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Recombinant lactobacillus plantarum capable of secreting colicins V and preparation method thereof

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: 2015-05-06
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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  • Recombinant lactobacillus plantarum capable of secreting colicins V and preparation method thereof
  • Recombinant lactobacillus plantarum capable of secreting colicins V and preparation method thereof
  • Recombinant lactobacillus plantarum capable of secreting colicins V and preparation method thereof

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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]The sequences were obtained from GenBank: the sequences of the pediocin guide peptide gene and the mature colibactin V gene, after codon modification and optimization, the pediocin guide peptide gene sequence of the present invention is shown in SEQ ID NO. The bacteriocin V gene sequence is shown in SEQ ID NO.3. The pediocin guide peptide gene and the mature colicin V gene are connected, and the fusion gene after the connection is synthesized by the biological company as follows, a total of 338bp, as shown in SEQ ID NO.1 (Nco I and Eco R I are added at both ends respectively. Enzyme cleavage sites and protected bases) synthetic fragments were 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, respectively, and then ligated overnight with DNA ligase....

Embodiment 2

[0036] Example 2 Construction of recombinant Lactobacillus plantarum strains

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

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

[0039] 2. Electrotransformation of recombinant plasmids to Lactococcus lactis NZ9000

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

Embodiment 3

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

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

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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 Lactobacillus plantarum secreting colicin V and a preparation method thereof. Background technique [0002] In the past 50 years, antibiotics, as the most commonly used disease prevention and growth-promoting feed additives, have brought benefits to the aquaculture industry, but also brought fatal disadvantages: animal intestinal flora imbalance, superinfection and bacterial resistance. Sexual and other adverse reactions have gradually become prominent, posing a serious threat to the aquaculture and feed industries. Therefore, the development of non-toxic, non-polluting and non-residue antibiotic alternatives has become a top priority for scientific researchers. Since the 1980s, microecological preparations based on lactic acid bacteria have begun to attract attention, and their functions of promoting the growth of intestinal beneficial bacteria, inhibiti...

Claims

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Application Information

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IPC IPC(8): C12N15/74C12N15/31C12N15/66C12N1/21A23K1/18A23K1/17A23L3/3571C12R1/25A23K20/195
Inventor 郝彦玲马夏吟
Owner CHINA AGRI UNIV
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