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Recombinant bacteriocin and preparation method and application thereof

A technology of bacteriocin and Escherichia coli, applied in the biological field, can solve the problems of affecting the activity, unable to obtain amidation modification, etc., and achieve the effects of low cost, good antibacterial activity and stability, and high yield

Active Publication Date: 2015-10-07
NORTHEAST AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

On the other hand, the recombinant bacteriocin protein in the E. coli system cannot be amidated after translation to affect its activity.

Method used

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  • Recombinant bacteriocin and preparation method and application thereof
  • Recombinant bacteriocin and preparation method and application thereof
  • Recombinant bacteriocin and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Cloning and prokaryotic expression vector construction of embodiment 1 bacteriocin LacAB gene

[0064] 1 Materials and methods

[0065] 1.1 Materials and reagents

[0066] The pMD18-T vector was used to construct a cloning vector and was purchased from Dalian Bao Biotechnology Co., Ltd.; pGEX-4T-1 was used to construct an expression vector and was purchased from Dalian Bao Biotechnology Co., Ltd. Bacterial DNA Extraction Kit and Agarose Gel DNA Recovery Kit were purchased from Beijing Tiangen Biochemical Technology Co., Ltd.; ExTaq, 10×ExTaqBuffer, DNA Marker and dNTP MTXture were purchased from Dalian Bao Biological Company.

[0067] 1.2 Solution preparation

[0068] Ampicillin: Dissolve 1g ampicillin dry powder in 20ml ddH 2 In O, filter through a 0.22m filter membrane, dispense into sterilized EP tubes, and freeze at -20°C.

[0069] Primer dilution: The primers were synthesized in Invitrogen Company. According to the nM number of the primers, the primers were dil...

Embodiment 2

[0126] Induced expression and purification of embodiment 2 bacteriocin

[0127] 1 Materials and methods

[0128] 1.1 Materials and solution preparation

[0129] Competent Escherichia coli (E.coli BL21), competent Escherichia coli (E.coli TOP10), and competent Escherichia coli (E.coli DH5α) were purchased from Treasure Bio (Dalian) Co., Ltd.

[0130] Bacterial lysate: 50mM Tris-HCl (pH8.5-9.0), 2mM EDTA, 100mMNaCl, 0.5% TritonX-100, 1mg / mL lysozyme.

[0131] 1.2 Method

[0132] 1.2.1 Induced expression of prokaryotic recombinant expression vector pGEX-4T-1-LacAB

[0133] The prokaryotic recombinant expression vector pGEX-4T-1-LacAB constructed in Example 1 was transformed into competent Escherichia coli cells (E.coliBL21), and the correct monoclonal colonies identified by double enzyme digestion were picked and placed in 700ul LB medium, and 0.7ulAmp (100mg / mL), cultured on a shaker at 37°C and 200r / min, and activated overnight. At a ratio of 1:50 (200ul), add the activate...

Embodiment 3

[0187] Example 3 Research on Antibacterial Properties of Recombinant Bacteriocins

[0188] 1 Experimental method

[0189] 1.1 Antibacterial spectrum detection of bacteriocins

[0190] 1) Oxford cup agar diffusion method

[0191] Select typical Gram-positive bacteria - Staphylococcus aureus, Micrococcus luteus, Listeria monocytogenes, Bacillus, Lactococcus lactis, Enterococcus, Lactobacillus and Gram-negative bacteria - Escherichia coli, Pseudomonas and Salmonella were indicator bacteria. The inhibitory effect of the expression product on the bacteria on the plate was observed by the Oxford cup agar diffusion method (the concentration of the added recombinant bacteriocin solution was 1 mg / mL), and its antibacterial activity was measured by the diameter of the inhibition zone, and the test was repeated 3 times.

[0192] 2) Hemocytometer counting method

[0193] Cultivate the indicator bacteria in a suitable medium at 37°C until the number of colonies reaches 10 6 cfu / mL. T...

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Abstract

The invention relates to a recombinant bacteriocin and a preparation method and an application thereof, and belongs to the technical field of biology. A nucleotide sequence of a LacAB gene of the recombinant bacteriocin is shown in an SEQ ID NO:1, and a recombinant expression vector pGEX-4T-1-LacAB comprises the nucleotide sequence shown in the SEQ ID NO:1. Escherichia coli BL21 containing the recombinant expression vector pGEX-4T-1-LacAB is induced by IPTG to express objective protein, and a bacterium solution is collected, recombinant protein in the bacterium solution is extracted; an obtained recombinant protein solution passes through a GST purification column, a GST label is combined with GST rabphilin rab on the column, and purified GST-LacAB protein is obtained through an eluant; the GST label is cut and removed by thrombin, purification is conducted through a GST purification kit, and the single recombinant bacteriocin LacAB is obtained. According to the recombinant bacteriocin and the preparation method and the application thereof, the antimicrobial activity and the stability of the recombinant bacteriocin are good, the antimicrobial spectrum is wide, and the recombinant bacteriocin can serve as an ideal antibiotic succedaneum, a human and animal biological drug, a feed supplement, an antiseptic substance, a disinfection agent and the like.

Description

technical field [0001] The invention relates to a recombinant bacteriocin and its preparation method and application, in particular to a recombinant bacteriocin LacAB and its preparation method and application, belonging to the field of biotechnology. Background technique [0002] The definition of bacteriocin was first proposed by Jocob in 1953 (Jack RW, Tagg JR, Ray B. Bacteriocins of gram-positive bacteria. Microbiol Rev, 1995, 59: 171-200.), currently, the literature and research community adopted The definition of bacteriocin is: a polypeptide, protein or protein complex with antibacterial activity produced by bacteria through ribosomes in the metabolic process, which has an inhibitory effect on bacteria with close parentage, and can kill or inhibit bacteria in similar habitats. Microorganisms, bacteriocin-producing strains have self-specific immunity and will not kill the producing bacteria (Datta V, Myskowski SM, Kwinn LA, Chiem DN, Varki N, Kansal RG, Kotb M, Nizet V...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/31C12N15/70C12N15/66C12N1/21C12P21/02C12R1/245
Inventor 于微张明辉高学军王琦
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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