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MAPWA fusion antibacterial peptide, preparation method and application thereof

A technology of antibacterial activity and recombinant bacteria, applied in the direction of biochemical equipment and methods, applications, hybrid peptides, etc., can solve problems of immeasurable practical significance and economic value

Inactive Publication Date: 2011-09-28
成都市金之源生物技术有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Therefore, referring to the successful methods, transfecting the antimicrobial peptide gene into specific cells of livestock and poultry to express it, and producing new transgenic animal species resistant to pathogen infection will become a new way to develop animal husbandry production, and its practical significance and economic value are immeasurable

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  • MAPWA fusion antibacterial peptide, preparation method and application thereof
  • MAPWA fusion antibacterial peptide, preparation method and application thereof
  • MAPWA fusion antibacterial peptide, preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0095] Example 1. Construction of novel fusion antimicrobial peptide gene MAPWA and construction of prokaryotic expression plasmid

[0096] The cDNA of Magainin (MA) and the cDNA of Phytoacetin (PWA) were transformed by Gene Splicing by Overlap Extension (GSOE) to construct the MAPWA fusion gene, and the pG-MAPWA prokaryotic expression vector was constructed. PCR and BamHI / EcoRI double enzyme digestion identification, sequencing results confirmed that the MA / PWA fusion gene has been successfully cloned into the prokaryotic expression vector pGEX-4T-1. Specific steps are as follows:

[0097] 1. Design and synthesis of overlapping primers

[0098]According to the cDNA base sequences of the mature peptides encoding Magainin and PWA respectively, five primers were designed and artificially synthesized. PF1 contains BamHI and XhoI restriction sites; the 3' end of PF2 contains a sequence complementary to the 3' end of PF1; The 3' end of PF3 contains a sequence complementary to the...

Embodiment 2

[0131] Example 2, Prokaryotic expression and expression activity research of MAPWA fusion gene

[0132] The MAPWA fusion gene expression plasmid was transformed into Escherichia coli BL21(DE3), and the expression of fusion protein was induced by IPTG. The expressed protein with GST tag was analyzed by SDS-PAGE, and its molecular weight was in line with the expected 33.9KDa. The expression product of the fusion protein exists in a soluble form in the supernatant of broken cells, and a large amount of expressed protein is purified by affinity chromatography; the fusion protein significantly inhibits Escherichia coli, Staphylococcus aureus, and pneumococcus in in vitro antibacterial experiments and growth of Pseudomonas aeruginosa. Specific steps are as follows:

[0133] 1. Induced expression of fusion protein and SDS-PAGE electrophoresis analysis

[0134] 1. Experimental steps: Pick a single colony containing pG-MAPWA and pGEX-4T-1 transformants and inoculate it in 2ml of LB l...

Embodiment 3

[0158] Example 3. Construction of Yeast Expression Vector Fused with Antimicrobial Peptide MAPWA and Study on its Expression Activity

[0159] The fusion antimicrobial peptide MAPWA gene was cloned into the baker's yeast secretion expression vector pYCa (pYES2 / CT was inserted into the a-factor secretion signal peptide), transformed into yCY3 baker's yeast, and high-efficiency expression strains were screened out; after the recombinant strain was induced to express the fusion antimicrobial peptide, The expression supernatant was analyzed for antibacterial activity and purified protein electrophoresis, and it was found that the fusion antimicrobial peptide existed in the expression supernatant, with a molecular weight of approximately 7.9kDa; the secretion and expression of the antimicrobial peptide was realized; the produced fusion antimicrobial peptide was effective against E. Pseudomonas, Staphylococcus aureus, and Streptococcus pneumoniae all had significant antibacterial act...

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Abstract

The invention discloses a MAPWA fusion antibacterial peptide, its preparation method and application. The fusion antibacterial peptide is a protein as in 1) or 2) or 3) as follows: 1) a protein composed of amino acid sequences shown in sequence 2 of the sequence table; 2) a protein from the fusion of a GST tag and the protein in 1); 3) a protein derived from 1) or 2) with antibacterial activity, specifically obtained by substituting and / or deleting and / or adding to the amino acid residue sequences comprising the protein in 1) or 2) by one or several amino acid residues. In the invention, an antibacterial peptide with strong antibacterial effect is obtained through genetic engineering technology, thus finishing a high expression study in yeast cells. The study shows that the antibacterial peptide can endure the high temperature of feed pelleting. With a creative production process for recombinant yeast fermentation provided in the invention, a further in-depth study is expected to develop the antibacterial peptide into an efficient, safe and multifunctional biological feed so as to replace or reduce antibiotic additives and improve the nutritional value of the feed. Therefore, a new technical support is provided for the sustainable development of animal husbandry of our country.

Description

technical field [0001] The present invention relates to MAPWA fusion antibacterial peptide and its preparation method and application. Background technique [0002] my country is the world's largest pig-raising country, with more than 700 million pigs slaughtered each year and more than 500 million pigs in stock; the pig-based animal husbandry accounts for about 34% of the large agricultural sector, and the contribution rate of animal husbandry to the growth of the total agricultural output value Up to 60% or more. However, in recent years, with the intensive and large-scale development of the pig industry, the problem of disease transmission has become an important problem restricting the development of the pig industry. In the breeding industry, there are more than 30 important infectious diseases (viruses, bacteria and parasitic diseases). Now not only the old diseases have not been eliminated, but also new diseases are constantly emerging. The average morbidity and morta...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/63C12N5/10C12N1/21C12N1/19C12P21/02A61K38/16A61P31/04A61P31/14A61P31/20A23K1/16C12R1/91C12R1/19A23K20/147
Inventor 高荣苏向东王海燕贾贤齐万小平杨发秀严俊勇白光明
Owner 成都市金之源生物技术有限公司
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