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Photobacterium damselae hemolysin Hlych protein having immunoprotection effect

A luminescent bacteria, immune protection technology, applied in the biological field, can solve the problems of high mortality, economic loss, rapid onset and other problems

Active Publication Date: 2018-07-27
HEBEI NORMAL UNIVERSITY OF SCIENCE AND TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] Photobacterium damselae is an important member of halophilic pathogenic bacteria. The diseased fish infected with this bacterium are clinically characterized by hemorrhagic sepsis, with rapid onset and high mortality. loss

Method used

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  • Photobacterium damselae hemolysin Hlych protein having immunoprotection effect
  • Photobacterium damselae hemolysin Hlych protein having immunoprotection effect
  • Photobacterium damselae hemolysin Hlych protein having immunoprotection effect

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Bacterial hemolytic activity observation

[0052] Prepare sheep blood and rabbit blood plates respectively, pick a single colony of Photobacterium mermaidus MCCC 1K03226, streak on the sheep blood and rabbit blood plates, incubate at 30°C for 24 hours, and observe the hemolytic activity. The results are as follows: figure 1 and figure 2 shown, where as figure 1 for the blood of sheep, figure 2 For rabbit blood. Depend on figure 1 and figure 2 It can be seen that the bacterium exhibits β-type hemolysis on the sheep blood and rabbit blood plates, showing that the bacterium has very strong hemolytic activity.

Embodiment 2

[0053] Example 2 Recombinant vector pMD18T-hly ch build

[0054] Download the whole genome sequence of Photobacterium damselae subsp.Damselae CIP 102761 (PhddCIP102761, gene accession number: NZ_ADBS00000000) from NCBI, find dly, hlyA pl and hlyA ch Gene sequence, designed primers, the primer sequence is shown in Table 1; wherein the dly gene sequence is SEQ ID NO: 1; hlyA pl The gene sequence is SEQ ID NO: 2; hlyA ch The gene sequence is SEQ ID NO:3. Genomic DNA of bacterial strain MCCC 1K03226 DNA was extracted using a bacterial genome extraction kit, using genomic DNA as a template, using the primers in Table 1, and amplifying by touchdown PCR. After the reaction, electrophoresed on 1% agarose gel. image 3 , wherein primer Dly-f checks, Dly-r checks and amplifies the length of Dly gene part, is used for detecting hemolysin Dly protein; Primer Hly pl -f check, Hly pl -r detection amplification Hly pl Gene portion length for detection of plasmid-encoded hemolysin; pri...

Embodiment 3

[0061] Example 3 Recombinant expression vector pET32a-hly ch Construction and prokaryotic expression

[0062] Use restriction endonuclease BamH I and Sal I to pMD18T-hly ch Carry out double digestion with the expression vector pET32a(+), recover the digested products by gel electrophoresis and mix them at a ratio of 3:1, connect at 22°C for 2 hours, transform the conjugate into E. coli competent DH5α, and spread it on the Incubate overnight at 37°C on LB plates. Pick a single colony and shake it, and use the vector universal primers for PCR identification. Pick positive colonies to expand culture, save the strain and extract the plasmid, the recombinant plasmid is marked as pET32a-hly ch .

[0063] The extracted recombinant plasmid was transformed into the expression strain BL21, spread on the LB solid plate containing Amp, and cultured overnight at 37°C. Pick a single colony on the Amp plate and shake overnight, inoculate 1mL of the bacterial solution into 100mL liquid L...

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Abstract

The invention discloses a photobacterium damselae hemolysin Hlych protein having immunoprotection effect. By constructing a recombinant vector pMD18T-hlych, a recombination expression vector pET32a-hlych and a prokaryotic expression hlych gene, the recombinant Hlych protein is prepared, SDS-PAGE analysis and Western blot are used for verifying the Hlych protein, and an animal protection experimentis used for evaluating the protection force. The Hlych protein of photobacterium damselae has good immunoprotection force, and establishes a base for a gene engineering subunit vaccine of photobacterium damselae.

Description

technical field [0001] The present invention relates to the field of biotechnology, more specifically to mermaid Photobacterium hemolysin Hly ch Protein preparation and identification methods. Background technique [0002] Photobacterium damselae is an important member of halophilic pathogens. The diseased fish infected with this bacterium are clinically characterized by hemorrhagic sepsis, with rapid onset and high mortality. loss. Infection can also occur in humans and other mammals, with a pathogenic bacterium that is common to humans, animals, and fish. The known high pathogenicity of Photobacter mermaidus usually carries a virulence plasmid of about 150kb, encoding two key virulence factors - hemolysin Dly protein and HlyA pl protein, and the chromatin also carries hemolysin Hly ch Genes, these three hemolysins all have varying degrees of influence on the virulence of bacteria. Hemolysin is one of the important virulence factors of Photobacterium mermaidus, which c...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N15/31A61K39/106A61P31/04G01N33/68
CPCA61K39/107A61K2039/552C07K14/28C12N15/70G01N33/68G01N2333/28
Inventor 吴同垒高桂生张志强史秋梅肖丽荣李巧玲
Owner HEBEI NORMAL UNIVERSITY OF SCIENCE AND TECHNOLOGY
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