CN116042536B - 一种牛疱疹病毒4型毒株及其培养方法和应用 - Google Patents
一种牛疱疹病毒4型毒株及其培养方法和应用 Download PDFInfo
- Publication number
- CN116042536B CN116042536B CN202210920690.7A CN202210920690A CN116042536B CN 116042536 B CN116042536 B CN 116042536B CN 202210920690 A CN202210920690 A CN 202210920690A CN 116042536 B CN116042536 B CN 116042536B
- Authority
- CN
- China
- Prior art keywords
- virus
- strain
- bovine
- bhv
- serum
- 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
Links
- 241000701066 Bovine gammaherpesvirus 4 Species 0.000 title claims abstract description 24
- 238000012136 culture method Methods 0.000 title abstract description 7
- 241000700605 Viruses Species 0.000 claims abstract description 60
- 241000283690 Bos taurus Species 0.000 claims abstract description 24
- 238000002360 preparation method Methods 0.000 claims abstract description 4
- 238000012258 culturing Methods 0.000 claims description 8
- 238000000034 method Methods 0.000 claims description 8
- 241001529453 unidentified herpesvirus Species 0.000 claims description 7
- 108020004414 DNA Proteins 0.000 claims description 6
- 102000053602 DNA Human genes 0.000 claims description 6
- 239000007788 liquid Substances 0.000 claims description 6
- 229960005486 vaccine Drugs 0.000 claims description 6
- 208000015181 infectious disease Diseases 0.000 claims description 5
- 238000003306 harvesting Methods 0.000 claims description 3
- 238000009472 formulation Methods 0.000 claims description 2
- 239000000203 mixture Substances 0.000 claims description 2
- 239000002773 nucleotide Substances 0.000 claims description 2
- 125000003729 nucleotide group Chemical group 0.000 claims description 2
- 210000002966 serum Anatomy 0.000 abstract description 18
- 210000000056 organ Anatomy 0.000 abstract description 6
- 230000005847 immunogenicity Effects 0.000 abstract description 4
- 210000004072 lung Anatomy 0.000 abstract description 4
- 210000000952 spleen Anatomy 0.000 abstract description 4
- 244000309466 calf Species 0.000 abstract description 3
- 210000003734 kidney Anatomy 0.000 abstract description 3
- 210000004185 liver Anatomy 0.000 abstract description 3
- 230000007918 pathogenicity Effects 0.000 abstract description 3
- 238000004321 preservation Methods 0.000 abstract description 3
- 210000004291 uterus Anatomy 0.000 abstract description 3
- 210000001215 vagina Anatomy 0.000 abstract description 3
- 231100000915 pathological change Toxicity 0.000 abstract 1
- 230000036285 pathological change Effects 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 22
- 230000003053 immunization Effects 0.000 description 13
- 238000002649 immunization Methods 0.000 description 12
- 238000006386 neutralization reaction Methods 0.000 description 7
- 238000001514 detection method Methods 0.000 description 6
- 238000003745 diagnosis Methods 0.000 description 6
- 238000010790 dilution Methods 0.000 description 6
- 239000012895 dilution Substances 0.000 description 6
- 238000012360 testing method Methods 0.000 description 6
- 239000000243 solution Substances 0.000 description 5
- 238000011081 inoculation Methods 0.000 description 4
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 3
- 241000204031 Mycoplasma Species 0.000 description 3
- 239000002671 adjuvant Substances 0.000 description 3
- 230000000120 cytopathologic effect Effects 0.000 description 3
- 201000010099 disease Diseases 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 230000002779 inactivation Effects 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
- 210000004698 lymphocyte Anatomy 0.000 description 3
- 210000002540 macrophage Anatomy 0.000 description 3
- 210000004877 mucosa Anatomy 0.000 description 3
- 230000017074 necrotic cell death Effects 0.000 description 3
- 230000003472 neutralizing effect Effects 0.000 description 3
- 210000000440 neutrophil Anatomy 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- 238000003756 stirring Methods 0.000 description 3
- 241000710780 Bovine viral diarrhea virus 1 Species 0.000 description 2
- 241000283977 Oryctolagus Species 0.000 description 2
- 241000283973 Oryctolagus cuniculus Species 0.000 description 2
- 206010046793 Uterine inflammation Diseases 0.000 description 2
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 2
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 2
- 235000011130 ammonium sulphate Nutrition 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 239000006285 cell suspension Substances 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- 238000002955 isolation Methods 0.000 description 2
- 238000012423 maintenance Methods 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 239000013642 negative control Substances 0.000 description 2
- 210000004180 plasmocyte Anatomy 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 208000024891 symptom Diseases 0.000 description 2
- 201000004813 Bronchopneumonia Diseases 0.000 description 1
- 206010010741 Conjunctivitis Diseases 0.000 description 1
- 201000004624 Dermatitis Diseases 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 208000004232 Enteritis Diseases 0.000 description 1
- 241000700586 Herpesviridae Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 206010035664 Pneumonia Diseases 0.000 description 1
- 238000012300 Sequence Analysis Methods 0.000 description 1
- 210000002718 aborted fetus Anatomy 0.000 description 1
- 206010000210 abortion Diseases 0.000 description 1
- 231100000176 abortion Toxicity 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 208000037883 airway inflammation Diseases 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000000740 bleeding effect Effects 0.000 description 1
- 238000010241 blood sampling Methods 0.000 description 1
- 230000036760 body temperature Effects 0.000 description 1
- 210000000481 breast Anatomy 0.000 description 1
- 239000006143 cell culture medium Substances 0.000 description 1
- 230000007910 cell fusion Effects 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 210000002808 connective tissue Anatomy 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000007850 degeneration Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 239000012154 double-distilled water Substances 0.000 description 1
- 238000001493 electron microscopy Methods 0.000 description 1
- 210000002919 epithelial cell Anatomy 0.000 description 1
- 210000003743 erythrocyte Anatomy 0.000 description 1
- 230000003203 everyday effect Effects 0.000 description 1
- 230000002550 fecal effect Effects 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 230000001434 glomerular Effects 0.000 description 1
- 210000003128 head Anatomy 0.000 description 1
- 210000003494 hepatocyte Anatomy 0.000 description 1
- 206010020718 hyperplasia Diseases 0.000 description 1
- 230000002163 immunogen Effects 0.000 description 1
- 230000000415 inactivating effect Effects 0.000 description 1
- 230000036512 infertility Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 210000005075 mammary gland Anatomy 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 238000005497 microtitration Methods 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 210000003928 nasal cavity Anatomy 0.000 description 1
- 238000011587 new zealand white rabbit Methods 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 239000001044 red dye Substances 0.000 description 1
- 230000000405 serological effect Effects 0.000 description 1
- 239000002356 single layer Substances 0.000 description 1
- 206010040882 skin lesion Diseases 0.000 description 1
- 231100000444 skin lesion Toxicity 0.000 description 1
- 239000002002 slurry Substances 0.000 description 1
- 230000007863 steatosis Effects 0.000 description 1
- 231100000240 steatosis hepatitis Toxicity 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 239000003053 toxin Substances 0.000 description 1
- 231100000765 toxin Toxicity 0.000 description 1
- 210000004926 tubular epithelial cell Anatomy 0.000 description 1
- 238000002255 vaccination Methods 0.000 description 1
- 230000009723 vascular congestion Effects 0.000 description 1
- 230000009385 viral infection Effects 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
- C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/12—Viral antigens
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/20—Antivirals for DNA viruses
- A61P31/22—Antivirals for DNA viruses for herpes viruses
-
- 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
- A61K2039/53—DNA (RNA) vaccination
-
- 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
- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/16011—Herpesviridae
- C12N2710/16711—Varicellovirus, e.g. human herpesvirus 3, Varicella Zoster, pseudorabies
- C12N2710/16721—Viruses as such, e.g. new isolates, mutants or their genomic sequences
-
- 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
- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/16011—Herpesviridae
- C12N2710/16711—Varicellovirus, e.g. human herpesvirus 3, Varicella Zoster, pseudorabies
- C12N2710/16734—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
-
- 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
- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/16011—Herpesviridae
- C12N2710/16711—Varicellovirus, e.g. human herpesvirus 3, Varicella Zoster, pseudorabies
- C12N2710/16751—Methods of production or purification of viral material
-
- 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)
- Virology (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Genetics & Genomics (AREA)
- Pharmacology & Pharmacy (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Immunology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Animal Behavior & Ethology (AREA)
- Biomedical Technology (AREA)
- Epidemiology (AREA)
- Mycology (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Molecular Biology (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)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
一种牛疱疹病毒4型毒株及其培养方法和应用，命名为DN01，该病毒已于2021年12月28日在中国典型培养物保藏中心进行的保藏，保藏编号为CCTCC NO:V202193。该病毒对犊牛具有较强致病力，能引起肾脏、肝脏、肺脏、脾脏、阴道和子宫等器官的病变；同时具备良好的免疫原性，可诱发母牛产生抗体；根据本发明中的病毒制备的高滴度的血清抗体，具有较好的血清效价。
Description
技术领域
本发明涉及分子生物技术领域，特别是一种牛疱疹病毒4型毒株及其培养方法和应用。
背景技术
牛疱疹病毒(Bovine herpesvirus，BHV-4)属于疱疹病毒科，丙型疱疹病毒亚科，猴病毒属，在国内和国外牛场中均有蔓延。这种病毒在患有结膜炎、肺炎和上呼吸道炎症、肠炎、皮肤病损、乳房皮炎、产后子宫炎、慢性子宫炎的牛身上皆有分离到。另外，BHV-4还与流产相关，在流产胎儿中有同时发现牛病毒性腹泻病毒(BVDV)和BHV-4。该病多为牛的隐性感染，结合其他病原一起发病，可能使临床症状加剧，且表现多样化，给诊断和治疗带来较大困难，对养牛业构成严重威胁。
由于该病感染多为隐性感染，合并其他病原感染后的临床表现多样化，难以通过临床观察作出诊断，因此，需要依靠实验室检测进行确诊，如病毒的分离鉴定，血清学诊断，以及分子生物学检测。目前对于BHV-4的诊断研究的较少，因此寻找一个具有较好免疫原性的毒株对于BHV-4的预防措施及诊断方法的研制有重大的意义。
发明内容
解决的技术问题：为了克服现有技术的不足，本发明提供一种牛疱疹病毒4型毒株及其培养方法和应用。该病毒可在MAC-T细胞中增殖，通过该病毒制得的灭活和纯化病毒可诱发母牛产生抗BHV-4的抗体；且制备的血清抗体，具有较好的血清效价。
技术方案：一种牛疱疹病毒4型毒株，命名为DN01，该病毒已于2021年12月28日在中国典型培养物保藏中心进行的保藏，保藏编号为CCTCC NO:V202193。
编码所述病毒的DNA分子，核苷酸序列如SEQ ID NO.1所示。
上述牛疱疹病毒4型毒株的培养方法，所述病毒感染MAC-T细胞后，37℃，5％ CO2培养箱中培养，当细胞病变达80％以上时，收获病毒液。
一种牛疱疹病毒BHV-4疫苗，含有上述毒株。
一种牛疱疹病毒BHV-4疫苗，含有上述DNA分子。
如下(1)-(2)任一项在制备疫苗制剂中的用途：(1)所述的牛疱疹病毒4型毒株；(2)权利要求2所述的DNA分子。
有益效果：本发明中的病毒DN01可在MAC-T细胞中增殖，病毒浓度可达107TCID50/mL，无外源病毒污染；本发明中的病毒DN01对牛具有致病力，病毒感染了犊牛之后产生了肾脏、肝脏、肺脏、脾脏、阴道和子宫等器官的病变；根据本发明中的病毒DN01制备的灭活和纯化病毒可诱发母牛产生抗BHV-4的抗体；根据本发明中的病毒DN01制备的血清抗体，具有较好的血清效价。
附图说明
图1为本发明中BHV-4的电镜观察图。
图2为本发明中各器官组织病理学观察图。
具体实施方式
下面结合附图和具体实施方式对本发明作进一步详细说明。
若未特别指明，实施例中所用的技术手段为本领域技术人员所熟知的常规手段，所用原料均为市售商品。
实施例1病毒的分离培养和鉴定
(1)病毒的分离
2020年底采集自河北某养殖场发病牛的粪便样本，低温运输至实验室。用MAC-T细胞(牛乳腺上皮细胞)培养法对样本进行病毒分离，培养三代后细胞出现细胞融合，进行病毒分离，获得病毒DN01,该病毒已于2021年12月28日在中国典型培养物保藏中心进行的保藏，保藏编号为CCTCC NO:V202193，保藏地址为中国湖北省武汉市武昌区八一路299号，武汉大学。
(2)病毒的鉴定
1.基因组序列分析
对该病毒的基因组序列进行分析，全基因序列为SEQ ID No.1所示的序列(见序列表)。
2.电镜检查
在电镜下观察牛疱疹病毒4型DN01，如图2所示，病毒颗粒直径范围在150nm左右，表面有囊膜。
3.无菌、支原体检测
按照《中国兽药典》方法对该病毒进行无菌、支原体检查，结果表明无支原体和其它微生物污染。
4.病毒滴度检测
采用微量滴定法测定该株病毒的病毒浓度，方法如下：用96孔细胞培养板培养MAC-T细胞，将病毒用细胞维持液从10-1倍比稀释至10-8，分别将每个稀释度的病毒液接种100μL/孔，每个稀释度接种8孔，将对数生长期的MAC-T细胞稀释至3×106个细胞/mL，每个细胞孔中接种100μL，同时设细胞对照孔。置37℃，5％CO2培养箱中培养，4-6天观察细胞感染病毒情况，计算病毒浓度，病毒浓度达107TCID50/mL。
实施例2DN01毒株的致病力
将4头试验犊牛随机分成2组，检测其牛疱疹病毒4型均为阴性。第1组接种107TCID50的病毒DN01，第2组接种4mL DMEM作为阴性对照组，接种方式为鼻腔接种。
接种后14天内，每天测量体温2次，记录临床症状，采集鼻拭子、眼拭子和肛门拭子进行病毒检测。在接种后第18天确定鼻腔停止排毒后，安乐死所有的实验牛，采集组织器官等样本，作组织病理学观察。结果显示对照组各器官正常，而实验组各器官病变程度如下(图2)：
肾脏：肾小球囊腔浆液和红细胞渗出，肾小管蛋白管型，间质结缔组织增生，肾小管上皮细胞变性坏死，淋巴细胞为主的炎性细胞浸润。
肝脏：肝细胞脂肪变性。
肺脏：肺脏支气管性肺炎，部分支气管黏膜坏死脱落，腔内大量细胞堆积。
脾脏：脾脏出血，并见有大量中性细胞浸润。
阴道：阴道黏膜坏死，血管充血，大量中性细胞、浆细胞、巨噬细胞、淋巴细胞等炎性细胞浸润。
子宫：子宫黏膜坏死，大量中性细胞、浆细胞、巨噬细胞、淋巴细胞等炎性细胞浸润，部分巨噬细胞空泡化，胞浆见有红染圆形蛋白物质。
实施例3母牛免疫原性试验
(1)病毒培养
复苏MAC-T细胞，经扩增，至细胞长至薄单层后，将病毒DN01接种至细胞瓶内，37℃，5％CO2培养箱中培养，每天观察细胞病变，当细胞病变达80％以上时，收获病毒液，-20℃保存备用。
(2)病毒灭活和纯化
病毒灭活前测定TCID50。将上述制备的病毒液用甲醛灭活24-48h。将病毒液按100mL加入42.5g硫酸铵的量加入硫酸铵，搅匀后至4℃冰箱搅拌沉淀过夜后，5000r/min离心40min，弃上清，沉淀用灭菌双蒸水悬浮，装于透析袋中，于PBS中搅拌透析过夜，即为纯化BHV-4病毒。病毒纯化后进行离心：10000rpm下10min，取上清液并将其病毒浓度调整为107TCID50/mL，即可用于后续免疫原性试验。
(3)抗体中和实验
将上述制备的纯化后病毒免疫荷斯坦母牛，4mL/头/次，在整个试验过程中观察记录母牛的健康情况，第一次免疫后20天进行第二次免疫，并于一免前、二免前(一免后20天)、一免后40天、一免后60天采集母牛的血液，收集血清，进行抗体中和测定。
方法如下：在96孔细胞培养板中加入分离获得的母牛血清，用细胞生长液倍比稀释至一定浓度后(每个稀释孔4个重复)，加入事先稀释至200TCID50/mL的病毒液，混合均匀后放入37℃，5％CO2培养箱中中和1h-2h，然后加入细胞悬液，细胞浓度为2-3×105个/mL，置于37℃，5％CO2培养箱中进行培养。5-7天后观察细胞病变情况，其中以半数细胞出现病变的血清的最高稀释倍数为血清的中和效价，阳性判定指标为：中和效价大于1:8；阴性血清效价小于1:8。结果见下表1。
表1为母牛免疫试验中和抗体检查结果
| 采血时间 | 中和效价 |
| 免疫前(0d) | ＜8 |
| 免疫后(20d) | 8 |
| 免疫后(40d) | 32 |
| 免疫后(60d) | 16 |
从表1可以看出，使用本发明病毒DN01制备的灭活和纯化病毒可诱发母牛产生抗BHV-4的抗体，并随着免疫次数的增加而呈上升趋势。
实施例4以病毒DN01为免疫原制备多抗血清
病毒DN01经过感染细胞、培养、收获病毒液、测定TCID50、灭活和纯化后，与弗氏完全佐剂充分乳化，免疫3只新西兰大白兔。采取颈背部皮下多点注射，2mL/只。免疫两周后加强免疫，佐剂换成弗氏不完全佐剂，共加强免疫3次，最后一次免疫2周后采血，分离血清进行中和抗体效价测定。
中和抗体效价测定方法：在96孔细胞板内加入分离的兔血清，用细胞维持液进行倍比稀释，加入稀释好的100TCID50/mL的病毒DN01病毒液，中和2-3h后加入细胞悬液，细胞浓度为2-3×105个/mL，置37℃，5％CO2培养箱中进行培养。5-7天后观察细胞病变情况，其中以出现细胞病变的血清最高稀释度为血清中和效价，阳性判断标准为：中和效价大于1:8，阴性对照血清效价小于1:8。试验结果显示3只新西兰兔的抗DN01血清的效价均大于1:1024。该结果证实使用本发明的病毒DN01免疫新西兰兔可获得血清效价较好的兔抗DN01多抗血清。
显然，上述实施例仅仅是为清楚地说明所作的举例，而并非对实施方式的限定。对于所属领域的普通技术人员来说，在上述说明的基础上还可以做出其它不同形式的变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引伸出的显而易见的变化或变动仍处于本发明创造的保护范围之中。
Claims (6)
1.一种牛疱疹病毒4型毒株，命名为DN01，该病毒已于2021年12月28日在中国典型培养物保藏中心进行的保藏，保藏编号为CCTCC NO:V202193。
2.编码权利要求1 所述病毒的DNA分子，其特征在于，核苷酸序列如SEQ ID NO.1 所示。
3.权利要求1所述牛疱疹病毒4型毒株的培养方法，其特征在于，所述病毒感染MAC-T细胞后，37℃，5% CO2培养箱中培养，当细胞病变达80%以上时，收获病毒液。
4.一种牛疱疹病毒BHV-4疫苗，其特征在于，含有权利要求1所述毒株。
5.一种牛疱疹病毒BHV-4疫苗，其特征在于，含有权利要求2所述DNA分子。
6.如下（1）-（2）任一项在制备疫苗制剂中的用途：（1）权利要求1所述的牛疱疹病毒4型毒株；（2）权利要求2所述的DNA分子。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210920690.7A CN116042536B (zh) | 2022-08-02 | 2022-08-02 | 一种牛疱疹病毒4型毒株及其培养方法和应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210920690.7A CN116042536B (zh) | 2022-08-02 | 2022-08-02 | 一种牛疱疹病毒4型毒株及其培养方法和应用 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN116042536A CN116042536A (zh) | 2023-05-02 |
| CN116042536B true CN116042536B (zh) | 2023-12-29 |
Family
ID=86118740
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210920690.7A Active CN116042536B (zh) | 2022-08-02 | 2022-08-02 | 一种牛疱疹病毒4型毒株及其培养方法和应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116042536B (zh) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117070476B (zh) * | 2023-10-13 | 2023-12-19 | 金宇保灵生物药品有限公司 | 一株牛疱疹病毒4型毒株及其在灭活疫苗制备中的应用 |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113061581A (zh) * | 2021-03-22 | 2021-07-02 | 华中农业大学 | 一种山羊源牛疱疹病毒1型毒株及其应用 |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2011026882A1 (en) * | 2009-09-03 | 2011-03-10 | Intervet International B.V. | Bovine herpesvirus vaccine |
-
2022
- 2022-08-02 CN CN202210920690.7A patent/CN116042536B/zh active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113061581A (zh) * | 2021-03-22 | 2021-07-02 | 华中农业大学 | 一种山羊源牛疱疹病毒1型毒株及其应用 |
Non-Patent Citations (3)
| Title |
|---|
| Cellular Targeting of Engineered Heterologous Antigens Is a Determinant Factor for Bovine Herpesvirus 4-Based Vaccine Vector Development;DONOFRIO, G.等;《ASM Journals》;第16卷(第11期);全文 * |
| Evaluation of Newly Developed Immunoperoxidase Monolayer Assays for Detection of Antibodies against Bovine Herpesvirus 4;WELLENBERG, G.J.等;《ASM Journals》;第6卷(第4期);全文 * |
| 牛疱疹病毒4研究进展;陈弟诗等;《动物医学进展》;第32卷(第6期);127-133 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN116042536A (zh) | 2023-05-02 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN112481154B (zh) | 一种绵羊肺炎支原体疫苗株、由该疫苗株制备得到的疫苗组合物及其应用 | |
| CN104099301B (zh) | 柯萨奇病毒a16型病毒株、用途、疫苗及其制备方法 | |
| CN112779193B (zh) | 一株滑液囊支原体强毒株及其应用 | |
| CN107375922B (zh) | 一种水溶性复合免疫佐剂及猪圆环病毒病疫苗 | |
| CN116042536B (zh) | 一种牛疱疹病毒4型毒株及其培养方法和应用 | |
| CN111690554B (zh) | 用于制备羊支原体肺炎疫苗的组合菌株、羊支原体肺炎三价灭活疫苗及其制备方法 | |
| CN111073863B (zh) | 猪流行性腹泻、猪德尔塔冠状病毒二联弱毒疫苗及其制备方法 | |
| WO2023045426A1 (zh) | 柯萨奇病毒a16型毒株及其免疫原性组合物和应用 | |
| CN113957007B (zh) | 一种鸡滑液囊支原体灭活疫苗 | |
| CN110314228B (zh) | 猪流行性腹泻、猪δ冠状病毒二联灭活疫苗以及其制备方法 | |
| CN111537714B (zh) | 一种用于检测鸡滑液囊支原体的冻干血凝抑制试验抗原及抗原组合、制备方法 | |
| CN116926022B (zh) | 一种鸽源基因Ⅵ型新城疫病毒mGZ08、用途、制备方法、培养方法、疫苗 | |
| CN101220351A (zh) | 利用Nsp2基因缺失构建PRRSV基因缺失疫苗毒株的方法及其应用 | |
| CN102805862B (zh) | 一种vero细胞培养新布尼亚病毒纯化灭活疫苗的制备方法 | |
| CN109402066B (zh) | 一株7型腺病毒及其制备的疫苗制品 | |
| CN109402067B (zh) | 一株55型腺病毒及其制备的疫苗制品 | |
| CN109207577B (zh) | Marco在筛选抗蓝耳病猪中的应用 | |
| CN104099269B (zh) | 猪肺炎支原体强毒株及其应用 | |
| CN112280748B (zh) | 绵羊源羊副流感病毒3型疫苗株、由该疫苗株制备的疫苗组合物及其制备方法和应用 | |
| CN109010814A (zh) | 副猪嗜血杆菌和猪肺炎支原体二联灭活疫苗的生产方法 | |
| CN107686833B (zh) | 一种猪细小病毒毒株及其应用 | |
| WO2024148700A1 (zh) | 一株赤羽病病毒毒株及其应用 | |
| CN112725288B (zh) | 犬腺病毒2型弱毒疫苗株及其应用 | |
| CN115161211B (zh) | 一种牛溶血性曼氏杆菌菌株及由其制备的灭活疫苗和疫苗制备方法 | |
| CN114015658B (zh) | 一种h9n2亚型禽流感和鸡滑液囊支原体的二联灭活疫苗 |
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 | ||
| CB03 | Change of inventor or designer information | ||
| CB03 | Change of inventor or designer information |
Inventor after: Pan Zihao Inventor after: Du Ju Inventor after: Wang Juanfang Inventor after: Yu Juxian Inventor before: Du Ju Inventor before: Wang Juanfang Inventor before: Yu Juxian |
|
| TA01 | Transfer of patent application right | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20230912 Address after: 210000 Wei Gang 1, Xuanwu District, Nanjing, Jiangsu Applicant after: NANJING AGRICULTURAL University Address before: Room 1104-2, spark maker space, Nanjing National Agricultural Innovation Park, No. 20, Yuhe Road, Pukou District, Nanjing, Jiangsu 210000 Applicant before: Daneng Biotechnology (Nanjing) Co.,Ltd. |
|
| GR01 | Patent grant | ||
| GR01 | Patent grant |