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Preparation method of antalgic active polypeptide

A technology of active peptides and plant antimicrobial peptides, applied in the field of genetic engineering, can solve the problems of low content and limited quantity of PaAMP, and achieve the effect of abundant resources, meeting drug research and development and clinical needs, and efficient soluble expression

Inactive Publication Date: 2015-09-16
JIANGSU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, whether the antimicrobial peptide PaAMP1 has analgesic activity has not been reported at home and abroad.
Using techniques such as gel filtration chromatography to isolate and purify PaAMP from Pokeweed seeds, but the content is low, and the amount of PaAMP obtained is limited, which is not conducive to in-depth functional research on PaAMP.

Method used

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  • Preparation method of antalgic active polypeptide
  • Preparation method of antalgic active polypeptide
  • Preparation method of antalgic active polypeptide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1: Synthesis and identification of pokeweed antimicrobial peptide PaAMP1 gene

[0025] The codons of the PaAMP1 gene (GenBank: AF048745) were optimized according to codon usage frequency in E. coli (see figure 1 ), the codon-optimized PaAMP1 gene sequence is SEQ ID NO.1, and the encoded protein sequence is SEQ ID NO.2.

[0026] SEQ ID NO.1:

[0027] gcaggctgta ttaagaacgg tggccgctgc aatgcgagcg caggtccgcc gtattgttgc

[0028] agcagctact gttttcagat tgcgggtcaa agctatggtg tgtgtaaaaa ccgttaa

[0029] SEQ ID NO.2:

[0030] Ala Gly Cys Ile Lys Asn Gly Gly Arg Cys Asn Ala Ser Ala Gly Pro

[0031] Pro Tyr Cys Cys Ser Ser Ser Tyr Cys Phe Gln Ile Ala Gly Gln Ser Tyr

[0032] Gly Val Cys Lys Asn Arg

[0033] The PaAMP1 gene (synthesized by Shanghai Jierui Biotechnology Co., Ltd.) was synthesized by chemical method, and the Bam HI / Hind After the double enzyme digestion of III, it was connected into the prokaryotic expression vector pET-32a (purchased from Novagen...

Embodiment 2

[0034] Example 2: Expression and separation and purification of recombinant PaAMP1

[0035] The verified correct recombinant plasmid pET-32a-PaAMP1 was introduced into the competent cells of Escherichia coli Origami (DE3) strain (purchased from Novagen) by heat shock method to obtain the genetically engineered bacterium Origami-PaAMP1.

[0036] Inoculate the Origami-PaAMP1 strain in LB medium (containing ampicillin 100 μg / mL), culture with shaking at 37°C until OD 600The value is about 0.8. Cool the cultured Origami-PaAMP1 cells to 28°C, add IPTG with a final concentration of 1mmol / L, and culture with shaking at 28°C for 12 hours. Cells were collected by centrifugation, frozen and thawed five times at 37°C / -20°C, and ultrasonically disrupted at 200w. After high-speed centrifugation, soluble recombinant PaAMP1 was isolated from the supernatant using nickel ion resin. The purified recombinant PaAMP1 was dialyzed to remove impurities such as imidazole, and then the recombinan...

Embodiment 3

[0038] Example 3: Analysis of the analgesic activity of recombinant PaAMP1

[0039] The experimental group was injected with different doses of the recombinant antimicrobial peptide PaAMP1 solution obtained in Example 2 (2.0mg / kg, 5.0mg / kg), the negative control group was injected with normal saline (0.9% NaCl), and the positive control group was injected with recombinant conus Toxin ω-MVIIA. Each group was injected with 20 male mice, each with a body weight of 20 ± 0.2 g. 45 minutes after administration, each mouse was injected with 0.2 mL of 0.6% glacial acetic acid to stimulate writhing behavior, and the writhing movements of each mouse within 20 minutes were counted, and then the experimental data were statistically analyzed.

[0040] The results showed that when the dose of PaAMP1 was 2.0 mg / kg, the average number of writhing times in mice was 20.95 times, 21.10 times in the positive control group, and 37.20 times in the negative control group; The frequency was 1.60 ...

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Abstract

The invention discloses a preparation method of an antalgic active polypeptide and belongs to the genetic engineering field. The preparation method comprises the following steps: optimizing the gene sequence of the pokeweed antibacterial peptide PaAMP1 according to the frequency of use of the escherichia coli codon, linking the gene sequence to a prokaryotic expression vector pET-32a by virtue of chemical synthesis; next, importing the recombinant plasmid to an escherichia coli Origami (DE3) strain and realizing high-efficiency solution expression of recombinant PaAMP1 after IPTG induction; separating and purifying the recombinant PaAMP1 by use of nickel ion affinity chromatography; according to the preparation method, a mouse acetic acid body torsion test is performed to prove that the recombinant PaAMP1 has remarkable analgesic activity and has potential application value in the analgesia field for the first time; as the PaAMP1 is from the plant pokeweed which is easy to plant at a large scale and is far abundant than maritime cone shell resources, natural antalgic polypeptides are expected to be industrially extracted from the large-scale planted pokeweeds to meet the requirements for medicine research and development and clinic use.

Description

technical field [0001] The invention discloses a preparation method of a polypeptide with analgesic activity, belonging to the field of genetic engineering. Background technique [0002] During long-term evolution, plants have developed different defense systems against plant pathogenic microorganisms, usually producing small molecular substances to inhibit the growth of microorganisms. [0003] In recent years, it has been discovered that thionin, defensin, chitinase, and antimicrobial peptides play an important role in plant defense against pathogenic microorganisms. The antimicrobial peptide PaAMP was obtained from Pokeweed (Pokeweed) by Liu et al. Phytolacca americana ) antimicrobial peptides (Liu Y, Luo J, Xu C, Ren F, Peng C, Wu G, and Zhao J. Purification, characterization, and molecular cloning of the gene of a seed-specific antimicrobial protein from pokeweed. Plant Physiology. 2000, 122: 1015-1024), and its expression is seed-specific. During germination, plant ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N15/29C07K14/415A61P29/00
Inventor 朱姗颖何华纲楚鹰魏斌李云亮马海乐
Owner JIANGSU UNIV
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