CN113717973B - Japanese eel liver expression antibacterial peptide LEAP2 gene promoter and application thereof - Google Patents
Japanese eel liver expression antibacterial peptide LEAP2 gene promoter and application thereof Download PDFInfo
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Classifications
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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
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/461—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from fish
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New breeds of animals
- A01K67/027—New breeds of vertebrates
- A01K67/0275—Genetically modified vertebrates, e.g. transgenic
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- 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
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- 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
- C12N15/746—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for lactic acid bacteria (Streptococcus; Lactococcus; Lactobacillus; Pediococcus; Enterococcus; Leuconostoc; Propionibacterium; Bifidobacterium; Sporolactobacillus)
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- 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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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- 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
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
- C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- 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
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2227/00—Animals characterised by species
- A01K2227/40—Fish
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2267/00—Animals characterised by purpose
- A01K2267/01—Animal expressing industrially exogenous proteins
Abstract
The invention relates to a Japanese eel liver expression antibacterial peptide LEP2 gene promoter and application thereof. The invention successfully clones the Japanese eel liver expressed antibacterial peptide LEP2 gene promoter sequence, and successfully constructs a Japanese eel liver expressed antibacterial peptide LEAP2 gene promoter pGL3-LEAP2-pro luciferase reporter plasmid; experiments prove that the Japanese eel liver expressed antibacterial peptide LEAP2 gene promoter can be induced and activated by virus mimics polyI: C, gram negative bacteria important surface antigen LPS and aeromonas hydrophila. The invention provides a good experimental system for researching the expression regulation mechanism of the Japanese eel liver expression antibacterial peptide LEAP2 gene and the natural immune response mechanism of fish against pathogenic bacteria infection, in particular for researching important fish inflammation related NF-kappaB and MAPK signal pathway network regulation mechanism, and creates conditions for constructing an expression vector to efficiently express exogenous genes or applying the promoter to transgenic fish construction by using the promoter.
Description
Technical Field
The invention relates to the technical field of genetic engineering, in particular to a Japanese eel liver expression antibacterial peptide LEP2 gene promoter and application thereof.
Background
Antibacterial peptides (antimicrobial peptides, AMPs) are a class of bioactive small molecule polypeptides, are important components of the innate immune defense system of the organism, can have better inhibition or killing effects on gram-negative bacteria, gram-positive bacteria, fungi, viruses, parasites and the like, and play an important role in natural immune response [ Chung, C.—R., J.— H.Jhong, Z.Wang, W.Chen, wan, horng and T.—Y.Lee (2020), "Characterization and Identification of Natural Antimicrobial Peptides on Different organics." International Journal of Molecular Sciences 21:986.]. Fish, like higher vertebrates such as mammals, have an innate and adaptive immune system. Antibacterial peptides, which are important components of the innate immune system of fish, are rapidly produced and spread in the body to play a defensive and killing role when the fish body is injured or affected by pathogenic microorganisms.
Fish antimicrobial peptides consist essentially of Cathelicidins, liver expressed antimicrobial peptide-2 (Liverexpressed Antimicrobial Peptide, leap-2), piscidins, beta-defensins, hepcidins, and NKlysins [ Ji Zhitao, xu Yang, jun, nie Pin (2020), "aquatic animal antimicrobial peptide research progress," aquatic journal 44 (09): 1572-1583 ]. Liver expressed antimicrobial peptide is a small molecule cationic protein, mainly expressed in the liver, with four highly conserved cysteines forming two pairs of disulfide bonds, playing a key role in killing bacteria (Ma et al 2009). Have been identified in a variety of fish species such as Japanese eel, golden pomfret (Trachinotus ovatus) rainbow trout (Oncorhynchus mykiss), channel catfish (Ictalurus punctatus), marchant (Megalobrama amblycephala), grass carp (Ctenopharyngodon idella), large yellow croaker (Larimichthys crocea), carp (Cyprinus carpio L.).
However, studies on the expression of an antimicrobial peptide promoter by the liver of Fish have been reported only in golden pomfret [ Liu, b., g.—d.liu, h.—y.guo, k.— C.Zhu, L.Guo, N.Zhang, b.—s.liu, s.—g.jiang and d.—c.zhang (2020), "Characterization and functional analysis of liver-expressed antimicrobial peptide-2 (LEAP-2) from golden pompano Trachinotus ovatus (Linnaeus 1758)," fishe & Shellfish Immunology 104:419-430; the research on gene functions and transcriptional regulation of two antibacterial peptides of trachinotus ovatus, such as a master paper, liu Andong, tianjin institute 2019, and English paper, is a part of the master paper, the content of which only relates to the research on transcriptional activity of the promoter in human cells 293 cells, and the immune regulation mechanism of whether the liver expressed antibacterial peptide promoter has transcriptional activity in fish cells and can be induced to be expressed by pathogenic microorganisms such as viruses and bacteria has not been reported yet.
Expression regulation of genes has become a hotspot in the field of molecular biology research, and promoters are important elements of gene expression regulation. In view of the importance of fish LEAP2 in antibacterial and viral immunity and disease control thereof, research of a gene expression regulation mechanism thereof provides a new idea for controlling fish bacterial and viral diseases by regulating the expression of LEAP 2.
The promoter is a key factor for determining gene expression and regulation thereof, and in order to research the expression regulation mechanism of fish LEAP2 gene, we obtain possible 5' flanking regulatory region sequences of the Japanese eel LEAP2 gene through comparing Japanese eel LEAP2 open reading frame sequences with genome sequences, and obtain Japanese eel LEAP2 gene promoter sequences through primer design PCR cloning verification, and analysis shows that the transcription binding sites of HNF-3B, C/EBPalp, sp1, USF, NF-kappa B, GATA and the like in addition to the transcription binding sites of HNF-3B, C/EBPalp, sp1, USF, NF-kappa B, GATA in the prior reported golden pomfret LEAP2 promoter also exist specific IRF1, ICSBP, NF-EM5, SRY, lyF-1, c-Jun, MATAalpha 2, elf-1, zen-1 and the like.
The report gene detection proves that the Japanese eel LEAP2 gene promoter has stronger promoter activity, and can be expressed by gram negative bacteria indicating important antigen LPS, aquatic organism important pathogenic bacteria aeromonas hydrophila and artificially synthesized double-stranded RNA polyI: C induction. Therefore, the cloning of the Japanese eel liver expression antibacterial peptide LEAP2 gene promoter and the induced expression analysis of the strong promoter activity thereof provide a good experimental system for researching the expression regulation mechanism of the Japanese eel liver expression antibacterial peptide LEAP2 gene and the natural immune response mechanism of fish against pathogenic bacteria infection, particularly the research of important fish inflammation related NF- κB and MAPK signal pathway network regulation mechanism, and create conditions for constructing an expression vector by using the promoter to efficiently express exogenous genes or applying the promoter to transgenic fish construction in application, thereby having important theoretical and practical significance.
Disclosure of Invention
The invention aims to overcome the defects of the prior art, provides a Japanese eel liver expression antibacterial peptide LEAP2 gene promoter and application thereof, and solves the problems in the background art.
One of the technical schemes adopted for solving the technical problems is as follows: provides a Japanese eel liver expressed antibacterial peptide LEAP2 gene promoter, the nucleotide sequence of which is shown in SEQ ID NO: 1.
The second technical scheme adopted by the invention for solving the technical problems is as follows: an expression cassette, recombinant vector, transgenic cell line, recombinant bacterium or recombinant virus containing the promoter is provided.
Preferably, the expression cassette is composed of the above-described promoter, a target gene whose transcription is promoted by the above-described promoter, and a terminator.
Preferably, the recombinant vector is pGL3-Basic, pGL2-Basic, pGL4.10, pGLuc.
Preferably, the recombinant bacteria are escherichia coli, bacillus subtilis, lactobacillus and saccharomycetes.
The third technical scheme adopted by the invention for solving the technical problems is as follows: the application of the Japanese eel liver expression antibacterial peptide LEAP2 gene promoter in constructing eukaryotic expression vectors, fish cells or mammalian cells for efficiently expressing exogenous genes is provided.
The fourth technical scheme adopted for solving the technical problems is as follows: provides the application of the Japanese eel liver expression antibacterial peptide LEAP2 gene promoter in constructing transgenic fish.
The invention has the following beneficial effects: according to the invention, a Japanese eel liver expression antibacterial peptide LEAP2 gene open reading frame first exon sequence is compared and analyzed to obtain a Japanese eel genome, and a landing PCR method is adopted to clone a 5' flanking region sequence of the liver expression antibacterial peptide LEAP2 gene, so as to obtain a Japanese eel liver expression antibacterial peptide LEAP2 gene promoter sequence; the report gene analysis experiment proves that the Japanese eel liver expressed antibacterial peptide LEAP2 gene promoter can be synthesized into double-stranded RNA poly I: C, gram negative bacteria escherichia coli important surface antigen LPS and aquatic animal important pathogenic bacteria aeromonas hydrophila induced activation. Therefore, the cloning of the Japanese eel liver expression antibacterial peptide LEAP2 gene promoter and the verification of the strong promoter activity thereof theoretically provide a good experimental system for researching the expression regulation mechanism of the fish liver expression antibacterial peptide LEAP2 gene and the antibacterial infection mechanism of important fish inflammation related functional genes, particularly the research of important fish inflammation related NF-kappa B and MAPK signal pathway network regulation mechanism, and create conditions for constructing an expression vector to efficiently express exogenous genes or applying the promoter to transgenic fish construction in application, thereby having important theoretical and practical significance.
Drawings
FIG. 1 is a schematic diagram 1 of the transcription factor binding site of the gene promoter of the Japanese eel liver expressed antibacterial peptide LEAP 2.
FIG. 2 is a schematic diagram 2 of the transcription factor binding site of the gene promoter of the liver expressed antibacterial peptide LEAP2 of Japanese eel.
FIG. 3 is a graph showing the quantitative analysis of the activity of the Japanese eel liver expressed antibacterial peptide LEAP2 gene promoter using a dual-luciferase reporter gene detection system.
The abscissa pGL3 represents the relative luciferase activity of the empty vector pGL3-Basic transfected EPC cells (as control group);
pGL3-LEAP2-pro luciferase relative activity of recombinant vector pGL3-LEAP2-pro transfected EPC cells (as experimental group).
As shown in FIG. 3, the relative activity of luciferase in EPC transfected cells transfected by recombinant vector pGL3-LEAP2-pro is 10.1 times that of EPC transfected by empty vector pGL3-Basic, which shows that the Japanese eel liver expressed antibacterial peptide LEAP2 gene promoter can better start transcription of luciferase reporter gene.
Three replicates were set for each experiment, each replicate being set in three parallels; error bars represent standard error of the mean. Statistical analysis of the significance differences between the experimental and control groups using the two-tailed group T-test, "x" p <0.05, "x" p <0.01.
FIG. 4 is a graph showing the change in activity of the promoter of the Japanese eel liver expressed antibacterial peptide LEAP2 gene under stimulation of the gram-negative E.coli important surface antigen LPS (30. Mu.g/mL).
The abscissa pGL3-Basic represents the relative luciferase activity of the empty vector pGL3-Basic transfected EPC cells (as control group);
pGL3-LEAP2-pro luciferase relative activity of recombinant vector pGL3-LEAP2-pro transfected EPC cells (as experimental group).
As shown in FIG. 4, the relative activity of luciferase in EPC transfected cells transfected with 24h recombinant vector pGL3-LEAP2-pro stimulated by LPS was 16.0 times that of EPC transfected with empty vector pGL3-Basic, indicating that the Japanese eel liver expression antibacterial peptide LEAP2 gene promoter can be induced and activated by LPS.
Three replicates were set for each experiment, each replicate being set in three parallels; error bars represent standard error of the mean. Statistical analysis of the significance differences between the experimental and control groups using the two-tailed group T-test, "x" p <0.05, "x" p <0.01.
FIG. 5 shows the pathogenic bacteria Aeromonas hydrophila (10) 6 cfu/mL) of the activity change pattern of the Japanese eel liver expressed antibacterial peptide LEAP2 gene promoter under stimulation.
The abscissa pGL3-Basic represents the relative luciferase activity of the empty vector pGL3-Basic transfected EPC cells (as control group);
pGL3-LEAP2-pro luciferase relative activity of recombinant vector pGL3-LEAP2-pro transfected EPC cells (as experimental group).
As shown in FIG. 5, the relative activity of luciferase in EPC transfected cells transfected with recombinant vector pGL3-LEAP2-pro for 6h stimulated by Aeromonas hydrophila is 6.9 times that of EPC transfected with empty vector pGL3-Basic, which shows that the promoter of Japanese eel liver expressed antibacterial peptide LEAP2 gene can be induced and activated by Aeromonas hydrophila.
Three replicates were set for each experiment, each replicate being set in three parallels; error bars represent standard error of the mean. Statistical analysis of the significance differences between the experimental and control groups using the two-tailed group T-test, "x" p <0.05, "x" p <0.01.
FIG. 6 is a graph showing the change in activity of the promoter of the Japanese eel liver expressed antibacterial peptide LEAP2 gene under stimulation of the artificial synthesis of double-stranded RNA polyI: C (50. Mu.g/mL) by a virus mimetic.
The abscissa pGL3-Basic represents the relative luciferase activity of the empty vector pGL3-Basic transfected EPC cells (as control group);
pGL3-LEP2-pro luciferase relative activity of recombinant vector pGL3-LEP2-pro transfected EPC cells (as experimental group).
As shown in FIG. 6, the relative activity of luciferase in EPC transfected cells transfected with recombinant vector pGL3-LEP2-pro for 24h by poly I: C was 3.8 times that of EPC transfected with empty vector pGL3-Basic, indicating that the Japanese eel liver expression antibacterial peptide LEAP2 gene promoter can be induced and activated by poly I: C.
Three replicates were set for each experiment, each replicate being set in three parallels; error bars represent standard error of the mean. Statistical analysis of the significance differences between the experimental and control groups using the two-tailed group T-test, "x" p <0.05, "x" p <0.01.
Detailed Description
For a better understanding of the present invention, reference will now be made in detail to the following examples and accompanying drawings, which are included to provide a further understanding of the invention, and it is to be understood by those skilled in the art that the following examples are not intended to limit the scope of the invention.
The experimental methods used in the following examples are conventional methods unless otherwise specified.
Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
EXAMPLE 1 cloning of the Japanese eel liver expressed antibacterial peptide LEAP2 Gene promoter
1. The genomic DNA of the muscle tissue of Japanese eel was extracted and purified using TaKaRa MiniBEST Universal Genomic DNA Extraction Kit Ver.5.0 kit. The specific operation is as follows:
1. 10mg of Japanese eel musculature was minced with a blade and placed in a 2mL centrifuge tube, 180. Mu.L of Buffer GL, 20. Mu.L of protease K and 10. Mu.L of RNase A (10 mg/mL) were added, and the mixture was lysed overnight in a 56℃water bath.
2. 200 mu L Buffer GB and 200 mu L100% ethanol are added into the lysate, and the mixture is fully sucked and uniformly mixed. Spin Column was mounted on a Collection Tube, the solution was transferred to Spin Column, centrifuged at 12,000rpm for 2 minutes, and the filtrate was discarded.
3. mu.L of BufferWA was added to Spin Column and centrifuged at 12,000rpm for 1 min, and the filtrate was discarded.
4. 700. Mu.L of BufferWB (with 100% ethanol added to the indicated volume before) was added to Spin Column around the tube wall, centrifuged at 12,000rpm for 1 min and the filtrate was discarded. 700. Mu.L of BufferWB was again added to Spin Column around the tube wall and centrifuged at 12,000rpm for 1 min, and the filtrate was discarded.
5. Spin Column was mounted on a Collection Tube and centrifuged at 12,000rpm for 2 minutes. The Spin Column was placed on a new 1.5mL centrifuge tube, 150. Mu.L of sterilized water heated to 65℃was added to the center of the Spin Column membrane, and the mixture was allowed to stand at room temperature for 5 minutes. The DNA was eluted by centrifugation at 12,000rpm for 2 minutes.
6. The genomic DNA thus extracted was subjected to absorbance measurement to determine its concentration.
2. The Japanese eel liver expressed antibacterial peptide LEAP2 gene promoter sequence is amplified by adopting a two-round touchdown PCR method. The method comprises the following specific steps:
1. the Japanese eel genome is compared and analyzed by a Japanese eel liver expression antibacterial peptide LEAP2 gene open reading frame first exon sequence (SEQ ID NO: 2), and an upstream primer ' 5'-CCTCACAAGGTTCACCGCAAGAC-3' (SEQ ID NO: 3) ' is a liver expression antibacterial peptide LEAP2 gene 5' flanking region sequence, and a downstream primer ' 5'-CAACACCAAACCGCAGGCAGT-3' (SEQ ID NO: 4) ' is a liver expression antibacterial peptide LEAP2 gene open reading frame first exon sequence, which is synthesized by Shanghai bioengineering company.
2. The first round of PCR was performed using Takara high fidelity enzyme 2XGC Buffer(Mg 2+ plus), the reaction system: 2X PrimeSTAR HS DNA Polymerase 12.5.5. Mu.L, upstream primer 0.5. Mu.L, downstream primer 0.5. Mu. L, gDNA 0.5.5. Mu.L, sterilized water 11. Mu.L; the touchdown PCR reaction procedure was 95℃for 5min;95 ℃ for 30s,60 ℃ for 30s,72 ℃ for 4min,4 cycles; 95 ℃ for 30s,58 ℃ for 30s,72 ℃ for 4min,4 cycles; 95 ℃ for 30s,56 ℃ for 30s,72 ℃ for 4min,30 cycles; 72 ℃ for 10min; 5min at 4 ℃.
3. The second PCR was performed using Takara 10 XEx Taq Buffer (Mg 2+ plus), the reaction system: ex Taq 0.13. Mu.L, 10 XEx Taq Buffer (Mg 2+ plus) 2.5. Mu.L, dNTP mix (2.5 mM each) 2. Mu.L, upstream primer 0.5. Mu.L, downstream primer 0.5. Mu.L, first round PCR product 0.5. Mu.L, sterile water 18.87. Mu.L; the touchdown PCR reaction procedure was 95℃for 5min;95 ℃ for 30s,60 ℃ for 30s,72 ℃ for 4min,4 cycles; 95 ℃ for 30s,58 ℃ for 30s,72 ℃ for 4min,4 cycles; 95 ℃ for 30s,56 ℃ for 30s,72 ℃ for 4min,30 cycles; 72 ℃ for 10min; 5min at 4 ℃.
And connecting the PCR product obtained by the second round of PCR amplification to a pMD19T-Simple vector of TaKaRa company for sequence determination and analysis, thereby obtaining the pMD19T-LEAP2-pro recombinant plasmid containing the Japanese eel liver expression antibacterial peptide LEAP2 gene promoter sequence.
The nucleotide sequence of the Japanese eel liver expressed antibacterial peptide LEAP2 gene promoter is shown in SEQ ID NO:1 is shown as follows:
the first exon sequence of the open reading frame of the Japanese eel liver expressed antibacterial peptide LEAP2 gene is shown as SEQ ID NO:2 is shown as follows:
ATGCACAAACAGGCCTGCAGTAGGGCAACAACTGCCTGCGGTTTGGTGTTG
example 2 prediction of the transcription factor binding site of the liver-expressed antibacterial peptide LEAP2 Gene of Japanese eel
On-line prediction software of the transcription factor binding site of the 5' flanking region of the Internet login gene, namely, alibaba2 (http:// gene-regulation.com/pub/programs/Alibaba 2/index.html), copies the promoter sequence of the liver expression antibacterial peptide LEAP2 gene obtained through cloning test verification, then pastes the copied promoter sequence in a dialog box in a fasta format, and clicks START to perform prediction analysis of the transcription factor binding site. The results are shown in FIG. 1:
the main transcription factor binding site of the Japanese eel liver expressed antibacterial peptide LEAP2 gene promoter is as follows:
example 3 analysis of Activity of the Japanese eel liver expressed antibacterial peptide LEAP2 Gene promoter
1. Construction of recombinant luciferase reporter vector pGL3-LEAP2-pro containing Japanese eel liver expression antibacterial peptide LEAP2 gene promoter fragment.
1. The Japanese eel liver expressed antibacterial peptide LEAP2 gene promoter fragment is inserted into a Luciferase reporter gene vector pGL3-Basic of Promega company, so that the expression of a firefly Luciferase (Luciferase) reporter gene is controlled by the Japanese eel liver expressed antibacterial peptide LEAP2 gene promoter, and the recombinant vector constructed is named pGL3-LEAP2-pro. The method comprises the following specific steps:
2. the upstream primer with MluI cleavage site was synthesized:
5'-CGACGCGTCCTCACAAGGTTCACCGCAAGAC-3' (SEQ ID NO: 5),
downstream primer with XhoI cleavage site:
5'-CCGCTCGAGCTTGAGGGAAGACGGATGGAGTG-3' (SEQ ID NO: 6).
3. High-fidelity enzyme from Takara 2X was usedGC Buffer(Mg 2+ plus), the reaction system: 2X PrimeSTAR HS DNA Polymerase 12.5.5. Mu.L, upstream primer 0.5. Mu.L, downstream primer 0.5. Mu. L, pMD19T-LEAP2-pro recombinant plasmid 0.5. Mu.LL, sterilized water 11. Mu.L; the touchdown PCR reaction procedure was 95℃for 5min;95 ℃ for 30s,60 ℃ for 30s,72 ℃ for 4min,4 cycles; 95 ℃ for 30s,58 ℃ for 30s,72 ℃ for 4min,4 cycles; 95 ℃ for 30s,56 ℃ for 30s,72 ℃ for 4min,30 cycles; 72 ℃ for 10min; 5min at 4 ℃. PCR product recovery was performed using Omega gel recovery kit.
4. The recovered PCR product and the vector pGL3-Basic were subjected to MluI/XhoI double digestion (Thermo Scientific Fermentas Fast Digest), respectively. The total of 40. Mu.L of the double enzyme digestion reaction system comprises 4. Mu.L of 10X FastDigest GreenBuffer, 2. Mu.L of each of enzyme MluI and enzyme XhoI, 1.5. Mu.g of each of carrier/PCR product and 40. Mu.L of sterilized water, and the enzyme digestion reaction is carried out after the above systems are uniformly mixed in a PCR tube, wherein the reaction procedures are as follows: 37 ℃ for 60min;80 ℃ for 20min;4 ℃ for 5min. The above-mentioned MluI/XhoI double-digested PCR product and vector pGL3-Basic were recovered by using Omega gel recovery kit, and the double-digested PCR product and vector pGL3-Basic were ligated with Takara T4 ligase in a ligation reaction system of 20. Mu.L, comprising 2. Mu.L of 10 XT 4 Buffer, 1. Mu. L T4 DNA ligase, 40ng of double-digested vector pGL3-Basic,300ng of double-digested PCR product, sterilized water to 20. Mu.L, and the above-mentioned systems were mixed in PCR tubes and ligated overnight at 16 ℃.
5. E.coli DH5 alpha competent cells are transformed by the above connection products, positive clones are screened by colony PCR, plasmids are extracted by using a small amount of endotoxin-free plasmid kit of Omega company, and the accuracy of inserting the promoter fragments is confirmed by sequencing, so that a recombinant luciferase reporter gene vector pGL3-LEAP2-pro containing the promoter fragments of the Japanese eel liver expressed antibacterial peptide LEAP2 gene is obtained.
2. And analyzing the basic activity of the gene promoter of the liver expressed antibacterial peptide LEAP2 of the Japanese eel by adopting a double-luciferase reporter gene detection system.
1. The EPC cells with better status were seeded into 48-well cell plates (1X 10) 5 And (3) adding an L15 culture medium (the L15 basal medium contains 10% Gibco Australian foetus calf serum), transferring into a constant temperature incubator at 28 ℃ for overnight culture, attaching the culture medium to a logarithmic phase, and carrying out transfection experiments when the attaching amount reaches about 80%. Cell culture medium was changed 2h before transfection.
For transfection, a transfection Reagent dilution was prepared with 0.5. Mu.L of Lipofectamine 3000Reagent transfection Reagent per well and 20. Mu.L of Opti-MEM low serum medium, and incubated at room temperature for 5min after mixing. Then 20 mu L of Opti-MEM low serum culture medium per well is fully mixed with the plasmids required per well, wherein a control group contains 20ng of Renilla luciferase reference reporter vector pRL-TK and 300ng of luciferase reporter vector pGL3-Basic vector, an experimental group contains 20ng of Renilla luciferase reference reporter vector pRL-TK and 300ng of recombinant luciferase reporter vector pGL3-LEAP2-pro, and then 0.5 mu L P3000 is added TM And (5) mixing the reagents uniformly. The prepared plasmid diluent is dropwise added into the transfection reagent diluent, the transfection complex solution is obtained after uniform mixing, the transfection complex solution is incubated for 15min at room temperature, EPC cell culture holes are slowly added, and the culture is carried out in a constant temperature incubator (28 ℃).
2.24h later, collecting transfected cells, respectively reading enzyme activity values of firefly luciferase and Renilla luciferase by using a double luciferase reporter gene detection system, and calculating the ratio of the enzyme activity values of the firefly luciferase and Renilla luciferase to obtain the relative activity of the luciferase in the transfected cells. The method for measuring the luciferase activity is carried out by referring to the instruction book of a dual-luciferase reporter gene detection system of Promega company, and comprises the following specific steps:
(1) Preparing reagents required by experiments: 1 XPLB lysate: 1 volume of 5X Passive Lysis Buffer is added with 4 volumes of double distilled water to be evenly mixed and prepared; start reagent (LAR i): completely dissolving Luciferase Assay Substrate powder in 10mL Luciferase Assay Buffer II solution, subpackaging with 1.5mL centrifuge tube, and storing in refrigerator at-80deg.C; stop reagent: 1 volume of 50X Stop was used as the experimental amount&GloSubstrate uses 49 volumes of Stop&Glo/>Buffer dilution.
(2) Cell culture medium in 48 well cell culture plates was slowly aspirated and 65. Mu.L of 1 XPLB lysate was added to each well.
(3) The 48-well cell culture plate was placed on a cell shaker and lysed by shaking for 15min.
(4) The lysed cell fluid was transferred to a 1.5mL centrifuge tube and centrifuged (13000 rpm,4 ℃,10 min).
(5) The supernatant after centrifugation was taken in 3. Mu.L in a 1.5mL centrifuge tube having good light transmittance.
(6) 10. Mu.L of Start reagent was added and the firefly luciferase activity value in the sample was measured using a GloMax 20/206 luminescence detector. Then 10 mu L Stop reagent is added to detect the luciferase activity value of the sea cucumber in the sample. The ratio of the two activities is the relative activity of luciferase of each sample.
The relative activities of pGL3-LEAP2-pro promoter were calculated using EPC cells co-transfected with empty vector pGL3-Basic and pRL-TK as control (see FIG. 3).
As shown in FIG. 3, the relative activity of luciferase in EPC transfected cells transfected by recombinant vector pGL3-LEAP2-pro is 10.1 times that of EPC transfected by empty vector pGL3-Basic, which shows that the Japanese eel liver expressed antibacterial peptide LEAP2 gene promoter can better start transcription of luciferase reporter gene.
3. Immunostimulatory experiments
EPC cells were transfected with pGL3-Basic and Japanese eel liver-expressed antibacterial peptide LEAP2 gene promoter recombinant vector pGL3-LEAP2-pro, respectively, together with Renilla luciferase internal reference reporter gene vector pRL-TK, respectively, after 12 hours of transfection, LPS (30. Mu.g/mL), poly I: C (50. Mu.g/mL) and Aeromonas hydrophila (10) were added to the cell culture solution, respectively 6 cfu/mL), transfected cells were collected for luciferase relative activity assays after 12h, and 6h of stimulation, respectively.
The change in activity of the Japanese eel liver expressed the antibacterial peptide LEAP2 gene promoter under LPS (30. Mu.g/mL) stimulation is shown in FIG. 4. The relative activity of luciferase in EPC cells transfected by recombinant vector pGL3-LEAP2-pro is 16.0 times that of EPC cells transfected by empty vector pGL3-Basic, which shows that the gene promoter of the Japanese eel liver expressed antibacterial peptide LEAP2 can be induced to activate by LPS, "x" p <0.05, "x" p <0.01.
In aeromonas (10) 6 cfu/mL) under stimulation condition, japanese eel liver expresses antibacterial peptide LEThe change in the activity of the promoter of the AP2 gene is shown in FIG. 5. The relative activity of luciferase in EPC cells transfected by pGL3-LEAP2-pro is 6.9 times that of EPC cells transfected by empty vector pGL3-Basic, which shows that the eel LEAP2 gene promoter of Japanese eel can be induced to activate by aeromonas hydrophila, "<0.05，“**”p<0.01。
The change in activity of the Japanese eel liver expressed the antibacterial peptide LEAP2 gene promoter under stimulation of poly I: C (50. Mu.g/mL) is shown in FIG. 6. The relative activity of luciferase in EPC cells transfected by recombinant vector pGL3-LEP2-pro is 3.8 times that of EPC cells transfected by empty vector pGL3-Basic, which shows that the gene promoter of the Japanese eel liver expressed antibacterial peptide LEAP2 can be induced and activated by poly I: C, "x" p <0.05, "x" p <0.01.
While specific embodiments of the invention have been described above, it will be appreciated by those skilled in the art that the specific embodiments described are illustrative only and not intended to limit the scope of the invention, and that equivalent modifications and variations of the invention in light of the spirit of the invention will be covered by the claims of the present invention.
Sequence listing
<110> university of beauty set
<120> a Japanese eel liver expressed antibacterial peptide LEP2 gene promoter and application thereof
<160> 6
<170> SIPOSequenceListing 1.0
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<211> 1742
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<213> Anguilla japonica
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cctcacaagg ttcaccgcaa gacacagagg atttgcaacc aagtactaaa tatgacaact 60
ttatttctga ttatgttaat ttgtccaatt aattttactt ccctaaaatg ggggctgtgt 120
ataaaaatag ctgcaattct tagacagatc tcccaatgtg gatgtaaata ccctaaaatt 180
taagctggca gttcgcactt ttaacctaat attcattgtt ttatttcaaa ttcattgtgc 240
tggagtatag taccaaaaat aaaaaataga taaaaaatat tatgtcactg tcctgataat 300
tttgtgtatc tgtatttcat atgaaatgta ttcttaatgc cacaaagcat tctgttggtg 360
atcaatattg aaactactga aagtgaaaca catgtgaaaa aacgaacaca tttgcactgt 420
tggaccactg ggtatttttg gtctgacctt tgtaacactt cagcaatctc cataaattgt 480
gaccaaaagg ctatctccac agctatcttt tgggtgggca aatttgattg gaagtgtctg 540
ttactggaaa ttgcactgtg ctcagtgaaa agatttgtag gattttgtct ctattctgct 600
tctttaattt agaaatttgt gtaaatgaat acatttaaat agttttgaaa atacttaaat 660
catgcagaaa attctttctc aaagcaaagt tttatttgat tggtgttgtt gaaactgttc 720
ctgctgcaaa tgacaagaat acactgtgtg ttgggtgaaa aaaggatttg ttttagacaa 780
gcttaaaaca ttttgctgaa atggacagta aggcactaaa tcaatgtgag atgggctgcg 840
gggcagtgtc acatagtttg catgtgtttg aacatttatc attcaaaaat atcttttgca 900
aaaaatgaaa ccagaatctg gtggaatatt ttggctttca gttttggtgc tttacaccaa 960
atttgcattc ctgtagtgta agtcctcttg gatcaaagta tctgctaagt aaatactaat 1020
atactaaata ctaaaacttc caagtaaagt acattgcgtt ctggaaactc aatattttgg 1080
atcaacctgg acttacattt tggatccaga caggttttca gcttcattct tattccctgt 1140
agccacagtt ttcaaaatcg aggttcatga ttcctgaaat tttttcagtt cccattcagt 1200
ccaaatcatg ccttctccct taatgaaatg ctaagggaat tttcataact gtaatgattg 1260
aatttcatta agaaatgtag tggcagccat ttcaggctgg tccacagtgg tcaaacagtt 1320
ttgaaacagt gattggtgca tgttaacagg atactgttta tacactctta ttaacagtgc 1380
ataatattga cggccttttt attctggact attattcaga gactggactg gagatggtgg 1440
gatggcaatg atgtactcgt tccatcttat cagcgttcgg tgccagaaag ctcagagctc 1500
ttggctcaca ccagagccta tgggacagtt tatctgtgcg gcgttcaaat atttactaaa 1560
cacgtgacat caccagggca caggaaagct gacttgggag ttgaagggga gcagatagcc 1620
tgcctgcgtc tgcggatttg gacaggaata taaaaggagg aacacagtgc caccgcttct 1680
ccagtctctc cagaaactct ccccaagcct cgttcatctc actccatccg tcttccctca 1740
ag 1742
<210> 2
<211> 51
<212> DNA
<213> Anguilla japonica
<400> 2
atgcacaaac aggcctgcag tagggcaaca actgcctgcg gtttggtgtt g 51
<210> 3
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 3
cctcacaagg ttcaccgcaa gac 23
<210> 4
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 4
caacaccaaa ccgcaggcag t 21
<210> 5
<211> 31
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 5
cgacgcgtcc tcacaaggtt caccgcaaga c 31
<210> 6
<211> 32
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 6
ccgctcgagc ttgagggaag acggatggag tg 32
Claims (7)
1. A promoter, which is characterized in that the promoter is a Japanese eel liver expressed antibacterial peptide LEAP2 gene promoter, and the nucleotide sequence of the promoter is shown in SEQ ID NO: 1.
2. An expression cassette, recombinant vector, transgenic cell line, recombinant bacterium or recombinant virus comprising the promoter of claim 1.
3. The expression cassette of claim 2, consisting of the promoter of claim 1, a gene of interest transcribed from the promoter of claim 1, and a terminator.
4. The recombinant vector of claim 2, wherein the recombinant vector is pGL3-Basic, pGL2-Basic, pGL4.10 or pGLuc.
5. The recombinant bacterium according to claim 2, wherein: the recombinant bacteria are escherichia coli, bacillus subtilis, lactobacillus or saccharomycetes.
6. The use of the Japanese eel liver expression antibacterial peptide LEAP2 gene promoter according to claim 1 in constructing eukaryotic expression vector or in efficiently expressing exogenous gene in fish cells or mammalian cells.
7. Use of the gene promoter of the antimicrobial peptide LEAP2 expressed in the liver of Japanese eel according to claim 1 for constructing transgenic fish.
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