CN113238053B - Plasmid for detecting STAT3 dimerization - Google Patents

Plasmid for detecting STAT3 dimerization Download PDF

Info

Publication number
CN113238053B
CN113238053B CN202110487845.8A CN202110487845A CN113238053B CN 113238053 B CN113238053 B CN 113238053B CN 202110487845 A CN202110487845 A CN 202110487845A CN 113238053 B CN113238053 B CN 113238053B
Authority
CN
China
Prior art keywords
stat3
flag
plasmid
tag
myc
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN202110487845.8A
Other languages
Chinese (zh)
Other versions
CN113238053A (en
Inventor
王紫静
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
West China Hospital of Sichuan University
Original Assignee
West China Hospital of Sichuan University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by West China Hospital of Sichuan University filed Critical West China Hospital of Sichuan University
Priority to CN202110487845.8A priority Critical patent/CN113238053B/en
Priority to KR1020210072639A priority patent/KR20210076887A/en
Publication of CN113238053A publication Critical patent/CN113238053A/en
Application granted granted Critical
Publication of CN113238053B publication Critical patent/CN113238053B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Immunology (AREA)
  • Genetics & Genomics (AREA)
  • Biotechnology (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Cell Biology (AREA)
  • General Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Food Science & Technology (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Plant Pathology (AREA)
  • Biophysics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Hospice & Palliative Care (AREA)
  • Oncology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Peptides Or Proteins (AREA)

Abstract

The invention discloses a plasmid for detecting STAT3 dimerization, and belongs to the technical field of molecular biology. The plasmid of the invention comprises the following 3 fragments: (1) a promoter; (2) a first STAT3 gene, the 5' end of which is linked to the sequence of tag protein A; (3) a second STAT3 gene, the 3' end of which is linked to the sequence of tag protein B; wherein (1) and (2) are linked by a gene of the 2A peptide; the tag protein A and the tag protein B are different in sequence. The plasmid can translate STAT3 proteins with different labels in a cell at a ratio of 1: 1, and after the proteins are purified by one label, the other label is detected, so that the STAT3 dimerization level can be objectively reflected.

Description

Plasmid for detecting STAT3 dimerization
Technical Field
The invention belongs to the technical field of molecular biology.
Background
Signal transducer and activator of transcription 3 (STAT3) is an transcriptionally active oncogene whose monomers are first phosphorylated in the cytoplasm and the phosphorylated STAT3 monomer dimerizes to form a dimer, becoming active STAT 3. The activated STAT3 dimer translocates to the nucleus to regulate the processes of tumor proliferation, survival, metastasis, angiogenesis, immune response and the like.
STAT3 dimer has become one of the important targets for screening new antitumor drugs, and studies have shown that G-quadruplet oligonucleotides (G-quartz oligo-nucleotides) can form a complex tertiary structure to inhibit dimerization of STAT3, thereby inhibiting growth of squamous carcinoma in vitro and in mouse models (liujun et al. study progress of STAT3 signaling pathway and its tumor molecule targeted therapy. journal of neuropsychiatric disease in china, vol 37, No.7, 2011). In order to improve the screening efficiency of the drugs for inhibiting STAT3 dimerization, a relevant cell model needs to be established to reflect the capacity of the drugs for inhibiting STAT3 dimerization.
Chenjunsheng et al respectively construct fluorescent reporter vectors of pECFP-N1-STAT3 and pEYFP-N1-STAT3, co-transfer the two into HEK-293T cells by using a liposome transfection technology, and detect the dimerization level of STAT3 molecules by using energy resonance transfer between ECFP (serving as a donor molecule) and EYFP (serving as an acceptor molecule) (Chenjunsheng et al. establishment of STAT3 dimerization inhibitor screening model based on the fluorescence resonance energy transfer technology. Chinese marine drugs, Vol.37, No. 3 of 2018). However, this method requires the construction of 2 vectors, and the amounts of both vectors are difficult to be kept uniform after co-transfection into cells, so that dimer formation may be inhibited due to an insufficient amount of one of the vectors, resulting in false positives. In addition, although the non-denaturing polyacrylamide gel electrophoresis is combined with the immunoblotting to detect the protein polymer, the detection sensitivity is low, and the experimental conditions are not easy to control.
Disclosure of Invention
The invention aims to solve the problems that: a plasmid for detecting STAT3 dimerization, and a method of detecting STAT3 dimerization are provided.
The technical scheme of the invention is as follows:
a plasmid for detecting STAT3 dimerization, the plasmid comprising the following 3 fragments:
(1) a promoter;
(2) a first STAT3 gene, the 5' end of which is linked to the sequence of tag protein A;
(3) a second STAT3 gene, the 3' end of which is linked to the sequence of tag protein B;
wherein (2) and (3) are linked by a 2A peptide;
the 2A peptide is called 2A self-shearing peptide, is a peptide fragment with the length of 18-22 amino acid residues and can induce the self-shearing of recombinant protein containing the 2A peptide in cells. The peptides all have a sequence motif, which often leads to the situation that at the final joint of glycine (G) and proline (P), ribosome can not be connected, and protein translation is terminated early, thereby causing the effect of 'cutting'.
The tag protein A and the tag protein B are different in sequence;
in the plasmid, the fragments (1) to (3) are arranged in the order of (1) - (2) - (3) or (1) - (3) - (2).
Further, the tag protein A is a FLAG tag, and the tag protein B is a Myc tag;
or, the tag protein A is a Myc tag, and the tag protein B is a FLAG tag.
Furthermore, the plasmid is obtained by using pCDH-CMV-MCS-EF1-CopGFP-T2A-Puro vector as an empty vector skeleton and inserting the fragments (1) to (3) into a multiple cloning site.
The preparation method of the plasmid comprises the following steps:
1) carrying out PCR amplification on cDNA of STAT3 by using primers with sequences shown as SEQ ID NO. 1-2 to obtain a Flag-STAT3 fragment;
2) inserting Flag-STAT3 into an empty vector skeleton to obtain pCDH-CMV-Flag-Stat 3-GFP-Puro;
3) carrying out PCR amplification on eDNA of STAT3 by using primers with sequences shown in SEQ ID NO. 6-7 to obtain a Myc-STAT3-T2A fragment;
4) the fragment Myc-STAT3-T2A is cut by NheI enzyme and inserted into pCDH-CMV-Flag-Stat3-GFP-Puro, and the fragment is obtained.
Further, the step 2) is as follows: the Flag-STAT3 fragment and the empty vector skeleton are cut by NheI and NotI enzyme and then connected by T4 ligase.
Further, the step 4) is as follows: after the pCDH-CMV-Flag-Stat3-GFP-Puro is cut by NheI enzyme, the pCDH-CMV-Flag-Stat3-GFP-Puro is connected with the fragment Myc-STAT3-T2A into a ring through homologous recombination.
A method of detecting the level of intracellular STAT3 dimerization, comprising:
a) transfecting the plasmid of any one of claims 1 to 3 into a cell;
b) isolating the protein according to tag protein a;
c) and (3) taking the separated protein as a sample, and detecting the tag protein B.
Further, the separation of step b) is a separation using an immunoprecipitation method.
Further, the detection in the step c) is carried out by using Western blot.
Use of the aforementioned plasmid for screening STAT3 dimerization inhibitors.
The invention has the beneficial effects that:
the plasmid of the invention can translate STAT3 protein with different labels 1: 1 in cells, and if STAT3 dimer is formed, the dimer simultaneously has two different labels (as shown in FIG. 1). After the protein is purified by one tag, the protein is detected by the other tag, and the STAT3 dimerization level can be objectively reflected.
Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.
The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.
Drawings
FIG. 1: schematic representation of STAT3 dimers with different tags.
FIG. 2 is a schematic diagram: the pCDH-CMV-MCS-EF1-CopGFP-T2A-Puro vector structure is shown schematically.
FIG. 3: pCDH-CMV-Flag-Stat3-GFP-Puro plasmid schematic.
FIG. 4: pCDH-CMV-Myc-STAT3-T2A-Flag-STAT3-GFP-Puro plasmid schematic.
FIG. 5: band of PCR product of Flag-STAT 3. The first lane from the left is DL15000DNA Marker (15000, 10000, 7500, 5000, 2500, 1000, 250bp from top to bottom), the second lane is the target band, and the third lane is Spark 2000DNA Marker (2000, 1000, 750, 500, 250, 100bp from top to bottom).
FIG. 6: the result of Flag antibody Immunoblotting (IB) detection and the result of Flag immunoblotting detection after Myc antibody co-Immunoprecipitation (IP).
FIG. 7: myc antibody Immunoblotting (IB) detection results and Myc immunoblotting detection results after Flag antibody co-Immunoprecipitation (IP).
Detailed Description
EXAMPLE 1 construction of the plasmid of the present invention
The construction process comprises the following steps: inserting a Flag-STAT3 fragment into a Multiple Cloning Site (MCS) of a pCDH-CMV-MCS-EF1-CopGFP-T2A-Puro (shown in figure 3) as an empty vector skeleton (Lifescience-market, hong Kong) by double enzyme digestion to obtain pCDH-CMV-Flag-Stat3-GFP-Puro (shown in figure 2); on the basis of pCDH-CMV-Flag-Stat3-GFP-Puro, a fragment of Myc-STAT3-T2A is inserted into the upstream adjacent position of a fragment of Flag-STAT3 to obtain the plasmid pCDH-CMV-Myc-STAT3-T2A-Flag-STAT3-GFP-Puro (shown in figure 4).
T2A is the gene for the 2A peptide.
The detailed steps are as follows:
firstly, construction of pCDH-CMV-Flag-Stat3-GFP-Puro
1. Primer design
Based on the sequence of NM-139276.2 in GenBank, PCR primers were designed, wherein the upstream primer carries NheI cleavage site and adds Kozak sequence (translation enhancing sequence), initiation codon, and Flag tag coding sequence, and STAT3 initiation sequence; the downstream primer carries NotI cleavage site, stop codon and STAT3 terminal sequence. Then, using cDNA of STAT3 from the DNA library as a template, PCR was performed to amplify the desired fragment. (the primer sequences are as follows)
Flag-STAT3 forward primer (SEQ ID N0.1):
CTA (enzyme-cleaved protection base) GCTAGC (NheI sequence) ACC (Kozac sequence: translation enhancing sequence) ATG (initiation codon) GACTACAAGGACGACGATGATAAG (Flag sequence) GCCCAATGGAATCAGCTACAGC (sequence for amplifying STAT3)
Flag-STAT3 reverse primer (SEQ ID N0.2):
AAT (restriction enzyme-cleaved protection base) GCGGCCGC (Not SEQ ID NO: TCA (stop codon) CATGGGGGAGGTAGCGCACTC (sequence for amplifying STAT3)
PCR reaction
The reaction system is shown in Table 1.
TABLE 1 PCR reaction System
Figure BDA0003050344180000041
Note: DDW, double distilled water, the same below.
The reaction conditions were as follows:
Figure BDA0003050344180000042
after the reaction was completed, the desired fragment was identified and recovered by electrophoresis using 1.0% Agarose, as shown in FIG. 5.
3. Enzyme digestion experiment
The expression vector pCDH-CMV-MCS-EF1-CopGFP-T2A-Puro and the PCR recovery product are subjected to double enzyme digestion by NheI and NotI, the system is shown in tables 2 and 3, and the enzyme digestion reaction conditions are as follows: 37 ℃ for 1 h.
TABLE 2 vector restriction reaction System
Figure BDA0003050344180000051
TABLE 3 STAT3 recovery product enzyme digestion reaction System
Figure BDA0003050344180000052
Add 6 × Loading Buffer 3 μ l, identify by 1% agarose gel electrophoresis and gel recover the band.
4. Connection experiment
Carrying out T4 DNA ligase connection on the vector fragment and the STAT3 fragment after enzyme digestion recovery, wherein the system is shown in Table 4, and the reaction conditions are as follows: after gentle mixing, ligation was carried out at 22 ℃ for 1 h.
TABLE 4 ligation reaction System
Figure BDA0003050344180000053
5. Transformation experiments
The ligation mixture was directly transferred to Stb13 competent cells by the following transformation procedure:
(1) adding 20 μ l of the ligation solution into 100 μ l of competent bacteria, and placing on ice for 30 min;
(2) thermally shocking at 42 deg.C for 90sec, and rapidly placing in ice for 5 min;
(3) all were plated on Amp-resistant LB plates and cultured overnight at 37 ℃ in an inverted manner.
6. Positive clone identification
Randomly picking 4 single clones into an EP tube containing Amp resistant 400 mul LB culture solution, shaking at 37 ℃ and 220rpm for 4h, taking 1 mul bacterial solution as a template, and carrying out PCR identification (selecting one section of sequence (about 1KB in size) of STAT3 to design a positive primer and a negative primer, carrying out PCR, wherein the positive band is a positive clone).
The primer sequence is as follows:
Forward：CTAGCTAGCACCATGGACTACAAGGACGACGATGATAAGGCCCAATGGAATCAGCTACAGC(SEQ ID NO.3)
Reverse：ATGGGCATGCAGGGCTGCCG(SEQ ID NO.4)
DNA sequencing
Sequencing clones identified as positive by colony PCR.
And (3) sequencing results:
Figure BDA0003050344180000061
Figure BDA0003050344180000071
the sequencing result shows that the pCDH-CMV-Flag-Stat3-GFP-Puro is successfully constructed.
Second, construction of pCDH-CMV-Myc-STAT3-T2A-Flag-STAT3-GFP-Puro
1. Primer design
Based on the sequence of NM-139276.2, PCR primers were designed, wherein the upstream and downstream primers both had NheI cleavage sites, and homology arms with pCDH-CMV-Flag-STAT3-GFP-Puro after NheI linearization, and Kozak sequence, initiation codon, Myc tag coding sequence, initiation sequence of STAT3, T2A sequence downstream, and terminal sequence of STAT3 were added upstream. Then, using cDNA of STAT3 from the DNA library as a template, PCR was performed to amplify the desired fragment. (the primer sequences are as follows)
Myc-STAT3 forward primer (SEQ ID NO. 6):
atagaagattctaga (homology arms) GCTAGC (NheI sequence) GCCACC (Kozak enhancer sequence) ATG (initiation codon) GAGCAAAAGCTCATTTCTGAAGAGGACCTG (Myc sequence) GCCCAATGGAATCAGCTACAGC (STAT3)
Myc-STAT3 reverse primer (SEQ ID NO. 7):
CTTGTAGTCCATGGT (homology arm) GCTAGC (NheI sequence) AGGGCCGGGATTCTCCTCCACGTCACCGCATGTTAGAAGACTTCCTCTGCCCTC (T2A sequence) CATGGGGGAGGTAGCGCACTCC (STAT3 sequence)
PCR reaction
The PCR reaction system and reaction time were the same as those in section 2 of the first section.
3. And (4) carrying out enzyme digestion reaction.
The expression vector pCDH-CMV-Flag-STAT3-GFP-Puro was subjected to a single cleavage with NheI, and the system was referred to Table 2 above (except for NotI in Table 2).
4. Homologous recombination reaction
The fragment of Myc-STAT3-T2A and linearized pCDH-CMV-Flag-STAT3-GFP-Puro are subjected to recombination reaction, the reaction system is shown in Table 5, and the reaction conditions are as follows: after mixing gently, the mixture was reacted at 50 ℃ for 30 min.
TABLE 5 homologous recombination reaction System
Figure BDA0003050344180000072
5. Transformation experiments
See above.
6. Positive clone identification
Randomly picking 2 monoclonals into an EP tube containing Amp resistant 400 mul LB culture solution, shaking at 37 ℃ and 220rpm for 4h, then transferring to 4ml of Amp resistant LB culture solution, shaking at 37 ℃ and 220rpm overnight, extracting plasmids the next day, then performing NheI single enzyme digestion, selecting the clone with a band of about 2300bp cut off, and sequencing.
The sequence obtained by sequencing (SEQ ID NO.8) is:
Figure BDA0003050344180000081
Figure BDA0003050344180000091
sequencing results show that the pCDH-CMV-Myc-STAT3-T2A-Flag-STAT3-GFP-Puro is successfully constructed.
Example 2 use of the plasmids of the invention for detecting STAT3 dimerization
The pCDH-CMV-Myc-STAT3-T2A-Flag-STAT3-GFP-Puro plasmid of example 1 was transfected into HEK293T cells, total cell protein was extracted 48 hours later, Myc or Flag was subjected to co-Immunoprecipitation (IP), proteins obtained from both total protein and IP were subjected to denaturing polyacrylamide gel electrophoresis, and then Flag or Myc was detected by Immunoblotting (IB).
As shown in FIG. 6, direct immunoblotting with Myc antibody revealed a band (input), which indicated that Myc-STAT3 expressed a protein; the input lane is a single band, since denaturing polyacrylamide gel electrophoresis will denature dimers into monomers. After the Flag is subjected to co-immunoprecipitation, a Myc antibody is used for immunoblotting, a band (IP) is shown, if no dimer is formed in the cell, STAT3 with Myc cannot be collected by co-immunoprecipitation, and no band appears; this result indicates that STAT3 proteins with Myc and Flag are linked together inside the cell, forming a STAT3 dimer.
Similarly, as shown in FIG. 7, the direct immunoblot detection with Flag antibody showed a band (input), and it can be seen that Flag-STAT3 expresses protein; following co-immunoprecipitation of Myc, immunoblotting with Flag antibody showed a band (IP) indicating that STAT3 proteins with Myc and Flag were linked together to form a STAT3 dimer.
The result of example 2 shows that after entering a target cell, the pCDH-CMV-Myc-STAT3-T2A-Flag-STAT3-GFP-Puro plasmid constructed by the invention can form two STAT3 monomers (respectively provided with Myc and Flag labels) under the action of 2A peptide, the molar ratio is 1: 1, and for a cell with high STAT3 dimerization level, the STAT3 with the Myc and Flag labels expressed by the plasmid can form a dimer, so that the plasmid is easy to detect by co-immunoprecipitation and immunoblotting.
The plasmid can be transferred into cells to establish a cell model, so that the change of the cell dimerization level can be conveniently monitored, and the plasmid can be used for screening STAT3 dimerization inhibitors or researching the influence of a certain factor on STAT3 dimerization. The invention has the advantages that the STAT3 dimerization related research can be realized only by transfecting one plasmid, the transfection efficiency can be greatly improved, and meanwhile, the experimental process is simplified, and the method is convenient and feasible.
SEQUENCE LISTING
<110> Sichuan university Hospital in western China
<120> a plasmid for detecting STAT3 dimerization
<130> GYKH1891-2021P0112721CC
<160> 8
<170> PatentIn version 3.5
<210> 1
<211> 61
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
ctagctagca ccatggacta caaggacgac gatgataagg cccaatggaa tcagctacag 60
c 61
<210> 2
<211> 35
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
aatgcggccg ctcacatggg ggaggtagcg cactc 35
<210> 3
<211> 61
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
ctagctagca ccatggacta caaggacgac gatgataagg cccaatggaa tcagctacag 60
c 61
<210> 4
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
atgggcatgc agggctgccg 20
<210> 5
<211> 2489
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
acgccatcca cgctgttttg acctccatag aagattctag agctagcacc atggactaca 60
aggacgacga tgataaggcc caatggaatc agctacagca gcttgacaca cggtacctgg 120
agcagctcca tcagctctac agtgacagct tcccaatgga gctgcggcag tttctggccc 180
cttggattga gagtcaagat tgggcatatg cggccagcaa agaatcacat gccactttgg 240
tgtttcataa tctcctggga gagattgacc agcagtatag ccgcttcctg caagagtcga 300
atgttctcta tcagcacaat ctacgaagaa tcaagcagtt tcttcagagc aggtatcttg 360
agaagccaat ggagattgcc cggattgtgg cccggtgcct gtgggaagaa tcacgccttc 420
tacagactgc agccactgcg gcccagcaag ggggccaggc caaccacccc acagcagccg 480
tggtgacgga gaagcagcag atgctggagc agcaccttca ggatgtccgg aagagagtgc 540
aggatctaga acagaaaatg aaagtggtag agaatctcca ggatgacttt gatttcaact 600
ataaaaccct caagagtcaa ggagacatgc aagatctgaa tggaaacaac cagtcagtga 660
ccaggcagaa gatgcagcag ctggaacaga tgctcactgc gctggaccag atgcggagaa 720
gcatcgtgag tgagctggcg gggcttttgt cagcgatgga gtacgtgcag aaaactctca 780
cggacgagga gctggctgac tggaagaggc ggcaacagat tgcctgcatt ggaggcccgc 840
ccaacatctg cctagatcgg ctagaaaact ggataacgtc attagcagaa tctcaacttc 900
agacccgtca acaaattaag aaactggagg agttgcagca aaaagtttcc tacaaagggg 960
accccattgt acagcaccgg ccgatgctgg aggagagaat cgtggagctg tttagaaact 1020
taatgaaaag tgcctttgtg gtggagcggc agccctgcat gcccatgcat cctgaccggc 1080
ccctcgtcat caagaccggc gtccagttca ctactaaagt caggttgctg gtcaaattcc 1140
ctgagttgaa ttatcagctt aaaattaaag tgtgcattga caaagactct ggggacgttg 1200
cagctctcag aggatcccgg aaatttaaca ttctgggcac aaacacaaaa gtgatgaaca 1260
tggaagaatc caacaacggc agcctctctg cagaattcaa acacttgacc ctgagggagc 1320
agagatgtgg gaatgggggc cgagccaatt gtgatgcttc cctgattgtg actgaggagc 1380
tgcacctgat cacctttgag accgaggtgt atcaccaagg cctcaagatt gacctagaga 1440
cccactcctt gccagttgtg gtgatctcca acatctgtca gatgccaaat gcctgggcgt 1500
ccatcctgtg gtacaacatg ctgaccaaca atcccaagaa tgtaaacttt tttaccaagc 1560
ccccaattgg aacctgggat caagtggccg aggtcctgag ctggcagttc tcctccacca 1620
ccaagcgagg actgagcatc gagcagctga ctacactggc agagaaactc ttgggacctg 1680
gtgtgaatta ttcagggtgt cagatcacat gggctaaatt ttgcaaagaa aacatggctg 1740
gcaagggctt ctccttctgg gtctggctgg acaatatcat tgaccttgtg aaaaagtaca 1800
tcctggccct ttggaacgaa gggtacatca tgggctttat cagtaaggag cgggagcggg 1860
ccatcttgag cactaagcct ccaggcacct tcctgctaag attcagtgaa agcagcaaag 1920
aaggaggcgt cactttcact tgggtggaga aggacatcag cggtaagacc cagatccagt 1980
ccgtggaacc atacacaaag cagcagctga acaacatgtc atttgctgaa atcatcatgg 2040
gctataagat catggatgct accaatatcc tggtgtctcc actggtctat ctctatcctg 2100
acattcccaa ggaggaggca ttcggaaagt attgtcggcc agagagccag gagcatcctg 2160
aagctgaccc aggtagcgct gccccatacc tgaagaccaa gtttatctgt gtgacaccaa 2220
cgacctgcag caataccatt gacctgccga tgtccccccg cactttagat tcattgatgc 2280
agtttggaaa taatggtgaa ggtgctgaac cctcagcagg agggcagttt gagtccctca 2340
cctttgacat ggagttgacc tcggagtgcg ctacctcccc catgtgagcg gccgcgaagg 2400
atctgcgatc gctccggtgc ccgtcagtgg gcagagcgca catcgcccac agtccccgag 2460
aagttggggg gaggggtcgg caatgaacc 2489
<210> 6
<211> 82
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
atagaagatt ctagagctag cgccaccatg gagcaaaagc tcatttctga agaggacctg 60
gcccaatgga atcagctaca gc 82
<210> 7
<211> 97
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
cttgtagtcc atggtgctag cagggccggg attctcctcc acgtcaccgc atgttagaag 60
acttcctctg ccctccatgg gggaggtagc gcactcc 97
<210> 8
<211> 2506
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
tcggagggga tatagcagag ctcgtttagt gaaccgtcag atcgcctgga gacgccatcc 60
acgctgtttt gacctccata gaagattcta gagctagcgc caccatggag caaaagctca 120
tttctgaaga ggacctggcc caatggaatc agctacagca gcttgacaca cggtacctgg 180
agcagctcca tcagctctac agtgacagct tcccaatgga gctgcggcag tttctggccc 240
cttggattga gagtcaagat tgggcatatg cggccagcaa agaatcacat gccactttgg 300
tgtttcataa tctcctggga gagattgacc agcagtatag ccgcttcctg caagagtcga 360
atgttctcta tcagcacaat ctacgaagaa tcaagcagtt tcttcagagc aggtatcttg 420
agaagccaat ggagattgcc cggattgtgg cccggtgcct gtgggaagaa tcacgccttc 480
tacagactgc agccactgcg gcccagcaag ggggccaggc caaccacccc acagcagccg 540
tggtgacgga gaagcagcag atgctggagc agcaccttca ggatgtccgg aagagagtgc 600
aggatctaga acagaaaatg aaagtggtag agaatctcca ggatgacttt gatttcaact 660
ataaaaccct caagagtcaa ggagacatgc aagatctgaa tggaaacaac cagtcagtga 720
ccaggcagaa gatgcagcag ctggaacaga tgctcactgc gctggaccag atgcggagaa 780
gcatcgtgag tgagctggcg gggcttttgt cagcgatgga gtacgtgcag aaaactctca 840
cggacgagga gctggctgac tggaagaggc ggcaacagat tgcctgcatt ggaggcccgc 900
ccaacatctg cctagatcgg ctagaaaact ggataacgtc attagcagaa tctcaacttc 960
agacccgtca acaaattaag aaactggagg agttgcagca aaaagtttcc tacaaagggg 1020
accccattgt acagcaccgg ccgatgctgg aggagagaat cgtggagctg tttagaaact 1080
taatgaaaag tgcctttgtg gtggagcggc agccctgcat gcccatgcat cctgaccggc 1140
ccctcgtcat caagaccggc gtccagttca ctactaaagt caggttgctg gtcaaattcc 1200
ctgagttgaa ttatcagctt aaaattaaag tgtgcattga caaagactct ggggacgttg 1260
cagctctcag aggatcccgg aaatttaaca ttctgggcac aaacacaaaa gtgatgaaca 1320
tggaagaatc caacaacggc agcctctctg cagaattcaa acacttgacc ctgagggagc 1380
agagatgtgg gaatgggggc cgagccaatt gtgatgcttc cctgattgtg actgaggagc 1440
tgcacctgat cacctttgag accgaggtgt atcaccaagg cctcaagatt gacctagaga 1500
cccactcctt gccagttgtg gtgatctcca acatctgtca gatgccaaat gcctgggcgt 1560
ccatcctgtg gtacaacatg ctgaccaaca atcccaagaa tgtaaacttt tttaccaagc 1620
ccccaattgg aacctgggat caagtggccg aggtcctgag ctggcagttc tcctccacca 1680
ccaagcgagg actgagcatc gagcagctga ctacactggc agagaaactc ttgggacctg 1740
gtgtgaatta ttcagggtgt cagatcacat gggctaaatt ttgcaaagaa aacatggctg 1800
gcaagggctt ctccttctgg gtctggctgg acaatatcat tgaccttgtg aaaaagtaca 1860
tcctggccct ttggaacgaa gggtacatca tgggctttat cagtaaggag cgggagcggg 1920
ccatcttgag cactaagcct ccaggcacct tcctgctaag attcagtgaa agcagcaaag 1980
aaggaggcgt cactttcact tgggtggaga aggacatcag cggtaagacc cagatccagt 2040
ccgtggaacc atacacaaag cagcagctga acaacatgtc atttgctgaa atcatcatgg 2100
gctataagat catggatgct accaatatcc tggtgtctcc actggtctat ctctatcctg 2160
acattcccaa ggaggaggca ttcggaaagt attgtcggcc agagagccag gagcatcctg 2220
aagctgaccc aggtagcgct gccccatacc tgaagaccaa gtttatctgt gtgacaccaa 2280
cgacctgcag caataccatt gacctgccga tgtccccccg cactttagat tcattgatgc 2340
agtttggaaa taatggtgaa ggtgctgaac cctcagcagg agggcagttt gagtccctca 2400
cctttgacat ggagttgacc tcggagtgcg ctacctcccc catggagggc agaggaagtc 2460
ttctaacatg cggtgacgtg gaggagaatc ccgtctgctg cagcac 2506

Claims (10)

1. A plasmid for detecting STAT3 dimerization, characterized by: the plasmid comprises the following 3 fragments:
(1) a promoter;
(2) a first STAT3 gene, the 5' end of which is linked to the sequence of tag protein A;
(3) a second STAT3 gene, the 3' end of which is linked to the sequence of tag protein B;
wherein (2) and (3) are linked by T2A, and T2A is a nucleotide encoding a 2A peptide; the tag protein A and the tag protein B are different in sequence;
in the plasmid, the fragments (1) to (3) are arranged in the order of (1) - (2) - (3) or (1) - (3) - (2).
2. The plasmid of claim 1, wherein: the tag protein A is a FLAG tag, and the tag protein B is a Myc tag;
or, the tag protein A is a Myc tag, and the tag protein B is a FLAG tag.
3. The plasmid of claim 1, wherein: the plasmid is obtained by inserting the fragments (1) to (3) into a multiple cloning site by taking a pCDH-CMV-MCS-EF1-CopGFP-T2A-Puro vector as an empty vector skeleton.
4. The method for producing the plasmid according to claim 3, wherein: the method comprises the following steps:
1) carrying out PCR amplification on cDNA of STAT3 by using primers with sequences shown as SEQ ID NO. 1-2 to obtain a Flag-STAT3 fragment;
2) inserting Flag-STAT3 into an empty vector skeleton to obtain pCDH-CMV-Flag-Stat 3-GFP-Puro;
3) carrying out PCR amplification on cDNA of STAT3 by using a primer with a sequence shown as SEQ ID NO. 6-7 to obtain a Myc-STAT3-T2A fragment;
4) the fragment of Myc-STAT3-T2A is cut by NheI enzyme and then inserted into pCDH-CMV-Flag-Stat3-GFP-Puro, thus obtaining the Myc-STAT gene.
5. The method of claim 4, wherein: the step 2) is as follows: the Flag-STAT3 fragment and the empty vector skeleton are cut by NheI and NotI enzyme and then connected by T4 ligase.
6. The method of claim 4, wherein: the step 4) is as follows: after the pCDH-CMV-Flag-Stat3-GFP-Puro is cut by NheI enzyme, the pCDH-CMV-Flag-Stat3-GFP-Puro is connected with the fragment Myc-STAT3-T2A into a ring through homologous recombination.
7. A method of detecting the level of intracellular STAT3 dimerization, comprising: the method comprises the following steps:
a) transfecting the plasmid of any one of claims 1 to 3 into a cell;
b) isolating the protein according to tag protein a; c) And (3) taking the separated protein as a sample, and detecting the tag protein B.
8. The method of claim 7, wherein: the isolation of step b) is an isolation using immunoprecipitation.
9. The method of claim 7, wherein: the detection in the step c) is carried out by using Western blot.
10. Use of the plasmid of any one of claims 1 to 3 for screening for inhibitors of STAT3 dimerization.
CN202110487845.8A 2021-04-30 2021-04-30 Plasmid for detecting STAT3 dimerization Active CN113238053B (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
CN202110487845.8A CN113238053B (en) 2021-04-30 2021-04-30 Plasmid for detecting STAT3 dimerization
KR1020210072639A KR20210076887A (en) 2021-04-30 2021-06-04 A plasmid for detecting stat3 dimerization

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202110487845.8A CN113238053B (en) 2021-04-30 2021-04-30 Plasmid for detecting STAT3 dimerization

Publications (2)

Publication Number Publication Date
CN113238053A CN113238053A (en) 2021-08-10
CN113238053B true CN113238053B (en) 2022-05-13

Family

ID=76607398

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202110487845.8A Active CN113238053B (en) 2021-04-30 2021-04-30 Plasmid for detecting STAT3 dimerization

Country Status (2)

Country Link
KR (1) KR20210076887A (en)
CN (1) CN113238053B (en)

Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5834247A (en) * 1992-12-09 1998-11-10 New England Biolabs, Inc. Modified proteins comprising controllable intervening protein sequences or their elements methods of producing same and methods for purification of a target protein comprised by a modified protein
CN101993945A (en) * 2009-08-10 2011-03-30 芮屈生物技术（上海）有限公司 In situ hybridization detection kit for Stat3 gene, detection method and application thereof
CN103882060A (en) * 2012-12-24 2014-06-25 苏州中同生物科技有限公司 Construction and identification of STAT3-targeting lentivirus interference vector
CN104805119A (en) * 2014-01-23 2015-07-29 杭州纽贝生物科技有限公司 Novel endogenous gene expression substituting lentivirus vector and construction method thereof
CN106380522A (en) * 2016-08-30 2017-02-08 无锡市人民医院 Eukaryotic recombinant protein for specifically inhibiting activity of STAT3, and preparation method and application thereof
CN109777827A (en) * 2018-12-26 2019-05-21 四川大学华西医院 A method of activation Notch1 signal path
CN110636836A (en) * 2017-04-19 2019-12-31 拜奥-帕斯控股股份有限公司 P-ethoxy nucleic acids for STAT3 inhibition
CN111655734A (en) * 2019-04-28 2020-09-11 广州市雷德生物科技有限公司 Zipper fastener structure for promoting formation of protein dimer and application thereof
CN112063643A (en) * 2020-09-09 2020-12-11 华中农业大学 Expression vector and method for detecting interaction of membrane proteins in bacteria
CA3145662A1 (en) * 2019-07-02 2021-01-07 M6P Therapeutics Vector compositions and methods of using same for treatment of lysosomal storage disorders

Family Cites Families (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7402398B2 (en) * 2003-07-17 2008-07-22 Monogram Biosciences, Inc. Measuring receptor homodimerization
ES2296423B1 (en) * 2003-07-31 2009-03-16 Consejo Sup. Investig. Cientificas CONSTRUCTION OF DNA FOR THE PRODUCTION OF DIMERIC FUSION PROTEINS AND ITS APPLICATIONS.
US7527970B2 (en) * 2004-10-15 2009-05-05 The United States Of America As Represented By The Department Of Health And Human Services Method of identifying active chromatin domains
FR2890446B1 (en) * 2005-09-05 2008-04-18 Cis Bio Internat Sa METHOD FOR DETECTING INTRACELLULAR INTERACTION BETWEEN BIO-MOLECULES
US20100093018A1 (en) * 2007-04-26 2010-04-15 Achillion Pharmaceuticals, Inc. Cells expressing chimeric proteins and assays using such cells
US8501925B2 (en) * 2009-01-05 2013-08-06 The United States Of America, As Represented By The Secretary, Department Of Health And Human Resources Nucleic acid modules for expression and tagging of membrane proteins and methods of use
US20160040186A1 (en) * 2014-08-07 2016-02-11 Xiaoyun Liu Dna construct and method for transgene expression
CA3107002A1 (en) * 2018-08-15 2020-04-30 Zymergen Inc. Applications of crispri in high throughput metabolic engineering

Patent Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5834247A (en) * 1992-12-09 1998-11-10 New England Biolabs, Inc. Modified proteins comprising controllable intervening protein sequences or their elements methods of producing same and methods for purification of a target protein comprised by a modified protein
CN101993945A (en) * 2009-08-10 2011-03-30 芮屈生物技术（上海）有限公司 In situ hybridization detection kit for Stat3 gene, detection method and application thereof
CN103882060A (en) * 2012-12-24 2014-06-25 苏州中同生物科技有限公司 Construction and identification of STAT3-targeting lentivirus interference vector
CN104805119A (en) * 2014-01-23 2015-07-29 杭州纽贝生物科技有限公司 Novel endogenous gene expression substituting lentivirus vector and construction method thereof
CN106380522A (en) * 2016-08-30 2017-02-08 无锡市人民医院 Eukaryotic recombinant protein for specifically inhibiting activity of STAT3, and preparation method and application thereof
CN110636836A (en) * 2017-04-19 2019-12-31 拜奥-帕斯控股股份有限公司 P-ethoxy nucleic acids for STAT3 inhibition
CN109777827A (en) * 2018-12-26 2019-05-21 四川大学华西医院 A method of activation Notch1 signal path
CN111655734A (en) * 2019-04-28 2020-09-11 广州市雷德生物科技有限公司 Zipper fastener structure for promoting formation of protein dimer and application thereof
CA3145662A1 (en) * 2019-07-02 2021-01-07 M6P Therapeutics Vector compositions and methods of using same for treatment of lysosomal storage disorders
CN112063643A (en) * 2020-09-09 2020-12-11 华中农业大学 Expression vector and method for detecting interaction of membrane proteins in bacteria

Non-Patent Citations (11)

* Cited by examiner, † Cited by third party
Title
A conditional two-hybrid (C2H) system for the detection of protein-protein interactions that are mediated by post-translational modification;Erce M A 等;《Proteomics》;20130430(第7期);第1059-1064页 *
A low-molecular-weight compound discovered through virtual database screening inhibits Stat3 function in breast cancer cells;Song H 等;《 Proceedings of the National Academy of Sciences》;20051231(第13期);第4700-4705页 *
A Two-Plasmid System for Independent Genetic Manipulation of Subunits of Homodimeric Proteins and Selective Isolation of Chimeric Dimers;Skowronek K 等;《 Analytical Biochemistry》;20020115(第2期);第185-191页 *
Decreased H2B monoubiquitination and overexpression of ubiquitin-specific protease enzyme 22 in malignant colon carcinoma;Wang Z 等;《Human Pathology》;20151231;第46卷(第7期);第1006-1014页 *
Engineering a de novo-designed coiled-coil heterodimerization domain off the rapid detection, purification and characterization of recombinantly expressed peptides and proteins;Tripet B 等;《Protein Eng》;19970301(第11期);第1029-1042页 *
GST-His双标签原核表达载体的构建及应用;赵静等;《生物技术通讯》;20090730(第04期);第520-522页 *
One-step affinity tag purification of full-length recombinant human AP-1 complexes from bacterial inclusion bodies using a polycistronic expression system;Wang W M 等;《Protein Expression & Purification》;20080205;第59卷(第1期);第144-152页 *
人KRAS真核表达载体的构建及生物学功能鉴定;王艺霖 等;《军事医学》;20200125;第44卷;第60-63页 *
基于PHA颗粒-PhaP标签和内含肽元件的极端嗜盐古菌蛋白的表达纯化系统;伍锦花 等;《微生物学报》;20141231(第9期);第998-1009页 *
基于荧光共振能量转移技术的STAT3二聚化抑制剂筛选模型的建立;陈俊生等;《中国海洋药物》;20180615(第03期);第13-21页 *
组蛋白去泛素化修饰的研究进展及其与肿瘤的关系;王紫静 等;《华西医学》;20151231(第10期);第1979-1982页 *

Also Published As

Publication number Publication date
KR20210076887A (en) 2021-06-24
CN113238053A (en) 2021-08-10

Similar Documents

Publication Publication Date Title
Wang et al. RNA binding by the novel helical domain of the influenza virus NS1 protein requires its dimer structure and a small number of specific basic amino acids
CN1416467B (en) Zinc finger domains and methods of identifying same
JP2005529590A5 (en)
NO165201B (en) PROCEDURE FOR THE PREPARATION OF HYBRID MUNAMLE LEUCOCYT INTERFERONS OF 165-166 AMINO ACIDS INCLUDING A HUMAN LEUKOCYT INTERFERON A SUBSEQUENCE AND A HUMAN LEUKOCYT INTERFERON B OR SEQUENCE
WO2015106584A1 (en) Tat-il-24-kdel fusion protein, and preparation method therefor and use thereof
CN113549634B (en) Gene for coding soluble HPV58L1 protein and construction and application of recombinant plasmid thereof
CN113238053B (en) Plasmid for detecting STAT3 dimerization
CN116987166B (en) Rubber tree HbSTRAP2 gene and application thereof
EP0416673A1 (en) Method for the expression of heterologous proteins produced in fused form in E. coli, use thereof, expression vectors and recombinant strains
CN110684794B (en) Method for preparing alpha, beta unsaturated fatty acid by using fatty acid as raw material and utilizing escherichia coli engineering bacteria
CN106084063B (en) Genetic engineering recombinant TRAIL fusion protein, preparation method and application thereof
CN116478262B (en) Application of RACK1 in resisting silkworm nuclear polyhedrosis virus BmNPV
CN107746432B (en) A β 42 modified protein and expression and purification method thereof
US5976864A (en) Expression and secretion of heterologous polypeptides from caulobacter
CN112481286B (en) Amino acid sequence for improving heterologous expression efficiency of recombinant milk protein
CN101775404A (en) Method for highly expressing basic protein with prokaryotic expression system
CN101880314B (en) Bursin-like peptide of gene engineering as well as expression gene and application thereof
KR100843634B1 (en) Transmembrane Delivery Peptide and Bio-material Comprising the Same
KR100803095B1 (en) Gene coding chitosanase from bacillus subtilis ch2 expression vector comprising the gene transformant transfected with the expression vector and method for purification of protein produced by thereof
CN114057861A (en) Bio-PROTAC artificial protein targeting UBE2C
CN102277327B (en) Colon bacillus for over-expressing RimL and application on preparing N-extrasin alpha acetylate
CN111850022A (en) Method for quickly adding or replacing protein tag of target gene
CN110616230A (en) Method for promoting secretory expression of zearalenone degrading enzyme ZHD518 protein and application
CN105198999A (en) Fusion protein and its preparation method and use
KR890004807B1 (en) Dna coding for antigen protein of rinderpest virus

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant