CN116574166A - 一种鱼类β防御素和外膜蛋白C的双顺反子DNA疫苗 - Google Patents
一种鱼类β防御素和外膜蛋白C的双顺反子DNA疫苗 Download PDFInfo
- Publication number
- CN116574166A CN116574166A CN202310484676.1A CN202310484676A CN116574166A CN 116574166 A CN116574166 A CN 116574166A CN 202310484676 A CN202310484676 A CN 202310484676A CN 116574166 A CN116574166 A CN 116574166A
- Authority
- CN
- China
- Prior art keywords
- fish
- dna vaccine
- vaccine
- outer membrane
- edwardsiella tarda
- 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
- 101710203379 Outer membrane porin C Proteins 0.000 title claims abstract description 26
- 101710160101 Outer membrane protein C Proteins 0.000 title claims abstract description 26
- 108050002883 beta-defensin Proteins 0.000 title claims abstract description 25
- 108010041986 DNA Vaccines Proteins 0.000 title claims abstract description 21
- 102000012265 beta-defensin Human genes 0.000 title claims abstract description 20
- 229940021995 DNA vaccine Drugs 0.000 title claims abstract description 19
- 241000251468 Actinopterygii Species 0.000 title abstract description 34
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 33
- 241000607471 Edwardsiella tarda Species 0.000 claims abstract description 24
- 241000269979 Paralichthys olivaceus Species 0.000 claims description 23
- 238000003259 recombinant expression Methods 0.000 claims description 10
- 239000013604 expression vector Substances 0.000 claims description 8
- 239000003910 polypeptide antibiotic agent Substances 0.000 claims description 8
- 239000002773 nucleotide Substances 0.000 claims description 7
- 125000003729 nucleotide group Chemical group 0.000 claims description 7
- 102000004169 proteins and genes Human genes 0.000 claims description 6
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 5
- 239000012634 fragment Substances 0.000 claims description 3
- 238000002360 preparation method Methods 0.000 claims description 3
- 239000012646 vaccine adjuvant Substances 0.000 claims description 2
- 229940124931 vaccine adjuvant Drugs 0.000 claims description 2
- 238000007792 addition Methods 0.000 claims 1
- 125000003277 amino group Chemical group 0.000 claims 1
- 230000037430 deletion Effects 0.000 claims 1
- 238000012217 deletion Methods 0.000 claims 1
- 238000006467 substitution reaction Methods 0.000 claims 1
- 238000002347 injection Methods 0.000 abstract description 23
- 239000007924 injection Substances 0.000 abstract description 23
- 230000003053 immunization Effects 0.000 abstract description 16
- 238000002649 immunization Methods 0.000 abstract description 16
- 229960005486 vaccine Drugs 0.000 abstract description 13
- 230000014509 gene expression Effects 0.000 abstract description 11
- 210000000952 spleen Anatomy 0.000 abstract description 11
- 208000015181 infectious disease Diseases 0.000 abstract description 9
- 210000003734 kidney Anatomy 0.000 abstract description 9
- 210000004969 inflammatory cell Anatomy 0.000 abstract description 8
- 210000003719 b-lymphocyte Anatomy 0.000 abstract description 6
- 230000028993 immune response Effects 0.000 abstract description 6
- 230000000694 effects Effects 0.000 abstract description 5
- 108091007433 antigens Proteins 0.000 abstract description 4
- 239000000427 antigen Substances 0.000 abstract description 3
- 102000036639 antigens Human genes 0.000 abstract description 3
- 230000003399 chemotactic effect Effects 0.000 abstract description 3
- 239000003053 toxin Substances 0.000 abstract description 3
- 231100000765 toxin Toxicity 0.000 abstract description 3
- 239000013612 plasmid Substances 0.000 description 40
- 210000003205 muscle Anatomy 0.000 description 14
- 210000004027 cell Anatomy 0.000 description 10
- 239000000047 product Substances 0.000 description 8
- 108020004414 DNA Proteins 0.000 description 7
- 238000011160 research Methods 0.000 description 7
- 210000001744 T-lymphocyte Anatomy 0.000 description 6
- 125000000129 anionic group Chemical group 0.000 description 6
- 238000002474 experimental method Methods 0.000 description 6
- 210000003128 head Anatomy 0.000 description 6
- 239000000243 solution Substances 0.000 description 6
- 241000269908 Platichthys flesus Species 0.000 description 5
- 239000002299 complementary DNA Substances 0.000 description 5
- 238000011534 incubation Methods 0.000 description 5
- 210000000265 leukocyte Anatomy 0.000 description 5
- 238000000034 method Methods 0.000 description 5
- 239000003242 anti bacterial agent Substances 0.000 description 4
- 229940088710 antibiotic agent Drugs 0.000 description 4
- 230000001580 bacterial effect Effects 0.000 description 4
- 238000003776 cleavage reaction Methods 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
- 230000007017 scission Effects 0.000 description 4
- 238000012163 sequencing technique Methods 0.000 description 4
- 238000013518 transcription Methods 0.000 description 4
- 230000035897 transcription Effects 0.000 description 4
- 238000001890 transfection Methods 0.000 description 4
- 241000283707 Capra Species 0.000 description 3
- 102000000541 Defensins Human genes 0.000 description 3
- 108010002069 Defensins Proteins 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 238000011529 RT qPCR Methods 0.000 description 3
- 241000700605 Viruses Species 0.000 description 3
- 201000010099 disease Diseases 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 210000004698 lymphocyte Anatomy 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 238000011084 recovery Methods 0.000 description 3
- 238000003757 reverse transcription PCR Methods 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 2
- 208000035143 Bacterial infection Diseases 0.000 description 2
- 230000004721 adaptive immunity Effects 0.000 description 2
- 238000000246 agarose gel electrophoresis Methods 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 230000001174 ascending effect Effects 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 238000005520 cutting process Methods 0.000 description 2
- 210000002308 embryonic cell Anatomy 0.000 description 2
- 230000002708 enhancing effect Effects 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- 238000000227 grinding Methods 0.000 description 2
- 230000008105 immune reaction Effects 0.000 description 2
- 230000036039 immunity Effects 0.000 description 2
- 238000010166 immunofluorescence Methods 0.000 description 2
- 230000008595 infiltration Effects 0.000 description 2
- 238000001764 infiltration Methods 0.000 description 2
- 238000007857 nested PCR Methods 0.000 description 2
- 239000008188 pellet Substances 0.000 description 2
- 230000001681 protective effect Effects 0.000 description 2
- 210000005084 renal tissue Anatomy 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 239000013598 vector Substances 0.000 description 2
- 101150102326 1.1 gene Proteins 0.000 description 1
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 1
- 241000269981 Bothidae Species 0.000 description 1
- 102100035793 CD83 antigen Human genes 0.000 description 1
- 102000012410 DNA Ligases Human genes 0.000 description 1
- 108010061982 DNA Ligases Proteins 0.000 description 1
- 101100347633 Drosophila melanogaster Mhc gene Proteins 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 101001066288 Gallus gallus GATA-binding factor 3 Proteins 0.000 description 1
- 101000946856 Homo sapiens CD83 antigen Proteins 0.000 description 1
- 239000012124 Opti-MEM Substances 0.000 description 1
- 101710160107 Outer membrane protein A Proteins 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 108010019160 Pancreatin Proteins 0.000 description 1
- 206010057249 Phagocytosis Diseases 0.000 description 1
- 108010013381 Porins Proteins 0.000 description 1
- 108020004511 Recombinant DNA Proteins 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 230000001464 adherent effect Effects 0.000 description 1
- 239000002671 adjuvant Substances 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 238000009360 aquaculture Methods 0.000 description 1
- 244000144974 aquaculture Species 0.000 description 1
- 210000000649 b-lymphocyte subset Anatomy 0.000 description 1
- 244000052616 bacterial pathogen Species 0.000 description 1
- 230000035605 chemotaxis Effects 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- 238000000684 flow cytometry Methods 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 238000012215 gene cloning Methods 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 230000001571 immunoadjuvant effect Effects 0.000 description 1
- 239000000568 immunological adjuvant Substances 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 229940031551 inactivated vaccine Drugs 0.000 description 1
- 230000015788 innate immune response Effects 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 238000007918 intramuscular administration Methods 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 210000003292 kidney cell Anatomy 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 238000003801 milling Methods 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 229940055695 pancreatin Drugs 0.000 description 1
- 239000012188 paraffin wax Substances 0.000 description 1
- 230000008782 phagocytosis Effects 0.000 description 1
- 102000007739 porin activity proteins Human genes 0.000 description 1
- 230000002516 postimmunization Effects 0.000 description 1
- 238000012257 pre-denaturation Methods 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 230000000384 rearing effect Effects 0.000 description 1
- 230000007115 recruitment Effects 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 229940031626 subunit vaccine Drugs 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 241001148471 unidentified anaerobic bacterium Species 0.000 description 1
- 238000002255 vaccination Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- 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
- A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
-
- 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
- A61K39/025—Enterobacteriales, e.g. Enterobacter
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/04—Immunostimulants
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/461—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from fish
-
- 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
-
- 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
-
- 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
-
- 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
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55511—Organic adjuvants
- A61K2039/55516—Proteins; Peptides
-
- 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)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Immunology (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Zoology (AREA)
- Engineering & Computer Science (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Microbiology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Wood Science & Technology (AREA)
- General Engineering & Computer Science (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Epidemiology (AREA)
- Mycology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Physics & Mathematics (AREA)
- Gastroenterology & Hepatology (AREA)
- Toxicology (AREA)
- Communicable Diseases (AREA)
- Plant Pathology (AREA)
- Oncology (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
本发明提供一种鱼类β防御素和外膜蛋白C的双顺反子DNA疫苗,从而获得一种能引起鱼体更强、更持久免疫反应、增强鱼体对迟缓爱德华氏菌感染的免疫保护率,降低鱼体感染死亡率的具有良好免疫保护效果的疫苗产品。本发明制备的迟缓爱德华氏菌的双顺反子DNA疫苗成功在鱼体内进行表达,并能够趋化鱼体炎性细胞在免疫后进入疫苗注射部位。相较于单一抗原注射组,双顺反子DNA疫苗能够引起鱼体脾脏和头肾中免疫相关基因更高的表达量和T、B淋巴细胞比例的上升。同时提高了在迟缓爱德华氏菌攻毒后的免疫保护效果。
Description
技术领域
本发明属于鱼类免疫学技术领域,具体涉及一种鱼类β防御素和外膜蛋白C的双顺反子DNA疫苗。
背景技术
迟缓爱德华氏菌(Edwardsiellatarda)是一种革兰氏阴性细菌,属于兼性厌氧细菌。它是一种常见的水生致病细菌,这种细菌能够在水体中迅速传播,使多种鱼类(包括牙鲆)感染并造成大量死亡,因此这对水产养殖业造成了巨大的经济损失。在养殖生产中,治疗迟缓爱德华氏菌所引起的疾病的常用方法是使用抗生素,但大量使用抗生素所造成的耐药性和残留问题给环境卫生及人类健康造成了极大威胁,人们迫切需要寻找其他方法来防治该疾病。
疫苗能够通过激活宿主的特异性免疫反应来预防疾病,近年来,许多研究表明接种疫苗是防治鱼类细菌性疾病的有效方法,为细菌性疾病的防治找到了新的思路。而与灭活疫苗和亚单位疫苗等传统疫苗相比,DNA疫苗具有安全性高、高效、易于生产、稳定性强等优点,是未来具有较大商业价值和研究潜力的研究方向。
目前鱼类DNA疫苗的大部分研究工作是针对于单一的抗原基因对宿主产生的免疫保护反应进行研究,研究表明DNA疫苗能够迅速激起鱼类的早期先天免疫反应,并通过启动细胞免疫反应来调节适应性免疫。外膜蛋白C是一种位于革兰氏阴性菌外膜上的孔蛋白,它是一种主要的细胞表面抗原,其在感染期间的表达和它在细胞表面表现出异源表位的能力使它成为疫苗开发中具有吸引力的候选抗原。Liu等人用编码迟缓爱德华氏菌外膜蛋白C(OmpC)的重组DNA质粒免疫牙鲆(Paralichthys olivaceus),诱发了特异性抗体的产生,虽然OmpC DNA疫苗可以诱导先天性和获得性免疫,但它们产生的免疫保护率并不理想(55%)。
发明内容
本发明提供一种鱼类β防御素和外膜蛋白C的双顺反子DNA疫苗,从而获得一种能引起鱼体更强、更持久免疫反应,增强鱼体对迟缓爱德华氏菌感染的免疫保护率,降低鱼体感染死亡率,具有良好免疫保护效果的疫苗产品。
本发明首先提供一种牙鲆抗菌肽β防御素蛋白,其氨基酸序列为SEQ ID NO:2;
其编码基因的一种核苷酸序列为SEQ ID NO:1;
所述的迟缓爱德华氏菌外膜蛋白C,其编码基因的核苷酸序列为
ATGATGAAACGCAATATTCTTGCAGTGGTAATCCCGGCTCTGCTGGTTGCTGGCGCGGCCAACGCGGCCGAGATGTACAACAAAGACGGCAACAAAGTTAGCCTGTACGGTAAAGTCGACGCCCGTCACGTATTCAGCTCTGACAAGTCTGAAGACGGCGATGCTACCTATGCCCGCTTTGGCTTCAAAGGTGAAACCCAGATCAACAGCGAACTGACCGGTTACGGCCAGTGGGAATACAACTTCCAGGCTAACAACTCCGAAGACAGCAACCCGGCTATCGGCCAGGAAGGCAACAAGACCCGTCTGGGCTTCGCCGGTCTGAAATATGGCGAGTTCGGCTCCCTGGATTACGGCCGTAACTACGGCGTAGTCTACGACGTCGAAGCTTGGACCGACGTACTGCCGGTCTTCGGTGGTGACTCCTACACCTACACCGACAACTTCATGAACGGCCGTACCAACGGCGTGGCCACCTACCGTAACAACGGCTTCTTCGGCCTGGTTGACGGCCTGAACGTCGCCCTGCAGTATCAGGGTAAAAACGGCAACAGCGACGAGAGCAACAACGGTCGTGACAAGCTGTCCAAGCAGAATGGCGACGGCTTCGGTATGTCTGCCTCCTACGATCTGGGCTGGGGCGTAAGCGCCGCGGCGGCCTTCTCCTCCTCTAACCGTACCCTGGATCAGAAACGTGGATATCTCAAGCCTAACACTAGCGGTGTAGCTACTGGTGATAAGGCTCAGGCCTGGACCACTGGTCTGAAGTATGACGCCAACAACGTCTACGTAGCCGCCATGTACGCCGAAACCCTGAACATGACCCCGTATGGTAACGGCGGTATCGCTAACAAGACCCAGAACTTCGAAGCTGTGGCTCAGTACCAGTTCGACTTCGGCCTGCGTCCGTCCATCGCCTACCTGCAGTCTAAGGGCAAGCAGCTGGGTACTGCCGCTAACGTAGACAAAGATCTGGTTAAATACCTGGATCTGGGTTCCTACTACTACTTCAACAAGAACATGTCCGCCTACGTTGACTACAAGATCAACCTGCTGGATGGCAACGACGCGTTCTACAAAGACAACGGTATCAGCACCGACAATATCGTTGGCGTTGGTATGATCTACCAGTTCTAA(SEQ ID NO:3);
其蛋白的氨基酸序列如下:
MMKRNILAVVIPALLVAGAANAAEMYNKDGNKVSLYGKVDAR
HVFSSDKSEDGDATYARFGFKGETQINSELTGYGQWEYNFQANNSED
SNPAIGQEGNKTRLGFAGLKYGEFGSLDYGRNYGVVYDVEAWTDVL
PVFGGDSYTYTDNFMNGRTNGVATYRNNGFFGLVDGLNVALQYQG
KNGNSDESNNGRDKLSKQNGDGFGMSASYDLGWGVSAAAAFSSSN
RTLDQKRGYLKPNTSGVATGDKAQAWTTGLKYDANNVYVAAMYAE
TLNMTPYGNGGIANKTQNFEAVAQYQFDFGLRPSIAYLQSKGKQLGT
AANVDKDLVKYLDLGSYYYFNKNMSAYVDYKINLLDGNDAFYKDN
GISTDNIVGVGMIYQF(SEQ ID NO:4);
本发明所提供的牙鲆阴离子抗菌肽β防御素可作为疫苗佐剂来制备疫苗;
所述的疫苗,作为本发明一个实施例的具体记载,为DNA疫苗;
本发明另一个方面还提供一种重组表达载体,所述的重组表达载体携带有用于编辑牙鲆阴离子抗菌肽β防御素和迟缓爱德华氏菌外膜蛋白C的核苷酸片段;
本发明提供的重组真核表达载体用于制备双顺反子DNA疫苗。
本发明再一个方面还提供一种双顺反子DNA疫苗,所述的疫苗包含有上述的重组表达载体。
本发明制备的迟缓爱德华氏菌的双顺反子DNA疫苗成功在鱼体细胞内进行表达,并能够趋化鱼体炎性细胞在免疫后进入疫苗注射部位。相较于单一抗原注射组,双顺反子DNA疫苗能够引起鱼体脾脏和头肾中免疫相关基因更高的表达量和T、B淋巴细胞比例的上升。同时提高了在迟缓爱德华氏菌攻毒后的免疫保护效果,免疫保护率由48.33%提升至74.17%。所制备的DNA双顺反子疫苗在注射后能够引起鱼体的免疫反应,增强了鱼体的先天及后天免疫;增强鱼体的抗迟缓爱德华氏菌感染能力,降低了感染后死亡率,给鱼体提供了良好的免疫保护效果。
附图说明
图1为三组质粒转染后牙鲆胚胎细胞的间接免疫荧光结果图。
图2为RT-PCR检测三组DNA质粒在注射部位牙鲆肌肉组织中的转录情况图。
图3为免疫后第5天注射部位肌肉炎性细胞募集情况及免疫相关基因表达情况图。
图4为免疫后0、12、24小时,3、5、9、28天脾脏中免疫相关基因表达变化图。
图5为免疫后0、12、24小时,3、5、9、28天头肾中免疫相关基因表达变化图。
图6为免疫后第2周脾脏T淋巴细胞和第5周B淋巴细胞流式分析结果以及第0、1、2、3、4、5、6周T淋巴细胞和淋巴细胞比例变化图。
图7为免疫后第2周头肾T淋巴细胞和第5周B淋巴细胞流式分析结果以及第0、1、2、3、4、5、6周T淋巴细胞和淋巴细胞比例变化图。
图8为攻毒后各组牙鲆的生存率曲线图。
具体实施方式
本发明在牙鲆中发现了一种阴离子β防御素,研究显示其具有广谱的抗菌活性和趋化及促吞噬活性的功能。其能够作为适应性免疫和先天性免疫之间的桥梁,有潜力成为有效的免疫佐剂。
本发明将阴离子β防御素和迟缓爱德华氏菌外膜蛋白C联合来制备双顺反子DNA疫苗,从而高效的增强鱼体的抗迟缓爱德华氏菌感染能力,降低了感染后死亡率。
下面结合实施例和附图对本发明进行详细的描述。
实施例1:从牙鲆中分离阴离子β防御素
1.1基因克隆
根据实验预测β防御素基因的全长,设计巢氏PCR内(fBD-OF,fBD-OR)外(fBD-IF,fBD-IR)引物,引物信息如下表1。以牙鲆头肾cDNA为模板,用上述引物通过巢式PCR扩增牙鲆β防御素基因。
表1:扩增β防御素基因的引物信息表
1.2构建克隆菌株及测序
将PCR产物进行琼脂糖凝胶电泳,切下预期条带并回收。取1μL回收产物与1μL T1载体于离心管中,使目的基因与载体连接。将连接产物转化入大肠杆菌DH5α感受态中,涂布于含有Amp抗生素的LB固体培养基中并置于细菌培养箱中过夜培养。挑取单菌落送去公司测序。
测序结果表明牙鲆抗菌肽β防御素的基因的核苷酸序列如下:ATGTCTCGTTATCGTGTGGCTGTTTTGGCGCTGGTCGTTGTCTTGCTTGTTTTTGTTGCAGAGAATGAAGCAGATCGACCTAAGGCAGACCGACCTAAGCCAGACCGACCTAAGGCAGATTGCTCCACCATACAGGGAGTCTGTAAAGACAGTTGCCTCTCAACAGAATTCTCTATTGGAGCTCTCGGCTGTTCCGCAGAGAGTTCAACTGTATGTTGCATAACCAAACCATAA(SEQ ID NO:1),
其编码蛋白的氨基酸序列如下:
MSRYRVAVLALVVVLLVFVAENEADRPKADRPKPDRPKADCSTIQ GVCKDSCLSTEFSIGALGCSAESSTVCCITKP(SEQ ID NO:2)。
实施例2:制备重组表达载体
一、重组真核质粒的制备
1、以实施例1中经测序构建好的克隆菌株和迟缓爱德华氏菌悬液作为模板扩增β防御素基因和迟缓爱德华氏菌外膜蛋白C基因,涉及到的引物信息如表2所示。PCR扩增反应程序为95℃预变性5min;95℃,30s;55、60℃,30s;72℃,40s,35个循环;72℃延伸10min。PCR产物进行琼脂糖凝胶电泳,利用DNA回收试剂盒切胶回收预期条带,将产物置于-20℃保存。
表2:构建重组真核质粒用到的引物信息表
2、按照下表3配制酶切体系,在37℃下酶切4h,利用DNA回收试剂盒回收酶切产物,调整pBudCE4.1酶切片段浓度为0.03pmol,β-defensin酶切片段浓度为0.3pmol后用T4 DNA连接酶于16℃连接过夜。
表3:酶切体系组分表
3、将连接产物转化至DH5α感受态中,冰上静置30min后,将感受态体系放入金属浴中42℃热激50s,冰上静置5min后将600μL的LB液体培养基加入感受态中。在超净台中将复苏1h后的感受态均匀涂布在含有抗生素的LB固体培养基上培养12h。挑取单菌落后经菌落PCR及测序检测获得插入片段正确的p-βdefensin质粒。再按照上述做法将迟缓爱德华氏菌外膜蛋白C基因插入质粒中制备p-βdefensin-OmpC双顺反子质粒以及p-OmpC质粒。
实施例3:双顺反子DNA质粒的体内与体外表达
1.细胞转染与注射:用6孔细胞培养板培养牙鲆胚胎细胞,使用无菌PBS清洗,加入Opti-MEM培养液。将转染体系加入孔中,混匀后置于细胞培养箱内培养。48h后使用胰酶消化细胞并收集。将细胞浓度调整至1x106 cells/mL,并转移至粘附载玻片上固定,将载玻片冻存于-20℃。
将健康牙鲆暂养一周后随机分为四组,每组120尾鱼。将制备好的pBudCE4.1(未连接目的基因的空质粒),p-OmpC(只连接了迟缓爱德华氏菌外膜蛋白C的质粒),p-OmpC-βdefensin(连接了牙鲆β防御素和迟缓爱德华氏菌外膜蛋白C的双顺反子质粒)质粒经肌肉免疫牙鲆,每尾鱼注射100μL(200ng/μL)质粒。
2.间接免疫荧光与RT-PCR检测:向固定好的细胞滴片上滴加BSA,于37℃封闭1.5h,使用PBST洗涤三次。将牙鲆β防御素和迟缓爱德华氏菌外膜蛋白C多克隆抗体稀释1000倍后滴在载玻片上于37℃孵育1.5h。使用PBS洗涤三次后,羊抗兔或羊抗鼠IgG作为二抗按说明书稀释后,滴加到载玻片上37℃孵育1h。使用PBS洗涤三次,按照说明书稀释DAPI,滴到载玻片上于室温孵育15min。孵育结束后滴加甘油封片,置于显微镜下观察。结果如图1显示,p-OmpC质粒转染组可以看到明亮的绿色荧光未见到红色荧光表明迟缓爱德华氏菌OmpC基因正常表达、β-defensin基因未表达;p-OmpC-βdefensin质粒转染组可见到明亮的绿色和红色荧光表明OmpC和β-defensin基因均正常表达;而pBudCE4.1空质粒转染组未见到绿色和红色荧光表明OmpC和β-defensin基因均未表达。
3.在免疫后第3、4、5、7、14、21、28天分别取三组鱼的注射部位肌肉组织提取总RNA,并反转录为cDNA。以18s作为内参基因进行RT-PCR实验。结果如图2A显示,在p-OmpC-βdefensin质粒注射组的肌肉组织中免疫后三周内均能检测到OmpC和β-defensin基因的转录;如图2B显示在p-OmpC质粒注射组的肌肉组织中免疫后三周内能检测到OmpC基因的转录;如图2C显示pBudCE4.1空质粒注射组的肌肉组织在任何时间均未检测到OmpC和β-defensin基因的转录。
4.免疫5天后,取各组牙鲆注射部位肌肉,修剪大小为边长0.5cm的正方体,置于固定液中。制备石蜡切片后进行苏木精-伊红染色,封片后置于显微镜下观察。如图3A所示,p-OmpC-βdefensin质粒注射组的肌肉组织具有明显的炎性细胞浸润情况;而p-OmpC质粒注射组的肌肉组织也有炎性细胞浸润情况但相较于p-OmpC-βdefensin质粒注射组的肌肉组织的炎性细胞数量明显减少;pBudCE4.1空质粒注射组的肌肉组织几乎见不到炎性细胞。说明作为佐剂的β防御素能够趋化炎性细胞进入注射部位并富集。
在免疫5天后,再分别取三组鱼的注射部位肌肉组织置于保护液中,提取总RNA。将提取的RNA反转录为cDNA,用于后续的qRT-PCR实验。使用的引物如下表4所示。
表4:免疫相关基因的引物信息表
以反转录后的cDNA为模板,以18s作为内参基因进行qRT-PCR实验。结果如图3B表明,p-OmpC-βdefensin质粒注射组肌肉组织中,GATA-3、CD83、MHCⅠ基因的表达量均高于p-OmpC质粒和pBudCE4.1空质粒注射组肌肉组织中的表达量。
5.脾脏与头肾中免疫相关基因表达量的动态变化和T、B淋巴细胞亚群比例的动态变化;
1)在免疫后第0、12、24小时,3、5、9、28天,分别取三组鱼的脾脏和头肾组织置于保护液中,提取总RNA,并反转录为cDNA。设计实验所用到的免疫相关基因引物,引物如表6。
表6:免疫相关基因的序列信息表
以18s基因作为内参基因,使用qRT-PCR仪进行实验。结果如图4和图5,表明免疫后p-OmpC-βdefensin质粒注射组以及p-OmpC质粒注射组头肾、脾脏组织中IL6、TNFα、CD4-1、MHCⅡ基因的表达量均呈现向上升后下降的趋势,且均显著高于pBudCE4.1空质粒注射组。说明,疫苗免疫后引起了鱼体的免疫反应。
2)在注射后第0、1、2、3、4、5、6周提取各组牙鲆脾脏和头肾白细胞,使用制备好的牙鲆IgM、CD4-1、CD4-2单克隆抗体作为一抗,荧光标记的商品化抗体作为二抗,借助流式细胞仪进行分析。简言之,将牙鲆脾脏和头肾组织的研磨液于4℃条件下100g离心25min。将组织研磨液上清缓慢滴入1.07g/mL与1.02g/mL的percoll溶液上方,4℃条件下840g离心30min。离心结束后,使用注射器将液面中间层的白细胞吸出,置于干净的离心管中并调整浓度为1×107cells/mL。以1:1000的比例将CD4-1、CD4-2、IgM单克隆抗体加入到提纯并调整好浓度的白细胞中,37℃孵育1.5h。使用PBS将白细胞清洗三次,以1:1000的比例将荧光标记的羊抗鼠IgG抗体加入到白细胞内,37℃孵育50分钟。孵育结束后将白细胞用PBS清洗三次,使用流式细胞仪分析脾脏和头肾中CD4-1+、CD4-2+、IgM+的比例。流式细胞分析结果如图6和7表明,免疫后牙鲆脾脏和头肾细胞中CD4-1+、CD4-2+T淋巴细胞,IgM+B淋巴细胞的比例均呈现先上升后下降的趋势,同时p-OmpC-βdefensin质粒注射组CD4-1+、CD4-2+T淋巴细胞和IgM+B淋巴细胞峰值时的比例明显高于p-OmpC质粒注射组和pBudCE4.1空质粒注射组。
3)相对免疫保护率的检测
攻毒实验在免疫第六周后进行,从各组中随机挑选120尾牙鲆:每组3个重复,每个重复40尾。使用LB液体培养基扩大培养攻毒使用的迟缓爱德华氏菌毒力株,在30℃条件下培养至对数期,5000g离心收集后,使用PBS将菌液浓度调整至1×107cfu/mL,每尾鱼注射100μL菌悬液。在攻毒后14天内统计死亡尾数,计算免疫保护率。统计显示,pBudCE4.1空质粒注射组牙鲆在攻毒后第9天后全部死亡,死亡率为100%,免疫保护率为0%;p-OmpC质粒注射组在攻毒两周后死亡21、20、21条,死亡率为52.67%,免疫保护率为48.33%;p-OmpC-βdefensin质粒注射组在攻毒两周后死亡10、10、11条,死亡率为25.83%,免疫保护率为74.17%。
综上,本发明构建的DNA双顺反子疫苗p-OmpC-βdefensin在免疫后引起的免疫相关基因的表达量和淋巴细胞比例显著高于空质粒组和只表达单个蛋白的p-OmpC质粒组,说明DNA双顺反子疫苗引起了宿主更强的免疫反应。此外,其免疫保护率相对于p-OmpC质粒组提高了25.84%,拥有更好的免疫效果,显著提高了免疫保护率。
Claims (8)
1.一种牙鲆抗菌肽β防御素蛋白,其特征在于,所述的牙鲆抗菌肽β防御素蛋白包含有:
1)氨基酸序列为SEQ ID NO:2的蛋白;
2)在1)中的蛋白上取代、缺失、添加一个或数个氨基,由1)所衍生的蛋白。
2.编码权利要求1所述的牙鲆抗菌肽β防御素蛋白的基因,其特征在于,所述的基因的核苷酸序列为SEQ ID NO:1。
3.权利要求1所述的牙鲆抗菌肽β防御素蛋白作为疫苗佐剂的应用。
4.一种用于重组表达权利要求1所述的牙鲆抗菌肽β防御素蛋白的重组表达载体,其特征在于,所述的重组表达中插入有权利要求2所述的基因的核苷酸片段。
5.权利要求4所述的重组表达载体在制备DNA疫苗中的应用。
6.一种双顺反子DNA疫苗,其特征在于,所述的双顺反子DNA疫苗包含有权利要求4所述的重组表达载体和用于重组表达迟缓爱德华氏菌外膜蛋白C的重组表达载体。
7.如权利要求6所述的双顺反子DNA疫苗,其特征在于,所述的迟缓爱德华氏菌外膜蛋白C的氨基酸序列为SEQ ID NO:4。
8.如权利要求7所述的双顺反子DNA疫苗,其特征在于,所述的迟缓爱德华氏菌外膜蛋白C的编码基因的核苷酸序列为SEQ ID NO:4。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202310484676.1A CN116574166A (zh) | 2023-05-04 | 2023-05-04 | 一种鱼类β防御素和外膜蛋白C的双顺反子DNA疫苗 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202310484676.1A CN116574166A (zh) | 2023-05-04 | 2023-05-04 | 一种鱼类β防御素和外膜蛋白C的双顺反子DNA疫苗 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN116574166A true CN116574166A (zh) | 2023-08-11 |
Family
ID=87538920
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202310484676.1A Pending CN116574166A (zh) | 2023-05-04 | 2023-05-04 | 一种鱼类β防御素和外膜蛋白C的双顺反子DNA疫苗 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN116574166A (zh) |
Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2008023453A1 (fr) * | 2006-08-24 | 2008-02-28 | Kawasakimitaka K.K. | Maladies de type edwardsiella induites chez le poisson et vaccin contre les maladies induites par des streptocoques |
KR20110018646A (ko) * | 2009-08-18 | 2011-02-24 | 대한민국(관리부서:국립수산과학원) | 넙치 유래의 항균성 펩타이드인 베타디펜신을 암호화하는 신규한 유전자 및 그의 용도 |
CN101991844A (zh) * | 2009-08-21 | 2011-03-30 | 中国科学院海洋研究所 | 一种迟缓爱德华氏菌重组亚单位疫苗及其应用 |
CN102206257A (zh) * | 2011-04-15 | 2011-10-05 | 华东理工大学 | 迟钝爱德华氏菌免疫保护性抗原、相关表达载体、疫苗和应用 |
CN103690942A (zh) * | 2013-12-20 | 2014-04-02 | 中国水产科学研究院黄海水产研究所 | 一种迟缓爱德华氏菌亚单位疫苗及其制备与应用 |
CN112999341A (zh) * | 2021-03-01 | 2021-06-22 | 中国科学院海洋研究所 | 迟缓爱德华氏菌外膜蛋白疫苗及其制备和应用 |
-
2023
- 2023-05-04 CN CN202310484676.1A patent/CN116574166A/zh active Pending
Patent Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2008023453A1 (fr) * | 2006-08-24 | 2008-02-28 | Kawasakimitaka K.K. | Maladies de type edwardsiella induites chez le poisson et vaccin contre les maladies induites par des streptocoques |
KR20110018646A (ko) * | 2009-08-18 | 2011-02-24 | 대한민국(관리부서:국립수산과학원) | 넙치 유래의 항균성 펩타이드인 베타디펜신을 암호화하는 신규한 유전자 및 그의 용도 |
CN101991844A (zh) * | 2009-08-21 | 2011-03-30 | 中国科学院海洋研究所 | 一种迟缓爱德华氏菌重组亚单位疫苗及其应用 |
CN102206257A (zh) * | 2011-04-15 | 2011-10-05 | 华东理工大学 | 迟钝爱德华氏菌免疫保护性抗原、相关表达载体、疫苗和应用 |
CN103690942A (zh) * | 2013-12-20 | 2014-04-02 | 中国水产科学研究院黄海水产研究所 | 一种迟缓爱德华氏菌亚单位疫苗及其制备与应用 |
CN112999341A (zh) * | 2021-03-01 | 2021-06-22 | 中国科学院海洋研究所 | 迟缓爱德华氏菌外膜蛋白疫苗及其制备和应用 |
Non-Patent Citations (6)
Title |
---|
BO-HYE NAM等: "Multiple β-defensin isoforms identified in early developmental stages of the teleost Paralichthys olivaceus.", FISH & SHELLFISH IMMUNOLOGY., vol. 28, no. 2, pages 267 - 274, XP026873020 * |
EDWARDSIELLA TARDA: "porin OmpC [Edwardsiella tarda]", NCBI REFERENCE SEQUENCE:WP_097363995.1 * |
FUGUO LIU等: "Construction and evaluation of an Edwardsiella tarda DNA vaccine encoding outer membrane protein C", MICROBIAL PATHOGENESIS, vol. 104, pages 238 - 247 * |
HAO,X等: "beta-defensin [Paralichthys olivaceus]", NCBI GENBANK UXX39609.1 * |
KHURSHID JALAL等: "Pan-Genome Reverse Vaccinology Approach for the Design of Multi-Epitope Vaccine Construct against Escherichia albertii.", INT. J. MOL. SCI., vol. 22, pages 1 - 17 * |
XIAOKAI HAO等: "The Functions of -Defensin in Flounder (Paralichthys olivaceus): Antibiosis, Chemotaxis and Modulation of Phagocytosis", BIOLOGY, vol. 10, pages 1 - 14 * |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
JP2020512835A (ja) | 水産養殖動物におけるアエロモナス出血性疾患の予防及び制御のための弱毒生ワクチン | |
CN102276730B (zh) | 金黄色葡萄球菌IsdBid-TRAP融合蛋白制备方法及其应用 | |
Shah et al. | Cross immunity of DNA vaccine pVAX1-cSZ2-IL-2 to Eimeria tenella, E. necatrix and E. maxima | |
Wang et al. | A novel recombinant BCG vaccine encoding Eimeria tenella rhomboid and chicken IL-2 induces protective immunity against coccidiosis | |
EP2560985B1 (en) | Nucleic acid sequences of a fish virus and the use thereof | |
CN107099496A (zh) | 融合表达鸡传染性法氏囊病毒vp2蛋白和沙门菌属外膜蛋白的重组乳酸菌菌株及其用途 | |
Zhao et al. | Oral vaccination with recombinant Lactobacillus casei expressing Aeromonas hydrophila Aha1 against A. hydrophila infections in common carps | |
CA2760098C (en) | Mutants of francisella tularensis and uses thereof | |
CN108359682B (zh) | 一种具有免疫保护作用的迟缓爱德华菌外膜蛋白PagC | |
Ji et al. | Construction and immunological evaluation of live vector vaccine based on attenuated Listeria monocytogenes vector against Vibrio parahaemolyticus infection | |
CN114437237B (zh) | 金黄色葡萄球菌trap靶向重组蛋白抗原及其用途 | |
CN116574166A (zh) | 一种鱼类β防御素和外膜蛋白C的双顺反子DNA疫苗 | |
CN110669714B (zh) | 肠炎沙门菌减毒疫苗候选株的制备与应用 | |
CN110628796B (zh) | 鱼类诺卡氏菌病共同抗原dna疫苗及其制备和应用 | |
CN113980101A (zh) | 一种胸膜肺炎放线杆菌亚单位疫苗 | |
JP5038911B2 (ja) | マイコバクテリウム・アヴィウム・亜種パラチュバキュローシスに対する免疫反応を誘発するための組成物 | |
CN107245105B (zh) | HN-VP233-221aa融合蛋白及其制备方法和应用 | |
CN111484960B (zh) | 新型爱德华氏菌减毒靶点及其应用 | |
KR100940111B1 (ko) | 이리도 바이러스 감염증에 대한 백신 조성물 및 이의제조방법 | |
CN107267432B (zh) | 布鲁氏菌104M疫苗株敲除Per基因的重组菌及应用 | |
CN115044524B (zh) | 一种基因工程重组乳酸菌及在抗拟态弧菌感染方面的应用 | |
CN116983397B (zh) | 海豚链球菌dna疫苗、制备方法及应用 | |
CN103451231A (zh) | 一种以人类复制缺陷腺病毒为载体的日本血吸虫病疫苗的构建及应用 | |
CN116376795B (zh) | 一种表达hsp70的重组乳酸菌及其制备方法与应用 | |
CN112159479B (zh) | 一种鸡毒支原体多抗原表位融合蛋白pMG-mEA及其应用 |
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 | ||
WD01 | Invention patent application deemed withdrawn after publication | ||
WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20230811 |