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Antibacterial peptide PR39 and PG1 co-expression vector and method for preparing PR39 and PG1 transgenic mice

A technology of co-expression vector and antibacterial peptide, applied in the field of genetic engineering, can solve the problem of transgenic animals that do not co-express two antibacterial peptides, and achieve the effect of reducing the use of antibiotics and solving food safety

Inactive Publication Date: 2017-08-25
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, transgenic animals expressing antimicrobial peptides mainly express a single antimicrobial peptide, and there are no reports on transgenic animals co-expressing two antimicrobial peptides

Method used

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  • Antibacterial peptide PR39 and PG1 co-expression vector and method for preparing PR39 and PG1 transgenic mice
  • Antibacterial peptide PR39 and PG1 co-expression vector and method for preparing PR39 and PG1 transgenic mice
  • Antibacterial peptide PR39 and PG1 co-expression vector and method for preparing PR39 and PG1 transgenic mice

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1: Construction of eukaryotic co-expression vector pCMS-PR39-T2A-PG1-EGFP

[0051] (1) Co-expression vector construction strategy

[0052] The two antimicrobial peptide genes PR39 and PG1 were connected in series through a T2A linking peptide, and then cloned into the eukaryotic expression vector pCMS-EGFP with two independent expression cassettes regulated by the two promoters (purchased from Youbao Biotechnology Co., Ltd.) Above, the co-expression of two antimicrobial peptide genes is regulated by the CMV promoter alone, and the sequence of the elements is CMV-Kozak-PR39-T2A-PG1-SV40pA; the expression of the EGFP gene is regulated by the SV40 promoter alone, and the sequence of the elements is arranged is SV40-Kozak-EGFP-bGHpA. The two expression systems are relatively independent and complete without interfering with each other, and can achieve the purpose of simultaneously and efficiently expressing two target proteins and one marker protein on the same euk...

Embodiment 2

[0064] Example 2: Large-scale extraction and enzyme digestion identification of PR39 and PG1 co-expression vector without endotoxin

[0065] (1) In order to reduce the cytotoxicity of the plasmid transfection to 293 cells, a large amount of co-expression vectors were extracted using OMEGA’s endotoxin removal kit Endo-free Plasmid Mini Kit before transfection. The operation steps are detailed in the instruction manual.

[0066] (2) Enzyme digestion identification of PR39 and PG1 co-expression vector

[0067] The extracted plasmids were identified by single- and double-enzyme digestion using FastDigest rapid restriction endonucleases.

[0068] The results showed that after a large amount of co-expression vectors were extracted, linearization (6847bp) was carried out at a single site (2368) of the vector with restriction endonuclease Not I, and two sites of the vector with restriction endonucleases Xho I and Bgl II were A single site (1092 and 6843) was double digested, cut into...

Embodiment 3

[0069] Example 3: PR39 and PG1 co-expression vector transfected 293 cells

[0070] The specific operation of PR39 and PG1 co-expression vector transfection into 293 cells is as follows:

[0071] 1. Cells were subcultured evenly in a 6-well plate after thawing, cultured with 10% FBS complete medium, and transfected when the cells grew to a confluence of 70% to 90%;

[0072] 2. In a separate test tube, dilute 7.5 μL Lipofectamine 3000 and 7.5 μg plasmid DNA in 125 μL Opti-MEM, add 15 μL P3000 reagent to the diluted DNA and mix well;

[0073] 3. Mix the diluted plasmid DNA with the diluted Lipofectamine 3000 reagent 1:1, and incubate at room temperature for 5 minutes;

[0074] 4. Carefully and evenly add the above mixture to the cells containing 1% double antibody and 10% FBS complete medium, avoid blowing up the 293 cells that are not firmly attached, gently cross-mix and put the cells back to 37°C , 5% CO2, and saturated humidity in a cell incubator to continue culturing;

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Abstract

The invention discloses an antibacterial peptide PR39 and PG1 co-expression vector and a method for preparing PR39 and PG1 transgenic mice. The antibacterial peptide PR39 and PG1 co-expression vector comprises a PR39 and PG1 co-expression control system and a screening gene EGFP expression control system. The method comprises the following steps: performing micro-injection on the PR39 and PG1 co-expression vector in mouse fertilized eggs, thereby obtaining the PR39 and PG1 transgenic mice. The transgenic mice are capable of realizing co-expression of the PR39 and PG1 and can achieve the effects of resisting attack of most of bacteria such as Gram negative bacteria and the like as well fungi, reducing usage of antibiotics and laying a foundation for obtaining transgenic livestock with PR39 and PG1 co-expression genes. If the livestock is capable of simultaneously expressing the antibacterial peptides PR39 and PG1, the usage of the antibiotics can be greatly reduced, and possibility is provided for solving antibiotics abuse and food safety problems.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a PR39 and PG1 co-expression vector and a construction method thereof, and a preparation method for PR39 and PG1 gene-transferred mice. Background technique [0002] my country is a big country in pig production and consumption, but diseases frequently occur in the pig industry, which seriously restricts the development of my country's pig industry. Antibiotics are widely used in animal husbandry in the form of feed additives and veterinary drugs because of their functions of preventing, treating diseases and promoting animal growth. It is estimated that the use of antibiotics in animals in recent years has accounted for more than half of the total global antibiotic use. Studies have shown that livestock animals are an important source of drug-resistant pathogens in humans. The long-term abuse of antibiotics leads to the emergence of drug-resistant pathogenic bacteria and dru...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N15/65C12N15/66A01K67/027
CPCA01K67/0278A01K2207/15A01K2217/072A01K2227/105A01K2267/02C12N15/65C12N15/66C12N15/8509
Inventor 李紫聪黄晓灵吴珍芳刘德武蔡更元郑恩琴
Owner SOUTH CHINA AGRI UNIV
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