Genetically engineered bacterium for efficiently expressing recombinant bacteriocin and construction method and application thereof

A construction method, bacteriocin technology, applied in the direction of genetic engineering, microorganism-based methods, medical preparations containing active ingredients, etc., can solve the problems of restricted application, difficult separation and purification, and low yield of strains, and achieve low cost, Less drug resistance and simple purification steps

Active Publication Date: 2013-08-07
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, at present, the yield of naturally occurring strains is low, and the separation and purification are difficult, which seriously restricts its application in production.

Method used

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  • Genetically engineered bacterium for efficiently expressing recombinant bacteriocin and construction method and application thereof
  • Genetically engineered bacterium for efficiently expressing recombinant bacteriocin and construction method and application thereof
  • Genetically engineered bacterium for efficiently expressing recombinant bacteriocin and construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] The preparation of embodiment 1 recombinant bacteriocin E50-52

[0051] 1. Design and synthesize the E50-52 gene sequence

[0052] According to the amino acid sequence of bacteriocin E50-52 published by GenBank and the codon usage preference of Escherichia coli (refer to http: / / www.kazusa.or.jp / codon), the gene sequence was designed to synthesize the bacteriocin E50-52 gene, and the synthesized bacteriocin E50 The nucleotide sequence of the -52 gene is shown in SEQ ID No.1, and the amino acid sequence of its encoded protein is shown in SEQ ID No.2.

[0053] The designed sequence was submitted to Invitrogen Company for gene synthesis. After synthesis, it was connected into T vector and transformed into E. coli DH5α to obtain T-E50-52 vector, which was verified by sequencing.

[0054] 2. Construction of pET-SUMO-E50-52 recombinant plasmid

[0055] Since the expression vector pET-SUMO used is linearized and contains prominent T bases, the T-E50-52 vector is used as a tem...

Embodiment 2

[0076] Example 2 Recombinant bacteriocin E50-52 MALDI-TOF / TOF mass spectrometry molecular weight detection

[0077] Take 0.5 μL of the purified recombinant bacteriocin sample, mix it with the matrix required for mass spectrometry detection at a ratio of 1:3, and spot it on the target. After drying naturally, use the AB4700MALDI-TOF / TOF mass spectrometer to scan the molecular weight of the recombinant bacteriocin. Mass spectrometry conditions: ion source acceleration voltage of 20kV, N2 laser, laser wavelength of 337nm, laser frequency of 200Hz, ion delay extraction time (Pulse ion extraction, PIE) of 330ns, mass spectrometry signal single-scan accumulation 2000 times, using peptide II standard kit (Bruker) ion Peak correction (m / z1000-m / z7000). The test results were analyzed by Data Explorer V4.5 software.

[0078] As a result of the analysis, it was found that the molecular weight of the recombinant bacteriocin E50-52 was 3339.72Da (such as Figure 7 shown), the molecular w...

Embodiment 3

[0079] The antibacterial activity verification of embodiment 3 recombinant bacteriocin E50-52

[0080] (1) Antibacterial activity detected by cup and saucer method

[0081] Using the cup and saucer method, Staphylococcus aureus ATCC25923 was used as the test strain, and 200 μL of the suspension of the test bacteria was spread on the nutrient gravy solid medium, and the Oxford cup was placed on it, and 100 μL of recombinant bacteriocin was added, and the same Undigested fusion protein or PBS was used as negative control, and cultured at 37°C for 8-12h. The antibacterial effect was observed on the second day.

[0082] Such as Figure 8 As shown, the fusion protein before digestion has no antibacterial activity, and the recombinant bacteriocin E50-52 after digestion and purification shows antibacterial activity against Staphylococcus aureus.

[0083] (2) Detection of minimum inhibitory concentration (MIC) of recombinant bacteriocin

[0084] The minimum inhibitory concentratio...

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Abstract

The invention relates to a genetically engineered bacterium for efficiently expressing recombinant bacteriocin and a construction method and an application thereof. The genetically engineered bacterium is escherichia coli BL21(DE3) carrying a Pet-sumo-e50-52 recombinant expressing carrier. The genetically engineered bacterium is constructed by steps of cloning bacteriocin E50-52 genes with a nucleotide sequence shown in SEQ ID No.1 to fused expression plasmid pET-SUMO and then converting the competent cell of the escherichia coli BL21(DE3). The genetically engineered bacterium is expressed through induction of IPTG (Isopropyl-beta-d-Thiogalactoside) so that SUMO-E50-52 fuse protein is expressed in escherichia coli; the fuse protein is cut by a specific SUMO protease to obtain the recombinant bacteriocin E50-52. The recombinant bacteriocin provided by the invention has good antimicrobial activity and stability, hardly generates drug tolerance, and can be used as ideal antibacterial agents, antiviral drugs, feed additives, preservatives, killing agents and the like.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a genetically engineered bacterium highly expressing recombinant bacteriocin, its construction method and application. Background technique [0002] In the past ten years, with the rapid progress of science and technology in our country, animal husbandry has also achieved sustained and stable development, and has become a pillar industry of the national economy. However, while veterinary drugs and pharmaceutical feed additives promote the production and development of the aquaculture industry, they also have serious negative effects due to their long-term and irregular use: First, the antibiotic resistance of pathogenic bacteria caused by the extensive use of antibiotics in the aquaculture industry is increasing. It is serious, causing the consumption of antibiotics to increase year by year, which not only leads to an increase in the cost of breeding, but also seriously threatens hum...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/31C12N15/70C12N1/21C12P21/02A61K38/16A23K1/16A61P31/00A61P31/12A61P1/00C12R1/19
Inventor 张日俊马青山王庆黄燕杨军香
Owner CHINA AGRI UNIV
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