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Antibacterial peptide as well as applications thereof to preparation of anti-infective drugs, antitumor drugs, immunopotentiators and feed additives

An anti-tumor drug and antibacterial peptide technology, applied in anti-tumor drugs, animal feed, antibacterial drugs, etc., can solve the problems of low natural yield of antibacterial peptides, expensive chemical synthesis, complicated natural extraction steps, etc., and achieve significant growth inhibition. effect, promote the secretion of IFNγ, and improve the effect of immune function

Active Publication Date: 2014-10-15
合肥迈可罗生物工程有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] However, the natural yield of antimicrobial peptides is low, the natural extraction steps are complex, and the yield is low; while chemical synthesis is quite expensive; therefore, the use of genetic engineering technology to produce antimicrobial peptides is the only way for the industrialization of the development and utilization of antimicrobial peptides, which is of great significance
However, antimicrobial peptides have small molecular weight, poor mRNA stability during recombinant expression, and antimicrobial peptides themselves have inhibitory effects on some engineering bacteria. Therefore, how to construct high-expression engineering bacteria is also a key issue.

Method used

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  • Antibacterial peptide as well as applications thereof to preparation of anti-infective drugs, antitumor drugs, immunopotentiators and feed additives
  • Antibacterial peptide as well as applications thereof to preparation of anti-infective drugs, antitumor drugs, immunopotentiators and feed additives
  • Antibacterial peptide as well as applications thereof to preparation of anti-infective drugs, antitumor drugs, immunopotentiators and feed additives

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1. Gene Cloning of Plutella xylostella antimicrobial peptide pxCA1

[0056] 1.1 Infection immunity of diamondback moth larvae

[0057] The larvae of Plutella xylostella were isolated and artificially raised to 4-7 days old. Escherichia coli JM109 cultured to the logarithmic phase was dipped in a needle to perform stress puncture on the abdomen of Plutella xylostella. After 24 hours of continuous feeding, the total RNA of the larvae was extracted.

[0058] 1.2 Extraction of total RNA

[0059] ① Grind the tissue specimen in a small grinder pre-cooled at -80°C, add Trizol reagent to the grinder, add 1ml TRIzol reagent per 50-100 mg of tissue (the volume of the tissue block should not exceed 10% of the volume of TRIzol), and fully grind the tissue into a homogenate; use a pipette gun to draw the homogenate into a 1.5ml EP tube, and place it on ice for 5 minutes.

[0060] ②After the nucleic acid and protein are fully dissociated, add 0.2ml chloroform to each 1ml TR...

Embodiment 2

[0083] Example 2 Expression and Extraction of Diamondback Moth Antimicrobial Peptide pxCA1 in Prokaryotic

[0084] 2.1 Construction and induced expression of genetic engineering fusion antimicrobial peptide prokaryotic expression engineering bacteria

[0085]According to the analysis of the online signal peptide prediction server and referring to the literature on the post-translational modification of cecropin family antimicrobial peptides, the amino acids in the signal peptide and N-terminal immature peptides in the full-length peptide (SEQ ID No.2) expressed by the pxCA1 gene were removed. Codon optimization was performed on the coding sequence of the mature peptide (25-65 amino acids) (SEQ ID No.3), and the coding gene suitable for expression in Escherichia coli was synthesized as shown in SEQ ID No.4.

[0086] The coding gene was constructed into the pET32a plasmid, the upstream and downstream cloning sites were KpnI / XhoI, and an enterokinase cleavage site (DDDDK▼) was in...

Embodiment 3

[0102] The mensuration (MTT method) of embodiment 3.pxCA1 antimicrobial peptide minimum antibacterial concentration

[0103] Escherichia coli (K12D31), Staphylococcus aureus (CMCC26003), Bacillus aeruginosa (CMCC10104), Bacillus subtilis (CMCC63501) and methicillin-resistant Staphylococcus aureus (MRSA5636) were cultured in LB liquid until OD 630 =0.6, and were diluted to 10 with LB 5 -2×10 5 CFU / ml.

[0104] Dilute the purified and concentrated pxCA1 antimicrobial peptide solution with LB. Take 10 μl of each dilution of the antimicrobial peptide solution and mix it with 90 μl of the diluted bacterial suspension; after culturing at 37°C for 6 hours, take Add 20 μl to a 96-well plate; add 10 μl of MTT to each well, incubate at 37°C for 1 hour, add 90 μl DMSO to each well to dissolve the insoluble matter formed in the well, and measure the absorbance of each well at 570 nm on a microplate reader. After that, 20 μl of the mixed culture solution was repeatedly sampled every 2 h...

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Abstract

The invention discloses a plutella xylostella antibacterial peptide as well as applications thereof to preparation of anti-infective drugs, antitumor drugs, immunopotentiators and feed additives. An amino acid sequence of the plutella xylostella antibacterial peptide is represented by SEQ ID No.3. The plutella xylostella antibacterial peptide has inhibition and killing effects on various bacteria, so that the peptide can be used for preparing the anti-infective drugs; the plutella xylostella antibacterial peptide has the extremely significant growth inhibition effect on breast cancer cells, hepatoma cells, colonic cancer cells and lung cancer cells and has the obvious antitumor activity in vitro, so that the peptide can be used for preparing the antitumor drugs; the plutella xylostella antibacterial peptide can promote CD4T cells to proliferate and secrete IFNgamma, can inhibit synthesis of IL4 and regulate balance of Th1 / Th2 and has the immunological enhancement activity, so that the peptide can be used for preparing the immunopotentiators; the plutella xylostella antibacterial peptide can significantly increase the survival rate of piglets, reduce the diarrhea rate of the piglets and growing and fattening pigs, increase the daily gain of the piglets and the growing and fattening pigs and reduce the feed conversion ratio of the piglets and the growing and fattening pigs when being used as the feed additives.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an antibacterial peptide and its application in the preparation of anti-infection drugs, anti-tumor drugs, immune enhancers and feed additives. Background technique [0002] Antibacterial peptide (antibacterial peptide) is a general term for a class of defensive polypeptides produced by specific coding genes of host cells under certain induction conditions to resist the pathogenic effect of exogenous pathogens. It has broad-spectrum antibacterial and antiviral effects. Is part of the natural immune system. [0003] Antimicrobial peptides have the characteristics of small molecular weight, high stability, and non-immunogenicity. Some antimicrobial peptides can still maintain their activity when heated at 100°C for 10-15 minutes; at the same time, antimicrobial peptides have strong resistance to higher ionic strength and higher or lower pH values, and some antimicrobial pe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/435C12N15/12C12N15/63C12N1/21C12N1/19A61K38/17A61P31/04A61P35/00A61P37/04A23K1/17A23K1/18A23K50/30A23K50/60
CPCA23K20/195A23K50/30A61K38/00C07K14/43563
Inventor 石应辉徐冬玲桂向东李晓祥
Owner 合肥迈可罗生物工程有限公司
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