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Alligator mississrppinsis Cathelicidin-AM antibacterial peptide as well as coded sequence and application thereof

An antibacterial peptide and alligator technology, applied in the field of antibacterial peptides, can solve the problems of complex methods of antibacterial peptides, high cost of antibacterial peptides, and low yield.

Active Publication Date: 2014-03-26
长沙巨龙生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The method of directly extracting antimicrobial peptides from crocodiles is complicated and the yield is very small; the cost of preparing antimicrobial peptides by chemical synthesis is too high, and there is no possibility of practical application

Method used

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  • Alligator mississrppinsis Cathelicidin-AM antibacterial peptide as well as coded sequence and application thereof
  • Alligator mississrppinsis Cathelicidin-AM antibacterial peptide as well as coded sequence and application thereof
  • Alligator mississrppinsis Cathelicidin-AM antibacterial peptide as well as coded sequence and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0075] Embodiment one: the solid-phase chemical synthesis method of Cathelicidin-AM antimicrobial peptide

[0076]The amino acid sequence of Cathelicidin-AM mature peptide was deduced according to the gene encoding American alligator Cathelicidin antimicrobial peptide, and the Cathelicidin-AM antimicrobial peptide was artificially synthesized according to the standard solid-phase peptide synthesis procedure. The synthesized peptide was desalted and purified by reverse phase-high pressure liquid chromatography (RP-HPLC, Column: X Bridge 4.6×250mm), and eluted with acetonitrile / water / trifluoroacetic acid system (Pump A: 0.07% trifluoroacetic in 100% water ; Pump B: 0.05% trifluoroacetic in 100% acetonitrile), and its molecular weight was determined by electrospray mass spectrometry (ESI-MS). The purity of the sample is ≥96%, the theoretical molecular weight is 4546, and the measured molecular weight is 4547.70, which are basically the same. The synthesized Cathelicidin-AM antib...

Embodiment 2

[0077] Example 2: Acquisition of American Alligator Cathelicidin-AM Antimicrobial Peptide cDNA

[0078] The inventors used TaKaRa Smart TM The cDNA library construction kit constructed the American alligator skin tissue cDNA library, sequenced randomly selected clones, and compared the measured sequence with the sequence in the international gene bank (GenBank) by BLAST to obtain the Cathelicidin-AM antibacterial Peptide cDNA.

[0079] 1. Extraction of total RNA from epithelial tissue of American alligator mucosa

[0080] The epithelial tissues of the American alligator mucosa were collected in cryopreservation tubes, and then the American alligators were released. The cryopreservation tubes were placed in liquid nitrogen and transported back to the laboratory, where they were stored in liquid nitrogen tanks for later use.

[0081] Take 0.1 g of mucosal epithelial tissue, grind it into powder at low temperature with liquid nitrogen. Add 2mL RNAiso Reagen and let it stand ...

Embodiment 3

[0123] Example 3: Recombinant expression of American alligator Cathelicidin antimicrobial peptide in Bacillus subtilis

[0124] 1. According to the sequence at both ends of the 17-139 polynucleotide in SQ ID NO: 1, the primers are calculated as follows:

[0125] P1: 5'-GTGGGGTTCAAAGAAAGGCCTG-3';

[0126] P2: 5'-CCCAAGCTT CTAGAACTTGCCAAGGAACTTGGC-3' (introduced Hind III restriction site).

[0127] Using the pET30a-cathAM plasmid as a template to amplify the Cathelicidin-AM gene, the PCR system is as follows:

[0128]

[0129] Reaction program: pre-denaturation at 94°C for 3min; denaturation at 94°C for 30s, annealing at 57°C for 30s, extension at 72°C for 1min, 35 cycles; extension at 72°C for 10min. 1% agarose gel electrophoresis to detect PCR products (see electrophoresis results image 3 ).

[0130] 2. Using the genomic DNA of Bacillus subtilis as a template, primers were set according to the sequence known in Genbank to amplify P 43 Promoter sequence, EcoR Ⅰ restr...

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Abstract

The invention provides an alligator mississrppinsis Cathelicidin-AM antibacterial peptide, a polynucleotide for coding the antibacterial peptide and an application of the antibacterial peptide. The antibacterial peptide has an amino acid sequence as shown in SEQ ID NO: 2. The polynucleotide for coding the antibacterial peptide has a nucleotide sequence containing the coded amino acid sequence as shown in SEQ ID NO: 2 or a complementary sequence of the nucleotide sequence. The antibacterial peptide has a remarkable inhibiting effect on various bacteria and has the minimal inhibitory concentration (MIC) of 62.5mug / mL for Echerichia coli (CMCC44102), Escherichia coli (ATCC25922) and Salmonella typhi (CMCC50115). The antibacterial peptide can be used for preparing feed additives, preservatives, agricultural biocontrol agents, therapeutic drug compositions for controlling microorganism infection and the like.

Description

technical field [0001] The invention relates to an antibacterial peptide of the Cathelicidin antibacterial peptide family, in particular to an American alligator Cathelicidin-AM antibacterial peptide. In addition, the present invention also relates to the nucleotide sequence encoding the antimicrobial peptide and the use of the antimicrobial peptide. Background technique [0002] Due to the indiscriminate use and abuse of antibiotics, many bacteria have produced drug-resistant strains, and the development of new antibiotics is not fast enough, so the contradiction between the development of new antibiotics and the demand for antibacterial is becoming increasingly prominent. In addition, the problem of antibiotic residues in animal products has attracted more and more attention, which directly endangers human health. The unique antibacterial mechanism of antimicrobial peptides provides the possibility to solve this problem, so it is very promising to be developed into a new ...

Claims

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Application Information

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IPC IPC(8): C07K14/46C12N15/75C12N1/21A23K1/16A01N47/44A61K38/17A61P31/00A23L3/3526
CPCA01N47/44A61K38/00C07K14/46Y02A50/30
Inventor 汪猜皮灿辉陈荣
Owner 长沙巨龙生物科技有限公司
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