Unlock instant, AI-driven research and patent intelligence for your innovation.

A kind of recombinant bacteriocin and its preparation method and application

A bacteriocin and Escherichia coli technology, which is applied in the biological field, can solve the problems of inability to obtain amidation modification and affect the activity, and achieve the effects of good antibacterial activity and stability, low cost and simple purification steps.

Active Publication Date: 2018-06-15
NORTHEAST AGRICULTURAL UNIVERSITY
View PDF0 Cites 0 Cited by
  • 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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • A kind of recombinant bacteriocin and its preparation method and application
  • A kind of recombinant bacteriocin and its preparation method and application
  • A kind of recombinant bacteriocin and its preparation method and application

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 dNTPMTxture 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 dilu...

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...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

PropertyMeasurementUnit
pore sizeaaaaaaaaaa
molecular weightaaaaaaaaaa
diameteraaaaaaaaaa
Login to View More

Abstract

The invention relates to a recombinant bacteriocin and its preparation method and application, belonging to the field of biotechnology. The nucleotide sequence of the recombinant bacteriocin LacAB gene of the present invention is shown in SEQ ID NO:1, and the recombinant expression vector pGEX-4T-1-LacAB includes the nucleotide sequence shown in SEQ ID NO:1. In the present invention, Escherichia coli BL21 containing the recombinant expression vector pGEX‑4T‑1‑LacAB is induced to express the target protein through IPTG, the bacterial liquid is collected, the recombinant protein in the bacterial liquid is extracted, and the obtained recombinant protein solution is passed through a GST purification column, GST label and The GST affinity protein on the column is bound, and the purified GST-LacAB protein is obtained through the eluent. After the GST tag is cut off with thrombin, it is purified with a GST purification kit to obtain a single recombinant bacteriocin LacAB. The recombinant bacteriocin of the invention has good antibacterial activity and stability, and a wide antibacterial spectrum, and can be used as an ideal antibiotic substitute, human and animal biological medicine, feed additive, preservative, disinfectant 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, RayB. Bacteriocins of gram-positive bacteria. Microbiol Rev, 1995, 59: 171-200.), at present, the bacteria adopted by literature and research groups The definition of element is: the 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 parental relationship, and can kill or inhibit microorganisms in similar habitats , the strains producing bacteriocin have self-specific immunity and will not kill the producing bacteria (DattaV, Myskowski SM, Kwinn LA, Chiem DN, Varki N, Kansal RG, Ko...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Patents(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