CN113699092B - Recombinant bacillus subtilis and construction method and application thereof - Google Patents
Recombinant bacillus subtilis and construction method and application thereof Download PDFInfo
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- CN113699092B CN113699092B CN202111251480.5A CN202111251480A CN113699092B CN 113699092 B CN113699092 B CN 113699092B CN 202111251480 A CN202111251480 A CN 202111251480A CN 113699092 B CN113699092 B CN 113699092B
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/335—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Lactobacillus (G)
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/12—Animal feeding-stuffs obtained by microbiological or biochemical processes by fermentation of natural products, e.g. of vegetable material, animal waste material or biomass
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
- A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
- A61K35/741—Probiotics
- A61K35/742—Spore-forming bacteria, e.g. Bacillus coagulans, Bacillus subtilis, clostridium or Lactobacillus sporogenes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
- C12N15/75—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Bacillus
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- Biomedical Technology (AREA)
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- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Wood Science & Technology (AREA)
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- Veterinary Medicine (AREA)
- Biophysics (AREA)
- Pharmacology & Pharmacy (AREA)
- Mycology (AREA)
- Physiology (AREA)
- Animal Husbandry (AREA)
- General Engineering & Computer Science (AREA)
- Food Science & Technology (AREA)
- Sustainable Development (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Epidemiology (AREA)
- Plant Pathology (AREA)
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Abstract
The invention discloses a recombinant bacillus subtilis with a preservation number of: CGMCC NO: 23102. the invention obtains a recombinant bacillus subtilis CGMCC NO capable of highly producing the plant lactobacillus papA by inserting the coding gene papA into a carrier and transforming a host bacillus subtilis: 23102. the recombinant bacillus subtilis CGMCC NO: 23102 can be used for producing recombinant plant lactobacillus papA, and has obvious inhibiting effect on Listeria monocytogenes.
Description
Technical Field
The invention relates to the field of microecology, in particular to recombinant bacillus subtilis and a construction method and application thereof.
Background
Pediocin (PA-1) is peptide bacteriocin secreted by Pediococcus acidilactici, and has inhibitory effect on food-borne pathogenic bacteria Listeria monocytogenes. A strain of Lactobacillus plantarum Zhang-LL is separated from preserved meat by professor Zhang hongxing of Beijing academy of agriculture, and the like, and has an inhibiting effect on Listeria monocytogenes and other bacteria (CN 104531562B). The purified active component is determined to be polypeptide through analysis, called plant lactobacillus element, the whole genome sequencing sequence analysis, the coding gene is papA, and the sequence is consistent with the sequence of pediocin PA-1. The precursor peptide of PA-1/papA comprises a leader peptide and a functional peptide, the mature functional peptide comprises 44 amino acids, has a molecular weight of 4.6 kDa, comprises four cysteine residues, and forms two disulfide bridges. The expression level of the plant lactobacillin/pediocin in natural host bacteria is limited, and the purification is difficult.
In order to increase the expression level of pediocin, researchers heterologously express PA-1. At present, the method for expressing PA-1/papA in Escherichia coli is complicated because PA-1 and papA are both located in one operon and comprise 4 coding frames, besides the functional genes of bacteriocin, an immunity protein, an ABC-transfer peptide with aminopeptidase and an auxiliary protein, namely three genes of PedBCD of Pediococcus and three genes of PapBCD of Lactobacillus plantarum. Pediocin is directly expressed in escherichia coli, has no bacteriostatic activity, and the whole operon must be added to a vector together to obtain an expression supernatant with bacteriostatic activity (Mesa-Pereira, O' Connor et al 2017). However, when the mature peptide of PA-1 is expressed alone, in order to maintain the effective folding of the mature peptide, it is necessary to add thioredoxin (Trx) gene (Beaulieu, Tolkatchev et al 2007, Liu, Han et al 2011), dihydrofolate reductase gene (DHFR) (Moon, pyrol et al 2006) and green fluorescent protein Gene (GFP) (Vermeulen, Van Staden et al 2020) before papA gene to obtain soluble fusion protein. The fusion protein has no bacteriostatic activity, and the bacteriostatic function of papA can be exerted only after the auxiliary peptides (Trx, DHFR and GFP) in the fusion peptide are cut off. Therefore, it is important to construct a new strain and a simple method for heterologous expression of PA-1/papA. Bacillus subtilis (A), (B) and (C)Bacillus subtilis) Is a food grade beneficial microorganism which is Generally Recognized As Safe (GRAS), and is selected as the starting strain of the research.
Disclosure of Invention
In order to solve the technical problems, the invention aims to provide a recombinant bacillus subtilis named as bacillus subtilisB. subtilisDBN-SKL-PA2, which is classified and named as bacillus subtilisBacillus subtilisThe microbial inoculum is preserved in China general microbiological culture Collection center with the preservation date of 2021, 8 months and 2 days, and the preservation numbers are as follows: CGMCC NO: 23102, the preservation address is No. 3 Xilu Beijing province, Chaoyang district, Beijing.
The invention also aims to provide a construction method of the recombinant bacillus subtilis for expressing the plant lactobacillus papA, which is obtained by connecting DNA with a nucleic acid sequence shown as SEQ ID number 1 with a linearized expression vector and transferring the DNA into host bacillus subtilis; the amino acid sequence of the nucleic acid sequence coding protein shown by the SEQ ID number 1 is shown by SEQ ID NO. 2.
The nucleic acid sequence SEQ ID number 1 specifically comprises: AAATACTACGGTAATGGGGTTACTTGTGGCAAACATTCCTGCTCTGTTGACTGGGGTAAGGCTACCACTTGCATAATCAATAATGGAGCTATGGCATGGGCTACTGGTGGACATCAAGGTAATCATAAATGCTAG, respectively;
the amino acid sequence SEQ ID number 2 of the encoded protein is specifically: KYYGNGVTCGKHSCSVDWGKATTCIINNGAMAWATGGHQGNHKC are provided.
Further, the host bacillus subtilis is bacillus subtilisB. subtilis WB800N；
Further, the expression vector of the recombinant protein is an escherichia coli-bacillus subtilis shuttle expression vector pHT 43.
The invention also provides a method for preparing the bacillus subtilis with the CGMCC NO: 23102A process for the preparation of bacteriocin papA, comprises:
streaking the recombinant bacillus subtilis on a 2YT solid culture medium containing chloramphenicol for activation, and performing inverted culture at 30-40 ℃ for overnight; selecting colonies, inoculating the colonies in a 2YT liquid culture medium, and culturing at 30-40 ℃ and 180-250 rpm overnight; then inoculating the strain into 2YT liquid culture medium containing chloramphenicol according to the volume percentage of 1-2%, culturing for 2-4 h at 30-40 ℃ and 180-250 rpm; adding IPTG with the final concentration of 0.1-1 mM, culturing at 28-30 ℃ and 180-; and after the culture is finished, taking the bacterial liquid, centrifuging for 15-25 min at 10000-.
The invention also provides the bacillus subtilis CGMCC NO: 23102 a large scale fermentation process wherein the media used are: 20 g/L peptone, 16 g/L yeast powder and 5 g/L NaCl2(ii) a The fermentation culture conditions are as follows: the fermentation temperature is 28-30 ℃, the rotation speed is 180-.
The invention also provides application of the recombinant bacillus subtilis in preparation of a drug or feed for inhibiting listeria monocytogenes.
The method specifically comprises the following steps:
the recombinant bacillus subtilis and/or the supernatant obtained after fermentation of the recombinant bacillus subtilis are/is prepared into a drug for inhibiting the listeria monocytogenes, or the recombinant bacillus subtilis and/or the supernatant obtained after fermentation of the recombinant bacillus subtilis are/is added into feed.
Compared with the prior art, the invention has the beneficial effects that:
the research constructs a PapA bacillus expression vector pHT43-papA2, and transforms a Bacillus subtilis recombinant strain; after induced expression and bacterial liquid centrifugation, the collected expression supernatant has inhibitory activity to Listeria monocytogenes, and subsequent treatments such as enzyme digestion of recombinant protein are omitted. The recombinant protein for inducing expression is named as PA 2; bacteriostatic experiments show that PA2 expressed by the bacillus subtilis has better inhibiting effect on Listeria monocytogenes than Lactobacillus plantarum Zhang-LL, and shows a large bacteriostatic zone.
Drawings
FIG. 1 is a schematic diagram of the construction of the recombinant shuttle expression plasmid pHT43-papA2 of Escherichia coli-Bacillus subtilis;
FIG. 2 is a diagram of the experiment of suppression of the supernatant of the DBN-SKL-PA2 induced expression of recombinant Bacillus subtilis to Listeria monocytogenes.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
Example 1: construction of recombinant shuttle expression plasmid pHT43-papA2
1. PCR amplification of the mature peptide gene fragment papA of phytolactobacillin.
Using Lactobacillus plantarum Zhang-LL genome as a template and papAF and papAR containing restriction enzyme sites as primers to carry out PCR amplification on target fragments of the Lactobacillus plantarum genepapADetecting the amplified product by 1% agarose gel electrophoresis, wherein the size of the PCR product is consistent with the expected size, and recovering and purifying the gel (the used reagent is from TaKaRa);
upstream primer (papAF: 5' -CG)GGATCCAAATACTACGGTAATGGGGT-3', underlinedBamH i cleavage site);
downstream primer (papAR: 5' -GC)TCTAGACTAGCATTTA TGATTACCTT G-3', underlinedXbaI cleavage site).
PCR reaction (50. mu.L): 5 XPrimerSTAR GXL Buffer 10. mu.L, dNTP 4. mu.L, primer papAF 1. mu.L, primer papAR 1. mu.L, DNA template 1. mu.L, PrimerSTAR DNA polymerase 1. mu.L, sterile water 32. mu.L.
PCR procedure: 3min at 95 ℃; 15sec at 98 ℃, 15sec at 55 ℃, 30sec at 72 ℃, 35 cycles; 7min at 72 ℃.
2、papAPCR products and cleavage of plasmid pHT 43.
Using restriction enzymesBamHI andXbapair vector pHT43 and purifiedpapAAnd (3) performing double enzyme digestion on the PCR products respectively, and purifying and recovering the enzyme digestion products.
3. Ligation and transformation
The gene fragment after enzyme digestionpapAAnd pHT43 were ligated by T4 DNA ligase (TaKaRa Co., Ltd.), the ligated product was transformed into E.coli Trans10 (all-gold) competent cells, single clones were selected on 2YT plates containing 100. mu.g/mL ampicillin, and positive transformants containing the recombinant shuttle expression plasmid pHT43-papA2 were obtained after colony PCR and sequencing verification. The construction of pHT43-papA2 is schematically shown in FIG. 1.
Example 2: construction of recombinant Bacillus subtilis containing recombinant shuttle expression plasmid pHT43-papA2
Plasmids were extracted from the E.coli Tans10 positive transformants containing the recombinant shuttle plasmid pHT43-papA2 constructed in example 1 using a plasmid extraction kit (Dalian TaKaRa); transforming the pHT43-papA2 into a Bacillus subtilis WB800N strain by adopting a Spizizizien transformation method, screening the strain on a 2YT plate with 5 mu g/mL chloramphenicol, and verifying the colony PCR to obtain the Bacillus subtilis containing the plasmid pHT43-papA2B. subtilisDBN-SKL-PA 2. The strain is preserved in China general microbiological culture Collection center (CGMCC) at 2021, 8 months and 2 days, the preservation date is 2021, 8 months and 2 days, and the preservation number is as follows: CGMCC NO: 23102, the preservation address is No. 3 Xilu Beijing province, Chaoyang district, Beijing.
Example 3: induced expression of Bacillus subtilis DBN-SKL-PA2
1. Preparation of seed bacteria suspension:
2YT liquid medium: yeast powder 10.0 g/L, tryptone 16.0 g/L, sodium chloride 5.0 g/L, pH7.0-7.2, liquid 50 mL/250mL triangular flask, 121 ℃ sterilization for 20 min.
2YT solid medium: 5.0 g/L yeast powder, 10.0 g/L tryptone, 10.0 g/L sodium chloride, 20.0 g/L agar and deionized water as a solvent, wherein the pH value is 7.0-7.2, the mixture is filled into a triangular flask with 100mL/250mL liquid, and the mixture is sterilized at 121 ℃ for 20 min.
The bacillus subtilis DBN-SKL-PA2 is streaked on a 2YT solid culture medium containing 5 mug/mL chloramphenicol resistance for activation, and inverted culture is carried out at 37 ℃ for 24; selecting single colony, inoculating to 2YT liquid culture medium containing 5 ug/mL chloramphenicol, culturing at 37 deg.C and 200rpm overnight;
2. inducible expression of recombinant PA2 protein
Inoculating 2% by volume of the culture medium into 2YT liquid culture medium containing 5 μ g/mL chloramphenicol, culturing at 37 deg.C and 200rpm for 3 h; adding IPTG with the final concentration of 0.1 mM, and inducing for 4 h at 28 ℃ and 200 rpm; and after the culture is finished, taking a bacterial liquid, centrifuging for 10 min at 10000 rpm, and taking a supernatant, namely an expression supernatant of a recombinant protein PA2 containing the mature peptide of the phytolactobacillin, wherein the PA2 contains a signal peptide of alpha-amyloase from a carrier and the mature peptide of papA.
Test example 1 study on the ability of Bacillus subtilis DBN-SKL-PA2 to inhibit Listeria monocytogenes by inducible expression of supernatant
Preparing lactobacillus plantarum Zhang-LL fermentation supernatant: inoculating lactobacillus plantarum Zhang-LL into an MRS culture medium, standing and culturing overnight at 37 ℃, and inoculating MRS according to a proportion of 2% for standing and culturing for 24 hours; collecting bacterial liquid, centrifuging, and separating supernatant to obtain Zhang-LL fermentation supernatant (Z) containing phytolactobacillin, wherein the fermentation supernatant (Z) is used as a control group. Preparing a listeria monocytogenes suspension: listeria monocytogenes ATCC 54003 is cultured by a TSBYE culture medium ((g/L) tryptone, 17.0; soybean peptone, 3.0; yeast extract powder, 6.0; sodium chloride, 5.0; dipotassium hydrogen phosphate, 2.5; glucose, 2.5; pH value 7.3 +/-0.1; 121 ℃, 15 min high-pressure sterilization), the bacterial suspension is diluted by 10 times by using a blank culture medium, and is an effective pathogenic bacteria suspension when OD600 is between 0.30 and 0.36, and the content of pathogenic bacteria in a pathogenic bacteria suspension stock solution is about 10^9 cfu/ml.
The test method comprises the following steps: placing a plurality of Oxford cups in each sterile plate at equal intervals, diluting the pathogenic bacteria stock solution to about 10^6 cfu/ml, adding 1 ml of acid hydrolyzed casein (1/9) into each 9 ml of MH semisolid nutrient agar ((g/L) beef powder, 2.0; soluble starch, 1.5; acid hydrolyzed casein, pH 7.4 +/-0.2; agar powder, 1%, 121 ℃, autoclaving for 15 min, keeping the temperature to 60 ℃), directly pouring the mixture into the sterile culture plate after shaking and mixing uniformly, and slowly rotating the plate to uniformly spread the culture medium. After the mixture is solidified, carefully clamping the oxford cup by using a sterile forceps, and keeping the oxford cup at 4 ℃ for later use; and adding 100 mu L of fermentation supernatant to be detected, namely expression supernatant of PA2 recombinant protein and fermentation supernatant of lactobacillus plantarum Zhang-LL into each hole, covering a dish cover, carefully moving to an incubator at 37 ℃, and placing and standing for culture on a flat dish. After 20 (18-24) hours of incubation, the capsule was opened, the Oxford cup removed, and the zone diameter was measured with a caliper, the results of which are shown in Table 1.
According to the test results, the PA2 has bacteriostatic activity on Listeria monocytogenes, and the bacteriostatic zone is larger than that of Zhang-LL, which shows that the effect is superior to that of fermentation supernatant of Zhang-LL. The bacteriostatic effect of PA2 and Zhang-LL on Listeria monocytogenes is shown in figure 2.
Sequence listing
<110> Zhejiang Dabei agriculture, agriculture and animal technology Co Ltd, Jiangxi Dabei agriculture, agriculture and animal technology Co Ltd
<120> recombinant bacillus subtilis and construction method and application thereof
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 135
<212> DNA
<213> Lactobacillus plantarum
<400> 1
aaatactacg gtaatggggt tacttgtggc aaacattcct gctctgttga ctggggtaag 60
gctaccactt gcataatcaa taatggagct atggcatggg ctactggtgg acatcaaggt 120
aatcataaat gctag 135
<210> 2
<211> 44
<212> PRT
<213> Lactobacillus plantarum (Lactobacillus plantarum)
<400> 2
Lys Tyr Tyr Gly Asn Gly Val Thr Cys Gly Lys His Ser Cys Ser Val
1 5 10 15
Asp Trp Gly Lys Ala Thr Thr Cys Ile Ile Asn Asn Gly Ala Met Ala
20 25 30
Trp Ala Thr Gly Gly His Gln Gly Asn His Lys Cys
35 40
<210> 3
<211> 28
<212> DNA
<213> Artificial sequences (artificial series)
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cgggatccaa atactacggt aatggggt 28
<210> 4
<211> 29
<212> DNA
<213> Artificial sequences (artificial series)
<400> 4
gctctagact agcatttatg attaccttg 29
Claims (7)
1. A recombinant Bacillus subtilis strain characterized by the following accession numbers: CGMCC NO: 23102.
2. a method for expressing the plant lactobacillus papA by using the recombinant bacillus subtilis as the claim 1, which is characterized in that the recombinant bacillus subtilis is streaked on a 2YT solid culture medium containing chloramphenicol for activation and is inversely cultured at 30-40 ℃ for overnight; selecting colonies, inoculating the colonies in a 2YT liquid culture medium, and culturing at 30-40 ℃ and 180-250 rpm overnight; then inoculating the strain into 2YT liquid culture medium containing chloramphenicol according to the volume percentage of 1-2%, culturing for 2-4 h at 30-40 ℃ and 180-250 rpm; adding IPTG with the final concentration of 0.1-1 mM, culturing at 28-30 ℃ and 180-; and after the culture is finished, taking the bacterial liquid, centrifuging for 15-25 min at 10000-.
3. The method of claim 2, wherein the 2YT solid medium comprises 5.0 g/L yeast powder, 10.0 g/L tryptone, 10.0 g/L sodium chloride, 20.0 g/L agar, deionized water as a solvent, pH7.0-7.2, 100mL/250mL triangular flask, and is sterilized at 121 ℃ for 20 min.
4. The method of claim 2, wherein the 2YT broth is prepared from yeast powder 10.0 g/L, tryptone 16.0 g/L, sodium chloride 5.0 g/L, pH7.0-7.2, 50 mL/250mL Erlenmeyer flask, sterilized at 121 ℃ for 20 min.
5. The large-scale fermentation process of recombinant Bacillus subtilis according to claim 1, wherein the culture medium used is: 20 g/L peptone, 16 g/L yeast powder and 5 g/L NaCl2Sterilizing at 121 deg.C for 20 min; the fermentation culture conditions are as follows: the fermentation temperature is 28-30 ℃, the rotation speed is 180-.
6. Use of the recombinant bacillus subtilis of claim 1 for the preparation of a medicament or feed for inhibiting listeria monocytogenes.
7. The use according to claim 6, wherein the recombinant Bacillus subtilis of claim 1 and/or a supernatant after fermentation thereof is prepared as a medicament for inhibiting Listeria monocytogenes, or the recombinant Bacillus subtilis of claim 1 and/or a supernatant after fermentation thereof is added to feed.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6451365B1 (en) * | 2000-07-14 | 2002-09-17 | Rhodia Inc. | Antibacterial composition for control of gram positive bacteria in food applications |
| CN104611286A (en) * | 2015-02-10 | 2015-05-13 | 南京财经大学 | Recombinant bacterium for preparing pediocin as well as preparation method and application thereof |
| CN110791508A (en) * | 2019-11-18 | 2020-02-14 | 深圳市圣西马生物技术有限公司 | Codon-optimized pediocin gene, expression vector and recombinant engineering strain |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6451365B1 (en) * | 2000-07-14 | 2002-09-17 | Rhodia Inc. | Antibacterial composition for control of gram positive bacteria in food applications |
| CN104611286A (en) * | 2015-02-10 | 2015-05-13 | 南京财经大学 | Recombinant bacterium for preparing pediocin as well as preparation method and application thereof |
| CN110791508A (en) * | 2019-11-18 | 2020-02-14 | 深圳市圣西马生物技术有限公司 | Codon-optimized pediocin gene, expression vector and recombinant engineering strain |
Non-Patent Citations (2)
| Title |
|---|
| 片球菌素pedA基因的原核表达、纯化及生物信息学分析;黄宇良等;《生物技术通报》;20171231;第33卷(第12期);摘要，图6-7，第146页右栏，第147页右栏第1-3段，第149页右栏第1-2段 * |
| 重组胰高血糖素样肽-1载体的构建及其在枯草芽孢杆菌中的表达;袁婧等;《江苏大学学报（医学版）》;20190531;第29卷(第3期);摘要，第199页左栏 * |
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