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Pseudosciaena crocea antitumor peptide piscidin 5 like and preparation method and application thereof

A technology of rlc-p5l and pet-28a, which is applied in the fields of antineoplastic drugs, botanical equipment and methods, biochemical equipment and methods, etc., can solve the problems of tumor or inflammation treatment failure, and achieve low production cost and simple expression system , strong biological activity

Pending Publication Date: 2021-01-26
福建福鼎海鸥水产食品有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In particular, the emergence of drug resistance has raised concerns around the world about the failure of tumor or inflammatory treatments

Method used

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  • Pseudosciaena crocea antitumor peptide piscidin 5 like and preparation method and application thereof
  • Pseudosciaena crocea antitumor peptide piscidin 5 like and preparation method and application thereof
  • Pseudosciaena crocea antitumor peptide piscidin 5 like and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] The construction of embodiment 1 large yellow croaker Lc-P5L prokaryotic recombinant expression vector

[0053] 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.

[0054] Upstream primer F1: 5′-CCG GAATTC GGAGACAACTACGGTACTTTC-3';

[0055] Downstream primer R1: 5′-CCG CTCGAG TTTGCTGCCGTCGTCCT-3'.

[0056] A fragment of the coding region of Lc-P5L was amplified. The PCR reaction conditions are: pre-denaturation at 94°C for 5 minutes; denaturation at 94°C for 45 seconds, and denaturation at 58°C

[0057] Anneal for 45s, extend for 45s at 72°C, repeat 35 cycles; extend for 10min at 72°C.

[0058] The PCR product wa...

Embodiment 2

[0060] 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) large intestine by heat shock method Bacillus and induce expression with IPTG.

[0061] 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

[0062] Example 3 Purification of expression products after IPTG induction in E.coli BL21(DE3) transformed with pET-28a-Lc-P5L recombinant plasmid

[0063] The rLc-P5L recombinant protein was purified by affinity chromatography. After a large amount of positive recombinant E.coliBL21 (DE3) was induced and expressed, it was centrifuged at 12000rpm at 4°C for 10min to remove the supernatant, and an appropriate amount of sonication solution (50mM Tris-HCl, 0.5M NaCl, 1 mM EDTA, pH 8.0), after crushing by high-pressure ultrasound, centrifuge at 12000 rpm at 4°C for 30 min to collect the precipitate. Add an appropriate amount of inclusion body washing solution (50mM Tris-HCl, 0.5M NaCl, 2M urea, 1% Triton X-100, pH7.8) to resuspend the bacteria, wash the inclusion body 3 times, add inclusion body lysis solution (100mM Triton -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 membran...

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Abstract

The invention provides pseudosciaena crocea antitumor peptide piscidin 5 like and a preparation method and application thereof, and relates to pseudosciaena crocea antibacterial peptide. On the basisof obtaining Lc-P5L through separation, a recombinant expression vector is successfully constructed according to Lc-P5L gene sequence characteristics, expression and purification are performed in an escherichia coli system to obtain recombinant rLc-P5L protein, a pET-28 a recombinant expression vector of Lc-P5L is constructed and converted into a host cell E.coli BL21(DE3), inducible expression isperformed in the host cell to obtain an expression product, and the expression product is separated and purified to obtain recombinant protein rLc-P5L. The pseudosciaena crocea antitumor peptide piscidin 5 like has the advantages of being high in expression yield, high in biological activity, simple in expression system, low in production cost, free of toxic substances and the like, is suitable for large-scale fermentation production and can provide theoretical data and support for artificial modification into substances with higher antitumor activity.

Description

technical field [0001] The invention relates to large yellow croaker antibacterial peptides, in particular to large yellow croaker antitumor peptide piscidin 5 like and its preparation method and application. Background technique [0002] With the development of research on antimicrobial peptides, scholars have found that antimicrobial peptides are the most promising candidates to completely or partially replace antibiotics. The unique mode of action of antimicrobial peptides targeting cell membranes hardly causes resistance. Therefore, in the past several years, a great deal of work and effort has been devoted to the study of antimicrobial peptides [1-3] , and more and more antimicrobial peptides have been isolated from bacteria, animals, and plants. Among them, marine organisms with high biodiversity are an important source of rich antimicrobial peptides. [0003] Study finds some antimicrobial peptides have strong antitumor activity [4] , and has great potential to be...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C07K14/46C12N15/70A61K38/17A61P35/00
CPCC07K14/461C12N15/70A61P35/00A61K38/00
Inventor 郑利兵苏永全迟长凤潘滢陈佳郑炜豪
Owner 福建福鼎海鸥水产食品有限公司
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