CN117660286A - Recombinant lactococcus lactis, preparation, construction method and application thereof - Google Patents
Recombinant lactococcus lactis, preparation, construction method and application thereof Download PDFInfo
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- CN117660286A CN117660286A CN202311696135.1A CN202311696135A CN117660286A CN 117660286 A CN117660286 A CN 117660286A CN 202311696135 A CN202311696135 A CN 202311696135A CN 117660286 A CN117660286 A CN 117660286A
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- 241000194035 Lactococcus lactis Species 0.000 title claims abstract description 35
- 235000014897 Streptococcus lactis Nutrition 0.000 title claims abstract description 35
- 238000002360 preparation method Methods 0.000 title claims abstract description 6
- 238000010276 construction Methods 0.000 title abstract description 11
- 108091043612 miR-146b stem-loop Proteins 0.000 claims abstract description 30
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- 206010061218 Inflammation Diseases 0.000 claims abstract description 10
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- 238000000034 method Methods 0.000 claims description 8
- 239000002299 complementary DNA Substances 0.000 claims description 6
- 239000013612 plasmid Substances 0.000 claims description 6
- 238000012216 screening Methods 0.000 claims description 5
- 239000013598 vector Substances 0.000 claims description 5
- 108090000623 proteins and genes Proteins 0.000 claims description 4
- 241000588724 Escherichia coli Species 0.000 claims description 3
- 230000029087 digestion Effects 0.000 claims description 3
- 238000004520 electroporation Methods 0.000 claims description 3
- 239000003814 drug Substances 0.000 claims description 2
- 238000009472 formulation Methods 0.000 claims description 2
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- 241000894006 Bacteria Species 0.000 abstract description 19
- 239000006041 probiotic Substances 0.000 abstract description 5
- 235000018291 probiotics Nutrition 0.000 abstract description 5
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- 210000000981 epithelium Anatomy 0.000 abstract description 2
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- 239000003112 inhibitor Substances 0.000 abstract description 2
- 244000005700 microbiome Species 0.000 abstract description 2
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- NVNLLIYOARQCIX-MSHCCFNRSA-N Nisin Chemical compound N1C(=O)[C@@H](CC(C)C)NC(=O)C(=C)NC(=O)[C@@H]([C@H](C)CC)NC(=O)[C@@H](NC(=O)C(=C/C)/NC(=O)[C@H](N)[C@H](C)CC)CSC[C@@H]1C(=O)N[C@@H]1C(=O)N2CCC[C@@H]2C(=O)NCC(=O)N[C@@H](C(=O)N[C@H](CCCCN)C(=O)N[C@@H]2C(NCC(=O)N[C@H](C)C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCSC)C(=O)NCC(=O)N[C@H](CS[C@@H]2C)C(=O)N[C@H](CC(N)=O)C(=O)N[C@H](CCSC)C(=O)N[C@H](CCCCN)C(=O)N[C@@H]2C(N[C@H](C)C(=O)N[C@@H]3C(=O)N[C@@H](C(N[C@H](CC=4NC=NC=4)C(=O)N[C@H](CS[C@@H]3C)C(=O)N[C@H](CO)C(=O)N[C@H]([C@H](C)CC)C(=O)N[C@H](CC=3NC=NC=3)C(=O)N[C@H](C(C)C)C(=O)NC(=C)C(=O)N[C@H](CCCCN)C(O)=O)=O)CS[C@@H]2C)=O)=O)CS[C@@H]1C NVNLLIYOARQCIX-MSHCCFNRSA-N 0.000 description 4
- 108010053775 Nisin Proteins 0.000 description 4
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 4
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention belongs to the technical field of recombinant microorganisms, and particularly relates to a recombinant lactococcus lactis, a preparation, a construction method and application thereof. According to the invention, probiotic lactococcus (lactococcus lactis NZ 9000) is taken as a carrier, double enzyme digestion sites of NcoI and XbaI are utilized to form recombinant bacteria (LL-miR-146 b) with special functions, and the recombinant bacteria can be implanted in intestinal tracts in an oral administration or clysis mode, so that intestinal flora can be changed on one hand; on the other hand, the miR-146b can be secreted to inhibit intestinal M1 type macrophage differentiation, so that intestinal inflammation is inhibited and intestinal mucosa epithelium repair is promoted, and the intestinal inflammation inhibitor is applied to various intestinal inflammations including inflammatory enteropathy, and the aim of promoting enteritis repair is fulfilled.
Description
Technical Field
The invention belongs to the technical field of recombinant microorganisms, and particularly relates to a recombinant lactococcus lactis, a preparation, a construction method and application thereof.
Background
Inflammatory Bowel Disease (IBD) is a lifelong recurrent intestinal chronic inflammation. It is a complex multi-factor disease with unknown etiology. It is thought that the interaction of dysbacteriosis in the intestinal tract with abnormal immune system in the intestinal mucosa of the host can lead to excessive immune activation and persistent chronic inflammation.
Current studies demonstrate that miR-146b (miRNA 146 b) can inhibit intestinal inflammation by remodelling intestinal macrophages. On the basis, the invention further provides a concept that recombinant bacteria LL-miR-146b carrying secretion miR-146b is constructed by taking probiotics as a carrier, so that intestinal flora can be remodeled, and M1 macrophage differentiation and inflammatory factor secretion can be inhibited.
Disclosure of Invention
The invention provides a recombinant lactococcus lactis, a preparation, a construction method and application thereof, and recombinant bacteria LL-miR-146b can be constructed to remodel intestinal flora through the lactococcus lactis and play a role of probiotics.
A recombinant lactococcus lactis transformed with a pre-miR-146b plasmid;
the gene sequence of the pre-miR-146b is shown as SEQ ID No. 1.
In particular, the carrier of the recombinant lactococcus lactis is pNZ-8148.
The original lactococcus lactis of the recombinant lactococcus lactis is lactococcus lactis NZ9000.
The method for preparing the recombinant lactococcus lactis component comprises the following steps:
(1) Performing double digestion on the constructed USP45-pre-miR-146b cDNA and a pNZ-8148 vector by using Ncol and XbaI; recovering the target bands, connecting, converting into MC1061 escherichia coli, screening positive clones, and extracting plasmids;
(2) Lactococcus lactis NZ9000 is prepared, pNZ-pre-miR-146b is transferred into NZ9000 by an electroporation method, and recombinant lactococcus lactis LL-miR-146b expressing miR-146b is constructed.
The application of the recombinant lactococcus lactis in preparing medicines for treating or improving intestinal inflammation.
A formulation comprising the recombinant lactococcus lactis described above.
The invention has the beneficial effects that:
according to the invention, probiotic lactococcus (lactococcus lactis NZ 9000) is taken as a carrier, double enzyme digestion sites of NcoI and XbaI are utilized to form recombinant bacteria (LL-miR-146 b) with special functions, and the recombinant bacteria can be implanted in intestinal tracts in an oral administration or clysis mode, so that intestinal flora can be changed on one hand; on the other hand, the miR-146b can be secreted to inhibit intestinal M1 type macrophage differentiation, so that intestinal inflammation is inhibited and intestinal mucosa epithelium repair is promoted, and the intestinal inflammation inhibitor is applied to various intestinal inflammations including inflammatory enteropathy, and the aim of promoting enteritis repair is fulfilled.
According to the invention, through successful construction of recombinant bacteria LL-miR-146b, intestinal flora can be remodeled through lactic acid coccus, so that the effect of probiotics is exerted; on the other hand, the recombinant bacterium LL-miR-146b can secrete miR-146b by itself, and the concentration of miR-146b in intestinal tracts is increased, so that the activation of M1 type macrophages is inhibited, and the aim of improving intestinal inflammation is fulfilled from multiple aspects. Meanwhile, the lactic acid coccus is used as a carrier, so that recombinant bacteria with special functions can be formed, and the recombinant bacteria have good biocompatibility and biodegradability and are harmless to human bodies, so that the recombinant bacteria have good application prospects.
Drawings
Fig. 1: a: a vector construction pattern diagram; B-C: miR-146b insertion site; d: the successful construction of LL-miR-146b and control bacterium LL-Scramble is confirmed by PCR; e: qPCR confirmed that LL-miR-146b expressed miR-146b.
Fig. 2: one generation of sequencing confirmed the successful construction of LL-miR-146b.
Fig. 3: one generation of sequencing confirmed the successful construction of the control bacteria LL-Scramble.
Fig. 4: a: detecting the extracted exosomes by using an electron microscope; b: western blot detection of exosome markers ALIX, CD9 and CD63 expression; c: detecting the grain size of exosomes by using a Markov laser particle sizer to be about 100-500nm; d: miR-146b expression in EV is detected by qPCR.
Detailed Description
For a better understanding of the present invention, reference will now be made to the following description of specific examples, which are included in the terminology used to describe specific embodiments of the invention and are not intended to limit the scope of the invention.
In the examples, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used, unless otherwise specified, are commercially available.
The sources of the raw materials used in the invention are as follows:
lactococcus lactis NZ9000 (VS-ELS 09000-01) was purchased from MoBiTec, germany.
Coli MC1061 (VS-ELS 10610-01) was purchased from MoBiTec, germany.
The pNZ-8148 vector is a plasmid vector available from MoBiTec, germany.
Pre-miR-146b cDNA, available from MoBiTec, germany.
Scramble cDNA, available from MoBiTec, germany.
In the invention, the following components are added:
nisin refers to Nisin.
PBS refers to PBS buffer.
Trizol refers to total RNA extraction reagent.
The gene sequence of pre-miR-146b is shown as SEQ ID No. 1.
The gene sequence of the Scramble is shown as SEQ ID No. 2.
Construction of recombinant bacteria
(1) Constructing recombinant bacteria LL-miR-146b: carrying out double digestion on the constructed pre-miR-146b cDNA and a control Scramble cDNA with a pNZ-8148 vector respectively, carrying out electrophoresis to recover corresponding target bands and connection, converting the target bands and connection into MC1061 escherichia coli, screening positive clones, extracting plasmids, and carrying out sequencing verification;
preparing lactococcus lactis NZ9000 competence, transferring pNZ-pre-miR-146b and pNZ-Scramble into NZ9000 by an electroporation method, constructing recombinant lactococcus lactis LL-miR-146b expressing miR-146b and control bacterium LL-Scramble, screening positive clones by a PCR method, and screening positive strains by double enzyme digestion verification of plasmids (shown in the accompanying drawings 1, 2 and 3);
(2) Detecting the expression of miR-146b in recombinant bacteria LL-miR-146b: in a 30 ℃ incubator, after LL-miR-146b and LL-Scramble grow to have Optical Density (OD) of about 0.4, two bacteria are treated by Nisin (10 ng/ml) for 4 hours, supernatants are removed by centrifugation (8000 rpm,10 min), washed twice by PBS, LL-miR-146b and LL-Scramble are lysed by lysozyme (25 mg/ml) at 37 ℃ for 30min, bacterial RNA is extracted by Trizol for qPCR to verify miR-146b expression, so that miR-146b expression in LL-miR-146b is verified;
(3) Collecting EV in the two bacterial supernatants, treating LL-miR-146b and LL-Scramble for 4 hours by nisin, centrifuging to remove bacterial sediment (8000 rpm,10 min), filtering bacterial culture medium for the first time by a vacuum filter of 0.45um, concentrating by Quix Stand Benchtop System, filtering the concentrated liquid for the second time by a vacuum filter of 0.22um, and finally centrifuging the obtained liquid at an ultra high speed (150000 g/min), and detecting EV characteristics of the collected product in the next step;
(4) EV identification and expression of miR-146b in EV: the morphology and diameter of EV are detected by a Markov laser particle sizer and an electron microscope, and miR-146b expression in EV is detected by a qPCR method.
As can be seen from fig. 4, part a is exosomes extracted by electron microscopy, and part B is expressed by Western blot detection exosome markers ALIX, CD9 and CD 63; the part C is the particle size of the exosome detected by a Markov laser particle sizer of about 100-500nm; part D is miR-146b expression in EV detected by qPCR method; it can be concluded that: recombinant bacterium LL-miR-146b can secrete a large amount of miR-146b.
The foregoing detailed description is directed to one of the possible embodiments of the present invention, which is not intended to limit the scope of the invention, but is to be accorded the full scope of all such equivalents and modifications so as not to depart from the scope of the invention.
Claims (6)
1. A recombinant lactococcus lactis, wherein the recombinant lactococcus lactis is a recombinant lactococcus lactis transformed with a pre-miR-146b plasmid;
the gene sequence of the pre-miR-146b is shown as SEQ ID No. 1.
2. The recombinant lactococcus lactis according to claim 1, wherein said recombinant lactococcus lactis carrier is pNZ-8148.
3. The recombinant lactococcus lactis according to claim 1, wherein the original lactococcus lactis of the recombinant lactococcus lactis is lactococcus lactis NZ9000.
4. A method of constructing a recombinant lactococcus lactis according to any one of claims 1 to 3, comprising the steps of:
(1) Performing double digestion on the constructed USP45-pre-miR-146b cDNA and a pNZ-8148 vector by using Ncol and XbaI; recovering the target bands, connecting, converting into MC1061 escherichia coli, screening positive clones, and extracting plasmids;
(2) Lactococcus lactis NZ9000 is prepared, pNZ-pre-miR-146b is transferred into NZ9000 by an electroporation method, and recombinant lactococcus lactis LL-miR-146b expressing miR-146b is constructed.
5. Use of a recombinant lactococcus lactis according to any one of claims 1-3 for the preparation of a medicament for the treatment or amelioration of intestinal inflammation.
6. A formulation comprising the recombinant lactococcus lactis of any one of claims 1-3.
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| CN102266571A (en) * | 2011-07-12 | 2011-12-07 | 中国农业大学 | Application of microRNA 302 in early embryonic development |
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2023
- 2023-12-12 CN CN202311696135.1A patent/CN117660286B/en active Active
Patent Citations (6)
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| CN102266571A (en) * | 2011-07-12 | 2011-12-07 | 中国农业大学 | Application of microRNA 302 in early embryonic development |
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| CN108938659A (en) * | 2017-05-19 | 2018-12-07 | 昆山彭济凯丰生物科技有限公司 | A kind of method and drug and their application of modulation of appetite and weight |
| CN107338262A (en) * | 2017-06-06 | 2017-11-10 | 中国水产科学研究院珠江水产研究所 | For expressing Streptococcusagalactiae plasmid and its construction method and the application of fluorescin |
| CN112143698A (en) * | 2019-06-28 | 2020-12-29 | 上海嘉慷生物工程有限公司 | Transmembrane IL-21 activated exosome secreted by CIK cells and application of related miRNA in antibiosis |
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Non-Patent Citations (2)
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