Larimichthys crocea antibacterial peptide piscidin 5 like, preparation method and applications thereof

A technology of rlc-p5l and pet-28a, which is applied in botany equipment and methods, biochemical equipment and methods, applications, etc., can solve the problem of no obvious antibacterial activity and achieve strong biological activity, high expression yield, and production low cost effect

Inactive Publication Date: 2019-02-01
宁德市富发水产有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] A novel piscidins-like gene (Lc-pis) in large yellow croaker (Larimichthyscrocea) is a typical gill-expressed polypeptide, which can induce the up-regulation of Lc-pis expression during the stage of stimulating Cryptocaryon larvae infection and trophozoite shedding stage, and synthesize the peptide Lc- pis has a broad-spectrum antibacterial activity, and it has strong resistance to stimulating Cryptocaryon trophozoites at a concentration of 6 μM. The results of treating trophozoites with different concentrations of Lc-pis show concentration and time dependence, suggesting that Lc-pis is not only a Antimicrobial peptide is also an insect-resistant peptide; similar to Lc-pis is the piscidins-like gene (So-pis) isolated from Sciaenopsocellatus, chemically synthesized So-pis has no obvious antibacterial activity, However, it has inhibitory activity against stimulatory Cryptocaryon, 12μM So-pis can kill more than 20% of trophozoites within 1 hour, and it has obvious time dependence; 6μM So-pis can cause cell membrane rupture to kill a part of stimulatory cryptonuclei Worm larva [3]

Method used

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  • Larimichthys crocea antibacterial peptide piscidin 5 like, preparation method and applications thereof
  • Larimichthys crocea antibacterial peptide piscidin 5 like, preparation method and applications thereof
  • Larimichthys crocea antibacterial peptide piscidin 5 like, preparation method and applications thereof

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Experimental program
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Effect test

Embodiment 1

[0046] The construction of embodiment 1 large yellow croaker Lc-P5L prokaryotic recombinant expression vector

[0047] According to the multiple cloning site of the pET-28a vector, specific primers F1 / R1 with restriction endonuclease sites were designed to amplify the sequence encoding the signal peptide in the ORF of the piscidin 5 like gene of large yellow croaker. An EcoR I restriction site was added to the 5' end of the forward primer F1; an Xho I restriction site was added to the 5' end of the downstream primer R1.

[0048] Upstream primer F1: 5′-CCG GAATTC GGAGACAACTACGGTACTTTC-3';

[0049] Downstream primer R1: 5′-CCG CTCGAG TTTGCTGCCGTCGTCCT-3'.

[0050] A fragment of the coding region of Lc-P5L was amplified. The PCR reaction conditions were: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 45 s, annealing at 58°C for 45 s, extension at 72°C for 45 s, repeating 35 cycles; extension at 72°C for 10 min.

[0051] The PCR product was recovered using an ...

Embodiment 2

[0053] Example 2 Induced expression of pET-28a-Lc-P5L recombinant plasmid in E.coli BL21(DE3): The correctly sequenced plasmid pET-28a-Lc-P5L was transformed into E.coli BL21(DE3) Escherichia coli by heat shock method and induced expression with IPTG.

[0054] The results show that, compared with before induction, E.coliBL21 (DE3) transformed with pET-28a-Lc-P5L recombinant plasmid has obvious induced expression of recombinant protein, and the protein band is around 10KDa (see figure 2 ).

Embodiment 3

[0055] Example 3 Purification of expression product after IPTG induction in E.coli BL21 (DE3) transformed with pET-28a-Lc-P5L recombinant plasmid

[0056] The rLc-P5L recombinant protein was purified by affinity chromatography, and after a large amount of positive recombinant E.coliBL21 (DE3) was induced and expressed, it was centrifuged at 12000rpm / min at 4°C for 10min to remove the supernatant, and an appropriate amount of sonication solution (50mM Tris-HCl, 0.5M NaCl, 1mM EDTA, pH8.0), after crushing by high-pressure ultrasound, centrifuge at 12000rpm / min at 4°C for 30min to collect the precipitate. Add an appropriate amount of inclusion body washing solution (50mMTris-HCl, 0.5M NaCl, 2M urea, 1% Triton X-100, pH 7.8) to resuspend the bacteria, wash the inclusion body for 3 times, add inclusion body lysate (100mMTris- HCl, 500mM Nacl, 8M urea, 20mM imidazole, pH 7.4; 0.3mg / ml and 2% Triton-X 100 (added when used)), the supernatant was filtered through a 0.22μm filter membra...

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Abstract

The invention provides a Larimichthys crocea antibacterial peptide piscidin 5 like, a preparation method and applications thereof, relates to an antibacterial peptide, and discloses a Larimichthys crocea antibacterial peptide piscidin 5 like gene coding sequence, a Larimichthys crocea antibacterial peptide piscidin 5 like amino acid sequence, a Larimichthys crocea antibacterial peptide piscidin 5like preparation method, and applications of the Larimichthys crocea antibacterial peptide piscidin 5 like. The preparation method comprises: constructing a Lc-p5L recombinant expression vector; transforming the recombinant expression vector into host cells, and carrying out induced expression on the host cells to obtain an expression product; and separating and purifying the obtained expression product to obtain a recombinant protein rLc-p5L, wherein the recombinant protein rLc-p5L has inhibition and killing activity on the larvae and the trophozoite of Cryptocaryon irritans, can be used in the preparation of insect-resistant drugs, and can further be used as feed additives for preventing and controlling diseases in aquaculture.

Description

technical field [0001] The invention relates to large yellow croaker antimicrobial peptides, in particular to large yellow croaker antimicrobial peptide piscidin 5 like and its preparation method and application. Background technique [0002] Fish self-immune factors have always been a research hotspot. As an important part of the non-specific immune system, AMPs have attracted more and more attention and attention from scholars in recent years. Compared with traditional antibiotics, bacteria hardly develop resistance to AMPs, therefore, AMPs are expected to become a substitute for antibiotics [1] . AMPs have been isolated from bacteria, animals, and plants. Studies have shown that they play an important role in lower vertebrates and invertebrates, because these animals mainly or entirely rely on non-specific immune systems to resist the invasion and infection of pathogenic organisms. . It has been reported that some AMPs in fish have strong activity on stimulating Crypto...

Claims

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Application Information

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IPC IPC(8): C12N15/12C07K14/46C12N15/70A61K38/17A61P33/02A23K20/147A23K50/80
CPCA61K38/00A23K20/147A23K50/80A61P33/02C07K14/461C12N15/70
Inventor 郑利兵毛勇王军郑炜强陈佳潘滢
Owner 宁德市富发水产有限公司
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