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Plectasin mature polypeptide dimer fusion protein and preparation method thereof

A peptide dimer, fusion protein technology, applied in the field of genetic engineering

Inactive Publication Date: 2010-10-06
THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0013] At present, there is no report on optimizing the mature pseudosigalcin mature polypeptide gene and artificially synthesizing the fusion protein gene of the pseudosigalcin mature polypeptide dimer, and for the pseudosigalcin mature polypeptide two fused in a specific way Whether the polymeric protein still has biological activity, and whether the fusion protein gene can be introduced into Pichia pastoris for inducible expression and high-density fermentation production have not been reported.

Method used

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  • Plectasin mature polypeptide dimer fusion protein and preparation method thereof
  • Plectasin mature polypeptide dimer fusion protein and preparation method thereof
  • Plectasin mature polypeptide dimer fusion protein and preparation method thereof

Examples

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Effect test

Embodiment 1

[0059] Example 1: Artificial synthesis of pseudo-sigoldin mature polypeptide dimer fusion protein gene

[0060] According to the known full gene sequence (GenBank: AJ964941) of Pseudomycin, according to the codons preferred by Pichia pastoris, without changing its amino acid sequence, the gene encoding the mature polypeptide of Pseudosiglobin was analyzed. Codon optimization. Compared with the unoptimized mature polypeptide gene of the optimized pseudosigladin, 19 nucleotide bases have been changed, involving a total of 19 codons, and the G+C content has changed from the original 45% to 46%. , see the comparison before and after codon optimization of the mature polypeptide gene of pseudosigladin figure 1 , the optimized mature pseudosigantillin gene still encodes the same 40 amino acid residues (see SEQ ID NO: 1), and the molecular weight is about 4.4KDa.

[0061] When artificially synthesizing the fusion protein gene of the mature pseudo-sigantagactin polypeptide dimer, the...

Embodiment 2

[0062] Embodiment 2: Cloning of the fusion protein gene of the mature polypeptide dimer of pseudosigaldin

[0063] The artificially synthesized plpDNA fragment of the mature polypeptide dimer fusion protein gene plpDNA of the above artificially synthesized pEASY-T1 (purchased from Beijing TransGenic Company) was directly inserted into the T site of the plasmid, and according to the method provided by the company, the obtained Bacterial clones containing the plasmid vector plp-T. Through DNA sequencing analysis, it was determined that the pseudosigalcin mature polypeptide dimer fusion protein gene plp contained in it was correct and complete (DNA sequencing was completed by Beijing Biaokai Technology Co., Ltd.).

Embodiment 3

[0064] Embodiment 3: Construction of yeast expression vector plp-9k

[0065] Perform double digestion with restriction endonucleases Xho I and Not I, cut out the pseudo-alphacin mature polypeptide dimer fusion protein gene plp connected to the intermediate plasmid vector plp-T, and run through agarose gel electrophoresis The dimeric fusion protein gene fragment is isolated and recovered. The plasmid pPIC9K was treated with the same restriction endonuclease, and the linearized pPIC9K plasmid DNA was separated and recovered by agarose gel electrophoresis. After the above two DNA fragments are mixed, they are linked together with ligase to obtain the high-efficiency expression vector plp-9k of Pichia pastoris (see figure 2 ). Then, the plasmid expression vector was used to transform Escherichia coli cells DH5α (purchased from GIBCO, USA) so as to replicate and preserve the plasmid.

[0066] Plasmid vector pPIC9K was purchased from Invitrogen, USA. It is a yeast-inducible expr...

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Abstract

The invention relates to a plectasin mature polypeptide dimer fusion protein, genes for coding the protein and an expression vector containing a DNA sequence for coding the protein. The invention also relates to a method for expressing the plectasin mature polypeptide dimer fusion protein in recombinant Pichia pastoris. The method comprises the following steps: using the expression vector containing the DNA sequence for coding the plectasin mature polypeptide dimer fusion protein to convert Pichia pastoris and obtain recombinant Pichia pastoris, and performing fermentation culture to secrete the plectasin mature polypeptide dimer fusion protein. The obtained plectasin mature polypeptide dimer fusion protein has bioactivity and can be widely used in the fields such as medical treatments, foods, feeds and scientific researches.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to a fusion protein of a pseudosigaldin mature polypeptide dimer and a preparation method thereof. Background technique [0002] Antimicrobial peptides (AMPs) refer to a class of small molecule polypeptides composed of 12-100 amino acids, with a net positive charge, amphipathic and antibacterial activity. The research on antimicrobial peptides has a history of more than 50 years. So far, about 880 antimicrobial peptides from different host sources have been identified (Brogden K.A., 2005). Antimicrobial peptides have attracted the attention of many researchers due to their broad-spectrum antibacterial activity (including against Gram-positive bacteria, Gram-negative bacteria, fungi, and even viruses). Especially in recent years, as the abuse of antibiotics has led to the continuous emergence of antibiotic-resistant pathogenic bacteria, it is urgent to develop a new ge...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/37C12N15/31C12N15/81C12N1/19C12P21/02A61K38/16A61P31/00A61P31/04C12R1/84
Inventor 王楠潘春刚李刚强刘德虎
Owner THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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