A kind of recombinant plant lactobacillus secreting colicin V and its preparation method

A technology of colibactin and Lactobacillus plantarum, applied in the field of microbial molecular biology, can solve problems such as animal intestinal flora imbalance, threats to the breeding industry and feed industry, and bacterial resistance to superinfection

Inactive Publication Date: 2018-11-13
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] In the past 50 years, antibiotics have been the most commonly used feed additives for disease prevention and growth promotion. While bringing benefits to the aquaculture industry, antibiotics have also brought fatal disadvantages: animal intestinal flora imbalance, superinfection and bacterial drug resistance Sexual and other adverse reactions gradually become prominent, posing a serious threat to the aquaculture and feed industries

Method used

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  • A kind of recombinant plant lactobacillus secreting colicin V and its preparation method
  • A kind of recombinant plant lactobacillus secreting colicin V and its preparation method
  • A kind of recombinant plant lactobacillus secreting colicin V and its preparation method

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Experimental program
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Effect test

Embodiment 1

[0034] Embodiment 1 carries the construction of the lactic acid bacteria expression vector of fusion gene fragment

[0035]Sequences obtained from GenBank: the sequences of the pediocin guiding peptide gene and the mature colibactin V gene, after codon transformation and optimization, the sequence of the pediocin guiding peptide gene of the present invention is shown in SEQ ID NO.2, mature large intestine The sequence of the bacitracin V gene is shown in SEQ ID NO.3. The pediocin guide peptide gene and the mature colicin V gene were connected, and the fusion gene after connection was synthesized by the biological company with the following sequence, a total of 338bp, as shown in SEQ ID NO.1 (NcoI and EcoRI enzymes were added at both ends respectively cutting site and protecting base) the synthetic fragment was constructed on the pUCK vector. The expression vector p118148 and the pUCK vector carrying the synthetic fragment were double-digested with Nco I and Eco R I respective...

Embodiment 2

[0036] Embodiment 2 constructs recombinant plant Lactobacillus strain

[0037] 1. Preparation of Lactococcus lactis NZ9000 competent cells

[0038] Lactococcus lactis NZ9000 was cultured with SGM17 liquid medium at 30°C until the OD value reached 0.2-0.7. Take 10ml of bacterial solution, centrifuge at 6000rpm, 4°C for 8min, collect the bacteria; add 2ml of pre-cooled 0.5mol / L sucrose solution (containing 10% glycerol) to wash twice, centrifuge at 6000rpm, 4°C for 8min, discard the supernatant; Suspend the cells with 1% pre-cooled 0.5 mol / L sucrose solution (containing 10% glycerol) corresponding to the volume of the cell culture medium, and dispense 40 μl for use.

[0039] 2. Electrotransformation of Lactococcus lactis NZ9000 with the recombinant plasmid

[0040] Take 40 μl of Lactococcus lactis NZ9000 competent cells, mix with 5 μl of the connection product obtained in Example 1, transfer to a 2mm electroporation cup, use Bio-Rad Gene Pulser Xcell TM Type electroporation i...

Embodiment 3

[0045] Embodiment 3 The activity detection method of colicin V produced by recombinant Lactobacillus plantarum of the present invention

[0046] The recombinant Lactobacillus plantarum prepared in Example 2 and the control strain (the strain obtained by electrotransforming p118148) were cultured in MRS liquid medium for 48 hours, centrifuged at 6000 rpm at 4° C., and the supernatant was taken. After adjusting the pH of the supernatant to 7.0, the supernatant was concentrated by the TCA method to 100 times. Use Escherichia coli DH5α and Listeria monocytogenes (ATCC54003) as the indicator bacteria, culture the indicator bacteria to the early logarithmic phase, take 100 μl and add it to 10ml of solid medium (Listeria Bacteria is TYPE medium; Escherichia coli is LB medium), mix evenly and pour it into a plate, and put an Oxford cup on the medium after cooling and solidifying. Add 100 μl of the supernatant of the concentrated recombinant strain and the control strain to the Oxford...

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Abstract

The invention relates to a recombinant lactobacillus plantarum capable of secreting colicins V and a preparation method thereof, belonging to the field of microbe molecule biology. The recombinant lactobacillus plantarum disclosed by the invention carries a free expression vector containing a pediocin PA-1 leading peptide-mature colicin V fused gene. The recombinant lactobacillus plantarum is prepared through connecting a pediocin PA-1 leading peptide-mature colicin V peptide fused gene to an expression vector, transferring a connecting product to lactococcus lactis by using an electrotransformation method, extracting a recombinant plasmid, and transferring the recombinant plasmid to lactobacillus plantarum BM-1 through electrotransformation. The recombinant lactobacillus plantarum disclosed by the invention can simultaneously secrete pediocin PA-1 and colicin V, and has a good inhibitory effect on colicins and listeria monocytogenes pathogenic bacteria; after being eaten by animals, the recombinant lactobacillus plantarum does not produce toxic and side effects and residues, so that the recombinant lactobacillus plantarum has no threat to the environment and food security, therefore, the recombinant lactobacillus plantarum has a good application prospect in the fields of feed industry and food industry.

Description

technical field [0001] The invention relates to the field of microbial molecular biology, in particular to a recombinant plant lactobacillus secreting colicin V and a preparation method thereof. Background technique [0002] In the past 50 years, antibiotics have been the most commonly used feed additives for disease prevention and growth promotion. While bringing benefits to the aquaculture industry, antibiotics have also brought fatal disadvantages: animal intestinal flora imbalance, superinfection and bacterial drug resistance Sexual and other adverse reactions gradually become prominent, posing a serious threat to the breeding industry and the feed industry. Therefore, the development of non-toxic, side-effect, pollution-free and residue-free alternatives to antibiotics has become a top priority for scientific researchers. Since the 1980s, microecological preparations based on lactic acid bacteria have attracted attention, and their functions of promoting the growth of ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/74C12N15/31C12N15/66C12N1/21A23K50/60A23K10/18A23L3/3571C12R1/25A23K20/195
Inventor 郝彦玲马夏吟
Owner CHINA AGRI UNIV
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