缓冲液，4℃过夜，吸取上清并用0.22μm滤器过滤，4℃保存噬菌体。
将噬菌体进行梯度稀释，取100μL各梯度噬菌体至新鲜培养的耻垢分枝杆菌mc2155菌液中，再与0.7％琼脂混匀，铺于7H10平板上，30℃培养箱避光培养2–3d，计算噬菌斑滴度。
1.4BCG△Mb2258重组卡介苗菌株的构建
制备野生型BCG(日本株)的感受态菌株，以感受态BCG：噬菌体＝1:10的比例混匀，避光孵育3–4h，补加10mL 7H9培养基(含OADC增菌液)，37℃孵育16–20h，离心收集BCG并涂布于含75μg/mL潮霉素的7H10平板上，37℃培养4–6周，挑取生长的阳性克隆。
1.5BCG△Mb2258重组卡介苗菌株的鉴定
挑取上述平板上的单克隆菌落于7H9培养基中37℃静置培养3周，收集菌液并抽提菌株的基因组DNA。以Mb2258基因左、右同源臂序列设计引物，Mb2258-upAACAGCTTGTTCGACGGGAT(SEQ ID No.：6)和Mb2258-down ACGTTTTGGCATTCTTGCCC(SEQ IDNo.：7)进行PCR扩增鉴定。PCR条件：95℃3min；95℃15s；58℃15s；72℃3min，30个循环；72℃5min。PCR鉴定正确的菌株送基因组DNA进行细菌基因组测序并确认重组卡介苗中的Mb2588基因片段被全部敲除。
结果如图1所示，通过PCR鉴定确认野生型BCG中可以鉴定到Mb2258基因(1012bp)，而重组BCG菌株BCG△Mb2258中检测不到Mb2258基因，而是被同源重组后的潮霉素抗性基因取代(4120bp)。同时基因测序结果确认重组卡介苗中的Mb2258基因被全部敲除。
实施例2小鼠水平检测重组BCG菌株BCG△Mb2258作为TB预防性疫苗的保护力
本实施例应用实施例1中构建的重组BCG菌株BCG△Mb2258，在小鼠水平验证其作为预防性疫苗对小鼠发生结核病的预防效果，具体的实验步骤和结果如下：
参考图2的实验流程图，采用皮内和黏膜两种接种的方式，分别将各组C57BL/6小鼠皮内接种2×106个或黏膜接种1×107个菌数的野生型BCG或重组BCG△Mb2258，未接种组作为对照。接种4周后在生物安全三级实验室中通过雾化吸入方式使各组小鼠感染结核分枝杆菌H37Rv菌株，感染菌量为100个细菌。
感染4周后，通过颈椎脱臼法处死小鼠，分离肺组织进行菌落计数，计算各组小鼠肺组织内的Mtb荷菌量。同时对各组小鼠的肺组织进行4％多聚甲醛固定和石蜡包埋，进一步通过苏木精-伊红染色和抗酸染色观察各组小鼠肺组织病理和荷菌量差异。
如图所示，与未接种的对照组小鼠相比，皮内接种和黏膜接种卡介苗或重组BCG△Mb2258都对抵御Mtb感染有效。其中黏膜接种比皮内接种效果更好。此外，无论是黏膜接种还是皮内接种，重组BCG△Mb2258菌株的保护效果均优于野生型卡介苗株。皮内接种条件下，接种重组BCG△Mb2258菌株的小鼠肺组织荷菌量比未接种卡介苗小鼠肺组织荷菌量下降1–2个数量级，比接种野生BCG菌株的小鼠肺组织荷菌量下降0.5–1.5个数量级；黏膜接种条件下，接种重组BCG△Mb2258菌株的小鼠肺组织荷菌量比未接种卡介苗小鼠肺组织荷菌量下降1.5–2个数量级，比接种野生BCG菌株的小鼠肺组织荷菌量下降0.5–1.5个数量级(如图3)。此外，无论是皮内还是黏膜接种条件下，接种重组BCG△Mb2258菌株的小鼠肺组织比未接种卡介苗以及接种了野生型BCG的小鼠肺组织表现为更少的炎性细胞浸润和更完好的肺泡组织结构(如图4)。
综上所述，与传统BCG菌株相比，重组BCG△Mb2258菌株可以更有效地提升机体免疫力抵御Mtb感染，因此具有优于传统BCG的预防结核病的效果。同时黏膜接种卡介苗的保护力优于皮内接种卡介苗。
实施例3小鼠水平检测重组BCG菌株BCG△Mb2258作为TB治疗性免疫调节药物的有效性
本实施例应用实施例1中构建的重组BCG菌株BCG△Mb2258，在小鼠水平验证其作为治疗性免疫调节药物对感染结核病小鼠的治疗效果，具体的实验步骤和结果如下：
参考图5的实验流程图，在生物安全三级实验室中通过雾化吸入方式使各组C57BL/6小鼠感染结核分枝杆菌H37Rv菌株。感染Mtb菌量为100个细菌。感染3天后，采用皮内接种的方式，分别将各组小鼠接种1×106个菌数的三种不同株型(上海D2株、日本株、巴斯德株)的野生型BCG或重组BCG△Mb2258，未接种组作为阴性对照，接种预防性微卡(智飞生物)组作为阳性对照。此后每两周皮内接种1次，共接种3次。
第3次免疫后2周，通过颈椎脱臼法处死小鼠，分离肺组织进行菌落计数，计算各组小鼠肺组织内的Mtb荷菌量。同时对各组小鼠的肺组织进行4％多聚甲醛固定和石蜡包埋，进一步通过苏木精-伊红染色和抗酸染色观察各组小鼠肺组织病理和荷菌量差异。
如图6所示，与未接种卡介苗的小鼠对比，皮内接种三种不同株型的野生型BCG菌株的小鼠肺组织荷菌量没有显著差异，提示野生型BCG菌株对于结核病治疗无明显效果。同时比较发现接种了三种不同株型的重组BCG△Mb2258菌株的小鼠肺组织荷菌量比未接种卡介苗组及接种野生型BCG的小鼠肺组织荷菌量均有明显下降，平均荷菌量下降幅度约为50％–90％，提示皮内接种不同株型的重组BCG△Mb2258菌株均对于结核病有明显的治疗效果(如图6)。此外，接种了重组BCG△Mb2258(日本株)和重组BCG△Mb2258(巴斯德株)的小鼠肺组织荷菌量与接种了预防性微卡的小鼠肺组织荷菌量相当，而接种了重组BCG△Mb2258(上海D2株)的小鼠肺组织荷菌量比接种了预防性微卡的小鼠肺组织荷菌量相比有所下降，平均荷菌量下降幅度约50％。
病理分析结果与上述结果一致：接种重组BCG△Mb2258菌株的小鼠肺组织比未接种卡介苗以及接种了野生型BCG的小鼠肺组织表现为更少的炎性细胞浸润和更完好的肺泡组织结构(如图7)。其中，在所有处理组中(包括阴性和阳性对照组、不同卡介苗和重组卡介苗处理组)接种了重组BCG△Mb2258(上海D2株)的小鼠肺组织表现为最少的炎性细胞浸润和最完好的肺泡组织结构。综上所述，重组BCG△Mb2258菌株可以更显著的提升宿主抵抗Mtb感染的能力，尤其可有效降低Mtb在肺部的存活水平并减轻组织病理损伤，因此具有良好的结核病治疗效果。以上研究数据提示重组卡介苗BCG△Mb2258作为结核病免疫治疗药物，其将通过提升宿主免疫反应发挥抗感染作用，与现有抗结核药物联用，将对活动性结核病(敏感菌和耐药菌感染)及结核潜伏感染具有治疗效果。
以上对本发明的具体实施例进行了详细描述，但其只作为范例，本发明并不局限于以上描述的具体实施例。对于本领域技术人员而言，任何对本发明进行同等修改和替代也都在本发明的范畴之中。因此，在不脱离本发明构思的前提下所做的变换和修改，均应涵盖在本发明的专利保护范围内。
Claims (10)
1. 一种治疗用途的重组卡介苗菌株，其特征在于，其中的基因Mb2258被完全敲除，所述基因Mb2258编码的氨基酸序列如SEQ ID No.：1所示。
2.根据权利要求1所述的治疗用途的重组卡介苗菌株，其特征在于，所述重组卡介苗菌株中的基因Mb2258通过噬菌粒介导的同源重组技术被全部敲除。
3.根据权利要求1或2所述的治疗用途的重组卡介苗菌株的构建方法，其特征在于，通过噬菌粒介导的同源重组技术敲除野生型卡介苗菌株中的基因Mb2258从而获得。
4.根据权利要求3所述的方法，其特征在于，是将野生型卡介苗菌株中的基因Mb2258的全长敲除。
5.一种治疗结核病的重组卡介苗药物，其含有根据权利要求1或2所述的治疗用途的重组卡介苗菌株作为有效成分。
6.根据权利要求5所述的重组卡介苗药物，其特征在于，所述药物包含所述治疗用途的重组卡介苗菌株的培养物或其组分。
7.根据权利要求6所述的应用，其特征在于，所述药物还包括药学上可接受的佐剂。
8.根据权利要求1或2所述的治疗用途的重组卡介苗菌株在制备治疗结核病的药物中的应用。
9.根据权利要求8所述的应用，其特征在于，所述药物包含所述治疗用途的重组卡介苗菌株的培养物或其组分。
10.根据权利要求9所述的应用，其特征在于，所述药物还包括药学上可接受的佐剂。。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310401070.7A CN116355827A (zh) | 2023-04-14 | 2023-04-14 | 一种治疗结核病的重组卡介苗药物及其应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310401070.7A CN116355827A (zh) | 2023-04-14 | 2023-04-14 | 一种治疗结核病的重组卡介苗药物及其应用 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN116355827A true CN116355827A (zh) | 2023-06-30 |
Family
ID=86933302
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310401070.7A Pending CN116355827A (zh) | 2023-04-14 | 2023-04-14 | 一种治疗结核病的重组卡介苗药物及其应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116355827A (zh) |
-
2023
- 2023-04-14 CN CN202310401070.7A patent/CN116355827A/zh active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| EP2040744B1 (en) | Live vaccine strains of francisella | |
| WO2009070700A1 (en) | Recombinant bcg tuberculosis vaccine for eliciting immune responses to mycobacterium tuberculosis | |
| US20230381224A1 (en) | Mutant Bordetella Strains and Methods of Use | |
| RU2760580C2 (ru) | РЕКОМБИНАНТНАЯ BCG, СВЕРХЭКСПРЕССИРУЮЩАЯ PhoP-PhoR | |
| JP2019513722A5 (zh) | ||
| CN116200347A (zh) | 一种gI、gE和TK三基因缺失株猫疱疹病毒疫苗及其应用 | |
| Gregory et al. | Epitope-based vaccination against pneumonic tularemia | |
| CN104685054A (zh) | Lsr2活性降低或敲除的改良BCG菌株及包含该菌株的药物组合物 | |
| CN115976091A (zh) | Pup基因敲除的结核分枝杆菌H37Ra菌株的构建方法及应用 | |
| EP2597151A2 (en) | Recombinant microorganisms, methods for preparing vaccine strains, antigens, and vector vaccine compositions of same, uses thereof, and related antibodies, diagnostic kit, and treatment and/or prophylactic methods | |
| Kamachi et al. | DNA vaccine encoding pertussis toxin S1 subunit induces protection against Bordetella pertussis in mice | |
| Robinson et al. | Vaccination with a live multi-gene deletion strain protects horses against virulent challenge with Streptococcus equi | |
| JP2023153868A (ja) | 結核の感染リスクがある人に対する予防剤として、又は感染した結核患者を治療するための二次薬として使用する組成物 | |
| CN116355827A (zh) | 一种治疗结核病的重组卡介苗药物及其应用 | |
| CN116286573A (zh) | 一株新型重组卡介苗菌株的构建及其应用 | |
| CN114438106B (zh) | Rv2779c基因及其表达产物作为抗结核分枝杆菌靶点的应用 | |
| CN115838732B (zh) | 一种重组卡介苗菌株及其构建方法和应用 | |
| Hosamani et al. | Cell culture adapted sheeppox virus as a challenge virus for potency testing of sheeppox vaccine | |
| WO2002066055A1 (en) | NOVEL MEDICINAL USE OF α ANTIGEN OR α ANTIGEN GENE | |
| WO2006065549A2 (en) | Attenuated vaccine useful for immunizations against coccidioides spp. infections | |
| WO2002102409A1 (en) | Inactivated mycobacterial ompatb and uses thereof | |
| Zhang et al. | Research Article Ag85A, As an S2 Vaccine Carrier, Reduces the Toxicity of the S2 Vaccine and Enhances the Protective Ability of Mice against Brucella | |
| CN117402798A (zh) | 一种防治结核病的重组卡介苗药物 | |
| Frey et al. | Molecular mechanisms of virulence and antigenicity of Mycoplasma mycoides subsp. mycoides SC: conclusions for prevention and control of CBPP [versão electrónica] |
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 |