CN117106613A - 一种鱼类维氏气单胞菌弱毒疫苗株及其应用 - Google Patents
一种鱼类维氏气单胞菌弱毒疫苗株及其应用 Download PDFInfo
- Publication number
- CN117106613A CN117106613A CN202310377122.1A CN202310377122A CN117106613A CN 117106613 A CN117106613 A CN 117106613A CN 202310377122 A CN202310377122 A CN 202310377122A CN 117106613 A CN117106613 A CN 117106613A
- Authority
- CN
- China
- Prior art keywords
- strain
- vaccine
- aeromonas veronii
- fish
- aeromonas
- 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
- 241000607574 Aeromonas veronii Species 0.000 title claims abstract description 70
- 229960005486 vaccine Drugs 0.000 title claims abstract description 48
- 241000251468 Actinopterygii Species 0.000 title claims abstract description 46
- -1 pplication Species 0.000 title description 2
- 239000003053 toxin Substances 0.000 title description 2
- 230000001580 bacterial effect Effects 0.000 claims abstract description 27
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 23
- 229940031551 inactivated vaccine Drugs 0.000 claims abstract description 21
- 229940031567 attenuated vaccine Drugs 0.000 claims abstract description 17
- 239000013505 freshwater Substances 0.000 claims abstract description 16
- 206010040047 Sepsis Diseases 0.000 claims abstract description 14
- 208000013223 septicemia Diseases 0.000 claims abstract description 14
- 101150033839 4 gene Proteins 0.000 claims abstract description 10
- 108091033409 CRISPR Proteins 0.000 claims abstract description 5
- 238000010354 CRISPR gene editing Methods 0.000 claims abstract description 5
- 108020004566 Transfer RNA Proteins 0.000 claims abstract description 5
- 108091032973 (ribonucleotides)n+m Proteins 0.000 claims abstract 2
- 241000607534 Aeromonas Species 0.000 claims description 18
- 230000003053 immunization Effects 0.000 claims description 12
- 238000002649 immunization Methods 0.000 claims description 11
- 241000252234 Hypophthalmichthys nobilis Species 0.000 claims description 10
- 230000000740 bleeding effect Effects 0.000 claims description 9
- 108091081062 Repeated sequence (DNA) Proteins 0.000 claims description 8
- 238000004458 analytical method Methods 0.000 claims description 8
- 238000012163 sequencing technique Methods 0.000 claims description 8
- 238000002791 soaking Methods 0.000 claims description 8
- 241000252230 Ctenopharyngodon idella Species 0.000 claims description 7
- 230000036039 immunity Effects 0.000 claims description 7
- 238000002360 preparation method Methods 0.000 claims description 7
- 210000000683 abdominal cavity Anatomy 0.000 claims description 6
- 239000007928 intraperitoneal injection Substances 0.000 claims description 6
- 230000008961 swelling Effects 0.000 claims description 6
- 241000404975 Synchiropus splendidus Species 0.000 claims description 5
- 206010003445 Ascites Diseases 0.000 claims description 4
- 241001275890 Megalobrama amblycephala Species 0.000 claims description 4
- 210000004185 liver Anatomy 0.000 claims description 4
- 210000000952 spleen Anatomy 0.000 claims description 4
- 241001519451 Abramis brama Species 0.000 claims description 3
- 210000000436 anus Anatomy 0.000 claims description 3
- 210000004369 blood Anatomy 0.000 claims description 3
- 239000008280 blood Substances 0.000 claims description 3
- 102000004169 proteins and genes Human genes 0.000 claims description 3
- 241001609213 Carassius carassius Species 0.000 claims description 2
- 208000020560 abdominal swelling Diseases 0.000 claims description 2
- 238000007654 immersion Methods 0.000 claims description 2
- 230000017074 necrotic cell death Effects 0.000 claims description 2
- 210000005084 renal tissue Anatomy 0.000 claims description 2
- 208000024891 symptom Diseases 0.000 claims description 2
- 241001098657 Pterois Species 0.000 claims 1
- 201000010099 disease Diseases 0.000 claims 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims 1
- 231100000636 lethal dose Toxicity 0.000 claims 1
- 230000002238 attenuated effect Effects 0.000 abstract description 13
- 230000004520 agglutination Effects 0.000 abstract description 10
- 241000283973 Oryctolagus cuniculus Species 0.000 abstract description 8
- 230000008092 positive effect Effects 0.000 abstract description 3
- 239000003814 drug Substances 0.000 abstract description 2
- 230000003115 biocidal effect Effects 0.000 abstract 1
- 229940079593 drug Drugs 0.000 abstract 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 10
- 241000607528 Aeromonas hydrophila Species 0.000 description 9
- 101150013736 gyrB gene Proteins 0.000 description 8
- 208000032843 Hemorrhage Diseases 0.000 description 7
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 7
- 239000007924 injection Substances 0.000 description 7
- 238000002347 injection Methods 0.000 description 7
- 230000004060 metabolic process Effects 0.000 description 7
- 239000000243 solution Substances 0.000 description 7
- 108020004465 16S ribosomal RNA Proteins 0.000 description 6
- 241000252211 Carassius Species 0.000 description 6
- 241000252212 Danio rerio Species 0.000 description 6
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 6
- 238000006243 chemical reaction Methods 0.000 description 6
- 101150012629 parE gene Proteins 0.000 description 6
- 238000012360 testing method Methods 0.000 description 6
- 238000000034 method Methods 0.000 description 5
- 239000011780 sodium chloride Substances 0.000 description 4
- 108700039887 Essential Genes Proteins 0.000 description 3
- 244000052616 bacterial pathogen Species 0.000 description 3
- 238000001784 detoxification Methods 0.000 description 3
- 238000011156 evaluation Methods 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 230000001018 virulence Effects 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 2
- 241000252229 Carassius auratus Species 0.000 description 2
- 230000002159 abnormal effect Effects 0.000 description 2
- 230000001154 acute effect Effects 0.000 description 2
- 239000003242 anti bacterial agent Substances 0.000 description 2
- 229940088710 antibiotic agent Drugs 0.000 description 2
- 238000009360 aquaculture Methods 0.000 description 2
- 244000144974 aquaculture Species 0.000 description 2
- 201000005008 bacterial sepsis Diseases 0.000 description 2
- 230000037396 body weight Effects 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 230000002354 daily effect Effects 0.000 description 2
- 230000034994 death Effects 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 230000003203 everyday effect Effects 0.000 description 2
- 210000002816 gill Anatomy 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 238000002955 isolation Methods 0.000 description 2
- 210000003734 kidney Anatomy 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 210000000056 organ Anatomy 0.000 description 2
- 230000001717 pathogenic effect Effects 0.000 description 2
- 238000004321 preservation Methods 0.000 description 2
- 108700022487 rRNA Genes Proteins 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 230000009182 swimming Effects 0.000 description 2
- 238000012070 whole genome sequencing analysis Methods 0.000 description 2
- 241000223600 Alternaria Species 0.000 description 1
- 206010003694 Atrophy Diseases 0.000 description 1
- 241001597062 Channa argus Species 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 108020004414 DNA Proteins 0.000 description 1
- 208000032969 Hemorrhagic Septicemia Diseases 0.000 description 1
- 241000252498 Ictalurus punctatus Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 208000014645 Pasteurella hemorrhagic septicemia Diseases 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 101100091878 Plasmodium falciparum (isolate 3D7) rpoC2 gene Proteins 0.000 description 1
- 231100000645 Reed–Muench method Toxicity 0.000 description 1
- 206010042674 Swelling Diseases 0.000 description 1
- 241000276707 Tilapia Species 0.000 description 1
- 241001482311 Trionychidae Species 0.000 description 1
- 206010052428 Wound Diseases 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 210000001015 abdomen Anatomy 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 239000010828 animal waste Substances 0.000 description 1
- 230000037444 atrophy Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 208000034158 bleeding Diseases 0.000 description 1
- 239000006161 blood agar Substances 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 239000005515 coenzyme Substances 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 208000027744 congestion Diseases 0.000 description 1
- 108091036078 conserved sequence Proteins 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000002224 dissection Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000035611 feeding Effects 0.000 description 1
- 208000010824 fish disease Diseases 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 238000001802 infusion Methods 0.000 description 1
- 229910001410 inorganic ion Inorganic materials 0.000 description 1
- 210000000936 intestine Anatomy 0.000 description 1
- 238000007912 intraperitoneal administration Methods 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 150000007523 nucleic acids Chemical group 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 244000039328 opportunistic pathogen Species 0.000 description 1
- 230000001706 oxygenating effect Effects 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 210000004303 peritoneum Anatomy 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 108020004418 ribosomal RNA Proteins 0.000 description 1
- 101150029016 rpo3 gene Proteins 0.000 description 1
- 101150102864 rpoD gene Proteins 0.000 description 1
- 238000011076 safety test Methods 0.000 description 1
- 238000007790 scraping Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 239000013535 sea water Substances 0.000 description 1
- 229930000044 secondary metabolite Natural products 0.000 description 1
- 239000010865 sewage Substances 0.000 description 1
- 101150117326 sigA gene Proteins 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000007671 third-generation sequencing Methods 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 238000002723 toxicity assay Methods 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 210000001835 viscera Anatomy 0.000 description 1
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
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
-
- 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
- A61K39/0208—Specific bacteria not otherwise provided for
-
- 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
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- 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
-
- 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
- A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
- A61K2039/552—Veterinary vaccine
-
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- Animal Behavior & Ethology (AREA)
- General Engineering & Computer Science (AREA)
- Virology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Biomedical Technology (AREA)
- Veterinary Medicine (AREA)
- Pharmacology & Pharmacy (AREA)
- Public Health (AREA)
- Immunology (AREA)
- Epidemiology (AREA)
- Mycology (AREA)
- Communicable Diseases (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
本发明公开了一种鱼类维氏气单胞菌弱毒疫苗株及其应用。本发明的维氏气单胞菌弱毒疫苗株,命名为FS12001,2023年3月7日保藏于湖北省武汉市武汉大学的中国典型培养物保藏中心,保藏编号为CCTCC NO:M 2023260。该弱毒疫苗株基因组的大小为4,711,745bp,GC含量为58.45%;具有4,128个编码蛋白质基因,128个转运RNA基因,31个核糖体RNA基因;具有4个CRISPR序列、4个基因岛、4个前噬菌体、4个基因簇、4个启动子和4个旁系同源基因。兔抗血清能与不同来源的维氏气单胞菌菌株产生良好的特异性凝集反应。弱毒活疫苗和灭活疫苗对淡水养殖鱼类安全性高,能有效预防维氏气单胞菌引起的细菌性败血症。对减少细菌性败血症防治中抗生素类药物的使用和保障水产品质量安全具有积极作用。
Description
技术领域
本发明涉及一种疫苗株及其应用,属于生物医药技术领域,具体涉及一种鱼类维氏气单胞菌弱毒疫苗株及其应用。
背景技术
淡水鱼细菌性败血症,是危害淡水鱼的种类最多、流行地域最广、流行季节最长、造成损失较大的一种急性传染病,其主要危害草鱼、鲢鱼、鳙鱼、鲤鱼、鲫鱼、鳊鱼、团头鲂、鳜鱼、罗非鱼、斑点叉尾鮰、鳖和蛙等淡水养殖水生动物,发病高峰期通常为每年的6~9月,常常出现急性暴发,死亡率高。病鱼大多有出血性败血症症状,如鳍条基部出血、肛门红肿、体表有出血点以及腹部肿胀,部分病鱼腹腔有血样腹水等。
引起淡水鱼细菌性败血症的主要病原为气单胞菌(Aeromonas spp.)。气单胞菌隶属于气单胞菌科(Aeromonadaceae)气单胞菌属(Aeromonas),其广泛分布于自然界的水体、土壤中,通常可以在淡水、海水、污水、健康或患病鱼类、食品以及动物粪便中分离到气单胞菌。气单胞菌被认为是一种机会致病菌,在外界条件如水温、水质、运输等条件影响下,会引起鱼类细菌性败血症的暴发。
2015年张德锋等对2009-2014年间我国南方地区70口池塘患病的草鱼、鲢鱼、鳙鱼、鲫鱼、鳜鱼、斑点叉尾和乌鳢等15种鱼类体内分离的143株气单胞菌为研究对象,通过细菌生理生化特征分析以及保守基因gyrB和rpoD序列测序分析,143株气单胞菌中有维氏气单胞菌80株,嗜水气单胞菌40株,简氏气单胞菌9株,舒氏气单胞菌10株和水生气单胞菌4株。表明近年来我国南方地区养殖鱼类气单胞菌病的病原具有较高的种类多样性,其中维氏气单胞菌(80/143,56%)的出现比例最高,该菌主要感染草鱼、鲢鱼和鳜鱼等;嗜水气单胞菌次之(40/143,28%),其主要感染鳙鱼、团头鲂和斑点叉尾等。
2021年刘小芳等通过PCR扩增管家基因gyrB,测序后构建系统发育进化树,把从患病草鱼、鲫鱼等淡水鱼分离的173株气单胞菌分类为维氏气单胞菌、嗜水气单胞菌和简氏气单胞菌3种。119株(68.8%,119/173)病原菌与维氏气单胞菌CECT 4486、CECT5761和ATCC35624等聚为一枝,这表明119株病原菌属于维氏气单胞菌类群。另外还发现,同一条患病鱼内脏中同时分离到维氏气单胞菌和嗜水气单胞菌,这说明维氏气单胞菌和嗜水气单胞菌能引起养殖鱼类复合感染,可能造成更大养殖损失。
目前国内已有针对嗜水气单胞菌的商品化灭活疫苗,也开展了维氏气单胞菌灭活疫苗和基因工程疫苗的相关研究,但还未有维氏气单胞菌天然弱毒活疫苗的。
发明内容
本发明目的在于拓展现有制备疫苗技术,提供维氏气单胞菌弱毒疫苗株及其应用。本发明提出了一种鱼类维氏气单胞菌弱毒疫苗株,2023年3月7日保藏于湖北省武汉市武汉大学的中国典型培养物保藏中心,保藏编号为CCTCC NO:M 2023260。
经测序分析,该维氏气单胞菌菌株FS12001基因组的大小为4,711,745bp,GC含量为58.45%。具有4,128个编码蛋白质基因(占基因组大小的86.65%),128个转运RNA基因,31个核糖体RNA基因。基因组有4个CRISPR序列、4个基因岛、4个前噬菌体、4个基因簇、4个启动子和4个旁系同源基因。基因组中重复序列总长度30,834bp,重复序列含量占0.65%。与已报道的不同来源的维氏气单胞菌有明显区别。
进一步而言,所述维氏气单胞菌菌株FS12001为弱毒株,对斑马鱼半数致死量为2.8×108CFU。
进一步而言,所述维氏气单胞菌弱毒株FS12001具有良好的凝集抗原性,其兔抗血清对不同来源的强毒的维氏气单胞菌菌株均能产生良好的特异性凝集反应。
本发明提供所述维氏气单胞菌弱毒株FS12001在制备疫苗中的应用,所述疫苗为活疫苗或灭活疫苗,对淡水鱼有良好的免疫效力,以安全剂量对鲫鱼进行高渗浸泡和注射免疫,可有效预防维氏气单胞菌引起的细菌性败血症。
进一步而言,所述疫苗可用于腹腔注射免疫或高渗浸泡免疫;
所述腹腔注射免疫中,使用所述维氏气单胞菌的活疫苗,菌株FS12001含量为3.0×107CFU/ml~3.0×108CFU/ml;使用所述维氏气单胞菌的灭活疫苗,菌株FS12001含量为3.0×109CFU/ml;
所述高渗浸泡免疫中,使用所述维氏气单胞菌的活疫苗,FS12001菌株含量为3.0×107CFU/ml~3.0×108CFU/ml;使用所述维氏气单胞菌的灭活疫苗,菌株FS12001含量为3.0×108CFU/ml。
进一步而言,所述维氏气单胞菌弱毒株FS12001在制备疫苗中的应用,所述疫苗应用于养殖草鱼、鲢鱼、鳙鱼、鲤鱼、鲫鱼、鳊鱼、团头鲂、鳜鱼等易发细菌性败血症的淡水鱼的免疫接种。
进一步而言,所述疫苗用于预防维氏气单胞菌引起的淡水鱼细菌性败血症。淡水鱼细菌性败血症以鳍条基部出血或充血、肛门红肿、体表有出血点以及腹部肿胀,肝脾肾组织有出血点或坏死、部分病鱼腹腔有血样腹水为主要症状。
借由上述技术方案,本发明具有如下优点和有益技术效果:
1)本发明通过攻毒斑马鱼,筛选出一株维氏气单胞菌天然弱毒株,该菌株分离自草鱼。该弱毒株的兔抗血清,能与不同来源的强毒的维氏气单胞菌菌株产生良好的特异性凝集反应,该弱毒株具有良好的抗原性;应用其制备的活疫苗和灭活疫苗,安全性高,腹腔注射和高渗浸泡免疫鲫鱼后,用强毒力的维氏气单胞菌菌株攻毒,分别有高达63%和46%的相对保护率。
2)本发明利用维氏气单胞菌天然弱毒株制备的弱毒疫苗和灭活疫苗,均能有效预防维氏气单胞菌引起的细菌性败血症,疫苗的应用对减少淡水鱼细菌性败血症防治中抗生素类药物使用、保障水产品质量安全具有积极作用。
附图说明
图1显示的是菌株FS12001的兔抗血清对维氏气单胞菌菌株的凝集反应结果;
图2显示的是菌株FS12001的16s RNA基因系统发育树;
图3显示的是菌株FS12001的gyrB基因系统发育树;
图4显示的是菌株FS12001的基因组圈图。
具体实施方式
本发明公开了一种鱼类维氏气单胞菌弱毒疫苗株及其应用。本发明的维氏气单胞菌Aeromonas veronii,2023年3月7日保藏于湖北省武汉市武汉大学的中国典型培养物保藏中心,保藏编号为CCTCC NO:M 2023260。经测序分析,该维氏气单胞菌FS12001基因组的大小为4,711,745bp,GC含量为58.45%。具有4,128个编码蛋白质基因(占基因组大小的86.65%),128个转运RNA基因,31个核糖体RNA基因。基因组有4个CRISPR序列、4个基因岛、4个前噬菌体、4个基因簇、4个启动子和4个旁系同源基因。基因组中重复序列总长度30,834bp,重复序列含量占0.65%。
本发明的鱼类维氏气单胞菌弱毒疫苗株与已报道的不同来源的维氏气单胞菌有明显区别。该弱毒株制备的兔抗血清,能与不同来源的强毒的维氏气单胞菌菌株产生良好的特异性凝集反应;应用其制备的弱毒活疫苗和灭活疫苗,对淡水养殖鱼类安全性高,能有效预防维氏气单胞菌引起的细菌性败血症,疫苗的应用对减少抗生素类药物使用、保障水产品质量安全具有积极作用。
以下分别通过具体较佳实施例结合效果试验例对本发明作进一步详细说明,但本发明并不仅限于以下的实施例。
实施例1维氏气单胞菌弱毒疫苗株的筛选
维氏气单胞菌菌株FS12001、AVCA07、GZ09007和YC170511均分离自患病的淡水养殖鱼,由本实验室鱼病室分离、鉴定和保存。将菌株分别接种TSB培养基,28℃、150rpm活化培养16~20h后,再划线接种绵羊血琼脂平板,28℃培养18~20h。分别刮取适量菌苔用灭菌0.65%盐水悬浮,活菌计数,同时10倍梯度稀释,制备不同浓度的菌液。每株菌的每个浓度菌液分别攻击20尾斑马鱼,每尾鱼腹腔注射20μl。注射前后各停喂饲料一日。注射后水温控制在27~29℃,每日观察并记录斑马鱼的死亡情况,连续观察记录7日。按Reed-Muench法计算半数致死量LD50。
结果如表1~4所示,菌株FS12001、AVCA07、GZ09007和YC170511对斑马鱼的半数致死量LD50分别为2.8×108CFU、1.5×103CFU、1.6×103CFU和1.1×103CFU。可见,菌株FS12001为弱毒株,AVCA07、GZ09007和YC170511为强毒株。
表1菌株FS12001的毒力测定
表2菌株AVCA07的毒力测定
表3菌株GZ09007的毒力测定
表4菌株的YC170511的毒力测定
实施例2维氏气单胞菌弱毒疫苗株抗原性评价
利用试管凝集试验来评价菌株FS12001的抗原性:将所有待检菌株FS12001、AVCA07、GZ09007和YC170511的细菌浓度调整为3.0×108CFU/ml。在不同试管中分别加入各个菌株的不同浓度的菌液2.7ml,然后再分别加入0.3ml的FS12001兔抗血清。将试管内的溶液轻微振荡混合后,在37℃恒温箱中静止反应3~6h,观察凝集结果。同时设FS12001菌株与阴性血清的对照。
结果如图1所示,FS12001兔抗血清对不同维氏气单胞菌菌株FS12001、AVCA07、GZ09007和YC170511均能产生良好的凝集反应,表明菌株FS12001的抗原性好。
(图1中试管1-4分别为菌株FS12001的兔抗血清对菌株AVCA07、GZ09007、YC170511、FS12001的凝集反应结果;试管5为阴性血清与菌株FS12001的凝集反应结果)
实施例3维氏气单胞菌FS12001株16s RNA和gyrB基因测序分析
将FS12001菌株接种在平板上,28℃培养过夜。利用细菌试剂盒提取病原菌基因组,扩增16S rRNA基因和看家基因gyrB。扩增16S rRNA基因的通用引物27F:5’-AGAGTTTGATCCTGGCTCAG-3',1492R:5’-GGTTACCTTGTTACGACTT-3'。扩增gyrB基因的上游引物gyrB-F:5’-TCCGGCGGTCTGCACGGCGT-3’,下游引物gyrB-R:5′-TTGTCCGGGTTGTACTCGTC-3′。PCR产物送公司测序,并将测得的基因序列在NCBI进行Blast同源性分析,调出与该序列相关性较高的核酸序列,通过ClustalX1.8软件进行多重比对分析,并采用MEGA7.0的邻接法构建系统发育树。
PCR扩增得到FS12001菌株16S rRNA基因序列大小约1500bp,看家基因gyrB大小约1200bp。在NCBI进行Blast同源性检索结果显示,该菌株16S rRNA和gyrB基因分别与维氏气单胞菌同源性高同源性达到99.9%以上。基于两种基因序列构建相应的系统发育树,两个序列均与维氏气单胞菌A.veronii聚为一支(图2、图3)。
实施例4全基因组测序
为了解维氏气单胞菌FS12001的基因组信息,应用试剂盒提取全基因组DNA,Illumina HISsq平台完成全基因组测序工作。三代测序产生的原始序列经过滤后获得68,610条高质量的测序序列、平均长度为7,763bp,共计532,635,333bp;用Hifiasm软件组装成4,711,745bp的基因组序列,该基因组组装程度达到单一长序列片段的水平,基因组内部没有缺失片段。经分析,FS12001基因组的大小为4,711,745bp,GC含量为58.45%。基因组含4,128个蛋白质编码基因,总长4,082,220bp,占基因组大小的86.65%;还具有128个转运RNA,31个核糖体RNA;基因组中重复序列总长度30,834bp,重复序列含量占0.65%。基因组含有4个CRISPR序列、4个基因岛、4个前噬菌体、4个基因簇、4个启动子和4个旁系同源基因(图4所示)。
将FS12001基因组预测的蛋白序列与eggNOG数据库比对,获得相应的功能注释结果,经统计共有21类3,077个基因(占编码基因总数74.54%)具有eggNOG功能分类,其中参与细菌转运与代谢相关过程(包括信号转导,转录,核苷酸转运和代谢,氨基酸转运和代谢,碳水化合物的运输和代谢,辅酶转运和代谢,脂类运输和新陈代谢,无机离子运输和代谢,次级代谢产物的合成、转运和分解代谢等)一共20类2,768个基因,占比达到eggNOG功能分类的72.82%;此外,还有724个(19.05%)未知功能基因。该菌株与已报道的不同来源的维氏气单胞菌有明显区别。
实施例5维氏气单胞菌弱毒活疫苗(FS12001株)的制备及安全性评价
将维氏气单胞菌菌株FS12001接种至TSB培养基,于28℃摇床活化18h;分别按1:100转接至500ml TSB培养基,28℃扩大培养18h。测定细菌培养液OD600值并进行活菌计数。
将上述新鲜培养的细菌培养液,离心后,弃上清液,用等体积的0.65%灭菌盐水重悬细菌沉淀,并依据活菌计数结果稀释成1.0×109CFU/ml、1.0×108CFU/ml和1.0×107CFU/ml 3个浓度的活疫苗。不同浓度的活疫苗,分别腹腔注射健康鲫鱼(体重18-20g,体长12-13cm)20尾,每尾注射0.2ml。连续观察14日。每日观察并记录试验池水温、水质、试验鱼的临床反应、摄食、游泳、活动情况及注射伤口的变化情况,统计死亡及健活鱼数。14天观察期结束后,随机选取3~5尾鲫解剖,与对照组比较,观察注射不同浓度活疫苗的鲫是否出现异常情况,观察鲫的体表和鳃、肝、脾、肾、肠道等组织器官的颜色和状态,并记录结果。
结果如表5所示,每日临床观察发现鲫鱼摄食、游动及体色、鱼便均正常。14天观察期结束后剖检,注射部位未出现结节或红肿;鳃无异常变化;腹腔无腹水;肝、脾、肾等器官无充血、出血、肿大或萎缩等改变;肠、腹膜无充血、出血。1.0×109CFU/ml、1.0×108CFU/ml和1.0×107CFU/ml 3个浓度的活疫苗对鱼均安全。
表5维氏气单胞菌FS12001活疫苗安全性试验结果
实施例6维氏气单胞菌弱毒活疫苗(FS12001株)的免疫效果评价
腹腔注射免疫:
制备菌株FS12001浓度为3.0×108CFU/ml和3.0×107CFU/ml的活疫苗、3.0×109CFU/ml的灭活疫苗(0.3%甲醛灭活)。各取适量的活疫苗和灭活疫苗,分别离心后,弃上清液,用等体积的0.65%灭菌盐水重悬细菌沉淀,分别腹腔注射免疫健康鲫鱼(体重18-20g,体长12-13cm)30尾,每尾注射0.2ml。另取30尾同批次同规格的鲫鱼作为对照组,腹腔注射0.65%灭菌盐水,0.2ml/尾。注射前后各停喂饲料一日。注射免疫后,放入不同鱼缸中,连续观察28日。免疫28日后,免疫组和对照组分别腹腔注射2LD50的维氏气单胞菌AVCA07菌液0.2ml(活菌数约1.2×106CFU)攻毒,攻毒前后分别禁食24h和72h。各组在水温27~29℃水环境隔离饲养,观察7日,每日记录各组的死鱼数,计算免疫组的相对保护率(RPS)。
结果如表6所示,对照组的死亡率90%,FS12001活疫苗组1(3.0×108CFU/ml)、FS12001活疫苗组2(3.0×107CFU/ml)和FS12001灭活疫苗组的死亡率分别为33%、50%和40%,相对保护率RPS分别为63%、44%和56%。
表6 FS12001活疫苗和灭活疫苗注射免疫鲫鱼的相对保护率
高渗浸泡免疫:
制备菌株FS12001浓度3.0×108CFU/ml和3.0×107CFU/ml的活疫苗、3.0×108CFU/ml的灭活疫苗(0.3%甲醛灭活)各3.0L,分别加入30.0g NaCL,NaCL终浓度为1.0%。每种疫苗溶液中各放入30尾鲫鱼(体重8-10g,体长8-9cm),充氧浸泡20~40min。另取30尾同批次同规格鲫鱼作为对照组,仅在3.0L的1.0%NaCL溶液中充氧浸泡20~40min。免疫前后各停喂饲料一日。浸泡免疫后,放入不同鱼缸中,连续观察28日。
免疫28日后,免疫组和对照组分别腹腔注射维氏气单胞菌AVCA07菌液0.2ml(活菌数约6.0×105CFU)攻毒,攻毒前后分别停饲24h和48h。各组在水温27~29℃水环境隔离饲养,观察7日,每日记录各组的死鱼数,计算免疫组的相对保护率(RPS)。
结果如表7所示,对照组的死亡率93%,FS12001活疫苗组1(3.0×108CFU/ml)、FS12001活疫苗组2(3.0×107CFU/ml)和FS12001灭活疫苗组的鲫鱼死亡率分别为50%、63%和60%,相对保护率RPS分别为46%、32%和35%。
表7 FS12001活疫苗和灭活疫苗高渗浸泡免疫鲫鱼的相对保护率
以上所述,仅是本发明的较佳实施例而已,并非对本发明作任何形式上的限制,故凡是未脱离本发明技术方案内容,依据本发明的技术实质对以上实施例所作的任何简单修改、等同变化与修饰,均仍属于本发明技术方案的范围内。
Claims (8)
1.一种鱼类维氏气单胞菌弱毒疫苗株,其特征在于:所述鱼类维氏气单胞菌弱毒疫苗株,其命名为维氏气单胞菌菌株FS12001,于2023年3月7日保藏于湖北省武汉市武汉大学的中国典型培养物保藏中心,保藏编号为CCTCC NO:M 2023260。
2.如权利要求1所述的鱼类维氏气单胞菌弱毒疫苗株,其特征在于:经测序分析,该维氏气单胞菌FS12001基因组的大小为4,711,745bp,GC含量为58.45%;具有4,128个编码蛋白质基因,占基因组大小的86.65%;具有128个转运RNA基因,31个核糖体RNA基因;基因组有4个CRISPR序列、4个基因岛、4个前噬菌体、4个基因簇、4个启动子和4个旁系同源基因;基因组中重复序列总长度30,834bp,重复序列含量占0.65%。
3.如权利要求2所述的鱼类维氏气单胞菌弱毒疫苗株,其特征在于:所述维氏气单胞菌菌株FS12001为弱毒株,对斑马鱼半数致死量为2.8×108CFU。
4.权利要求1-3中任一项所述的鱼类维氏气单胞菌弱毒疫苗株在制备疫苗中的应用,其特征在于:所述鱼类维氏气单胞菌弱毒疫苗株为维氏气单胞菌菌株FS12001;所述疫苗用于预防维氏气单胞菌引起的细菌性败血症。
5.如权利要求4所述的应用,其特征在于:所述疫苗为活疫苗或灭活疫苗。
6.如权利要求4所述的应用,其特征在于:所述疫苗可用于腹腔注射免疫或高渗浸泡免疫;
所述腹腔注射免疫中,使用所述维氏气单胞菌的活疫苗,菌株FS12001含量为3.0×107CFU/ml~3.0×108CFU/ml;使用所述维氏气单胞菌的灭活疫苗,菌株FS12001含量为3.0×109CFU/ml;
所述高渗浸泡免疫中,使用所述维氏气单胞菌的活疫苗,菌株FS12001含量为3.0×107CFU/ml~3.0×108CFU/ml;使用所述维氏气单胞菌的灭活疫苗,菌株FS12001含量为3.0×108CFU/ml。
7.如权利要求4所述的应用,其特征在于:所述疫苗应用于养殖草鱼、鲢鱼、鳙鱼、鲤鱼、鲫鱼、鳊鱼、团头鲂、鳜鱼等易发细菌性败血症的淡水鱼的免疫接种。
8.如权利要求7所述的应用,其特征在于:所述疫苗用于预防维氏气单胞菌引起的淡水鱼细菌性败血症;
所述淡水鱼细菌性败血症以鳍条基部出血或充血、肛门红肿、体表有出血点以及腹部肿胀,肝脾肾组织有出血点或坏死、部分病鱼腹腔有血样腹水为主要症状。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202310377122.1A CN117106613B (zh) | 2023-04-10 | 2023-04-10 | 一种鱼类维氏气单胞菌弱毒疫苗株及其应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202310377122.1A CN117106613B (zh) | 2023-04-10 | 2023-04-10 | 一种鱼类维氏气单胞菌弱毒疫苗株及其应用 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN117106613A true CN117106613A (zh) | 2023-11-24 |
CN117106613B CN117106613B (zh) | 2024-01-30 |
Family
ID=88797210
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202310377122.1A Active CN117106613B (zh) | 2023-04-10 | 2023-04-10 | 一种鱼类维氏气单胞菌弱毒疫苗株及其应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN117106613B (zh) |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN104784683A (zh) * | 2015-03-16 | 2015-07-22 | 西南大学 | 一种维氏气单胞菌渔用注射疫苗的制备及使用方法 |
CN105779370A (zh) * | 2016-01-07 | 2016-07-20 | 海南大学 | 一种鱼类败血病病原维氏气单胞菌减毒活疫苗及其应用 |
CN110862951A (zh) * | 2019-09-06 | 2020-03-06 | 海南大学 | 一种维氏气单胞菌减毒菌株的构建方法、菌株及其应用 |
-
2023
- 2023-04-10 CN CN202310377122.1A patent/CN117106613B/zh active Active
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN104784683A (zh) * | 2015-03-16 | 2015-07-22 | 西南大学 | 一种维氏气单胞菌渔用注射疫苗的制备及使用方法 |
CN105779370A (zh) * | 2016-01-07 | 2016-07-20 | 海南大学 | 一种鱼类败血病病原维氏气单胞菌减毒活疫苗及其应用 |
CN105950526A (zh) * | 2016-01-07 | 2016-09-21 | 海南大学 | 一种鱼类败血病病原维氏气单胞菌减毒活疫苗及其应用 |
CN110862951A (zh) * | 2019-09-06 | 2020-03-06 | 海南大学 | 一种维氏气单胞菌减毒菌株的构建方法、菌株及其应用 |
Non-Patent Citations (2)
Title |
---|
任燕等: "两株草鱼源维氏气单胞菌菌株的主要表型特征及致病力比较", 中国预防兽医学报, vol. 41, no. 2 * |
刘小芳: "鱼源气单胞菌的毒力基因检测、分型及致病力", 水产学报, vol. 45, no. 3 * |
Also Published As
Publication number | Publication date |
---|---|
CN117106613B (zh) | 2024-01-30 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Madetoja et al. | Occurrence of Flavobacterium psychrophilum in fish-farming environments | |
Liu et al. | First report of Vibrio vulnificus infection in grass carp Ctenopharyngodon idellus in China | |
Elbahnaswy et al. | Differential gene expression and immune response of Nile tilapia (Oreochromis niloticus) challenged intraperitoneally with Photobacterium damselae and Aeromonas hydrophila demonstrating immunosuppression | |
CN109735471B (zh) | 一株微小杆菌及其作为益生菌在水产上的应用 | |
Soto et al. | Co-infection of Acipenserid herpesvirus 2 (AciHV-2) and Streptococcus iniae in cultured white sturgeon Acipenser transmontanus | |
CN104560851B (zh) | 杀鲑气单胞菌活疫苗制剂和冻干疫苗制品的制备方法及应用 | |
DK181323B1 (en) | Method and composition comprising probiotic bacteria for increasing weight of fish and isolated strains of psychrobacter and pseudomonas | |
CN108939062A (zh) | 一种杀鲑气单胞菌疫苗及其应用 | |
CN113201501A (zh) | 一株具有跨种裂解能力的弧菌噬菌体及其应用 | |
CN114908013A (zh) | 一株产ddp-iv抑制剂的厦门希瓦氏菌及其应用 | |
CN112375712A (zh) | 一株乳酸乳球菌及其应用 | |
Yang et al. | A single WAP domain (SWD)-containing protein with antiviral activity from Pacific white shrimp Litopenaeus vannamei | |
Zhang et al. | Adaptively differential expression analysis in gill of Chinese mitten crabs (Eriocheir japonica sinensis) associated with salinity changes | |
CN111575243B (zh) | 坎氏弧菌噬菌体及其应用 | |
CN103014051B (zh) | 鱼用多效价活疫苗及其应用 | |
CN117106613B (zh) | 一种鱼类维氏气单胞菌弱毒疫苗株及其应用 | |
CN115197919B (zh) | 弧菌噬菌体组合物及其制备方法和应用 | |
CN105886429B (zh) | 一种无抗生素标记的嗜水气单胞菌减毒菌及应用 | |
Chen et al. | Aeromonas veronii infection remarkably increases expression of lysozymes in grass carp (Ctenopharyngodon idellus) and injection of lysozyme expression cassette along with QCDC adjuvant significantly upregulates immune factors and decreases cumulative mortality | |
CN110408573B (zh) | 一种鼠李糖乳杆菌yfi-6及其在抗大鲵虹彩病毒中的应用 | |
CN110713955A (zh) | 一株乳酸菌及其在水产养殖中的应用 | |
EP3273775B1 (en) | Process to infect crustaceans with infectious agents | |
CN111484960A (zh) | 新型爱德华氏菌减毒靶点及其应用 | |
CN109385406A (zh) | 一株副溶血弧菌噬菌体及其在增强水产动物免疫力方面的应用 | |
WO2023022080A1 (ja) | 新規微生物、新規微生物等を含有する抗ホワイトスポットウイルス剤およびその製造方法、並びに抗ホワイトスポットウイルスの防除方法 |
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 |