WO2022121109A1 - 一种减毒鰤鱼诺卡氏菌及其构建方法和应用 - Google Patents

一种减毒鰤鱼诺卡氏菌及其构建方法和应用 Download PDF

Info

Publication number
WO2022121109A1
WO2022121109A1 PCT/CN2021/075555 CN2021075555W WO2022121109A1 WO 2022121109 A1 WO2022121109 A1 WO 2022121109A1 CN 2021075555 W CN2021075555 W CN 2021075555W WO 2022121109 A1 WO2022121109 A1 WO 2022121109A1
Authority
WO
WIPO (PCT)
Prior art keywords
gluns
nocardia
amberjack
attenuated
gene
Prior art date
Application number
PCT/CN2021/075555
Other languages
English (en)
French (fr)
Inventor
夏立群
王文基
鲁义善
甘桢
侯素莹
Original Assignee
广东海洋大学
广东海洋大学深圳研究院
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by 广东海洋大学, 广东海洋大学深圳研究院 filed Critical 广东海洋大学
Priority to US17/428,791 priority Critical patent/US20220372500A1/en
Publication of WO2022121109A1 publication Critical patent/WO2022121109A1/zh

Links

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)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • 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
    • C12N15/76—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Actinomyces; for Streptomyces
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/05—Actinobacteria, e.g. Actinomyces, Streptomyces, Nocardia, Bifidobacterium, Gardnerella, Corynebacterium; Propionibacterium
    • 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
    • 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)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
    • 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
    • C12R2001/365—Nocardia
    • 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
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
    • Y02A40/81—Aquaculture, e.g. of fish

Definitions

  • the invention belongs to the technical field of genetic engineering, and in particular relates to an attenuated Nocardia amberjack and a construction method and application thereof.
  • Nocardia amberjack belongs to the Bacteria, Firmicutes, Actinobacteria, Actinobacterales, Nocardiaceae, Nocardia.
  • Nocardia amberjack grows slowly, the bacteria grow on the plate with yellow sand or granular bumps, and the surface forms folds. Under liquid culture conditions, bacteria tend to aggregate to form large particles [1] .
  • the prevention and control of Nocardia in amberjack is still based on prevention.
  • the attenuated strain of bacteria, as a kind of active bacteria will not cause harm to the host on the basis of maintaining the original immunogenicity, so the attenuated strain has attracted much attention.
  • Nocardia amberjack related studies have been exploring the construction of attenuated strains. However, the protective effect of related attenuated strains is still not ideal and lacks immunogenicity.
  • the object of the present invention is to provide a kind of attenuated Nocardia amberjack and its construction method and application, and the constructed Nocardia amberjack has lower pathogenicity and still retains a better immunogen It can be used as a candidate strain in the preparation of vaccines or other biological products.
  • the present invention provides an attenuated Nocardia amberjack, the attenuated Nocardia amberjack lacks GluNS gene.
  • amino acid of the protein encoded by the GluNS gene is shown in SEQ ID NO: 12 or a sequence with homology or coverage of 30% to 99% with SEQ ID NO: 12.
  • the deposit number of the attenuated Nocardia amberjack is GDMCC No: 61258.
  • the invention provides the construction method of described attenuated amberjack Nocardia, comprising the following steps:
  • the recombinant cells are cultured and screened to obtain attenuated Nocardia amberjack.
  • the recombinant homology arm of the GluNS gene in step 1) consists of an upstream fragment and a downstream fragment of the GluNS gene.
  • the length of the upstream fragment or the downstream fragment of the GluNS gene is independently 5 bp to 2000 bp.
  • the upstream fragment of the GluNS gene uses the upstream 389bp fragment of the GluNS gene as the upstream homology arm
  • the downstream fragment uses the downstream 407bp fragment of the GluNS gene as the downstream homology arm
  • the upstream homology arm-downstream homology arm is formed in the connection mode.
  • the recombinant homology arm of the GluNS gene uses the upstream 389bp fragment of the GluNS gene as the upstream homology arm
  • the downstream fragment uses the downstream 407bp fragment of the GluNS gene as the downstream homology arm
  • the upstream homology arm-downstream homology arm is formed in the connection mode.
  • the recombinant homology arm of the GluNS gene uses the upstream 389bp fragment of the GluNS gene as the upstream homology arm
  • nucleotide sequence of the recombinant homology arm of the GluNS gene described in step 1) is shown in SEQ ID NO: 1.
  • the vector in step 1) includes pRE112;
  • the recombination homology arm of the GluNS gene is inserted into the multiple cloning site of pRE112 as Sac I/Kpn I.
  • the conversion described in step 2) includes electroconversion
  • the parameters of the electrical conversion are preferably as follows: voltage 180-220V, pulse interval 800-1200ms, pulse duration 80-120 ⁇ s, and 25-35 square waves.
  • the culture solution in step 3) is a BHI solution containing 0.3 mM sucrose preheated at 26-30°C, the culture temperature is 26-30°C, and the culture time is 10-12 hours.
  • the screening comprises using PCR amplification to detect whether the bacterium contains the knockout plasmid pRE112- ⁇ GluNS;
  • the primer pair for PCR amplification is 112-F1/112-R1;
  • the nucleotide sequence of the 112-F1 is shown in SEQ ID NO: 2;
  • the nucleotide sequence of the 112-R1 is shown in SEQ ID NO: 3;
  • the PCR amplification product was sequenced to show the deletion of the GluNS gene fragment, and the bacteria contained the knockout plasmid pRE112- ⁇ GluNS, which was a positive clone.
  • the screening also includes identifying attenuated Nocardia amberjack;
  • the identification includes using PCR amplification method to identify whether the GluNS gene is missing;
  • the primer pairs for PCR amplification are GluNS-F1/GluNS-R1 and GluNS-F2/GluNS-R2;
  • the nucleotide sequence of the GluNS-F1 is shown in SEQ ID NO: 10;
  • the nucleotide sequence of the GluNS-R1 is shown in SEQ ID NO: 11;
  • the nucleotide sequence of the GluNS-F2 is shown in SEQ ID NO: 14;
  • the nucleotide sequence of the GluNS-R2 is shown in SEQ ID NO: 15;
  • GluNS-F1/GluNS-R1 primer pair for GluNS gene deletion strain could not amplify the target band; using GluNS-F2/GluNS-R2 primer pair for GluNS gene deletion strain can only amplify the upstream and downstream homologous 796bp band.
  • the present invention provides the application of the attenuated Nocardia amberjack in preparing a vaccine or a related biological product comprising the attenuated Nocardia amberjack.
  • the glutamate endopeptidase (GluNS) gene in the wild-type Nocardia amberjack is knocked out by means of genetic engineering to construct a strain with good protection to the host.
  • the attenuated strain can not only effectively reduce the pathogenicity of bacteria, but also retain better immunogenicity.
  • the LC50 of the wild strain is 4.74 ⁇ 10 5 CFU/mL
  • the LC50 of the attenuated strain is 3.41 ⁇ 10 6 CFU/mL, which is 1 order of magnitude lower than that of the wild strain. It showed that the virulence of this strain decreased significantly.
  • the results of the live bacteria challenge experiment show that the relative immune protection rate of the attenuated strain reaches 93.38%, indicating that the attenuated strain is an attenuated strain with better protection to the host (strain preservation number is GDMCC No: 61258).
  • the attenuated Nocardia amberjack constructed by the present invention also has high genetic stability.
  • the GluNS deletion strain Nocardia amberjack ZJ0503-6296 was continuously transmitted to 30 generations, and the GluNS gene fragment could not be detected, indicating that the GluNS deletion strain could inherit stably.
  • Fig. 1 is pRE112 plasmid map
  • Fig. 2 is the recombinant plasmid map constructed by the present invention
  • Figure 3 is the electrophoresis image obtained from the first screening of the GluNS gene deletion strain Nocardia amberjack ZJ0503-6296; M: DNA marker 2000, 1, 2: experimental group, 3: ZJ0503, 4: pRE112;
  • Figure 4 is the electrophoresis image obtained by the second screening of Nocardia amberjack ZJ0503-6296, a GluNS gene deletion strain; M: DNA marker 2000, 1: Nocardia amberjack ZJ0503-6296, 2: ZJ0503;
  • Figure 5 shows the stable genetic verification of Nocardia amberjack ZJ0503-6296, a GluNS gene deletion strain; M: DNA marker 10000, 1: Nocardia amberjack ZJ0503-6296, 2-4: ZJ0503;
  • Figure 6 is a schematic diagram of the construction method of the knockout plasmid pRE112- ⁇ GluNS;
  • Figure 7 shows the construction principle of Nocardia amberjack ZJ0503-6296, a GluNS gene deletion strain. Biomaterial deposit information
  • GDMCC Guangdong Provincial Microbial Culture Collection Center
  • the address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Institute of Microbiology , deposited on October 29, 2020, with the deposit number of GDMCC No: 61258.
  • the present invention provides an attenuated Nocardia amberjack, the attenuated Nocardia amberjack lacks GluNS gene.
  • the deposit number of the attenuated Nocardia amberjack is preferably GDMCC No: 61258.
  • the present invention also provides the construction method of described attenuated amberjack Nocardia, comprising the following steps:
  • the recombinant cells are cultured and screened to obtain attenuated Nocardia amberjack.
  • the invention provides the method for constructing the attenuated Nocardia amberjack.
  • the construction principle is to clone the upstream and downstream fragments of the target gene of about 500 bp as homology arms, connect the homology arms together by overlapping PCR method, and construct the deletion vector pRE112 superior.
  • the deletion vector was introduced into Nocardia amberjack, and the homologous arm fragment on the deletion vector was replaced by the homologous arm fragment contained in the bacteria and the target gene fragment by the method of homologous recombination.
  • the gene was deleted, and only the recombination homology arm fragment was contained, thereby constituting the attenuated Nocardia amberjack (see Figure 7).
  • the recombinant homology arm of the GluNS gene is inserted into the vector to construct the recombinant vector.
  • the recombinant homology arm of the GluNS gene preferably consists of an upstream fragment and a downstream fragment of the GluNS gene.
  • the present invention does not specifically limit the lengths of the upstream and downstream fragments of the GluNS gene, and the lengths of the upstream and downstream fragments of the GluNS gene well known in the art may be used, for example, 5 bp to 2000 bp.
  • the upstream fragment of the GluNS gene uses the upstream 389bp fragment of the GluNS gene as the upstream homology arm
  • the downstream fragment uses the downstream 407bp fragment of the GluNS gene as the downstream homology arm
  • the upstream homology arm-downstream homology arm uses the upstream homology arm-downstream homology arm.
  • the ligation forms the recombinant homology arms of the GluNS gene.
  • GluNS SEQ ID NO:1(GCCTGCACCCTGGGCGGTGACAGCGGCGGCGCGATCGTGTCGGGCACGCTGGCGCTGGGCATCACCAGCGGTTCGAACGCGGCCGACGCGCCGAACTGCAACGAGGCCAACACCGCGCTGGCAGTACGGTGGAACCGCGTCGCTGGGCATCCCGGTGCGCGATTCTCACCGAAATCGACGCCAATTCCGGTGGCGGCGTCGGCAGCGGAATTCAGGTGCGGACGGTCCAACGCCTGACGGCGAAGTGCTCCAGCACCCGACGGCAAAGCGGTCCAACGCCTGACGATGGCGACATCGGCGGTAAACGGACATCCTTCACATTGCCCCTGAATCCGACATGCCCGCTGTTTGCCCCGTGAATACCGAAACGTCACCTCGCAATAAGTAGGCATAACGGGCCCGACACCATATAGTCGGGGCCATGCTCCTGCCACGTATGCGCTTCCATGCCTGCCACG
  • the nucleotide sequence of the GluNS-UF is preferably shown in SEQ ID NO: 5 (CATATGGGGCATCACCAGCGGTTCG), and the nucleotide sequence of the GluNS-UR is preferably shown in SEQ ID NO: 6 (TGCATCCGAATTAGTGAGGTCAGAGGAGCATGGCCCCGACT).
  • SEQ ID NO: 7 TGCATCCGAATTAGTGAGGTCAGAGGAGCATGGCCCCGACT.
  • the nucleotide sequence of the GluNS-DF is preferably shown in SEQ ID NO: 8 (AGTCGGGGCCATGCTCCTCTGACCTCACTAATTCGGATGCA), and the nucleotide sequence of the GluNS-DR is preferably shown in SEQ ID NO: 9 (GAGCTCAATTCGCCGGGTAGTCGC).
  • the reaction system was: 2 ⁇ L of GluNS gene upstream fragment product, 2 ⁇ L of GluNS gene downstream fragment product, 1 ⁇ L each of GluNS-UF and GluNS-DR, 25 ⁇ L of rTaq enzyme, and 19 ⁇ L of sterile water.
  • the vector preferably includes the pRE112 plasmid, see Figure 1 .
  • the recombination homology arm of the GluNS gene is inserted into the multiple cloning site of pRE112, preferably Sac I/Kpn I.
  • the method for inserting the recombinant homology arms of the GluNS gene into the vector preferably includes enzyme cleavage and ligation.
  • the enzymatic cleavage includes enzymatic cleavage of the recombinant homology arm and the vector of the GluNS gene, respectively.
  • the present invention does not have a special restriction on the method of the enzymatic cleavage, and the enzymatic cleavage method well known in the art can be used.
  • the present invention has no particular limitation on the ligation method, and a ligation method well known in the art may be used, for example, T4 ligase is used for ligation.
  • the present invention has no special limitation on the ligation method of the T4 ligase, and the ligation scheme of the T4 ligase known in the art can be used.
  • the present invention also preferably includes identification; the identification preferably adopts the method of PCR amplification or the method of sequencing.
  • the constructed recombinant vector is shown in Figure 2.
  • the present invention transforms the recombinant vector into Nocardia amberjack competent cells to obtain recombinant cells.
  • the method for preparing the competent cells of Nocardia amberjack may adopt the method known in the art for preparing competent cells.
  • the source of the Nocardia amberjack is not specifically limited, and the Nocardia amberjack well-known in the art can be used.
  • the wild strain ZJ0503 of Nocardia amberjack is used as the material.
  • the conversion preferably includes electro-transformation.
  • the parameters of the electrical conversion are preferably as follows: voltage 180-220V, pulse interval 800-1200ms, pulse duration 80-120 ⁇ s, and 25-35 square waves.
  • the recombinant cells are cultured and screened to obtain attenuated Nocardia amberjack.
  • the culture solution is preferably a BHI solution containing 0.3 mM sucrose preheated at 26-30°C, the culture temperature is preferably 26-30°C, and the culture time is preferably 10-12 hours.
  • the screening is preferably performed by using the antibiotics corresponding to the resistance genes carried in the vector.
  • the BHI plate containing 25 mg/mL chloramphenicol resistance is preferably used for screening.
  • the screening preferably further includes identifying the attenuated Nocardia amberjack.
  • the identifying preferably includes identifying whether the GluNS gene is deleted using PCR amplification methods.
  • the primer pairs for PCR amplification are preferably GluNS-F1/GluNS-R1 and GluNS-F2/GluNS-R2.
  • the nucleotide sequence of the GluNS-F1 is preferably as shown in SEQ ID NO: 10 (CCGCCAAGGACGTGAG).
  • the nucleotide sequence of the GluNS-R1 is preferably as shown in SEQ ID NO: 11 (CTGTCACCGATGAGCTGGTA).
  • the nucleotide sequence of the GluNS-F2 is preferably as shown in SEQ ID NO: 14 (GGGCATCACCAGCGGTTCG).
  • the nucleotide sequence of the GluNS-R2 is preferably as shown in SEQ ID NO: 15 (AATTCGCCGGGTAGTCGC).
  • PCR amplification reaction program 94°C, 5min, 30 cycles: 94°C, 30s, (55°C (GluNS-F2/GluNS-R2), 58°C (GluNS-F1/GluNS-R1), 30s, 72°C , 1min. 72 °C, 5min.
  • the invention provides the attenuated Nocardia amberjack constructed by the method for constructing the attenuated Nocardia amberjack, and the genome of the attenuated Nocardia amberjack lacks the GluNS gene.
  • the LC50 of the attenuated Nocardia amberjack was 3.41 ⁇ 10 6 CFU/mL, while the LC50 of the wild strain was 4.74 ⁇ 10 5 CFU/mL.
  • the wild strain was reduced by an order of magnitude, indicating a significant reduction in the virulence of this strain.
  • the relative immune protection rate of the attenuated Nocardia amberjack was 93.38%, and it had high immunogenicity.
  • the attenuated Nocardia amberjack constructed by the present invention is directly knocked out on the genome of Nocardia amberjack, the bacteria does not contain resistance plasmids, conforms to biological safety, and will not be affected by the loss of plasmids Growth of Nocardia in amberjack.
  • the genetic stability of the attenuated Nocardia amberjack was measured after passage of the attenuated Nocardia amberjack for 30 generations.
  • the method is to use GluNS-F1 (SEQ ID NO:10) and GluNS-R1 (SEQ ID NO:11), GluNS-F2 (SEQ ID NO:14) and GluNS-R2 (SEQ ID NO:15) primers for amplification , screening for GluNS deletion strains.
  • the present invention provides the attenuated Nocardia amberjack in preparing a vaccine or comprising the attenuated Nocardia amberjack Application of Cardiomycosis-related biological products.
  • the present invention does not have a special limitation on the preparation method of the vaccine, and a vaccine preparation method well known in the art can be used.
  • the prepared vaccine can be used for preventing and treating fish nocardiosis caused by Nocardia amberjack, and has high application value.
  • the present invention does not specifically limit the types of the relevant biological products, and the types of biological products known in the art may be used, such as therapeutic products or diagnostic products.
  • Step 1 Construction of homologous recombination vector.
  • the preserved wild strain of Nocardia amberjack was aseptically inoculated into the brain heart infusion (Brain Heart Infusion, BHI) solid medium (purchased from Guangdong Huankai Microorganism Technology Co., Ltd.), Invert at 26-30°C.
  • BHI Brain Heart Infusion
  • BHI liquid medium purchased from Guangdong Huankai Microorganism Technology Co., Ltd.
  • a certain volume of bacterial liquid was taken to extract bacterial genome, which was used as a template, and the upstream and downstream fragments of the GluNS gene were cloned using the primers in Table 1, and then connected to the pRE112 vector.
  • the amplification procedure was as follows: the reaction procedure of PCR amplification was: 94°C, 5min; 30 cycles: 94°C, 30s, 55°C, 30s, 72°C, 1min; 72°C, 5min.
  • the amplification system was: 1 ⁇ L of Nocardia amberjack genomic DNA, 2 ⁇ L of upstream primer (F) and 2 ⁇ L of downstream primer (R), 25 ⁇ L of rTaq enzyme, and 20 ⁇ L of sterile water.
  • the underline is the restriction site, and the wavy line is the overlapping PCR site.
  • the method for obtaining overlapping PCR products using GluNS-UF and GluNS-DR primers, and using the upstream fragment of GluNS gene and the downstream fragment of GluNS gene as templates, overlapping PCR products can be obtained by PCR amplification.
  • the PCR amplification program was: 94°C, 5 min, 15 cycles: 94°C, 30s; 58°C, 30s, 72°C, 1min; 72°C, 5min.
  • the reaction system was: 2 ⁇ L of GluNS gene upstream fragment product, 2 ⁇ L of GluNS gene downstream fragment product, 1 ⁇ L each of GluNS-UF and GluNS-DR, 25 ⁇ L of rTaq enzyme, and 19 ⁇ L of sterile water.
  • Step 2 Preparation of Nocardia amberjack competent cells
  • the preserved wild strain of Nocardia amberjack was aseptically inoculated into Brain Heart Infusion (BHI) solid medium (purchased from Guangdong Huankai Microorganism Technology Co., Ltd.), invert at 28°C nourish.
  • BHI Brain Heart Infusion
  • knockout plasmid pRE112- ⁇ GluNS was added to the competent cells, so that the total amount of knockout plasmid was 1 ⁇ g, and mixed in an ice bath for 30 minutes. The mixture was then added to a 96-well microtiter plate, 100 ⁇ L per well. Set the parameters of the electrorotor: voltage 200V, frequency 30, interval 1000ms, duration 60ms. After electroporation, 100 ⁇ L of BHI liquid medium pre-warmed at 28°C was added. Incubate in a 28°C incubator for 2 hours.
  • Step 4 Screening of positive clones
  • Nocardia amberjack bacteria solution recovered after electroporation and spread it on a BHI plate containing chloramphenicol (25mg/mL) resistance
  • Nocardia amberjack bacteria solution without electroporation as a negative control
  • an equal amount was spread on the BHI plate containing chloramphenicol resistance, placed in a biochemical incubator, and incubated upside down at 28°C until colonies grew out of the plate.
  • a single colony was picked and cultured for 5 days in BHI liquid medium without 10% sucrose, and the primer pair 112-F1/112-R1 in Table 4 was used to detect whether the bacteria contained the knockout plasmid pRE112- ⁇ GluNS.
  • the PCR amplification reaction program was: 94°C, 5 min, 30 cycles: 94°C, 30s, 55°C, 30s, 72°C, 1 min. 72°C, 5min.
  • the amplification system was: template DNA 1 ⁇ L, 112-F1 and 112-R1 1 ⁇ L each, rTaq enzyme 12 ⁇ L, and sterile water 10 ⁇ L.
  • the bacterial liquid was inoculated into BHI liquid medium containing 10% sucrose to continue culturing, and multiple PCR detection was performed using the deletion strain verification primers in Table 5 to screen for GluNS gene deletion strains.
  • the PCR amplification reaction program was: 94°C, 5 min, 30 cycles: 94°C, 30s, (55°C (GluNS-F2/GluNS-R2), 58°C (GluNS-F1/GluNS-R1)), 30s, 72 °C, 1 min. 72°C, 5min.
  • the amplification system was: 1 ⁇ L of template DNA, 1 ⁇ L of F and R, 12 ⁇ L of rTaq enzyme, and 10 ⁇ L of sterile water.
  • the 463bp fragment within the GluNS gene of Nocardia amberjack wild strain (ZJ0503) could be amplified, but the target band could not be amplified by the deletion strain ZJ0503-6296.
  • the GluNS-F2/GluNS-R2 primer pair the GluNS gene of the wild strain of Nocardia amberjack (ZJ0503), and its upstream and downstream homology arms, a total of 2042bp bands can be amplified; but the deletion strain ZJ0503-6296 can only be amplified. Increase the upstream and downstream homologous 796bp bands.
  • Nocardia amberjack GluNS deletion strain Nocardia amberjack ZJ0503-6296 and Nocardia amberjack wild strain (ZJ0503) were picked from plates to EP containing BHI liquid medium, respectively. tube, and cultured for 3 to 4 days. Then continue to streak on the non-resistant BHI plate, invert at 28°C until colonies grow on the plate, and observe the colony morphology and growth characteristics. The GluNS deletion strains were continuously streaked and cultured to 30 generations, and the genetic stability of the corresponding strains was verified with the deletion strain primers (GluNS-F2 and GluNS-R2).
  • Fig. 5 The results are shown in Fig. 5.
  • the GluNS-deficient strain was continuously transmitted to 30 generations, and the GluNS gene fragment could not be detected, indicating that the GluNS-deficient strain could be inherited stably.
  • the results are shown in Tables 6 and 7.
  • the LC50 of the wild strain was 4.74 ⁇ 10 5 CFU/mL, and the LC50 of Nocardia amberjack ZJ0503-6296 was 3.41 ⁇ 10 6 CFU/mL.
  • the concentration was 1 order of magnitude lower than that of the wild strain, indicating that the virulence of the strain was significantly reduced.
  • Relative immune protection rate ⁇ 1 – [immune group mortality (%) / control group mortality (%)] ⁇ ⁇ 100% Formula I.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Biomedical Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Mycology (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Molecular Biology (AREA)
  • Oncology (AREA)
  • Communicable Diseases (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Epidemiology (AREA)
  • Immunology (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Biophysics (AREA)
  • Virology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

提供了一种减毒鰤鱼诺卡氏菌及其构建方法和应用。通过基因工程手段，将野生型鰤鱼诺卡氏菌中谷氨酸内肽酶(GluNS)基因部分或全部序列敲除，构建一株对宿主具有较好保护性的减毒株，既能有效降低细菌的致病性，又保留了较好的免疫原性。该减毒鰤鱼诺卡氏菌可作为疫苗候选株应用于制备防治诺卡氏菌病的疫苗或其他生物制品。

Description

一种减毒鰤鱼诺卡氏菌及其构建方法和应用
本申请要求于2020年12月07日提交中国专利局、申请号为202011430248.3、发明名称为“一种减毒鰤鱼诺卡氏菌及其构建方法和应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明属于基因工程技术领域，具体涉及一种减毒鰤鱼诺卡氏菌及其构建方法和应用。
背景技术
近年来鱼类诺卡氏菌病已经成为水产养殖业中的严重病害，当水产动物体质虚弱、免疫力低下时，病原菌可通过鳃、饲料或者伤口等进行感染。病鱼表现为腹部膨大，内脏器官常弥漫性分布有致密肉芽肿，该病的感染率和死亡率都比较高，目前尚缺乏有效的防治措施，给水产养殖行业带来巨大损失。近年来鰤鱼诺卡氏菌(Nocardia seriolae)已成为鱼类诺卡氏菌病的主要病原，超过39种海淡水养殖鱼种深受其害。
相关研究表明，导致鱼类诺卡氏菌病的主要病原菌是鰤鱼诺卡氏菌。鰤鱼诺卡氏菌在分类上属于细菌域(Bacteria)、厚壁菌门(Firmicutes)、放线菌纲(Actinobacteria)、放线菌目(Actinobacterales)、诺卡氏菌科(Nocardiaceae)、诺卡氏菌属(Nocardia)。鰤鱼诺卡氏菌生长缓慢，该菌在平板上生长呈现黄色砂粒或颗粒状凸起，表面形成褶皱。液体培养条件下，细菌容易聚集形成大颗粒 [1]。目前关于鰤鱼诺卡氏菌防治手段，仍然是以预防为主。细菌的减毒株作为一种活性细菌，在保持原有免疫原性的基础上，又不会对宿主造成危害，因此减毒株备受关注。然而在鰤鱼诺卡氏菌中，已有相关研究在探索减毒株的构建。但是相关的减毒株的保护效果依然不够理想，缺乏免疫原性。
发明内容
有鉴于此，本发明的目的在于提供一种减毒鰤鱼诺卡氏菌及其构建方法和应用，构建的鰤鱼诺卡氏菌具有较低致病性外仍保留了较好的免疫原性，作为候选株在制备疫苗或其他生物制品中应用。
本发明提供了一种减毒鰤鱼诺卡氏菌，所述减毒鰤鱼诺卡氏菌缺失GluNS基因。
优选的，所述GluNS基因编码的蛋白质的氨基酸如SEQ ID NO:12所示或与SEQ ID NO:12同源性或覆盖度达到30％～99％的序列。
优选的，所述减毒鰤鱼诺卡氏菌的保藏编号为GDMCC No:61258。
本发明提供了所述减毒鰤鱼诺卡氏菌的构建方法，包括以下步骤：
1)将GluNS基因的重组同源臂插入载体中构建重组载体；
2)将所述重组载体转化入鰤鱼诺卡氏菌感受态细胞中，得到重组细胞；
3)将所述重组细胞经培养，筛选，得到减毒鰤鱼诺卡氏菌。
优选的，步骤1)中所述GluNS基因的重组同源臂由GluNS基因上游片段和下游片段组成。
优选的，所述GluNS基因上游片段或下游片段的长度独立为5bp～2000bp。
优选的，所述GluNS基因上游片段以GluNS基因上游389bp片段作为上游同源臂，所述下游片段以GluNS基因下游407bp片段作为下游同源臂，以上游同源臂-下游同源臂的连接方式形成所述GluNS基因的重组同源臂。
优选的，步骤1)中所述GluNS基因的重组同源臂的核苷酸序列如SEQ ID NO:1所示。
优选的，步骤1)中所述载体包括pRE112；
所述GluNS基因的重组同源臂插入pRE112的多克隆位点为Sac I/Kpn I。
优选的，步骤2)中所述转化包括电转化；
所述电转化的参数优选如下：电压180～220V，脉冲间隔时间800～1200ms，脉冲持续时间80～120μs，方波25～35个。
优选的，步骤3)中所述培养用溶液为26～30℃预热的含0.3mM蔗糖的BHI溶液，所述培养的温度为26～30℃，所述培养的时间10～12h。
优选的，所述筛选包括采用PCR扩增方法检测细菌内是否含有敲除质粒pRE112-ΔGluNS；
所述PCR扩增用引物对为112-F1/112-R1；
所述112-F1的核苷酸序列如SEQ ID NO:2所示；
所述112-R1的核苷酸序列如SEQ ID NO:3所示；
PCR扩增产物经测序显示缺失GluNS基因片段，得到所述细菌中含有敲除质粒pRE112-ΔGluNS，为阳性克隆。
优选的，所述筛选后，还包括鉴定减毒鰤鱼诺卡氏菌；
所述鉴定包括采用PCR扩增方法鉴定GluNS基因是否缺失；
所述PCR扩增用引物对为GluNS-F1/GluNS-R1和GluNS-F2/GluNS-R2；
所述GluNS-F1的核苷酸序列如SEQ ID NO:10所示；
所述GluNS-R1的核苷酸序列如SEQ ID NO:11所示；
所述GluNS-F2的核苷酸序列如SEQ ID NO:14所示；
所述GluNS-R2的核苷酸序列如SEQ ID NO:15所示；
用GluNS-F1/GluNS-R1引物对对GluNS基因缺失株无法扩增出目的条带；用GluNS-F2/GluNS-R2引物对对GluNS基因缺失株只能扩增上下 游同源796bp条带。
本发明提供了所述的减毒鰤鱼诺卡氏菌在制备疫苗或包含所述减毒鰤鱼诺卡氏菌的相关生物制品中的应用。
本发明提供的减毒鰤鱼诺卡氏菌，通过基因工程手段，将野生型鰤鱼诺卡氏菌中谷氨酸内肽酶(GluNS)基因敲除，构建一株对宿主具有较好保护性的减毒株，该减毒株既能有效降低细菌的致病性，又保留了较好的免疫原性。实验表明，野生株的半致死浓度为4.74×10 5CFU/mL，所述减毒株的半致死浓度为3.41×10 6CFU/mL，其半致死浓度相较野生株降低了1个数量级，表明了该菌株毒力发生显著下降。活菌攻毒实验结果表明，所述减毒株的相对免疫保护率达到93.38％，说明所述减毒株是一株对宿主具有较好保护性的减毒株(菌株保藏编号为GDMCC No:61258)。
同时，本发明构建的减毒鰤鱼诺卡氏菌还具有较高的遗传稳定性。实验表明，GluNS缺失株鰤鱼诺卡氏菌ZJ0503-6296连续传至30代，无法检测出GluNS基因片段，表明GluNS缺失株能稳定遗传。
附图说明
图1为pRE112质粒图谱；
图2为本发明构建后的重组质粒图谱；
图3为GluNS基因缺失株鰤鱼诺卡氏菌ZJ0503-6296的第一次筛选得到的电泳图；M:DNA marker 2000，1，2：实验组，3：ZJ0503，4：pRE112；
图4为GluNS基因缺失株鰤鱼诺卡氏菌ZJ0503-6296的第二次筛选得到的电泳图；M:DNA marker 2000，1：鰤鱼诺卡氏菌ZJ0503-6296，2：ZJ0503；
图5为GluNS基因缺失株鰤鱼诺卡氏菌ZJ0503-6296的稳定遗传验证；M:DNA marker 10000，1：鰤鱼诺卡氏菌ZJ0503-6296，2-4：ZJ0503；
图6为敲除质粒pRE112-ΔGluNS构建方法示意图；
图7为GluNS基因缺失株鰤鱼诺卡氏菌ZJ0503-6296的构建原理。生物材料保藏信息
鰤鱼诺卡氏菌(Nocardia sp.)ZJ0503-6296，保藏于广东省微生物菌种保藏中心，单位简称GDMCC，地址为广州市先烈中路100号大院59号楼5楼，广东省微生物研究所，保藏时间2020年10月29日，保藏编号为GDMCC No：61258。
具体实施方式
本发明提供了一种减毒鰤鱼诺卡氏菌，所述减毒鰤鱼诺卡氏菌缺失GluNS基因。
本发明对GluNS基因的序列没有特殊限制，凡缺失本领域所熟知的 GluNS基因的全部核苷酸序列(SEQ ID NO:13，gtgctcgtcgccggacccctggcggcgagcgcacacgcggaacccgcgacccccgatctgcccgcccagctgatcgcggccatcacccgcgacctcaagatctccccgcaggactacctggcccgggccgacaccgcacagaaggtggccaccttcgccaccaccgcgcagcggcagttcccgcaggtcttcggcggcgcctggctggacgagaccggcaaggccgtcgtggcgctggccccgggcgagggcgtggacaaggcccgcaaggccgtgcaggacgccggtttcaccgccaaggacgtgagcaagagcgagaccacgctgcgcggcgagaagaacgccttccagcagtggctcaaggatcagcccgagtccgtggcgaaggccatccggggcgtggccatcgacaccttgaacaacagcatcgcggtgcgggtggacaagcccgatctgccgctgccgggcttcgtggacccggcccgcgtcatcgtcatgaccgcaccgccggtcggcggcgagggccagaacgtgccgcaggccaccgagatcgcgggcgcgggcccgcgcgccatcgccgccggcgaggcctacgcctcggtcgccggccgcatgtcgctgcgctgctcgctcggcttcaacggcaccgacggcaacggcaatgtcgtcaatatcaccgcgggccactgcaacccgaacatcgcggccaccggcggcgcgaactcgccgagcgtgtaccagctcatcggtgacagccgcggtcccgaggtcggccagttccagaagtcggtgctgggcaacgaggactactcgatcgtcggcatcaacgaccagttccgcgacgccttctccaacccgttcgtaaccgtcccgggctcggcctcgatcgccgtcaccggcgtggccgtgccggtggtcggcgcgccggtctgcaagtcgggtgcgcgcaccggcttcagctgcggcgtggtgaacgccgtggaccagaccgtccaggtcggcgaccgcctgctgacccagtccttctccgccaatatctgtgccctgcccggtgattcgggtggcccgctggtgaccggcacgctggccctgggcatcgccagcgcatcctcggtcgccgactacccgatctgcgagatcccgaacctgctcggcctgatcaccggcaacaccccgcagctgttcgcgcagccggtgagcaccgtgctctccgacaacccggggctgcgggtccgcaccacgtaa)或部分核苷酸序列(ORF6296)均能实现减毒目的，因此，缺失全部或部分GluNS基因的核苷酸序列和编码的氨基酸序列均在本发明保护的范围之内。
在本发明中，所述GluNS基因编码的蛋白质的氨基酸优选如SEQ ID NO:12所示(VLVAGPLAASAHAEPATPDLPAQLIAAITRDLKISPQDYLARADTAQKVATFATTAQRQFPQVFGGAWLDETGKAVVALAPGEGVDKARKAVQDAGFTAKDVSKSETTLRGEKNAFQQWLKDQPESVAKAIRGVAIDTLNNSIAVRVDKPDLPLPGFVDPARVIVMTAPPVGGEGQNVPQATEIAGAGPRAIAAGEAYASVAGRMSLRCSLGFNGTDGNGNVVNITAGHCNPNIAATGGANSPSVYQLIGDSRGPEVGQFQKSVLGNEDYSIVGINDQFRDAFSNPFVTVPGSASIAVTGVAVPVVGAPVCKSGARTGFSCGVVNAVDQTVQVGDRLLTQSFSANICALPGDSGGPLVTGTLALGIASASSVADYPICEIPNLLGLITGNTPQLFAQPVSTVLSDNPGLRVRTT)。本发明缺失的GluNS基因编码蛋白质还包括与SEQ ID NO:12同源性或覆盖度达到30％～99％的序列。
所述减毒鰤鱼诺卡氏菌的保藏编号优选为GDMCC No:61258。
本发明还提供了所述减毒鰤鱼诺卡氏菌的构建方法，包括以下步骤：
1)将GluNS基因的重组同源臂插入载体中构建重组载体；
2)将所述重组载体转化入鰤鱼诺卡氏菌感受态细胞中，得到重组细胞；
3)将所述重组细胞经培养，筛选，得到减毒鰤鱼诺卡氏菌。
本发明提供了所述减毒鰤鱼诺卡氏菌构建方法，构建原理为克隆目的基因上下游约500bp片段作为同源臂，通过重叠PCR方法将同源臂连接在一起，构建到缺失载体pRE112上。将缺失载体导入到鰤鱼诺卡氏菌中，利用同源重组的方法，将缺失载体上重组同源臂片段替换细菌中含有同源臂和目的基因片段，使得鰤鱼诺卡氏菌中目的基因缺失，只含有重组同源臂片段，以此构成减毒鰤鱼诺卡氏菌(见图7)。
本发明将GluNS基因的重组同源臂插入载体中构建重组载体。
在本发明中，所述GluNS基因的重组同源臂优选由GluNS基因上游片段和下游片段组成。本发明对所述GluNS基因上游片段和下游片段的长度不做具体限定，采用本领域所熟知的GluNS基因上游片段和下游片段的长度即可，例如5bp～2000bp均可。在本发明实施例中，所述GluNS基因上游片段以GluNS基因上游389bp片段作为上游同源臂，所述下游片段以GluNS基因下游407bp片段作为下游同源臂，以上游同源臂-下游同源臂的连接方式形成所述GluNS基因的重组同源臂。所述GluNS基因的重组同源臂的核苷酸序列优选如SEQ ID NO:1(GCCTGCACCCTGGGCGGTGACAGCGGCGGCGCGATCGTGTCGGGCACGCTGGCGCTGGGCATCACCAGCGGTTCGAACGCGGCCGACGCGCCGAACTGCAACGAGGCCAACACCGCGCTGGCGCAGTACGGTGGAACCGCGTCGCTGGGCATCCCGGTGCGCGCGATTCTCACCGAAATCGACGCCAATTCCGGTGGCGGCGTCGGCAGCGGAATTCAGGTGCGGACGCGGTCCAACGCCTGACGGCGAAGTGCTCCAGCACCCGACGGCAAAGCGGTCCAACGCCTGACGATGGCGACATCGGCGGTAAACGGACATCCTTCACATTGCCCCTGAATCCGACATGCCCGCTGTTTGCCCCGTGAATACCGAAACGTCACCTCGCAATAAGTAGGCATAACGGGCCCGACACCATATAGTCGGGGCCATGCTCCTGCCACGTATGCGCTTCTCCATGCCTGCCGCGGGCCGACGCCCGCGGTCCCTTGTCCGTACTGCGGCGATCGCGGCGACCTCTTCACGGCACCCTCGAAAAGGCCGGTAGCTCAACAGCTACCGGCCTTTTGTCGTCCCGGTGGGGAAGAGATACCGCTGACCTCACTAATTCGGATGCAGCGCTGGAAGTTCGTGGCCCGCCTCGGTCTGGACTGACGGAGCGGGGGAGCGAAGCGGAGGAGCGGAGGAGGGAAGACCGAGGTAACAGGGCCACCAACCGCCGTAGCGCAGCGGAGGCAAATTAAACAGAGCATGCTGGGGATATGTGTTTGGTGTTGCTGGGTTGGCGAGCGCATCCGGAGTACCGCTTGATCGTGGCCGCCAACCGGGACGAGTTCTTCACCCGCCCAACGGAATCGCTGCGCCGGTGGGACGAAGTGCCCGGGGTGCTGGCCGGGCGGGACCTGGGCGCGGCCGGACCGGTGCCCGGCACCTGGCTCGGTGCGCTGCCCGATCATCGCCGCTTCGCGACGGTCACCAATGTGCGACTACCCGGCGAATTCCGCGCCGATGTGCGCTCGCGC GGCGCGCTGCTGCTGG)。所述GluNS基因上游片段的核苷酸序列优选如SEQ ID NO:4所示(GCCTGCACCCTGGGCGGTGACAGCGGCGGCGCGATCGTGTCGGGCACGCTGGCGCTGGGCATCACCAGCGGTTCGAACGCGGCCGACGCGCCGAACTGCAACGAGGCCAACACCGCGCTGGCGCAGTACGGTGGAACCGCGTCGCTGGGCATCCCGGTGCGCGCGATTCTCACCGAAATCGACGCCAATTCCGGTGGCGGCGTCGGCAGCGGAATTCAGGTGCGGACGCGGTCCAACGCCTGACGGCGAAGTGCTCCAGCACCCGACGGCAAAGCGGTCCAACGCCTGACGATGGCGACATCGGCGGTAAACGGACATCCTTCACATTGCCCCTGAATCCGACATGCCCGCTGTTTGCCCCGTGAATACCGAAACGTCACCTCGCAATAAGTAGGCATAACGGGCCCGACACCATATAGTCGGGGCCATGCTCCTGCCACGTATGCGCTTCTCCATGCCTGCCGCGGGCCGACGCCCGCGGTCCCTTGTCCGTACTGCGGCGATCGCGGCGACCTCT)。所述GluNS基因上游片段的扩增用引物优选为GluNS-UF和GluNS-UR。所述GluNS-UF的核苷酸序列优选如SEQ ID NO:5所示(CATATGGGGCATCACCAGCGGTTCG)，所述GluNS-UR的核苷酸序列优选如SEQ ID NO:6所示(TGCATCCGAATTAGTGAGGTCAGAGGAGCATGGCCCCGACT)。所述GluNS基因下游片段的核苷酸序列优选如SEQ ID NO:7所示(TCACGGCACCCTCGAAAAGGCCGGTAGCTCAACAGCTACCGGCCTTTTGTCGTCCCGGTGGGGAAGAGATACCGCTGACCTCACTAATTCGGATGCAGCGCTGGAAGTTCGTGGCCCGCCTCGGTCTGGACTGACGGAGCGGGGGAGCGAAGCGGAGGAGCGGAGGAGGGAAGACCGAGGTAACAGGGCCACCAACCGCCGTAGCGCAGCGGAGGCAAATTAAACAGAGCATGCTGGGGATATGTGTTTGGTGTTGCTGGGTTGGCGAGCGCATCCGGAGTACCGCTTGATCGTGGCCGCCAACCGGGACGAGTTCTTCACCCGCCCAACGGAATCGCTGCGCCGGTGGGACGAAGTGCCCGGGGTGCTGGCCGGGCGGGACCTGGGCGCGGCCGGACCGGTGCCCGGCACCTGGCTCGGTGCGCTGCCCGATCATCGCCGCTTCGCGACGGTCACCAATGTGCGACTACCCGGCGAATTCCGCGCCGATGTGCGCTCGCGCGGCGCGCTGCTGCTGG)。所述GluNS基因下游片段的扩增用引物优选为GluNS-DF和GluNS-DR。所述GluNS-DF的核苷酸序列优选如SEQ ID NO:8所示(AGTCGGGGCCATGCTCCTCTGACCTCACTAATTCGGATGCA)，所述GluNS-DR的核苷酸序列优选如SEQ ID NO:9所示(GAGCTCAATTCGCCGGGTAGTCGC)。利用GluNS-UF和GluNS-DR引物，以GluNS基因上游片段和GluNS基因下游片段为模板，通过PCR扩增可获得重叠PCR产物。PCR扩增程序为：94℃，5min，15个循环：94℃，30s，58℃，30s，72℃，1min。72℃，5min。反应体系为：GluNS基因上游片段产物2μL，GluNS基因下游片段产物2μL，GluNS-UF 和GluNS-DR各1μL，rTaq酶25μL，无菌水19μL。
在本发明中，所述载体优选包括pRE112质粒，见图1。所述GluNS基因的重组同源臂插入pRE112的多克隆位点优选为Sac I/Kpn I。所述GluNS基因的重组同源臂插入载体的方法优选包括酶切和连接。所述酶切包括分别对所述GluNS基因的重组同源臂和载体进行酶切。本发明对所述酶切的方法没有特殊限制，采用本领域所熟知的酶切方法即可。本发明对所述连接的方法没有特殊限制，采用本领域所熟知的连接方法即可，例如利用T4连接酶进行连接。本发明对所述T4连接酶的连接方法没有特殊限制，采用本领域所熟知的T4连接酶的连接方案即可。连接后，本发明还优选包括鉴定；所述鉴定优选采用PCR扩增的方法或采用测序的方法均可。构建后的重组载体见图2。
得到重组载体后，本发明将所述重组载体转化入鰤鱼诺卡氏菌感受态细胞中，得到重组细胞。
在本发明中，所述鰤鱼诺卡氏菌感受态细胞的制备方法采用本领域公知的制备感受态细胞的方案即可。所述鰤鱼诺卡氏菌的来源不做具体限定，采用本领域所熟知的鰤鱼诺卡氏菌即可。为了举例说明减毒株的构建方法，本发明实施例中，以鰤鱼诺卡氏菌野生株ZJ0503为材料实施。
本发明对所述转化的方法没有特殊限制，采用本领域所熟知的转化方法即可。在本发明实施例中，所述转化优选包括电转化。所述电转化的参数优选如下：电压180～220V，脉冲间隔时间800～1200ms，脉冲持续时间80～120μs，方波25～35个。
得到重组细胞后，本发明将所述重组细胞经培养，筛选，得到减毒鰤鱼诺卡氏菌。
在本发明中，所述培养用溶液优选为26～30℃预热的含0.3mM蔗糖的BHI溶液，所述培养的温度优选为26～30℃，所述培养的时间优选为10～12h。所述筛选优选采用载体中所携带的抗性基因对应的抗生素进行筛选。在本发明实施例中，优选采用含25mg/mL氯霉素抗性的BHI平板进行筛选。
在本发明中，所述筛选后优选还包括鉴定减毒鰤鱼诺卡氏菌。所述鉴定优选包括采用PCR扩增方法鉴定GluNS基因是否缺失。所述PCR扩增用引物对优选为GluNS-F1/GluNS-R1和GluNS-F2/GluNS-R2。所述GluNS-F1的核苷酸序列优选如SEQ ID NO:10所示(CCGCCAAGGACGTGAG)。所述GluNS-R1的核苷酸序列优选如SEQ ID NO:11所示(CTGTCACCGATGAGCTGGTA)。所述GluNS-F2的核苷酸序列优选如SEQ ID NO:14所示(GGGCATCACCAGCGGTTCG)。所述GluNS-R2的核苷酸序列优选如SEQ ID NO:15所示(AATTCGCCGGGTAGTCGC)。PCR扩增反应程序为：94℃，5min，30个循环：94℃，30s，(55℃(GluNS-F2/GluNS-R2)，58℃(GluNS-F1/ GluNS-R1)，30s，72℃，1min。72℃，5min。
本发明提供了所述减毒鰤鱼诺卡氏菌的构建方法构建得到的减毒鰤鱼诺卡氏菌，所述减毒鰤鱼诺卡氏菌的基因组缺失GluNS基因。所述减毒鰤鱼诺卡氏菌的半致死浓度为3.41×10 6CFU/mL，而野生株的半致死浓度为4.74×10 5CFU/mL，所述减毒鰤鱼诺卡氏菌较野生株降低了1个数量级，表明了该菌株毒力发生显著下降。所述减毒鰤鱼诺卡氏菌的相对免疫保护率为93.38％，具有较高的免疫原性。同时本发明构建的减毒鰤鱼诺卡氏菌，是在鰤鱼诺卡氏菌基因组上直接进行敲除，菌内不含抗性质粒，符合生物安全，并且不会以为质粒的丢失而影响鰤鱼诺卡氏菌的生长。
在本发明中，为了测定减毒鰤鱼诺卡氏菌的遗传稳定性，将减毒鰤鱼诺卡氏菌传代30代后，测定减毒鰤鱼诺卡氏菌的遗传稳定性。方法为采用GluNS-F1(SEQ ID NO:10)和GluNS-R1(SEQ ID NO:11)，GluNS-F2(SEQ ID NO:14)和GluNS-R2(SEQ ID NO:15)引物进行扩增，筛选GluNS缺失株。
基于所述减毒鰤鱼诺卡氏菌的毒力下降且保持较理想的免疫原性，本发明提供了所述减毒鰤鱼诺卡氏菌在制备疫苗或包含所述减毒鰤鱼诺卡氏菌的相关生物制品中的应用。本发明对所述疫苗的制备方法没有特殊限制，采用本领域所熟知的疫苗的制备方法即可。制备的疫苗能够用于防治因鰤鱼诺卡氏菌引起的鱼类诺卡氏菌病，具有较高的应用价值。本发明对所述相关生物制品的种类不做具体限定，采用本领域所熟知的生物制品的种类即可，例如治疗制品或诊断制品等。
下面结合实施例对本发明提供的一种减毒鰤鱼诺卡氏菌及其构建方法和应用进行详细的说明，但是不能把它们理解为对本发明保护范围的限定。
实施例1
减毒鰤鱼诺卡氏菌的构建方法
步骤一：构建同源重组载体。
具体的，将保存的鰤鱼诺卡氏菌野生株(ZJ0503)无菌操作划线接种到脑心浸液(Brain Heart Infusion，BHI)固体培养基(购自广东环凯微生物科技有限公司)，26～30℃倒置培养。待平板上长出单菌落，无菌操作挑取单菌落至40～60ml BHI液体培养基中，26～30℃，100～150rpm培养至对数生长期。取一定体积的菌液提取细菌基因组，以此作为模板，利用表1引物分别克隆GluNS基因上下游各片段，然后连接到pRE112载体中。扩增程序为：PCR扩增的反应程序为PCR扩增程序为：94℃，5min；30个循环：94℃，30s，55℃，30s，72℃，1min；72℃，5min。扩增体系为：鰤鱼诺卡氏菌基因组DNA 1μL，上游引物(F)和下游引物(R)各2μL，rTaq酶25μL，无菌水20μL。
表1引物序列
Figure PCTCN2021075555-appb-000001
注：下划线为酶切位点，波浪线为重叠PCR位点。
从-80℃冰箱中取出含有pRE112质粒的E.coli S17-1菌种(购自武汉淼灵生物科技有限公司)，37℃水浴融化，划线于含有氯霉素抗性的LB平板中。挑取单菌落过夜扩大培养，随后使用质粒提取试剂盒提取质粒。按照表2体系对pRE112质粒和重叠PCR产物分别进行双酶切实验，37℃反应30min。随后按照表3，将重叠PCR产物与质粒pRE112连接。重叠PCR产物获得方法：利用GluNS-UF和GluNS-DR引物，以GluNS基因上游片段和GluNS基因下游片段为模板，通过PCR扩增可获得重叠PCR产物。PCR扩增程序为：94℃，5min，15个循环：94℃，30s；58℃，30s，72℃，1min；72℃，5min。反应体系为：GluNS基因上游片段产物2μL，GluNS基因下游片段产物2μL，GluNS-UF和GluNS-DR各1μL，rTaq酶25μL，无菌水19μL。
表2双酶切反应体系
Figure PCTCN2021075555-appb-000002
表3重叠PCR产物与质粒pRE112连接体系
Figure PCTCN2021075555-appb-000003
步骤二：鰤鱼诺卡氏菌感受态细胞的制备；
将保存的鰤鱼诺卡氏菌野生株(ZJ0503)无菌操作划线接种到脑心浸液(Brain Heart Infusion，BHI)固体培养基(购自广东环凯微生物科技有限公司)，28℃倒置培养。待平板上长出单菌落，无菌操作挑取单菌落至50ml BHI液体培养基中，28℃，120rpm培养至对数生长期。取达到对数生长期的30ml鰤鱼诺卡氏菌菌液于50ml离心管中，4℃，8000rpm收集细菌菌体；用10ml 10％无菌甘油分别洗涤菌体两次，再用10ml无菌甘油重悬细菌菌体。
步骤三：电转化；
吸取一定体积的敲除质粒pRE112-ΔGluNS加入到感受态细胞中，使得敲除质粒总量为1μg，冰浴混匀30min。随后将混合液加入到96孔酶标板中，每孔100μL。设置电转仪参数：电压200V、频率30、间隔时间1000ms、持续时间60ms。电转后，加入100μL 28℃预热的BHI液体培养基。置于28℃培养箱中静置复苏2小时。
步骤四：阳性克隆筛选；
取电转后复苏的鰤鱼诺卡氏菌菌液100μL涂布于含有氯霉素(25mg/mL)抗性的BHI平板上，另以没电转过的鰤鱼诺卡氏菌菌液作为阴性对照，等量涂布于含有氯霉素抗性的BHI平板上，放在生化培养箱中，28℃倒置培养至平板长出菌落。挑取单菌落到不含有10％蔗糖的BHI液体培养基中培养5d，用表4引物对112-F1/112-R1检测细菌内是否含有敲除质粒pRE112-ΔGluNS。PCR扩增反应程序为：94℃，5min，30个循环：94℃，30s，55℃，30s，72℃，1min。72℃，5min。扩增体系为：模板DNA 1μL，112-F1和112-R1各1μL，rTaq酶12μL，无菌水10μL。将菌液接种到含有10％蔗糖的BHI液体培养基中继续培养，用表5缺失株验证引物进行多重PCR检测，筛选GluNS基因缺失株。PCR扩增反应程序为：94℃，5min，30个循环：94℃，30s，(55℃(GluNS-F2/GluNS-R2)，58℃(GluNS-F1/GluNS-R1))，30s，72℃，1min。72℃，5min。扩增体系为：模板DNA 1μL，F和R各1μL，rTaq酶12μL，无菌水10μL。
用GluNS-F1/GluNS-R1引物对，可扩增鰤鱼诺卡氏菌野生株(ZJ0503)GluNS基因内部463bp片段，而缺失株ZJ0503-6296则无法扩增出目的条带。用GluNS-F2/GluNS-R2引物对，可扩增鰤鱼诺卡氏菌野生株(ZJ0503)GluNS基因，及其上下游同源臂共2042bp条带；但是对于缺失株ZJ0503-6296只能扩增上下游同源796bp条带。
结果如图3和图4所示，构建成功的GluNS基因缺失株命名为鰤鱼诺卡氏菌(Nocardia sp.)ZJ0503-6296。
表4 pRE112验证引物
Figure PCTCN2021075555-appb-000004
Figure PCTCN2021075555-appb-000005
表5 GluNS缺失株筛选引物
Figure PCTCN2021075555-appb-000006
实施例2
稳定性遗传分析
为测定鰤鱼诺卡氏菌GluNS缺失株的遗传稳定性，分别从平板挑取鰤鱼诺卡氏菌ZJ0503-6296和鰤鱼诺卡氏菌野生株(ZJ0503)至含有BHI液体培养基的EP管中，培养3～4天。然后继续划线于无抗性的BHI平板上，28℃倒置培养至平板上长出菌落、观察菌落形态和生长特性。连续划线接种培养GluNS缺失株至30代，用缺失株引物(GluNS-F2和GluNS-R2)验证相应菌株的遗传稳定性。
结果如图5所示，GluNS缺失株连续传至30代，无法检测出GluNS基因片段，表明GluNS缺失株能稳定遗传。
实施例3
1.半致死浓度测定
为了测定缺失株鰤鱼诺卡氏菌ZJ0503-6296和鰤鱼诺卡氏菌野生株(ZJ0503)的半致死浓度(LD 50)。参考文献方法 [2]，用无菌PBS溶液调整菌悬液浓度为10 4、10 5、10 6、10 7、10 8CFU/mL，实验组每尾杂交鳢腹腔注射100μL菌液，对照组注射等量的无菌PBS溶液，每个浓度设立三个平行组，每组30尾。连续观察14天，每天正常投喂商业饲料并记录死鱼情况。参考文献方法 [3]，采用SPSS17.0进行统计学分析并计算LD 50。
结果如表6和表7所示，野生株的半致死浓度为4.74×10 5CFU/mL，鰤鱼诺卡氏菌ZJ0503-6296的半致死浓度为3.41×10 6CFU/mL，其半致死浓度相较野生株降低了1个数量级，表明了该菌株毒力发生显著下降。
表6鰤鱼诺卡氏菌各菌株死亡数统计
Figure PCTCN2021075555-appb-000007
Figure PCTCN2021075555-appb-000008
2.免疫保护率的计算；
将缺失株鰤鱼诺卡氏菌ZJ0503-6296以10 6CFU/mL免疫杂交鳢35天后，进行鰤鱼诺卡氏菌野生株活菌攻毒实验，并计算免疫保护率，同时设置对照组，对照组用PBS代替鰤鱼诺卡氏菌ZJ0503-6296。根据公式I计算免疫保护率。
相对免疫保护率(RPS)＝{1–[免疫组死亡率(％)/对照组死亡率(％)]}×100％     公式I。
表8鰤鱼诺卡氏菌注射杂交鳢存活率与免疫保护率的计算结果
Figure PCTCN2021075555-appb-000009
由上述可知，缺失株鰤鱼诺卡氏菌ZJ0503-6296的相对免疫保护率达 到93.38％，取得了较理想的免疫原性，可用于后续疫苗的制备。
参考文献
[1]朱志东,吕莉,邓剑壕,等.鱼类诺卡氏菌病的研究进展[J].水产养殖,2018,39(01):48–52.
[2]王文基,陈建林,侯素莹,等.鰤鱼诺卡氏菌感染乌斑杂交鳢的组织病理学研究[J].基因组学与应用生物学,2019,38(10):4439–4446.
[3]熊浩明,魏柏青,魏荣杰,等.用SPSS软件计算鼠疫菌半数致死量(LD_(50))[J].中国人兽共患病学报,2013,29(11):1127–1130.
以上实施例的说明只是用于帮助理解本发明的方法及其核心思想。应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以对本发明进行若干改进和修饰，这些改进和修饰也落入本发明权利要求的保护范围内。对这些实施例的多种修改对本领域的专业技术人员来说是显而易见的，本文中所定义的一般原理可以在不脱离本发明的精神或范围的情况下在其它实施例中实现。因此，本发明将不会被限制于本文所示的这些实施例，而是要符合与本文所公开的原理和新颖特点相一致的最宽的范围。

Claims (14)

  1. 一种减毒鰤鱼诺卡氏菌，其特征在于，所述减毒鰤鱼诺卡氏菌缺失GluNS基因。
  2. 根据权利要求1所述减毒鰤鱼诺卡氏菌，其特征在于，所述GluNS基因编码的蛋白质的氨基酸如SEQ ID NO:12所示或与SEQ ID NO:12同源性或覆盖度达到30％～99％的序列。
  3. 根据权利要求1或2所述减毒鰤鱼诺卡氏菌，其特征在于，所述减毒鰤鱼诺卡氏菌的保藏编号为GDMCC No:61258。
  4. 权利要求1～3任意一项所述减毒鰤鱼诺卡氏菌的构建方法，其特征在于，包括以下步骤：
    1)将GluNS基因的重组同源臂插入载体中构建得到重组载体；
    2)将所述重组载体转化入鰤鱼诺卡氏菌感受态细胞中，得到重组细胞；
    3)将所述重组细胞经培养，筛选，得到减毒鰤鱼诺卡氏菌。
  5. 根据权利要求4所述减毒鰤鱼诺卡氏菌的构建方法，其特征在于，步骤1)中所述GluNS基因的重组同源臂由GluNS基因上游片段和下游片段组成。
  6. 根据权利要求5所述减毒鰤鱼诺卡氏菌的构建方法，其特征在于，所述GluNS基因上游片段或下游片段的长度独立为5bp～2000bp。
  7. 根据权利要求6所述减毒鰤鱼诺卡氏菌的构建方法，其特征在于，所述GluNS基因上游片段以GluNS基因上游389bp片段作为上游同源臂，所述下游片段以GluNS基因下游407bp片段作为下游同源臂，以上游同源臂-下游同源臂的连接方式形成所述GluNS基因的重组同源臂。
  8. 根据权利要求7所述减毒鰤鱼诺卡氏菌的构建方法，其特征在于，步骤1)中所述GluNS基因的重组同源臂的核苷酸序列如SEQ ID NO:1所示。
  9. 根据权利要求4所述减毒鰤鱼诺卡氏菌的构建方法，其特征在于，步骤1)中所述载体包括pRE112质粒；
    所述GluNS基因的重组同源臂插入pRE112质粒的多克隆位点为Sac I/Kpn I。
  10. 根据权利要求4所述减毒鰤鱼诺卡氏菌的构建方法，其特征在于，步骤3)中所述培养用溶液为26～30℃预热的含0.3mM蔗糖的BHI溶液，所述培养的温度为26～30℃，所述培养的时间10～12h。
  11. 根据权利要求4所述减毒鰤鱼诺卡氏菌的构建方法，其特征在于，步骤2)中所述转化包括电转化；
    所述电转化的参数优选如下：电压180～220V，脉冲间隔时间800～1200ms，脉冲持续时间80～120μs，方波25～35个。
  12. 根据权利要求4所述减毒鰤鱼诺卡氏菌的构建方法，其特征在于， 所述筛选包括采用PCR扩增方法检测细菌内是否含有敲除质粒pRE112-ΔGluNS；
    所述PCR扩增用引物对为112-F1/112-R1；
    所述112-F1的核苷酸序列如SEQ ID NO:2所示；
    所述112-R1的核苷酸序列如SEQ ID NO:3所示；
    PCR扩增产物经测序显示缺失GluNS基因片段，得到所述细菌中含有敲除质粒pRE112-ΔGluNS，为阳性克隆。
  13. 根据权利要求4～11任意一项所述减毒鰤鱼诺卡氏菌的构建方法，其特征在于，所述筛选后，还包括鉴定减毒鰤鱼诺卡氏菌；
    所述鉴定包括采用PCR扩增方法鉴定GluNS基因是否缺失；
    所述PCR扩增用引物对为GluNS-F1/GluNS-R1和GluNS-F2/GluNS-R2；
    所述GluNS-F1的核苷酸序列如SEQ ID NO:10所示；
    所述GluNS-R1的核苷酸序列如SEQ ID NO:11所示；
    所述GluNS-F2的核苷酸序列如SEQ ID NO:14所示；
    所述GluNS-R2的核苷酸序列如SEQ ID NO:15所示；
    用GluNS-F1/GluNS-R1引物对对GluNS基因缺失株无法扩增出目的条带；用GluNS-F2/GluNS-R2引物对对GluNS基因缺失株只能扩增上下游同源796bp条带。
  14. 权利要求1～3任意一项所述的减毒鰤鱼诺卡氏菌在制备疫苗或包含所述减毒鰤鱼诺卡氏菌的相关生物制品中的应用。
PCT/CN2021/075555 2020-12-07 2021-02-05 一种减毒鰤鱼诺卡氏菌及其构建方法和应用 WO2022121109A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US17/428,791 US20220372500A1 (en) 2020-12-07 2021-02-05 Attenuated nocardia seriolae and construction method and use thereof

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202011430248.3A CN112481183A (zh) 2020-12-07 2020-12-07 一种减毒鰤鱼诺卡氏菌及其构建方法和应用
CN202011430248.3 2020-12-07

Publications (1)

Publication Number Publication Date
WO2022121109A1 true WO2022121109A1 (zh) 2022-06-16

Family

ID=74940007

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2021/075555 WO2022121109A1 (zh) 2020-12-07 2021-02-05 一种减毒鰤鱼诺卡氏菌及其构建方法和应用

Country Status (3)

Country Link
US (1) US20220372500A1 (zh)
CN (1) CN112481183A (zh)
WO (1) WO2022121109A1 (zh)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008074783A1 (en) * 2006-12-18 2008-06-26 Intervet International B.V. Immune stimulant against fish pathogenic bacteria
CN104232535A (zh) * 2014-09-02 2014-12-24 广东海洋大学 鰤鱼诺卡氏菌诱变弱毒株及其应用

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110628796B (zh) * 2019-05-20 2023-03-24 广东海洋大学深圳研究院 鱼类诺卡氏菌病共同抗原dna疫苗及其制备和应用

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008074783A1 (en) * 2006-12-18 2008-06-26 Intervet International B.V. Immune stimulant against fish pathogenic bacteria
CN104232535A (zh) * 2014-09-02 2014-12-24 广东海洋大学 鰤鱼诺卡氏菌诱变弱毒株及其应用

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
CHEN JIANLIN, ET AL.: "Identification of a mitochondrial‐targeting secretory protein from Nocardia seriolae which induces apoptosis in fathead minnow cells", JOURNAL OF FISH DISEASES APR 2010, vol. 42, no. 11, 3 September 2019 (2019-09-03), pages 1493 - 1507, XP055941763, ISSN: 1365-2761, DOI: 10.1111/jfd.13062 *
DUO TIAN, ET AL.: "A Review of Research Progress of Nocardia Seriolae", FISHERIES SCIENCE, vol. 36, no. 3, 31 May 2017 (2017-05-31), pages 391 - 394, XP055941762, ISSN: 1003-1111, DOI: 10.16378/j.cnki.1003-1111.2017.03.023 *
WANG WENJI, ET AL.: "Characterization and function study of a glutamyl endopeptidase homolog from Nocardiaseriolae", JOURNAL OF FISH DISEASES APR 2010, vol. 44, 27 November 2020 (2020-11-27), pages 813 - 821, XP055941761, ISSN: 1365-2761, DOI: 10.1111/jfd.13311 *

Also Published As

Publication number Publication date
CN112481183A (zh) 2021-03-12
US20220372500A1 (en) 2022-11-24

Similar Documents

Publication Publication Date Title
CN104894075B (zh) CRISPR/Cas9和Cre/lox系统编辑伪狂犬病毒基因组制备疫苗方法和应用
CN109825464B (zh) 敲除t6ss-1基因簇的杀香鱼假单胞菌鱼用减毒疫苗
WO2021217959A1 (zh) 一种包含非洲猪瘟病毒免疫原性蛋白的重组载体、重组菌及其应用
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
CN113005069B (zh) 一种减毒鰤鱼诺卡氏菌及其制备方法和应用
WO2020135108A1 (zh) 鸭瘟病毒gE和gI双基因无痕缺失株DPV CHv-ΔgE+ΔgI及其构建方法
CN101979598B (zh) 一种携带荧光素酶报告基因的hsv-1 bac系统的构建方法
CN108441462A (zh) 一株枯草芽孢杆菌及其制备方法
CN109266593B (zh) 基于Ngpiwi蛋白介导的禽多杀性巴氏杆菌基因敲除菌株及其构建方法和应用
CN106939320B (zh) 一种伪狂犬病毒js-2012株感染性克隆质粒、构建方法与应用
WO2022121109A1 (zh) 一种减毒鰤鱼诺卡氏菌及其构建方法和应用
CN109468256B (zh) 整合四拷贝f18菌毛操纵子基因和双拷贝f4菌毛操纵子基因的益生菌克隆株、构建方法
CN109468255B (zh) 整合单拷贝功能性f4菌毛操纵子基因的益生菌克隆株、构建方法及应用
CN111454872A (zh) 禽致病性大肠杆菌VI型分泌系统clpV基因缺失株及其构建方法和应用
CN112746050B (zh) 一种鰤鱼诺卡氏菌减毒株及其制备方法和应用
CN116162637A (zh) 一种融合基因、其所编码的蛋白及其在鱼类虹彩病毒和弹状病毒二联口服疫苗的应用
CN109504643B (zh) 整合四拷贝功能性f18菌毛操纵子基因的益生菌克隆株、构建方法及应用
CN110669714B (zh) 肠炎沙门菌减毒疫苗候选株的制备与应用
CN109628361B (zh) 整合双拷贝功能性f4菌毛操纵子基因猪源益生菌ep1克隆株、构建方法及应用
JP6412865B2 (ja) ビフィドバクテリウム・ブレーベ株特異的遺伝子
CN112442473A (zh) 一种禽大肠杆菌疫苗株
CN104762244B (zh) 猪链球菌SBP_bac_5基因缺失株及其构建方法与应用
CN109608535A (zh) 一种优化的鸡α干扰素肽链及其重组表达工程菌株
CN112481185B (zh) 一种预防黄颡鱼“裂头病”浸泡疫苗株的构建和应用
CN108220217B (zh) 一种用于递送及表达外源抗原的减毒单核细胞增生李斯特氏菌及其应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21901826

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 21901826

Country of ref document: EP

Kind code of ref document: A1