CN113717973A

CN113717973A - Japanese eel liver expression antibacterial peptide LEP2 gene promoter and application thereof

Info

Publication number: CN113717973A
Application number: CN202110943773.3A
Authority: CN
Inventors: 冯建军; 彭欣慰; 林鹏; 王艺磊; 陈鹏云
Original assignee: Jimei University
Current assignee: Jimei University
Priority date: 2021-08-17
Filing date: 2021-08-17
Publication date: 2021-11-30
Anticipated expiration: 2041-08-17
Also published as: CN113717973B

Abstract

The invention relates to a Japanese eel liver expression antibacterial peptide LEP2 gene promoter and application thereof. According to the invention, a promoter sequence of the Japanese eel liver expression antibacterial peptide LEP2 gene is successfully cloned, and a Japanese eel liver expression antibacterial peptide LEAP2 gene promoter pGL3-LEAP2-pro luciferase report plasmid is successfully constructed; experiments prove that the promoter of the Japanese eel liver expression antibacterial peptide LEAP2 gene can be induced and activated by virus simulant poly I: C, gram-negative bacteria important surface antigen LPS and aeromonas hydrophila. The invention can provide a good experimental system for researching an expression regulation mechanism of the Japanese eel liver expression antibacterial peptide LEAP2 gene, a natural immune response mechanism of fish for resisting pathogenic bacteria infection, in particular an important research on a regulation mechanism of a NF-kB and MAPK signal path network related to fish inflammation, and creates conditions for constructing an expression vector to efficiently express a foreign gene by using the promoter or applying the promoter to the construction of transgenic fish.

Description

Japanese eel liver expression antibacterial peptide LEP2 gene promoter and application thereof

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

Antimicrobial Peptides (AMPs) are small Molecular polypeptides with biological activity, are important components of the innate immune defense system of the body, can well inhibit or kill gram-negative bacteria, gram-positive bacteria, fungi, viruses, parasites and the like, and play an important role in Natural immune response reaction [ Chung, C. -R., J. -H.Jhong, Z.Wang, W.Chen, Wan, Horng and T. -Y.Lee (2020). "chromatography and Identification of Natural Antimicrobial Peptides on Difference Organisms." International Journal of Molecular Sciences 21:986 ]. Fish, like higher vertebrates such as mammals, have innate and adaptive immune systems. The antibacterial peptide is an important component of the fish innate immune system, and can be rapidly produced and spread in vivo to play a role in defense and killing when fish bodies are damaged or invaded by pathogenic microorganisms.

The fish antibacterial Peptide mainly comprises Cathelicidins, liver-expressed antibacterial Peptide-2 (Lipex antibacterial Peptide 2, LEAP-2), Piscidins, beta-defensens, Hepcidins and NKlysins [ Zitao, xu Yang, Jun, Nees (2020). ] aquatic animal antibacterial Peptide research progress 44(09): 1572) 1583 ]. The liver-expressed antibacterial peptide is a small-molecule cationic protein, is mainly expressed in the liver, has four highly-conserved cysteines to form two pairs of disulfide bonds, and plays a key role in killing bacteria (Ma et al.2009). Has been identified in various fish species such as Japanese eels, golden pomfret (Trachinotus ovatus) rainbow trout (Oncorhynchus mykiss), channel catfish (Iculus puncatus), Megalobrama amblycephala (Megalobrama amblycephala), grass carp (Ctenophagogondon idella), large yellow croaker (Larimoches crocea), carp (Cyprinus carpio L.).

However, the research on the expression of antibacterial peptide promoter in Fish liver has 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). "chromatography and functional analysis of liver-expressed antibacterial peptide-2(LEAP-2) from gold and spleen catalysis, etc." Fish & Shell immunization 104: 419-430; the contents of the research on the gene functions and transcriptional regulation of two antibacterial peptides of trachinotus ovatus, a master paper, Liuguangdong, Tianjin agriculture academy 2019 and an English paper published as a part of the master paper only relate to the transcriptional activity research of the promoter in human cell 293 cells, and the mechanism of immune regulation on whether the liver expression antibacterial peptide promoter has transcriptional activity in fish cells and can be induced and expressed by pathogenic microorganisms such as viruses, bacteria and the like is not reported.

The regulation of gene expression has become a hotspot in the field of molecular biology research, and promoters are important elements for the regulation of gene expression. In view of the importance of the fish LEAP2 in resisting bacteria and virus immunity and preventing and treating diseases thereof, the research on a gene expression regulation mechanism of the fish LEAP2 provides a new idea for preventing and treating the bacteria and virus diseases of the fish by regulating the expression of LEAP 2.

A promoter is a key factor determining gene expression and regulation, in order to research an expression regulation mechanism of a fish LEAP2 gene, a possible sequence of a 5' flanking regulation region of a Japanese eel LEAP2 gene is obtained through comparison analysis of a Japanese eel LEAP2 open reading frame sequence and a genome sequence, a Japanese eel LEAP2 gene promoter sequence is obtained through primer design PCR cloning verification, and analysis shows that the specific transcription factor binding sites of IRF1, ICSBP, NF-EM5, SRY, LyF-1, c-Jun, MATalpha2, Elf-1, Zen-1 and the like exist besides the transcription binding sites of HNF-3B, C/EBPalp, Sp1, USF, NF-kappaB and GATA which are reported in a golden pompano LEAP2 promoter at present.

The reporter gene detection experiments prove that the Japanese eel LEAP2 gene promoter has stronger promoter activity, and can be expressed by gram-negative bacteria to show important antigen LPS, aquatic organism important pathogenic bacteria aeromonas hydrophila and artificially synthesized double-stranded RNA poly I: C for induced expression. Therefore, the cloning of the promoter of the anguilla japonica liver expression antibacterial peptide LEAP2 gene and the induced expression analysis of the strong promoter activity thereof provide a good experimental system for researching the expression regulation mechanism of the anguilla japonica liver expression antibacterial peptide LEAP2 gene, the natural immune response mechanism of fishes for resisting pathogenic bacteria infection, in particular the important research on the regulation mechanism of NF-kB and MAPK signal path network related to fish inflammation, and create conditions for constructing an expression vector to efficiently express an exogenous gene by using the promoter or applying the promoter to the construction of transgenic fishes in the application aspect, thereby having important theoretical and practical significance.

Disclosure of Invention

The invention aims to overcome the defects of the prior art, provides a promoter for expressing an antimicrobial peptide LEAP2 gene by liver of anguilla japonica and application thereof, and solves the problems in the background art.

One of the technical schemes adopted by the invention for solving the technical problems is as follows: provides a promoter of Japanese eel liver expression antibacterial peptide LEAP2 gene, the nucleotide sequence of which is shown as SEQ ID NO: 1 is shown.

The second technical scheme adopted by the invention for solving the technical problems is as follows: provides an expression cassette, a recombinant vector, a transgenic cell line, a recombinant bacterium or a recombinant virus containing the promoter.

Preferably, the expression cassette consists of the above-mentioned promoter, the target gene whose transcription is initiated by the above-mentioned promoter, and a terminator.

Preferably, the recombinant vector is pGL3-Basic, pGL2-Basic, pGL4.10 or pGLuc.

Preferably, the recombinant bacteria are escherichia coli, bacillus subtilis, lactic acid bacteria and yeast.

The third technical scheme adopted by the invention for solving the technical problems is as follows: provides the application of the promoter of the Japanese eel liver expression antibacterial peptide LEAP2 gene in constructing eukaryotic expression vectors, fish cells or mammalian cells to efficiently express exogenous genes.

The fourth technical scheme adopted by the invention for solving the technical problems is as follows: provides the application of the promoter of the Japanese eel liver expression antibacterial peptide LEAP2 gene in constructing transgenic fish.

The invention has the following beneficial effects: according to the invention, the Japanese eel genome is analyzed by comparing the first exon sequences of the open reading frame of the Japanese eel liver expression antibacterial peptide LEAP2 gene, and the 5' flanking region sequence of the liver expression antibacterial peptide LEAP2 gene is analyzed and predicted by adopting a touchdown PCR method, so that the Japanese eel liver expression antibacterial peptide LEAP2 gene promoter sequence is obtained; the reported gene analysis experiment proves that the promoter of the Japanese eel liver expression antibacterial peptide LEAP2 gene can be artificially 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 for inducing and activating. Therefore, the cloning of the promoter of the Japanese eel liver expression antibacterial peptide LEAP2 gene 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 an important fish inflammation related functional gene, in particular the important research of the fish inflammation related NF-kB and MAPK signal path network regulation mechanism, create conditions for constructing an expression vector to efficiently express an exogenous gene by using the promoter or applying the promoter to the construction of transgenic fish in the application aspect, and have important theoretical and practical significance.

Drawings

FIG. 1 is a schematic diagram 1 of Japanese eel liver expression antibacterial peptide LEAP2 gene promoter transcription factor binding site.

FIG. 2 is a schematic diagram of Japanese eel liver expression antibacterial peptide LEAP2 gene promoter transcription factor binding site 2.

FIG. 3 is a diagram of quantitative analysis of the activity of the promoter of Japanese eel liver expression antimicrobial peptide LEAP2 gene by using a dual-luciferase reporter gene detection system.

The abscissa pGL3 indicates the relative luciferase activity of the empty vector pGL3-Basic transfected EPC cells (as control);

pGL3-LEAP2-pro is the relative luciferase activity of recombinant vector pGL3-LEAP2-pro transfected EPC cells (as experimental group).

As shown in FIG. 3, the relative activity of luciferase in the EPC cell transfected by the recombinant vector pGL3-LEAP2-pro is 10.1 times that of the EPC cell transfected by the empty vector pGL3-Basic, which indicates that the promoter of the L.japonica liver expression antibacterial peptide LEAP2 gene can well promote the transcription of luciferase reporter gene.

Each experiment was performed in triplicate, three replicates each time; error bars represent standard error of the mean. The experimental group was statistically analyzed for significant differences from the control group using a two-tailed group T-test, "x" p <0.05, "x" p < 0.01.

FIG. 4 is a graph showing the activity change of the promoter of the Japanese eel liver expression antimicrobial peptide LEAP2 gene under the stimulation of the important surface antigen LPS (30. mu.g/mL) of gram-negative bacteria Escherichia coli.

The abscissa pGL3-Basic indicates the relative luciferase activity of the empty vector pGL3-Basic transfected EPC cells (as control);

As shown in FIG. 4, the relative activity of luciferase in EPC cells transfected by 24h recombinant vector pGL3-LEAP2-pro stimulated by LPS is 16.0 times that of EPC cells transfected by empty vector pGL3-Basic, which indicates that the promoter of the LEAP2 gene for expressing the antimicrobial peptide from the liver of Anguilla japonica can be induced and activated by LPS.

FIG. 5 shows Aeromonas hydrophila (10), an important pathogen of aquatic animals⁶cfu/mL) under the stimulation condition, the activity change diagram of the Japanese eel liver expression antibacterial peptide LEAP2 gene promoter.

As shown in FIG. 5, the relative activity of luciferase in EPC cells transfected by recombinant vector pGL3-LEAP2-pro stimulated by Aeromonas hydrophila 6h is 6.9 times that of EPC cells transfected by empty vector pGL3-Basic, which indicates that the promoter of the liver-expressed antimicrobial peptide LEAP2 gene of Anguilla japonica can be induced and activated by Aeromonas hydrophila.

FIG. 6 is a diagram showing the activity change of the promoter of Japanese eel liver expression antimicrobial peptide LEAP2 gene under the stimulation condition of virus simulant artificially synthesized double-stranded RNA poly I: C (50. mu.g/mL).

pGL3-LEP2-pro is the relative luciferase 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 cells transfected by 24h recombinant vector pGL3-LEP2-pro stimulated by poly I: C is 3.8 times that of EPC cells transfected by empty vector pGL3-Basic, which indicates that the promoter of the liver expression antibacterial peptide LEAP2 gene of anguilla japonica can be induced and activated by poly I: C.

Detailed Description

For better understanding of the present invention, the following embodiments and the accompanying drawings are used to describe the present invention in further detail, but those skilled in the art will appreciate that the following embodiments are not intended to limit the scope of the present invention, and any changes and modifications based on the present invention are within the scope of the present invention.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1 cloning of promoter of Japanese eel liver-expressed antimicrobial peptide LEAP2 Gene

Firstly, extracting and purifying the Japanese eel muscle tissue genome DNA by adopting a TaKaRa MiniBEST Universal Genomic DNA Extraction Kit Ver.5.0 Kit. The specific operation is as follows:

1. 10mg of Japanese eel muscle tissue was cut with a razor blade and placed in a 2mL centrifuge tube, and 180. mu.L of Buffer GL, 20. mu.L of protease K and 10. mu.L of RNase A (10mg/mL) were added thereto, followed by lysis in a 56 ℃ water bath overnight.

2. Add 200. mu.L Buffer GB and 200. mu.L 100% ethanol to the lysate, pipette well and mix well. The Spin Column was mounted on a Collection Tube, the solution was transferred to the Spin Column, centrifuged at 12,000rpm for 2 minutes, and the filtrate was discarded.

3. mu.L of BufferWA was added to Spin Column, centrifuged at 12,000rpm for 1 minute, and the filtrate was discarded.

4. mu.L of BufferWB (with the previously indicated volume of 100% ethanol added) was added to the Spin Column around the tube wall, centrifuged at 12,000rpm for 1 min, and the filtrate was discarded. mu.L of BufferWB was again added to the Spin Column around the tube wall, centrifuged at 12,000rpm for 1 minute, and the filtrate was discarded.

5. Spin Column was mounted on the Collection Tube and centrifuged at 12,000rpm for 2 minutes. Spin Column was placed on a new 1.5mL centrifuge tube, and 150 μ L of sterile water heated to 65 ℃ was added to the center of the Spin Column membrane and allowed to stand at room temperature for 5 minutes. DNA was eluted by centrifugation at 12,000rpm for 2 minutes.

6. The concentration of the extracted genomic DNA was measured by absorbance measurement.

Secondly, amplifying a promoter sequence of the Japanese eel liver expression antibacterial peptide LEAP2 gene by adopting a two-round touchdown PCR method. The method comprises the following specific steps:

1. the method comprises the steps of analyzing a genome of anguilla japonica by comparing a first exon sequence (shown as SEQ ID NO: 2) of an open reading frame of a liver expression antibacterial peptide LEAP2 gene, amplifying a 5 'flanking region sequence predicted to have the liver expression antibacterial peptide LEAP2 gene by analysis, wherein an upstream primer' 5'-CCTCACAAGGTTCACCGCAAGAC-3' (shown as SEQ ID NO: 3) 'is the 5' flanking region sequence of the liver expression antibacterial peptide LEAP2 gene, and a downstream primer '5'-CAACACCAAACCGCAGGCAGT-3'(shown as SEQ ID NO: 4)' is the first exon sequence of the open reading frame of the liver expression antibacterial peptide LEAP2 gene, and synthesizing the sequences by Shanghai bioengineering company.

2. The first round of PCR was extracted with Takara high-fidelity enzyme 2

GC Buffer(Mg²⁺plus), the reaction system: 2 XPrimeSTAR HS DNA Polymerase 12.5. 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 dropping PCR reaction program is 95 ℃ for 5 min; at 95 ℃ for 30s, at 60 ℃ for 30s, at 72 ℃ for 4min, for 4 cycles; at 95 ℃ for 30s, at 58 ℃ for 30s, at 72 ℃ for 4min, for 4 cycles; 30 cycles of 95 ℃ for 30s, 56 ℃ for 30s, and 72 ℃ for 4 min; 10min at 72 ℃; 5min at 4 ℃.

3. The second round of 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 (2.5mM each) 2. mu.L, forward primer 0.5. mu.L, reverse primer 0.5. mu.L, first round PCR product 0.5. mu.L, sterilized water 18.87. mu.L; the dropping PCR reaction program is 95 ℃ for 5 min; at 95 ℃ for 30s, at 60 ℃ for 30s, at 72 ℃ for 4min, for 4 cycles; 30s at 95 ℃, 30s at 58 ℃ and 4min at 72 ℃,4 cycles; 30 cycles of 95 ℃ for 30s, 56 ℃ for 30s, and 72 ℃ for 4 min; 10min at 72 ℃; 5min at 4 ℃.

And connecting the PCR product obtained by the second round of PCR amplification to a TaKaRa company pMD19T-Simple vector for sequence determination and analysis, thereby obtaining a pMD19T-LEAP2-pro recombinant plasmid containing a promoter sequence of the Japanese eel liver expression antibacterial peptide LEAP2 gene.

The nucleotide sequence of the promoter of the Japanese eel liver expression antibacterial peptide LEAP2 gene is shown as SEQ ID NO: 1, and the following components:

the sequence of the first exon of the open reading frame of Japanese eel liver expression antibacterial peptide LEAP2 gene is shown as SEQ ID NO: 2, as shown in the figure:

ATGCACAAACAGGCCTGCAGTAGGGCAACAACTGCCTGCGGTTTGGTGTTG

example 2 Japanese eel liver expression antimicrobial peptide LEAP2 gene promoter transcription factor binding site prediction

The online prediction software of transcription factor binding sites of the 5' flanking region of the internet access gene Alibaba2(http:// gene-regulation. com/pub/programs/Alibaba2/index. html), the promoter sequence of the liver expression antimicrobial peptide LEAP2 gene obtained by clone test verification is copied and then pasted in a dialog box in a fasta format, and START is clicked to perform prediction analysis of the transcription factor binding sites. The results are shown in FIG. 1:

the binding sites of the main transcription factors of the Japanese eel liver expression antibacterial peptide LEAP2 gene promoter are as follows:

example 3 Activity analysis of Japanese eel liver-expressed antimicrobial peptide LEAP2 Gene promoter

Firstly, constructing a recombinant luciferase reporter gene vector pGL3-LEAP2-pro containing a promoter fragment of a Japanese eel liver expression antibacterial peptide LEAP2 gene.

1. The promoter fragment of the Japanese eel liver expression antibacterial peptide LEAP2 gene is inserted into a Luciferase reporter gene vector pGL3-Basic of Promega company, so that the expression of the firefly Luciferase (Luciferase) reporter gene is controlled by the Japanese eel liver expression antibacterial peptide LEAP2 gene promoter, and the constructed recombinant vector is named as pGL3-LEAP 2-pro. The method comprises the following specific steps:

2. synthesizing an upstream primer with an MluI enzyme cutting site:

5'-CGACGCGTCCTCACAAGGTTCACCGCAAGAC-3' (SEQ ID NO: 5),

downstream primer with XhoI cleavage site:

5'-CCGCTCGAGCTTGAGGGAAGACGGATGGAGTG-3' (as shown in SEQ ID NO: 6).

3. Adopts high-fidelity enzyme 2 from Takara

GC Buffer(Mg²⁺plus), the reaction system: 2 XPrimeSTAR HS DNA Polymerase 12.5. mu.L, upstream primer 0.5. mu.L, downstream primer 0.5. mu. L, pMD19T-LEAP2-pro recombinant plasmid 0.5. mu.L, sterilized water 11. mu.L; the dropping PCR reaction program is 95 ℃ for 5 min; at 95 ℃ for 30s, at 60 ℃ for 30s, at 72 ℃ for 4min, for 4 cycles; at 95 ℃ for 30s, at 58 ℃ for 30s, at 72 ℃ for 4min, for 4 cycles; 30 cycles of 95 ℃ for 30s, 56 ℃ for 30s, and 72 ℃ for 4 min; 10min at 72 ℃; 5min at 4 ℃. PCR product recovery was performed using Omega gel recovery kit.

4. The recovered PCR product and the vector pGL3-Basic were digested with MluI/XhoI (Thermo Scientific Fermentas Fast Digest), respectively. The total amount of the double enzyme digestion reaction system is 40 mu L, comprising 4 mu L of 10 XFastdigest GreenBuffer, 2 mu L of each of enzyme MluI and enzyme XhoI, 1.5 mu g of each of vector/PCR product, and sterilized water is supplemented to 40 mu L, the system is uniformly mixed in a PCR tube for enzyme digestion reaction, and the reaction program is as follows: 60min at 37 ℃; 20min at 80 ℃; 4 ℃ for 5 min. Respectively recovering the MluI/XhoI double-enzyme-digested PCR product and the vector pGL3-Basic by using an Omega gel recovery kit, connecting the double-enzyme-digested PCR product and the vector pGL3-Basic by using Takara T4 ligase, wherein the connecting reaction system is 20 mu L and comprises 2 mu L of 10 XT 4 Buffer, 1 mu L T4 DNA ligase, 40ng of double-enzyme-digested vector pGL3-Basic and 300ng of double-enzyme-digested PCR product, supplementing sterile water to 20 mu L, uniformly mixing the systems in a PCR tube, and connecting at 16 ℃ for overnight.

5. Coli DH5 alpha competent cells are transformed by the ligation product, positive clone is screened by colony PCR, plasmids are extracted by an Omega small-amount endotoxin-free plasmid kit, and the correctness of the insertion of a promoter fragment is confirmed by sequencing, so that the recombinant luciferase reporter gene vector pGL3-LEAP2-pro containing the promoter fragment of the Japanese eel liver expression antibacterial peptide LEAP2 gene is obtained.

Secondly, analyzing the basic activity of the promoter of the Japanese eel liver expression antibacterial peptide LEAP2 gene by adopting a dual-luciferase reporter gene detection system.

1. The well-conditioned EPC cells were seeded into 48-well cell plates (1X 10)⁵And/well), adding an L15 culture medium (an L15 basic culture medium contains 10% Gibco Australia fetal bovine serum), transferring into a constant-temperature incubator at 28 ℃ for overnight culture, allowing the cells to adhere to the wall and recovering to the state of logarithmic growth phase, and performing a transfection experiment when the adherence amount reaches about 80%. Cell culture medium was changed 2h before transfection.

In transfection, 0.5. mu.L of Lipofectamine 3000Reagent transfection Reagent and 20. mu.L of Opti-MEM low serum medium per well were mixed to prepare a dilution of transfection Reagent, and the mixture was incubated at room temperature for 5 min. Then, 20 mu.L of Opti-MEM low-serum culture medium per well is fully mixed with the required plasmid per well, wherein a control group contains 20ng of renilla luciferase internal reference reporter gene vector pRL-TK and 300ng of luciferase reporter gene vector pGL3-Basic vector, an experimental group contains 20ng of renilla luciferase internal reference reporter gene vector pRL-TK and 300ng of recombinant luciferase reporter gene vector pGL3-LEAP2-pro, and then 0.5 mu L P3000 is added^TMAnd mixing the reagents. The prepared plasmid diluent is dropwise added into the transfection reagent diluent, mixed uniformly to form a transfection compound solution, incubated at room temperature for 15min, slowly added with EPC cell culture wells, and cultured in a constant-temperature incubator (28 ℃).

And 2.24h later, collecting transfected cells, respectively reading enzyme activity values of firefly luciferase and renilla luciferase by using a dual-luciferase reporter gene detection system, and calculating the ratio of the enzyme activity values of the firefly luciferase and the renilla luciferase to obtain the relative activity of the luciferase in the transfected cells. The luciferase enzyme activity determination method is carried out by referring to the specification of a dual-luciferase reporter gene detection system of Promega company, and comprises the following specific steps:

(1) preparing reagents required by the experiment: 1 XPLB lysate: 1 volume of 5 multiplied by Passive powders Buffer and 4 volumes of double distilled water are mixed evenly to prepare the product; start reagent (LARI): completely dissolving the Luciferase Assay Substrate powder in 10mL Luciferase Assay Buffer II solution, subpackaging by using 1.5mL centrifuge tubes, and storing in a refrigerator at-80 ℃; stop reagent: 1 volume of 50 × Stop according to experimental amount&Glo

Substrate uses a 49 volume Stop&Glo

And (4) diluting by using Buffer.

(2) Cell culture medium was slowly aspirated from 48 well cell culture plates 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 for 15min with shaking.

(4) The lysed cell fluid was transferred to a 1.5mL centrifuge tube and centrifuged (13000rpm, 4 ℃, 10 min).

(5) mu.L of the centrifuged supernatant was taken out and placed in a 1.5mL centrifuge tube having a good light transmittance.

(6) Add 10. mu.L of Start reagent and measure the amount of firefly luciferase activity in the sample using a GloMax 20/206 luminometer. Subsequently, 10. mu.L of Stop reagent was added to measure the luciferase activity value of sea cucumber in the sample. The ratio of the activities of the two is the relative activity of the luciferase in each sample.

The relative activity of the pGL3-LEAP2-pro promoter was calculated using EPC cells co-transfected with the empty vector pGL3-Basic and pRL-TK as a control (see FIG. 3).

Third, immunostimulation experiment

pGL3-Basic and Japanese eel liver expression antibacterial peptide LEAP2 gene promoter recombinant vectors pGL3-LEAP2-pro and a renilla luciferase reference reporter gene vector pRL-TK are used for transfecting EPC cells respectively, and after 12 hours of transfection, LPS (30 mu g/mL), poly I: C (50 mu g/mL) and aeromonas hydrophila (10 mu g/mL) are added into a cell culture solution respectively⁶cfu/mL), transfected cells were collected for luciferase relative activity assays 12h, 12h and 6h after stimulation, respectively.

The activity of the promoter of Japanese eel liver expression antimicrobial peptide LEAP2 gene under the stimulation condition of LPS (30. mu.g/mL) is shown in FIG. 4. The relative activity of luciferase in the EPC cells transfected by the recombinant vector pGL3-LEAP2-pro is 16.0 times that of the EPC cells transfected by the empty vector pGL3-Basic, which indicates that the promoter of the LEAP2 gene for expressing the antimicrobial peptide from the liver of the Japanese eel can be induced and activated by LPS, ". p <0.05,". p < 0.01.

In Aeromonas hydrophila (10)⁶cfu/mL) under the stimulation condition, the activity change of the promoter of the L.japonica liver expression antimicrobial peptide LEAP2 gene is shown in FIG. 5. The relative activity of luciferase in pGL3-LEAP2-pro transfected EPC cells was 6.9 times that of the empty vector pGL3-Basic transfected EPC cells, indicating that the promoter of the Japanese eel LEAP2 gene can be induced to be activated by Aeromonas hydrophila, ". multidot." p<0.05，“**”p<0.01。

The activity change of the promoter of Japanese eel liver expression antibacterial peptide LEAP2 gene under poly I: C (50. mu.g/mL) stimulation is shown in FIG. 6. The relative activity of luciferase in the EPC cells transfected by the recombinant vector pGL3-LEP2-pro is 3.8 times that of the EPC cells transfected by the empty vector pGL3-Basic, which indicates that the promoter of the LEAP2 gene for expressing the antibacterial peptide from the liver of the Japanese eel can be induced and activated by poly I: C, ". x" p <0.05, ". x" p < 0.01.

Although specific embodiments of the invention have been described above, it will be understood by those skilled in the art that the specific embodiments described are illustrative only and are not limiting upon the scope of the invention, and that equivalent modifications and variations can be made by those skilled in the art without departing from the spirit of the invention, which is to be limited only by the appended claims.

Sequence listing

<110> college university

<120> Japanese eel liver expression antibacterial peptide LEP2 gene promoter and application thereof

<160> 6

<170> SIPOSequenceListing 1.0

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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

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gaccaaaagg ctatctccac agctatcttt tgggtgggca aatttgattg gaagtgtctg 540

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tctttaattt agaaatttgt gtaaatgaat acatttaaat agttttgaaa atacttaaat 660

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ctgctgcaaa tgacaagaat acactgtgtg ttgggtgaaa aaaggatttg ttttagacaa 780

gcttaaaaca ttttgctgaa atggacagta aggcactaaa tcaatgtgag atgggctgcg 840

gggcagtgtc acatagtttg catgtgtttg aacatttatc attcaaaaat atcttttgca 900

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agccacagtt ttcaaaatcg aggttcatga ttcctgaaat tttttcagtt cccattcagt 1200

ccaaatcatg ccttctccct taatgaaatg ctaagggaat tttcataact gtaatgattg 1260

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tgcctgcgtc tgcggatttg gacaggaata taaaaggagg aacacagtgc caccgcttct 1680

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<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

1. The promoter is characterized by being a promoter of Japanese eel liver expression antibacterial peptide LEP2 gene, and the nucleotide sequence of the promoter is shown as SEQ ID NO: 1 is shown.

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 3, wherein the expression cassette consists of the promoter of claim 1, the gene of interest whose transcription is initiated by the promoter of claim 1, and a terminator.

4. The recombinant vector of claim 3, wherein the recombinant vector is pGL3-Basic, pGL2-Basic, pGL4.10, pGLuc.

5. The recombinant bacterium according to claim 3, wherein: the recombinant bacteria are escherichia coli, bacillus subtilis, lactobacillus and saccharomycetes.

6. The application of the Japanese eel liver expression antibacterial peptide LEP2 gene promoter according to claim 1 in constructing eukaryotic expression vectors, fish cells or mammalian cells to efficiently express exogenous genes.

7. The application of the Japanese eel liver expression antibacterial peptide LEP2 gene promoter according to claim 1 in constructing transgenic fish.