CN113406331A - Fungal immunomodulatory protein cell positioning method based on green fluorescent protein - Google Patents
Fungal immunomodulatory protein cell positioning method based on green fluorescent protein Download PDFInfo
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Abstract
A fungal immunomodulatory protein cell positioning method based on green fluorescent protein comprises the steps of cloning a vector pUC57-GFP containing green fluorescent protein gene shown as Seq ID No.1, recombining the vector pUC57-GFP with a vector sequence pET-30a to obtain recombinant plasmid pET-GFP, further recombining with a glu1 gene sequence shown as Seq ID No.5 to obtain recombinant plasmid pET-glu1-GFP, constructing a pET-glu1-GFP prokaryotic expression strain based on the recombinant plasmid, and culturing to obtain glu1-GFP recombinant protein for positioning fungal immunomodulatory protein cells. The invention uses the green fluorescent protein to carry out cell positioning on the fungal immunomodulatory protein, can accurately and quickly identify whether the protein enters the cell, and provides a wider prospect for the exploration of the biological activity of the fungal immunomodulatory protein.
Description
Technical Field
The invention relates to a technology in the field of bioengineering, in particular to a fungal immunomodulatory protein cell localization method based on green fluorescent protein.
Background
Green Fluorescent Protein (GFP) was originally a protein found in jellyfish when aequorin bound Ca2+This then fluoresces blue which further excites GFP to produce green fluorescence. The fluorescence emitted after GFP excitation has long duration, and does not need the action of any other accessory factors, and after the cDNA of GFP is successfully cloned and successfully expressed in colibacillus and mammalian cells, the application prospect is realized. The probe is applied to the processes of protein expression, pathogen infection monitoring, cell positioning and the like at present, and is an ideal probe for monitoring gene expression and protein positioning in living cells and tissues. Has been widely used as a reporter gene for cytological experimental study of living bodies.
Fungal Immunomodulatory Protein (FIP) is a small molecule protein with immunomodulatory activity isolated from higher basidiomycetes. The protein of FIP family has the functions of Anti-Tumor, Anti-allergy, and stimulating immune cells to generate various cytokines and other immune regulation effects, and has good medical health care value and application prospect (Li QZ et al, Fungal immunomodulation Proteins: Characteriostic, Potential Anti-Tumor Activities and therapeutic Molecular mechanisms. drug discovery today,2019,24: 307) and the like. Macrophages, which are mononuclear phagocytes, produce large amounts of proinflammatory or anti-inflammatory cytokines that elicit an immune response, and are often used as cellular models for evaluating natural product immunoregulatory activity. The existing research shows that the recombinant FIP-glu is a potential stimulating factor for proliferation and activation of mouse peritoneal macrophages, can regulate proinflammatory and anti-inflammatory mediators of the macrophages at the mRNA level, and can mediate the immunoregulation function of the macrophages through PI3K and MAPKs pathways. (Li QZ, et al, immunomodulating activity of biochemical protein via PI3K/Akt and MAPK signaling pathway in RAW264.7 cells. J Cell physiology.2019, 234: 23337-.
When the biological activity of the fungal immunomodulatory protein on the macrophage is researched, the identification of whether the protein enters the macrophage to play a role is the primary step. The existing protein cell positioning method mainly comprises an immunofluorescence technology, an immune colloidal gold marker, a fusion reporter gene and the like. The immunofluorescence technique is based on the principle of reaction between antigen and antibody, the known antigen or antibody is marked with fluorescein to prepare a fluorescent marker, and then the fluorescent antibody is used as a molecular probe to detect the corresponding antigen in cells or tissues. Although the approximate distribution of the target protein in the cell can be obtained by the method, the obtained image is the superposition of fluorescence signals in the whole cell, and higher resolution cannot be obtained, so that the method is not suitable for the research of intracellular protein localization. The immune colloidal gold labeling technology is to make colloidal gold attract an antibody by utilizing the property that the colloidal gold is negatively charged in an alkaline environment, so as to label the antibody. This method can very accurately reflect the precise location of different proteins in the cell structure, but it is rather complicated to operate, and requires a lot of time and cost.
The method for fusing the reporter gene is to fuse the target gene with the reporter gene such as gfp which is easy to monitor, regulate the expression of the fusion gene by using the expression regulation mechanism of the target gene, and monitor the existence state of the fusion protein in the cell under a fluorescent microscope. Many scholars construct fusion vectors by means of GFP for subcellular localization, for example, Staphylyu and the like use genome of the orf virus AH-F10 strain as a template to construct pEGFP (N1) ORFV127 fusion expression vectors, and use GFP to perform subcellular localization on ORFV127 protein (Staphylyu and the like, clone expression and subcellular localization analysis of the orf virus 127 gene, Jiangsu agricultural science, 2019,35(03): 646-. The Yang theory and others use GFP to construct Adv, CerS2, GFP recombinant adenovirus, and laser confocal microscope to locate the expression and cell location of CerS2-GFP fusion protein in liver cancer cell HepG2 (Yang theory and others, human CerS2 recombinant adenovirus construction and its effect on the cell cycle of liver cancer cell HepG 2. Chongqing medicine, 2018,47(31): 39397 8-.
From the existing research, gfp can be used as the most widely used cell marker gene at present, and can effectively perform cell localization on the fusion protein. Through the search of the prior art, no literature report on the cell localization of Fungal Immunomodulatory Protein (FIP) by using GFP has been found so far.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides a fungal immunomodulatory protein cell positioning method based on green fluorescent protein, which constructs a fusion expression vector of GFP and FIP-glu protein, expresses an FIP mutant with the green fluorescent protein in a prokaryotic expression system and transfers the expression vector into macrophage RAW264.7, and uses the green fluorescent protein to perform cell positioning on the fungal immunomodulatory protein, so that whether the protein enters cells can be accurately and quickly identified, and a wider prospect is provided for the exploration of the biological activity of the fungal immunomodulatory protein.
The invention is realized by the following technical scheme:
the invention clones a vector pUC57-GFP containing a green fluorescent protein gene shown as Seq ID No.1, recombines the vector pUC57-GFP with a pET-30a vector sequence to obtain a recombinant plasmid pET-GFP, further recombines with a glu1 gene sequence shown as Seq ID No.5 to obtain a recombinant plasmid pET-glu1-GFP, constructs a pET-glu1-GFP prokaryotic expression strain based on the recombinant plasmid, and obtains the glu1-GFP recombinant protein for positioning fungal immunomodulatory protein cells by culturing.
The nucleotide sequence of the recombinant plasmid pET-GFP is shown as Seq ID No. 2.
The recombinant plasmid pET-GFP is preferably transferred into a commercially available Escherichia coli DH5 alpha competent cell and subjected to colony PCR to identify the accuracy of the recombinant plasmid.
The nucleotide sequence of the recombinant plasmid pET-glu1-GFP is shown as Seq ID No. 8.
The prokaryotic expression strain is a commercially available escherichia coli BL21(DE3) competent cell.
The glu1-GFP recombinant protein is preferably subjected to purification treatment.
The positioning of the fungal immunomodulatory protein cells refers to: fungal immunomodulatory protein cells are cultured in a culture medium comprising a glu1-GFP recombinant protein, such that the glu1-GFP recombinant protein comprising fluorescence is subjected to the fungal immunomodulatory protein cells.
The fungal immunomodulatory protein cell is not limited to a macrophage.
The positioning method specifically comprises the following steps:
step 1) designing and synthesizing a green fluorescent protein gene according to a gene sequence of the green fluorescent protein, and cloning the gene into a pUC-57 vector to obtain pUC 57-GFP;
step 2) according to the gene sequences of the green fluorescent protein and the pET-30a vector, using BamH I and Not I to carry out enzyme digestion on pUC57-GFP and pET-30a vector plasmids, connecting enzyme digestion products to obtain a recombinant plasmid pET-GFP, and transferring the recombinant plasmid pET-GFP into a escherichia coli DH5 alpha competent cell;
step 3) carrying out colony PCR on the recombinant plasmid pET-GFP by using primers T7 and T7T to identify the accuracy of the recombinant plasmid;
step 4) digesting the pET-GFP recombinant vector and the glu1 gene by Nde I and BamH I according to the pET-GFP recombinant vector sequence and the glu1 gene sequence, and connecting digestion products to obtain a recombinant plasmid pET-glu 1-GFP;
step 5) transferring the recombinant plasmid into a competent cell of escherichia coli BL21(DE3) to construct a pET-glu1-GFP escherichia coli prokaryotic expression strain;
step 6) placing the Escherichia coli positive monoclonal bacteria on LBKanCulturing in liquid culture medium to obtain rFIP-glu1-GFP recombinant protein and purifying;
the LBKanThe content of the liquid culture medium comprises the following components: 10g/L Tryptone (Tryptone), 5g/L Yeast extract (Yeast extract), 10g/L sodium chloride (NaCl), 25 μ g/mL kanamycin (Kan).
And step 7) carrying out SDS-PAGE and Western blotting detection on the rFIP-glu1-GFP recombinant protein.
And 8) treating the macrophage by using rFIP-glu1-GFP recombinant protein, observing the recombinant protein under a fluorescence microscope, and carrying out cell localization.
Technical effects
The invention integrally solves the technical problem of realizing the cell localization of the protein when various immune cells or tumor cells are used for experiments through the fusion expression of the green fluorescent protein and the fungal immunomodulatory protein.
Compared with the prior art, the invention obtains the prokaryotic expression strain of the fungal immunomodulatory protein FIP-glu1-gfp (LZ-8) with a green fluorescent protein marker, and directly meets the requirement of the protein on the positioning in cells in subsequent FIP activity experiments.
Drawings
FIG. 1 shows the electrophoresis of plasmid pET-30a and GFP;
in the figure: (A) the plasmid pET-30a is digested, lane M: DNA Marker DL 15,000; lane 1: after the plasmid pET-30a is subjected to enzyme digestion; lane 2: before the enzyme digestion of the plasmid pET-30 a; (B) the restriction electrophoresis of the recombinant plasmid pUC57-GFP, lane 1: before the enzyme digestion of a recombinant plasmid pUC 57-GFP; lane 2: after the recombinant plasmid pUC57-GFP is cut by enzyme; GFP gene indicated by arrow;
FIG. 2 is a PCR electrophoretogram of recombinant plasmid pET-GFP colony;
in the figure: lane M: DNA Marker DL 2,000; lanes 1-9: detecting the result of the escherichia coli transformant containing the recombinant plasmid pET-GFP; lane-the most: a negative control is carried out, and the negative control,
FIG. 3 is a flow chart of the construction of the recombinant plasmid pET-glu 1-GFP;
FIG. 4 is a clone electrophoretogram of FIP-glu1(LZ-8) gene;
in the figure: lane M: DNA Marker DL 2,000; lane 1: FIP-glu1(LZ-8) gene; lane-the most: a negative control is carried out, and the negative control,
FIG. 5 is an electrophoretogram of recombinant plasmid pET-GFP;
in the figure: lane M: DNA Marker DL 15,000; lane 1: before the recombinant plasmid pET-GFP is digested; lane 2: after the recombinant plasmid pET-GFP is cut by enzyme;
FIG. 6 shows PCR electrophoresis of recombinant plasmid pET-glu1-GFP colonies;
in the figure: colony PCR assay, lane M: DNA Marker DL 2,000; lanes 1-12: the detection result of the E.coli transformant containing the recombinant plasmid pET-glu 1-GFP; lane-the most: negative control
FIG. 7 is a SDS-PAGE and Western blotting assay of glu1-GFP protein;
in the figure: (A) coli transformants SDS-PAGE detection, lane M: protein standard molecular weight; lane 1: e.coli transformant total protein without IPTG induction; lane 2-5: IPTG-induced total protein of e.coli transformants, (B) detection of purified recombinant glu1-GFP protein, lane M: protein standard molecular weight; lane 1: SDS-PAGE detection of the purified recombinant glu1-GFP protein; lane 2: western blotting detection is carried out on the purified recombinant glu1-GFP protein,
FIG. 8 is a map of the localization of recombinant glu1-GFP cells;
in the figure: (A) photographs of glu1-GFP treated cells under visible light; (B) photograph of glu1-GFP treated cells under UV light.
Detailed Description
The embodiment relates to a fungal immunomodulatory protein cell localization method based on green fluorescent protein, which comprises the following steps:
step 1) construction of pET-GFP recombinant plasmid
Amino acid sequences of a Linker and GFP are used as blueprints to synthesize a Linker + GFP gene sequence (synthesized by Shanghai biological engineering Co., Ltd.), a synthetic sequence is shown as Seq ID No.1, and the gene is cloned in a pUC-57 vector to obtain pUC 57-GFP. Prokaryotic expression vectors pET-30a and pUC57-GFP were extracted, and double digestion was carried out using restriction enzymes BamH I and Not I. As shown in FIG. 1, plasmid pET-30a was cleaved to give a single band, approximately 5,390bp in size, consistent with the expectation. The pUC57-GFP gene was double digested to give two bands, the larger band was the linearized vector pUC-57 and the smaller band was GFP, 779bp in size, consistent with the expectations.
pET-30a was ligated with GFP using T4 DNA ligase to construct a recombinant plasmid pET-GFP, which was transformed into E.coli DH 5. alpha. competent cells. Through LBKanAfter the solid medium is screened, a plurality of colonies are respectively picked for colony PCR detection, and the PCR reaction procedure is as follows: 5min at 94 ℃ (30 s at 94 ℃, 1min at 55 ℃, 40s at 72 ℃)35cycles, 10min at 72 ℃; the primer sequences used for PCR were T7 and T7T as shown in Seq ID No.3 and Seq ID No.4, respectively.
The amplified gene fragment should be 1,107 bp. As a result, as shown in FIG. 2, lanes 1, 2, 3, 5, 7, 8 and 9 each had a specific band at the corresponding position. The sequencing of Shanghai biological engineering Co., Ltd shows that the sequence is correct. The success of the construction of the recombinant plasmid pET-GFP is shown.
Step 2) construction of rFIP-glu1-GFP recombinant plasmid
As shown in FIG. 3, a prokaryotic expression vector pET-glu1-GFP was constructed. As shown in FIG. 4, a single band appeared at about 400bp after PCR amplification of glu1, and the size was consistent with that expected. The PCR amplification reaction program is as follows: 5min at 94 ℃ (30 s at 94 ℃, 1min at 55 ℃, 40s at 72 ℃)35cycles, 10min at 72 ℃; the primer sequences used for PCR are shown in Seq ID No.6 and Seq ID No.7, respectively.
And extracting a recombinant plasmid pET-GFP. The recombinant plasmid and the PCR purified product were digested with restriction enzymes Nde I and BamH I. Since the PCR product was double-digested and only several bases were reduced, no change before and after digestion could be observed on agarose gel electrophoresis, and no electrophoretic pattern was shown. As shown in FIG. 5, the recombinant plasmid pET-GFP was digested to a single band, which was 6016bp in size, and was identical to the expected band, as shown in Seq ID No. 2. After the corresponding bands are recovered, the recombinant plasmid pET-glu1-GFP is constructed by connecting the bands through T4 DNA ligase, and the recombinant plasmid is transferred into Escherichia coli DH5 alpha competent cells. Through LBKanAfter the solid medium is screened, a plurality of colonies are respectively picked to carry out colony PCR detection, and the amplified gene fragment is 1,323 bp. The PCR reaction program is: 5min at 94 ℃ (30 s at 94 ℃, 1min at 55 ℃, 40s at 72 ℃)35cycles, 10min at 72 ℃; the primer sequences used for PCR were T7 and T7T as shown in Seq ID No.3 and Seq ID No.4, respectively. The results are shown in FIG. 6, where 12 samples each had a specific band at the corresponding position. The sequencing result shows that the sequence is correct, namely as shown in Seq ID No. 8. The success of the construction of the recombinant plasmid pET-glu1-GFP is shown.
Step 3) expression and purification of rFIP-glu1-GFP protein
The recombinant plasmid pET-glu1-GFP is transformed into escherichia coli BL21(DE3) competent cells (purchased from Shanghai Biotechnology engineering Co., Ltd.), and after IPTG induction, the produced protein is subjected to 10% SDS-PADE and Western blotting detection. SDS-PAGE detection is carried out by preparing SDS-PAGE gel according to a method provided by a website of TaKaRa biotech (Beijing) Co Ltd, carrying out electrophoresis at 80V, and carrying out electrophoresis at 120V until the sample is migrated to the separation gel; western blotting detection is carried out by a semidry method, wherein the primary antibody is a mouse anti-6 XHis monoclonal antibody, and the secondary antibody is goat anti-mouse IgG marked by HRP. As a result of the detection, as shown in FIG. 7, a specific band, i.e., a fusion protein of recombinant FIP-glu1(LZ-8) and GFP, abbreviated as glu1-GFP, appeared at about 40kD in the sample. Dialyzing the supernatant with 3.5kD dialysis bag, freeze-drying the solution in the dialysis bag, dissolving with lysine Buffer, purifying with His60 Ni Superflow Resin column (purchased from Shanghai Biotech engineering Co., Ltd.), dialyzing the purified eluate with 3.5kD dialysis bag, freeze-drying the solution in the dialysis bag, and dissolving in PBS.
Step 4) cellular localization of rFIP-glu1-GFP protein
After washing macrophage RAW264.7 with PBS, cells were suspended in DMEM cell culture medium (GE Healthcare Life Sciences, HyClone) by trypsinizationTM) Count cells at 2X 105At a concentration of one/mL, cells were inoculated into a new cell culture flask containing DMEM medium and placed in CO2The incubator was incubated at 37 ℃. 2X 105Cell/well concentration cells were plated in 12-, 24-or 96-well cell culture plates, cultured for 24h, medium was discarded, medium containing recombinant glu1-GFP was added, and culture was continued for 24 h. The cells were observed by fluorescence microscopy, and the results are shown in FIG. 8, indicating that recombinant glu1-GFP entered the interior of macrophages.
The method utilizes protein fusion expression to construct protein prokaryotic expression strain with markers, and directly produces the fungal immunomodulatory protein capable of cell positioning. Compared with the prior art, the method directly produces the fungal immunomodulatory protein with the fluorescent label through the fusion expression construction of GFP and FIP-glu1, thereby more conveniently obtaining the cell localization result of the fungal immunomodulatory protein.
The foregoing embodiments may be modified in many different ways by those skilled in the art without departing from the spirit and scope of the invention, which is defined by the appended claims and all changes that come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.
Sequence listing
<110> Shanghai university of transportation
<120> fungal immunomodulatory protein cell localization method based on green fluorescent protein
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 779
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
ggatccggcg gtggtggttc tggtggtggt ggttccggtg gtggtggttc catggtttct 60
aagggtgagg agctgtttac cggtgtcgtc cctattctgg ttgagttgga cggtgacgtt 120
aacggtcaca agttctccgt ttctggagag ggtgagggtg acgctaccta cggtaagttg 180
accttgaagt tcatttgcac caccggaaag ttgccagtgc cttggccaac cttggttacc 240
accttgacct acggagttca gtgttttagc agataccctg accacatgaa gcagcacgat 300
ttcttcaagt ccgctatgcc agaaggctac gtccaagagc gtactatctt cttcaaggac 360
gacggtaact acaagactag agctgaggtc aagtttgagg gtgataccct ggttaacaga 420
atcgagttga agggtattga cttcaaagag gatggtaaca ttttgggtca caagctggag 480
tacaactaca actcccacaa cgtttacatt atggcagaca agcaaaagaa cggtatcaag 540
gtcaacttca agatcagaca caacatcgag gacggtagcg tccaactggc cgatcactac 600
caacaaaaca ccccaatcgg tgacggacct gtcctgttgc cagacaacca ctacttgtcc 660
acccaatccg ccttgtccaa ggacccaaac gagaagagag accacatggt tttgttggag 720
ttcgtcactg ccgctggtat tactttgggt atggacgagt tgtacaagta agcggccgc 779
<210> 2
<211> 6162
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa 60
ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt 120
tgttagcagc cggatctcag tggtggtggt ggtggtgctc gagtgcggcc gcttacttgt 180
acaactcgtc catacccaaa gtaataccag cggcagtgac gaactccaac aaaaccatgt 240
ggtctctctt ctcgtttggg tccttggaca aggcggattg ggtggacaag tagtggttgt 300
ctggcaacag gacaggtccg tcaccgattg gggtgttttg ttggtagtga tcggccagtt 360
ggacgctacc gtcctcgatg ttgtgtctga tcttgaagtt gaccttgata ccgttctttt 420
gcttgtctgc cataatgtaa acgttgtggg agttgtagtt gtactccagc ttgtgaccca 480
aaatgttacc atcctctttg aagtcaatac ccttcaactc gattctgtta accagggtat 540
caccctcaaa cttgacctca gctctagtct tgtagttacc gtcgtccttg aagaagatag 600
tacgctcttg gacgtagcct tctggcatag cggacttgaa gaaatcgtgc tgcttcatgt 660
ggtcagggta tctgctaaaa cactgaactc cgtaggtcaa ggtggtaacc aaggttggcc 720
aaggcactgg caactttccg gtggtgcaaa tgaacttcaa ggtcaactta ccgtaggtag 780
cgtcaccctc accctctcca gaaacggaga acttgtgacc gttaacgtca ccgtccaact 840
caaccagaat agggacgaca ccggtaaaca gctcctcacc cttagaaacc atggaaccac 900
caccaccgga accaccacca ccagaaccac caccgccgga tccgatatca gccatggcct 960
tgtcgtcgtc gtcggtaccc agatctgggc tgtccatgtg ctggcgttcg aatttagcag 1020
cagcggtttc tttcatacca gaaccgcgtg gcaccagacc agaagaatga tgatgatgat 1080
ggtgcatatg tatatctcct tcttaaagtt aaacaaaatt atttctagag gggaattgtt 1140
atccgctcac aattccccta tagtgagtcg tattaatttc gcgggatcga gatcgatctc 1200
gatcctctac gccggacgca tcgtggccgg catcaccggc gccacaggtg cggttgctgg 1260
cgcctatatc gccgacatca ccgatgggga agatcgggct cgccacttcg ggctcatgag 1320
cgcttgtttc ggcgtgggta tggtggcagg ccccgtggcc gggggactgt tgggcgccat 1380
ctccttgcat gcaccattcc ttgcggcggc ggtgctcaac ggcctcaacc tactactggg 1440
ctgcttccta atgcaggagt cgcataaggg agagcgtcga gatcccggac accatcgaat 1500
ggcgcaaaac ctttcgcggt atggcatgat agcgcccgga agagagtcaa ttcagggtgg 1560
tgaatgtgaa accagtaacg ttatacgatg tcgcagagta tgccggtgtc tcttatcaga 1620
ccgtttcccg cgtggtgaac caggccagcc acgtttctgc gaaaacgcgg gaaaaagtgg 1680
aagcggcgat ggcggagctg aattacattc ccaaccgcgt ggcacaacaa ctggcgggca 1740
aacagtcgtt gctgattggc gttgccacct ccagtctggc cctgcacgcg ccgtcgcaaa 1800
ttgtcgcggc gattaaatct cgcgccgatc aactgggtgc cagcgtggtg gtgtcgatgg 1860
tagaacgaag cggcgtcgaa gcctgtaaag cggcggtgca caatcttctc gcgcaacgcg 1920
tcagtgggct gatcattaac tatccgctgg atgaccagga tgccattgct gtggaagctg 1980
cctgcactaa tgttccggcg ttatttcttg atgtctctga ccagacaccc atcaacagta 2040
ttattttctc ccatgaagac ggtacgcgac tgggcgtgga gcatctggtc gcattgggtc 2100
accagcaaat cgcgctgtta gcgggcccat taagttctgt ctcggcgcgt ctgcgtctgg 2160
ctggctggca taaatatctc actcgcaatc aaattcagcc gatagcggaa cgggaaggcg 2220
actggagtgc catgtccggt tttcaacaaa ccatgcaaat gctgaatgag ggcatcgttc 2280
ccactgcgat gctggttgcc aacgatcaga tggcgctggg cgcaatgcgc gccattaccg 2340
agtccgggct gcgcgttggt gcggacatct cggtagtggg atacgacgat accgaagaca 2400
gctcatgtta tatcccgccg ttaaccacca tcaaacagga ttttcgcctg ctggggcaaa 2460
ccagcgtgga ccgcttgctg caactctctc agggccaggc ggtgaagggc aatcagctgt 2520
tgcccgtctc actggtgaaa agaaaaacca ccctggcgcc caatacgcaa accgcctctc 2580
cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg 2640
ggcagtgagc gcaacgcaat taatgtaagt tagctcactc attaggcacc gggatctcga 2700
ccgatgccct tgagagcctt caacccagtc agctccttcc ggtgggcgcg gggcatgact 2760
atcgtcgccg cacttatgac tgtcttcttt atcatgcaac tcgtaggaca ggtgccggca 2820
gcgctctggg tcattttcgg cgaggaccgc tttcgctgga gcgcgacgat gatcggcctg 2880
tcgcttgcgg tattcggaat cttgcacgcc ctcgctcaag ccttcgtcac tggtcccgcc 2940
accaaacgtt tcggcgagaa gcaggccatt atcgccggca tggcggcccc acgggtgcgc 3000
atgatcgtgc tcctgtcgtt gaggacccgg ctaggctggc ggggttgcct tactggttag 3060
cagaatgaat caccgatacg cgagcgaacg tgaagcgact gctgctgcaa aacgtctgcg 3120
acctgagcaa caacatgaat ggtcttcggt ttccgtgttt cgtaaagtct ggaaacgcgg 3180
aagtcagcgc cctgcaccat tatgttccgg atctgcatcg caggatgctg ctggctaccc 3240
tgtggaacac ctacatctgt attaacgaag cgctggcatt gaccctgagt gatttttctc 3300
tggtcccgcc gcatccatac cgccagttgt ttaccctcac aacgttccag taaccgggca 3360
tgttcatcat cagtaacccg tatcgtgagc atcctctctc gtttcatcgg tatcattacc 3420
cccatgaaca gaaatccccc ttacacggag gcatcagtga ccaaacagga aaaaaccgcc 3480
cttaacatgg cccgctttat cagaagccag acattaacgc ttctggagaa actcaacgag 3540
ctggacgcgg atgaacaggc agacatctgt gaatcgcttc acgaccacgc tgatgagctt 3600
taccgcagct gcctcgcgcg tttcggtgat gacggtgaaa acctctgaca catgcagctc 3660
ccggagacgg tcacagcttg tctgtaagcg gatgccggga gcagacaagc ccgtcagggc 3720
gcgtcagcgg gtgttggcgg gtgtcggggc gcagccatga cccagtcacg tagcgatagc 3780
ggagtgtata ctggcttaac tatgcggcat cagagcagat tgtactgaga gtgcaccata 3840
tatgcggtgt gaaataccgc acagatgcgt aaggagaaaa taccgcatca ggcgctcttc 3900
cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc 3960
tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat 4020
gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt 4080
ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg 4140
aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc 4200
tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt 4260
ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa 4320
gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta 4380
tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa 4440
caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa 4500
ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt 4560
cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt 4620
ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat 4680
cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat 4740
gaacaataaa actgtctgct tacataaaca gtaatacaag gggtgttatg agccatattc 4800
aacgggaaac gtcttgctct aggccgcgat taaattccaa catggatgct gatttatatg 4860
ggtataaatg ggctcgcgat aatgtcgggc aatcaggtgc gacaatctat cgattgtatg 4920
ggaagcccga tgcgccagag ttgtttctga aacatggcaa aggtagcgtt gccaatgatg 4980
ttacagatga gatggtcaga ctaaactggc tgacggaatt tatgcctctt ccgaccatca 5040
agcattttat ccgtactcct gatgatgcat ggttactcac cactgcgatc cccgggaaaa 5100
cagcattcca ggtattagaa gaatatcctg attcaggtga aaatattgtt gatgcgctgg 5160
cagtgttcct gcgccggttg cattcgattc ctgtttgtaa ttgtcctttt aacagcgatc 5220
gcgtatttcg tctcgctcag gcgcaatcac gaatgaataa cggtttggtt gatgcgagtg 5280
attttgatga cgagcgtaat ggctggcctg ttgaacaagt ctggaaagaa atgcataaac 5340
ttttgccatt ctcaccggat tcagtcgtca ctcatggtga tttctcactt gataacctta 5400
tttttgacga ggggaaatta ataggttgta ttgatgttgg acgagtcgga atcgcagacc 5460
gataccagga tcttgccatc ctatggaact gcctcggtga gttttctcct tcattacaga 5520
aacggctttt tcaaaaatat ggtattgata atcctgatat gaataaattg cagtttcatt 5580
tgatgctcga tgagtttttc taagaattaa ttcatgagcg gatacatatt tgaatgtatt 5640
tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgaaatt 5700
gtaaacgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt 5760
aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagataggg 5820
ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc 5880
aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc accctaatca 5940
agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga 6000
tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa 6060
ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc 6120
gccgcgctta atgcgccgct acagggcgcg tcccattcgc ca 6162
<210> 3
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
taatacgact cactataggg 20
<210> 4
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
gctagttatt gctcagcgg 19
<210> 5
<211> 648
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
gatccaaacg atgagatttc cttcaatttt tactgcagtt ttattcgcag catcctccgc 60
attagctgct ccagtcaaca ctacaacaga agatgaaacg gcacaaattc cggctgaagc 120
tgtcatcggt tactcagatt tagaagggga tttcgatgtt gctgttttgc cattttccaa 180
cagcacaaat aacgggttat tgtttataaa tactactatt gccagcattg ctgctaaaga 240
agaaggggta tctctcgaga aaagagaggc tgaagcttac gtagaattca tgtctgacac 300
cgctttgatc ttcagactgg cttgggacgt caagaagttg tccttcgact acaccccaaa 360
ctggggaaga ggtaacccaa acaacttcat cgacaccgtt accttcccaa aggtcttgac 420
tgacaaggcc tacacctaca gagttgccgt ttctggtcgt aacctgggtg tcaagccatc 480
ctacgctgtt gagtccgacg gttcccagaa ggtcaacttc ttggagtaca actctggtta 540
cggtatcgct gacactaaca ccatccaagt tttcgttgtc gacccagaca ccaacaacga 600
cttcatcatt gctcaatgga acgggcccca tcatcatcat catcatca 648
<210> 6
<211> 59
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
atatacatat gcaccatcat catcatcatg agctcatgtc tgacaccgct ttgatcttc 59
<210> 7
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
actcgagcgg atccgttcca ttgagcaatg atgaagtcg 39
<210> 8
<211> 6662
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa 60
ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt 120
tgttagcagc cggatctcag tggtggtggt ggtggtgctc gagtgcggcc gcttacttgt 180
acaactcgtc catacccaaa gtaataccag cggcagtgac gaactccaac aaaaccatgt 240
ggtctctctt ctcgtttggg tccttggaca aggcggattg ggtggacaag tagtggttgt 300
ctggcaacag gacaggtccg tcaccgattg gggtgttttg ttggtagtga tcggccagtt 360
ggacgctacc gtcctcgatg ttgtgtctga tcttgaagtt gaccttgata ccgttctttt 420
gcttgtctgc cataatgtaa acgttgtggg agttgtagtt gtactccagc ttgtgaccca 480
aaatgttacc atcctctttg aagtcaatac ccttcaactc gattctgtta accagggtat 540
caccctcaaa cttgacctca gctctagtct tgtagttacc gtcgtccttg aagaagatag 600
tacgctcttg gacgtagcct tctggcatag cggacttgaa gaaatcgtgc tgcttcatgt 660
ggtcagggta tctgctaaaa cactgaactc cgtaggtcaa ggtggtaacc aaggttggcc 720
aaggcactgg caactttccg gtggtgcaaa tgaacttcaa ggtcaactta ccgtaggtag 780
cgtcaccctc accctctcca gaaacggaga acttgtgacc gttaacgtca ccgtccaact 840
caaccagaat agggacgaca ccggtaaaca gctcctcacc cttagaaacc atggaaccac 900
caccaccgga accaccacca ccagaaccac caccgccgga tccaaacgat gagatttcct 960
tcaattttta ctgcagtttt attcgcagca tcctccgcat tagctgctcc agtcaacact 1020
acaacagaag atgaaacggc acaaattccg gctgaagctg tcatcggtta ctcagattta 1080
gaaggggatt tcgatgttgc tgttttgcca ttttccaaca gcacaaataa cgggttattg 1140
tttataaata ctactattgc cagcattgct gctaaagaag aaggggtatc tctcgagaaa 1200
agagaggctg aagcttacgt agaattcatg tctgacaccg ctttgatctt cagactggct 1260
tgggacgtca agaagttgtc cttcgactac accccaaact ggggaagagg taacccaaac 1320
aacttcatcg acaccgttac cttcccaaag gtcttgactg acaaggccta cacctacaga 1380
gttgccgttt ctggtcgtaa cctgggtgtc aagccatcct acgctgttga gtccgacggt 1440
tcccagaagg tcaacttctt ggagtacaac tctggttacg gtatcgctga cactaacacc 1500
atccaagttt tcgttgtcga cccagacacc aacaacgact tcatcattgc tcaatggaac 1560
gggccccatc atcatcatca tcatcatatg tatatctcct tcttaaagtt aaacaaaatt 1620
atttctagag gggaattgtt atccgctcac aattccccta tagtgagtcg tattaatttc 1680
gcgggatcga gatcgatctc gatcctctac gccggacgca tcgtggccgg catcaccggc 1740
gccacaggtg cggttgctgg cgcctatatc gccgacatca ccgatgggga agatcgggct 1800
cgccacttcg ggctcatgag cgcttgtttc ggcgtgggta tggtggcagg ccccgtggcc 1860
gggggactgt tgggcgccat ctccttgcat gcaccattcc ttgcggcggc ggtgctcaac 1920
ggcctcaacc tactactggg ctgcttccta atgcaggagt cgcataaggg agagcgtcga 1980
gatcccggac accatcgaat ggcgcaaaac ctttcgcggt atggcatgat agcgcccgga 2040
agagagtcaa ttcagggtgg tgaatgtgaa accagtaacg ttatacgatg tcgcagagta 2100
tgccggtgtc tcttatcaga ccgtttcccg cgtggtgaac caggccagcc acgtttctgc 2160
gaaaacgcgg gaaaaagtgg aagcggcgat ggcggagctg aattacattc ccaaccgcgt 2220
ggcacaacaa ctggcgggca aacagtcgtt gctgattggc gttgccacct ccagtctggc 2280
cctgcacgcg ccgtcgcaaa ttgtcgcggc gattaaatct cgcgccgatc aactgggtgc 2340
cagcgtggtg gtgtcgatgg tagaacgaag cggcgtcgaa gcctgtaaag cggcggtgca 2400
caatcttctc gcgcaacgcg tcagtgggct gatcattaac tatccgctgg atgaccagga 2460
tgccattgct gtggaagctg cctgcactaa tgttccggcg ttatttcttg atgtctctga 2520
ccagacaccc atcaacagta ttattttctc ccatgaagac ggtacgcgac tgggcgtgga 2580
gcatctggtc gcattgggtc accagcaaat cgcgctgtta gcgggcccat taagttctgt 2640
ctcggcgcgt ctgcgtctgg ctggctggca taaatatctc actcgcaatc aaattcagcc 2700
gatagcggaa cgggaaggcg actggagtgc catgtccggt tttcaacaaa ccatgcaaat 2760
gctgaatgag ggcatcgttc ccactgcgat gctggttgcc aacgatcaga tggcgctggg 2820
cgcaatgcgc gccattaccg agtccgggct gcgcgttggt gcggacatct cggtagtggg 2880
atacgacgat accgaagaca gctcatgtta tatcccgccg ttaaccacca tcaaacagga 2940
ttttcgcctg ctggggcaaa ccagcgtgga ccgcttgctg caactctctc agggccaggc 3000
ggtgaagggc aatcagctgt tgcccgtctc actggtgaaa agaaaaacca ccctggcgcc 3060
caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca 3120
ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtaagt tagctcactc 3180
attaggcacc gggatctcga ccgatgccct tgagagcctt caacccagtc agctccttcc 3240
ggtgggcgcg gggcatgact atcgtcgccg cacttatgac tgtcttcttt atcatgcaac 3300
tcgtaggaca ggtgccggca gcgctctggg tcattttcgg cgaggaccgc tttcgctgga 3360
gcgcgacgat gatcggcctg tcgcttgcgg tattcggaat cttgcacgcc ctcgctcaag 3420
ccttcgtcac tggtcccgcc accaaacgtt tcggcgagaa gcaggccatt atcgccggca 3480
tggcggcccc acgggtgcgc atgatcgtgc tcctgtcgtt gaggacccgg ctaggctggc 3540
ggggttgcct tactggttag cagaatgaat caccgatacg cgagcgaacg tgaagcgact 3600
gctgctgcaa aacgtctgcg acctgagcaa caacatgaat ggtcttcggt ttccgtgttt 3660
cgtaaagtct ggaaacgcgg aagtcagcgc cctgcaccat tatgttccgg atctgcatcg 3720
caggatgctg ctggctaccc tgtggaacac ctacatctgt attaacgaag cgctggcatt 3780
gaccctgagt gatttttctc tggtcccgcc gcatccatac cgccagttgt ttaccctcac 3840
aacgttccag taaccgggca tgttcatcat cagtaacccg tatcgtgagc atcctctctc 3900
gtttcatcgg tatcattacc cccatgaaca gaaatccccc ttacacggag gcatcagtga 3960
ccaaacagga aaaaaccgcc cttaacatgg cccgctttat cagaagccag acattaacgc 4020
ttctggagaa actcaacgag ctggacgcgg atgaacaggc agacatctgt gaatcgcttc 4080
acgaccacgc tgatgagctt taccgcagct gcctcgcgcg tttcggtgat gacggtgaaa 4140
acctctgaca catgcagctc ccggagacgg tcacagcttg tctgtaagcg gatgccggga 4200
gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc gcagccatga 4260
cccagtcacg tagcgatagc ggagtgtata ctggcttaac tatgcggcat cagagcagat 4320
tgtactgaga gtgcaccata tatgcggtgt gaaataccgc acagatgcgt aaggagaaaa 4380
taccgcatca ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg 4440
ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg 4500
gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag 4560
gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga 4620
cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct 4680
ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc 4740
tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg 4800
gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc 4860
tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca 4920
ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag 4980
ttcttgaagt ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct 5040
ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc 5100
accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga 5160
tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca 5220
cgttaaggga ttttggtcat gaacaataaa actgtctgct tacataaaca gtaatacaag 5280
gggtgttatg agccatattc aacgggaaac gtcttgctct aggccgcgat taaattccaa 5340
catggatgct gatttatatg ggtataaatg ggctcgcgat aatgtcgggc aatcaggtgc 5400
gacaatctat cgattgtatg ggaagcccga tgcgccagag ttgtttctga aacatggcaa 5460
aggtagcgtt gccaatgatg ttacagatga gatggtcaga ctaaactggc tgacggaatt 5520
tatgcctctt ccgaccatca agcattttat ccgtactcct gatgatgcat ggttactcac 5580
cactgcgatc cccgggaaaa cagcattcca ggtattagaa gaatatcctg attcaggtga 5640
aaatattgtt gatgcgctgg cagtgttcct gcgccggttg cattcgattc ctgtttgtaa 5700
ttgtcctttt aacagcgatc gcgtatttcg tctcgctcag gcgcaatcac gaatgaataa 5760
cggtttggtt gatgcgagtg attttgatga cgagcgtaat ggctggcctg ttgaacaagt 5820
ctggaaagaa atgcataaac ttttgccatt ctcaccggat tcagtcgtca ctcatggtga 5880
tttctcactt gataacctta tttttgacga ggggaaatta ataggttgta ttgatgttgg 5940
acgagtcgga atcgcagacc gataccagga tcttgccatc ctatggaact gcctcggtga 6000
gttttctcct tcattacaga aacggctttt tcaaaaatat ggtattgata atcctgatat 6060
gaataaattg cagtttcatt tgatgctcga tgagtttttc taagaattaa ttcatgagcg 6120
gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc 6180
gaaaagtgcc acctgaaatt gtaaacgtta atattttgtt aaaattcgcg ttaaattttt 6240
gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa 6300
aagaatagac cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa 6360
agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac 6420
gtgaaccatc accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga 6480
accctaaagg gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa 6540
aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc 6600
tgcgcgtaac caccacaccc gccgcgctta atgcgccgct acagggcgcg tcccattcgc 6660
ca 6662
Claims (8)
1. A fungal immunomodulatory protein cell positioning method based on green fluorescent protein is characterized in that after a vector pUC57-GFP containing green fluorescent protein gene shown as Seq ID No.1 is cloned, the vector pUC57-GFP is recombined with pET-30a vector sequence to obtain recombinant plasmid pET-GFP, the recombinant plasmid pET-glu1-GFP is further recombined with glu1 gene sequence shown as Seq ID No.5 to obtain recombinant plasmid pET-glu1-GFP prokaryotic expression strain, and glu1-GFP recombinant protein for positioning fungal immunomodulatory protein cell is obtained by culturing;
the nucleotide sequence of the recombinant plasmid pET-GFP is shown as Seq ID No. 2.
2. The method of claim 1, wherein the recombinant plasmid pET-GFP is transformed into E.coli DH5 a competent cells and subjected to colony PCR to determine the accuracy of the recombinant plasmid;
the nucleotide sequence of the recombinant plasmid pET-glu1-GFP is shown as Seq ID No. 8.
3. The method of claim 1, wherein the prokaryotic expression strain is E.coli BL21(DE3) competent cell.
4. The method of claim 1, wherein the glu1-GFP recombinant protein is purified.
5. The method of claim 1, wherein the step of localizing the fungal immunomodulatory protein cell comprises: fungal immunomodulatory protein cells are cultured in a culture medium comprising a glu1-GFP recombinant protein, such that the glu1-GFP recombinant protein comprising fluorescence is subjected to the fungal immunomodulatory protein cells.
6. The method of claim 1, wherein the fungal immunomodulatory protein cell is a macrophage.
7. A method according to any one of claims 1 to 6, wherein the method of localising a fungal immunomodulatory protein cell comprises:
step 1) designing and synthesizing a green fluorescent protein gene according to a gene sequence of the green fluorescent protein, and cloning the gene into a pUC-57 vector to obtain pUC 57-GFP;
step 2) according to the gene sequences of the green fluorescent protein and the pET-30a vector, using BamH I and Not I to carry out enzyme digestion on pUC57-GFP and pET-30a vector plasmids, connecting enzyme digestion products to obtain a recombinant plasmid pET-GFP, and transferring the recombinant plasmid pET-GFP into a escherichia coli DH5 alpha competent cell;
step 3) carrying out colony PCR on the recombinant plasmid pET-GFP by using primers T7 and T7T to identify the accuracy of the recombinant plasmid;
step 4) digesting the pET-GFP recombinant vector and the glu1 gene by Nde I and BamH I according to the pET-GFP recombinant vector sequence and the glu1 gene sequence, and connecting digestion products to obtain a recombinant plasmid pET-glu 1-GFP;
step 5) transferring the recombinant plasmid into a competent cell of escherichia coli BL21(DE3) to construct a pET-glu1-GFP escherichia coli prokaryotic expression strain;
step 6) placing the Escherichia coli positive monoclonal bacteria on LBKanCulturing in liquid culture medium to obtain rFIP-glu1-GFP recombinant protein and purifying;
step 7) carrying out SDS-PAGE and Western blotting detection on rFIP-glu1-GFP recombinant protein;
and 8) treating the macrophage by using rFIP-glu1-GFP recombinant protein, observing the recombinant protein under a fluorescence microscope, and carrying out cell localization.
8. The method of claim 7, wherein the LB molecules are as defined inKanThe content of the liquid culture medium comprises the following components: 10g/L of tryptone, 5g/L of yeast extract, 10g/L of sodium chloride and 25 mu g/mL of kanamycin.
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