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Antimicrobial peptide, coding sequence and preparation method

An antibacterial peptide and sequence technology, applied in the field of genetic engineering, can solve the problems of unstable plasmid transformants, limited expression, easy loss of target genes, etc. Effect

Inactive Publication Date: 2018-01-16
广州郡雅科技应用有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the expression of the target gene AM in Bacillus subtilis is limited, the plasmid transformant is unstable, and the target gene is easily lost, etc.

Method used

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  • Antimicrobial peptide, coding sequence and preparation method
  • Antimicrobial peptide, coding sequence and preparation method
  • Antimicrobial peptide, coding sequence and preparation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Example 1: Antibacterial peptide Cathelicidin- AM1 gene cloning

[0018] According to the needs of gene modification and cloning of antimicrobial peptide Cecropin DC1, Primer premier 5.0 software was used to design

[0019] Design and synthesis of primers:

[0020] P1: 5'-ATGGTGGGGTTCAAAGGC-3';

[0021] P2: 5'-TGTGTTGTGCTGACACACTA-3'.

[0022] Reaction conditions: pre-denaturation at 95°C for 5 min; 30 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 1 min; the reaction was terminated after extension at 72°C for 5 min.

[0023] The amplified products were analyzed by electrophoresis on 1.5% agarose gel at a voltage of 4 v / cm, electrophoresis for 1 h, and observed under a gel imager. Gel extraction kit (QIAquick Gel Extraction Kit, QIAGEN Company) was used to recover and purify the target gene. The randomly selected plasmids were sent to a sequencing company for sequencing.

Embodiment 2

[0024] Example 2: Obtaining recombinant virus

[0025] The constructed recombinant transfer vector was co-transfected with Sf21 insect eukaryotic cells with the polynucleated polyhedrosis virus AcNPV-DNA of Spodoptera californica, and the recombinant virus expressing the antimicrobial peptide Cathelicidin-AM1 was obtained as follows:

[0026] Add 5μg recombinant transfer vector DNA, 15μg AcNPV-DNA and 1.0ml co-transfection buffer to a 1.5ml sterilized Eppendorf tube, incubate the mixture at room temperature for 30min and co-transfect Sf21 cells in logarithmic phase; then After co-transfection, Sf21 cells were washed twice with serum-free medium Sf-900ⅡSFM medium, then cultured with complete medium containing 10% FBS at 27°C for 4-6 days, and the culture supernatant was collected as the virus stock solution The recombinant virus is screened, and the recombinant virus is subjected to plaque screening to obtain a virus solution, and the virus is named as the recombinant virus con...

Embodiment 3

[0027] Example 3: Extraction of recombinant baculovirus

[0028] 1) Take a 1.5 ml centrifuge tube and add 20 μl of Proteinase K solution.

[0029] 2) Add 200 μl of serum to the centrifuge tube, and then add 200 μl of Tiangen Buffer GB (the function of this buffer is to lyse cells and separate nucleic acid and polypeptide), and vortex for 15 seconds.

[0030] 3) Incubate at 55°C for 15 minutes, centrifuge briefly, and collect the solution on the tube wall to the bottom of the tube.

[0031] 4) Add 250 μl absolute ethanol, vortex for 15 seconds, place at room temperature for 5 minutes, centrifuge briefly, and collect the solution on the tube wall to the bottom of the tube.

[0032] 5) Put one Spin Columns CG* into Collection Tubes (2 ml), transfer the solution obtained in 4) to a spin column, centrifuge at 10 000 rpm for 30 seconds, and discard the waste in the collection tube.

[0033] 6) Add 500 μl of Tiangen Buffer GD (peptide impurity removal buffer) to the adsorption colu...

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Abstract

The invention provides an antimicrobial peptide cathelicidin-AM1, a gene coding the antimicrobial peptide and a preparation method of the antimicrobial peptide. The antimicrobial peptide comprises anamino acid sequence as shown in SEQ ID NO. 1; the gene coding the antimicrobial peptide comprises a nucleotide sequence coding the SEQ ID NO. 1; the gene of the antimicrobial cathelicidin-AM1 is designed and synthesized by utilizing the gene engineering technique, a recombinant virus comprising the antimicrobial peptide cathelicidin-AM1 gene is built, and Sf21 cells are used as a host to express and produce the recombinant antimicrobial cathelicidin-AM1 with the biological activity.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, in particular to an antimicrobial peptide, a nucleotide sequence encoding the antimicrobial peptide and a method for preparing the antimicrobial peptide. Background technique [0002] Antimicrobial peptides are a class of biologically active small molecule polypeptides, which have the advantages of strong bactericidal power, broad antibacterial spectrum, good stability and no drug resistance. More importantly, antimicrobial peptides have almost no killing effect on eukaryotic cells, and only act on prokaryotic cells. [0003] After reviewing the prior art, it was found that Chinese patent 201310553924.X disclosed an antibacterial peptide Cathelicidin-AM and its preparation method and application. The invention patent constructs a cDNA library, transforms the shuttle plasmid, and transfers it into Bacillus subtilis for expression. However, the expression of the target gene AM in Baci...

Claims

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Application Information

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IPC IPC(8): C07K14/46C12N15/12C12N15/866
Inventor 不公告发明人
Owner 广州郡雅科技应用有限公司
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