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An Extracellular Protein ef-tu from Photobacterium mermaidus

A photobacillus, extracellular protein technology, applied in the fields of fermentation, bacterial peptides, antibacterial drugs, etc., can solve the problems of unsatisfactory immune effect, low immune efficiency of inactivated vaccines, and safety issues that cannot be ignored, and achieve good immune protection. Effect

Active Publication Date: 2021-08-06
HEBEI NORMAL UNIVERSITY OF SCIENCE AND TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Vaccines are an important means to control the prevalence of infectious diseases. At present, the research on vaccines against Photobacterium mermaidi infection is still in the preliminary stage. In addition, there are insurmountable problems in the development of vaccines for this bacteria. On the one hand, the immune efficiency of inactivated vaccines is low, and the immune effect is not satisfactory; On the one hand, although genetically engineered attenuated vaccines have excellent immune effects, safety issues cannot be ignored

Method used

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  • An Extracellular Protein ef-tu from Photobacterium mermaidus
  • An Extracellular Protein ef-tu from Photobacterium mermaidus
  • An Extracellular Protein ef-tu from Photobacterium mermaidus

Examples

Experimental program
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Effect test

Embodiment 1

[0040] Embodiment 1 Construction of pMD18-T-EF-Tu recombinant vector

[0041] Download the whole genome of Photobacterium damselae ATCC3359 (ACCESSION NUMBER: NZ_CP018297), and design the primers as follows according to the gene sequence of EF-Tu:

[0042] EF-Tu-F: 5'-GC GGATCC ATGTCTAAAGAAAAAATTTGAACG-3', BamH I; SEQ ID NO: 1;

[0043] EF-Tu-R: 5'-GC GTC GAC TTAAGCGATGATTGTAGCAAC-3', SalI, SEQ ID NO: 2;

[0044] Extract Genomic DNA of Photobacterium damselae ATCC3359 as a template for PCR amplification: Prepare 50 μL reaction system: Taq DNA polymerase 1.0 μL, PCR Buffer 5 μL, DNA template 1.0 μL, dNTPs 4 μL, primers EF-Tu-F and EF-Tu-F 1.0 μL each, ddH2O 37 μL; amplification program: 94°C pre-denaturation for 5 min; 94°C denaturation for 40 s; 52°C annealing for 30 s; 72°C extension for 1 min; 72°C total extension for 5 min. The result is as figure 1 As shown, the size was as expected, and the PCR product was recovered from the gel. The obtained product was connected w...

Embodiment 2

[0045] Construction of embodiment 2 plasmid pET32a-EF-Tu

[0046] The expression plasmids pET32a and pMD18-T-EF-Tu were extracted, and the expression plasmids pET32a and pMD18-T-EF-Tu were digested with BamHI and SalI respectively, and the target band was recovered from the gel. Mix with pMD18-T-EF-Tu in a ratio of 3:1, connect overnight at 25°C, add the ligation product to DH5α E. coli competent cells, keep ice-bathed for 30 minutes, then transfer to 42°C and incubate for 90 seconds, then ice-bath again for 5 minutes , add 600 μL LB liquid medium and incubate at 37°C for 1 hour, apply the obtained bacteria on an Amp plate (50 μg / mL), and incubate overnight at 37°C; pick a single clone colony for shaking culture, use the carrier universal primer: T7 as the upstream Primers, Stag is the downstream primer for PCR identification, the amplification program is: 94°C pre-denaturation for 5min; 94°C denaturation for 45s; 54°C annealing for 30s; 72°C extension for 1min; 72°C total ext...

Embodiment 3

[0047] Example 3 Induced Expression of Extracellular Protein EF-Tu from Photobacterium mermaidus

[0048] Transform the recombinant expression plasmid pET32a-EF-Tu into the expression strain BL21, pick a single clone colony liquid LB (Amp100μg / mL, tryptone 1%, sodium chloride 1% and yeast extract 0.5%) culture overnight, suck 1mL the next day Inoculate 100mL liquid LB medium containing Amp with bacterial solution, shake the bacteria at 200rpm until OD 600 When the value reached 0.6, IPTG (concentration: 1 mM) was added to induce expression for 4 hours, and the cells were collected by centrifugation.

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Abstract

The invention discloses an application of an extracellular protein EF-Tu of Photobacterium mermaidi with immune protection function in the preparation of a subunit vaccine of Photobacterium mermaidi, wherein the preparation method of the extracellular protein EF-Tu of Photobacterium mermaidi comprises pMD18-T Construction of ‑EF‑Tu recombinant vector, construction of plasmid pET32a‑EF‑Tu, inducible expression of extracellular protein EF‑Tu of Photobacterium mermaida and verification of extracellular protein EF‑Tu of Photobacterium mermaidi. The present invention provides an extracellular protein EF-Tu of Photobacterium mermaidi with immune protection, which is expressed and purified by a prokaryotic expression system, and the immune protection of the extracellular protein EF-Tu of Photobacterium mermaidi is evaluated through animal experiments. It lays the foundation for the further development of photobacteria mermaid subunit vaccine and nucleic acid vaccine selection.

Description

technical field [0001] The invention relates to the field of biotechnology, and more specifically relates to an extracellular protein EF-Tu of Photobacterium mermaidus. Background technique [0002] Photobacterium damselae is an important member of halophilic pathogens. The diseased fish is clinically characterized by hemorrhagic sepsis, with rapid onset and high mortality, which can cause significant economic losses to the marine economic fish farming industry. At the same time, Photobacterium mermaid can infect humans and mammals, which has certain public health significance. Previous studies have found that Photobacterium mermaid can secrete a large amount of extracellular products to the outside of the bacteria, and has a good immune protection effect, and the transcription initiation factor Tu (elongation factor Tu, EF-Tu) is an important component of extracellular products, so the mermaid luminesces Whether the Bacillus extracellular protein EF-Tu has good immune prot...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K39/02C07K14/195C12N15/31C12N15/70A61P31/04
CPCA61K39/0208A61P31/04C07K14/195C12N15/70
Inventor 吴同垒史秋梅张志强于秀剑杜万年高桂生
Owner HEBEI NORMAL UNIVERSITY OF SCIENCE AND TECHNOLOGY
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