CN114836470A - Vector for expressing combined fluorescent protein fragment and application thereof - Google Patents
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Abstract
The invention belongs to the technical field of biology, and particularly relates to a group of vectors for expressing four fluorescent protein fragments, which can be used for realizing the complementation of fluorescent proteins and fluorescence resonance energy transfer, and can monitor the interaction of 4 proteins at the living cell level. The CENP-T/W/S/X complex fusion protein and CENPT, CENPW, SPC24 and SPC25 fusion proteins are selected in the embodiment of the invention to verify the system. The system becomes a leading-edge technology in the field of biophotonic in the age of functional proteomics, and has very considerable application prospect.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a vector for expressing four combined fluorescent protein fragments and application thereof, which can be used for generating fusion protein to realize four-molecule fluorescence resonance energy transfer analysis.
Background
The life activity is represented by the interaction between biological macromolecules on the molecular level. Protein-proteinQualitative interactions are important among them. FRET technology is a biophotonic approach to study protein-protein interactions. Fluorescence Resonance Energy Transfer (FRET) refers to the fact that, in two different fluorophores, if the emission spectrum of one fluorophore (Donor/Donor) overlaps with the absorption spectrum of the other fluorophore (Acceptor/Acceptor) to some extent, the distance between the two fluorophores is appropriate (generally smaller than that of the two fluorophores)
) The phenomenon of fluorescence energy transfer from the donor to the acceptor can be observed, i.e., fluorescence emitted from the former group is observed when excited at the excitation wavelength of the latter group. The fluorescent groups can be different small-molecule fluorescein or different fluorescent proteins.
The conventional FRET technique for studying the interaction between two proteins is to express protein a fused with cyan fluorescent protein (e.g. ecfp (enhanced cyan fluorescent protein), mTurquoise2, mCerulean) and protein B fused with yellow fluorescent protein (e.g. eyfp (enhanced yellow fluorescent protein), Venus). When two fusion proteins are expressed simultaneously in a cell, if the A/B two proteins can directly interact and the spatial distance of the fused cyan/yellow fluorescent protein after the A/B interaction is less than 10 nm, when the cell is irradiated with excitation light of the cyan fluorescent protein having a wavelength of 434nm, the emission light of the 527nm yellow fluorescent protein, that is, a FRET signal, can be detected.
For example, when the fluorescent protein mTurquoise2 is fused to protein a and the fluorescent protein EYFP is fused to protein B, if protein a is bound to protein B and the separation is timely, FRET will occur when mTurquoise2 is excited with 434nm light and the emission signal of EYFP can be detected. Because the fluorescent protein can be genetically labeled, FRET has become a powerful tool for detecting the interaction and activity change (such as kinase activity and acetyl transferase activity) of biomacromolecule at a nanometer distance in a living body, and has wide application in biomacromolecule interaction analysis, cell physiological research, immunoassay and the like.
However, existing FRET techniques are generally only capable of detecting interactions between 2 proteins or 3 proteins. Many protein complexes or protein-protein interactions are the binding between 4 proteins, but at present, there is no technical means to detect the interaction between 4 proteins directly by using a visualization method.
Disclosure of Invention
In order to develop a method capable of directly characterizing direct interaction between 4 proteins, the invention provides a vector for realizing four-molecule fluorescence resonance energy transfer analysis by expressing combined fluorescent protein fragments, and proves that four expressed fusion proteins can generate FRET signals in cells. This demonstrates that the method of the present invention is an effective imaging method to study the interaction between 4 proteins.
The embodiment of the invention utilizes a bimolecular fluorescence complementation strategy to divide mTurquoise2 fluorescent protein into two fragments of mTurquoise2N (AA1-155) and mTurquoise2C (AA156-239), divide EYFP fluorescent protein into two fragments of EYFP PN (AA1-155) and EYFP (AA156-239), amplify DNA sequences coding the four protein fragments by utilizing PCR, insert pcDNA3.1/Myc-His-B vector after enzyme digestion, and form recombinant vectors for expressing different fluorescent protein fragments. And then four target proteins of A/B/C/D are selected to construct recombinant plasmids which are respectively fused and expressed with the fluorescent protein fragments. Four plasmids were transfected into HeLa cells to achieve intracellular expression of four fusion proteins, mTurquoise2N-A, mTurquoise2C-B, EYFPN-C and EYFPC-D. If the four proteins A/B/C/D interact in the cell, A/B will mediate the complementation of mTurquoise2N/mTurquoise2C into a fluorescent mTurquoise2 protein, and C/D will mediate the complementation of EYFP/EYFP into a fluorescent EYFP protein. When the spatial distance of functional mTurquoise2 and EYFP is less than 10 nm, the emission light (indicated by CFPem) signal of Cyan Fluorescent Protein (CFP) decreases when the cell is irradiated with 434nm excitation light (indicated by CFPex), while the emission light (indicated by YFPem) of 527nm Yellow Fluorescent Protein (YFP) can be detected. Three image signals of CFPex/CFPem, YFP Pex/YFP, and CFPex/YFP are respectively collected under a microscope, and a FRET signal is divided by a CFP signal according to the following formula to obtain a FRET ratio. The detection of a FRET ratio greater than 1.0 confirms the direct intracellular interaction of the four proteins A/B/C/D.
Based on the research of the invention, in one aspect, the invention provides a recombinant vector combination, which comprises a first recombinant vector, a second recombinant vector, a third recombinant vector and a fourth recombinant vector,
wherein, the first recombinant vector contains a coding gene of a first segment of the first fluorescent protein, the second recombinant vector contains a coding gene of a second segment of the first fluorescent protein, and the first segment and the second segment of the first fluorescent protein form the first fluorescent protein;
the third recombinant vector contains a coding gene of a first fragment of the second fluorescent protein, the fourth recombinant vector contains a coding gene of a second fragment of the second fluorescent protein, and the first fragment and the second fragment of the second fluorescent protein form the second fluorescent protein;
wherein the first fluorescent protein and the second fluorescent protein are capable of generating a fluorescence resonance energy transfer signal.
In some embodiments, the first fluorescent protein is a cyan fluorescent protein and the second fluorescent protein is a yellow fluorescent protein.
In some embodiments, the first fluorescent protein is Enhanced Cyan Fluorescent Protein (ECFP), mTurquoise2, or mCerulean and the second fluorescent protein is Enhanced Yellow Fluorescent Protein (EYFP) or Venus.
In some embodiments, the first recombinant vector and the second recombinant vector are obtained by inserting the first fragment and the second fragment of the first fluorescent protein into the same site of the vector, respectively, and the third recombinant vector and the fourth recombinant vector are obtained by inserting the first fragment and the second fragment of the second fluorescent protein into the same site of the vector, respectively.
In some embodiments, the first fluorescent protein is mTurquoise2, the first fragment of which is SEQ ID NO: 11, and the second fragment is the amino acid sequence shown in SEQ ID NO: 12.
In some embodiments, the genes encoding the first and second fragments of the first fluorescent protein are inserted into the pcDNA3.1/myc-His-B vector, respectively, and preferably, the genes encoding the first and second fragments of the first fluorescent protein are inserted into the KpnI site or the Xba I site of the pcDNA3.1/myc-His-B vector, respectively.
In some embodiments, the second fluorescent protein is EYFP, the first fragment of which is SEQ ID NO: 13, and the second fragment is the amino acid sequence shown in SEQ ID NO: 14, or a pharmaceutically acceptable salt thereof.
In some embodiments, the genes encoding the first and second fragments of the second fluorescent protein are inserted into the pcDNA3.1/myc-His-B vector, respectively, and preferably, the genes encoding the first and second fragments of the second fluorescent protein are inserted into the Kpn I site or the Xba I site of the pcDNA3.1/myc-His-B vector, respectively.
On the other hand, the invention provides a method for detecting the interaction of four proteins to be detected in real time by using the recombinant vector combination, which comprises the following steps:
respectively inserting the coding genes of the four proteins to be detected into a first recombinant vector, a second recombinant vector, a third recombinant vector and a fourth recombinant vector to obtain recombinant vectors for respectively expressing the four fusion proteins;
transferring the recombinant vector expressing the four fusion proteins into the same host cell;
and detecting the fluorescence signal of the host cell, and judging whether the four proteins to be detected interact with each other.
In some embodiments, if a FRET signal is generated in the host cell, there are interactions between the four test proteins and the spatial distance of the interactions is less than 10 nanometers;
if no FRET signal is produced in the host cell, there is no interaction or the spatial distance between the four test proteins is greater than 10 nanometers.
Experiments prove that a set of fluorescent protein pairs which can generate FRET signals after complementation is created by using a molecular biological means, and the fluorescent protein pairs are cloned into different plasmid vectors so as to be fused and expressed with different proteins. When there is an interaction between four proteins coupled to different moieties, it is possible to first generate a fully functional cyan fluorescent protein and a fully functional yellow fluorescent protein in a complementary manner. When the two pairs of proteins interact in close proximity, a FRET signal may then be generated, demonstrating that the four proteins form a spatially close complex within the cell. The invention provides a fluorescent protein molecule pair capable of generating FRET signals, and two pairs of interacting proteins in a CENP-TWSX compound are respectively coupled on complementary combined protein fragments, so that when the constituent proteins of the CENP-TWSX compound interact, cyan fluorescent protein mTurquoise2 and yellow fluorescent protein EYFP molecule pair are complemented again, and when cells are irradiated by 434nm exciting light under a microscope, 474nm emitting light emitted by the cyan fluorescent protein mTurquoise2 can partially excite EYFP, and 527nm emitting light signals of the EYFP can be observed and collected. The traditional FRET technology can only analyze the interaction between two proteins, and the system can directly analyze the interaction between four proteins, can be widely applied to the dynamic process tracing of protein complexes in cells or outside cells, and has considerable application prospect.
The interaction among the four proteins is detected under the condition of living cell culture, so the invention can dynamically detect the space-time dynamics of the interaction of the target protein in the cell in real time, and has wide application in the aspects of cell cycle, cell division, autophagy, formation and dissociation of membrane-free organelles and other cell biology scientific problems and the screening of targeted drugs.
Drawings
The drawings are only for purposes of illustrating and explaining the present invention and are not to be construed as limiting the scope of the present invention. Wherein:
FIG. 1 is a schematic diagram of mTurquoise2N-CENPT (Bonsai), mTurquoise2C-CENPW, EYFPN-CENPS, EYFPC-CENPX fusion proteins and experimental results;
FIG. 2 is a diagram of mTurquoise2N-CENPT (Bonsai), mTurquoise2C-CENPW, Spc24-EYFPN, Spc25-EYFPC four fusion proteins and experimental results.
Detailed Description
In order that the objects, technical solutions and advantages of the present invention will become more apparent, the present invention will be further described in detail with reference to the accompanying drawings in conjunction with the following specific embodiments.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The primers and DNA sequences used in the following examples were synthesized by Shanghai Biotech.
The sequences in the following examples are as follows:
the nucleotide sequence of the PCR product mTurquoise2N-C1 is the sequence SEQ ID NO: 1, the encoded protein is mTurquoise2N, the amino acid sequence is the sequence SEQ ID NO: 11;
the nucleotide sequence of the PCR product mTurquoise2C-C1 is SEQ ID NO: 2, the encoded protein is mTurquoise2C, the amino acid sequence is the sequence SEQ ID NO: 12;
the nucleotide sequence of the PCR product EYFPN-C1 is shown as a sequence SEQ ID NO: 3, the coded protein is EYFPN, and the amino acid sequence is SEQ ID NO: 13;
the nucleotide sequence of the PCR product EYFPC-C1 is shown as a sequence SEQ ID NO: 4, the coded protein is EYFPC, and the amino acid sequence is SEQ ID NO: 14;
the amino acid sequence of CENPT-Bonsai is shown as a sequence SEQ ID NO: 15, the nucleotide sequence is the sequence of SEQ ID NO: 5;
the amino acid sequence of CENPW is the sequence SEQ ID NO: 16, the nucleotide sequence is the sequence of SEQ ID NO: 6;
the amino acid sequence of CENPS is the sequence SEQ ID NO: 17, the nucleotide sequence is the sequence of SEQ ID NO: 7;
the amino acid sequence of CENPX is the sequence SEQ ID NO: 18, the nucleotide sequence is the sequence SEQ ID NO: 8;
the amino acid sequence of Spc24 is the sequence SEQ ID NO: 19, the nucleotide sequence is the sequence of SEQ ID NO: 9;
the amino acid sequence of Spc25 is the sequence SEQ ID NO: 20, the nucleotide sequence is the sequence of SEQ ID NO: 10.
example 1 analysis of the interaction of CENP-T/W/S/X four proteins Using four-molecule FRET
One, acquisition of combined fluorescent proteins mTurquoise2 and EYFP
Four DNA fragments, namely mTurquoise2N-C1, mTurquoise2C-C1, mTurquoise2N-N1 and mTurquoise2C-N1, which respectively express two protein fragments, namely mTurquoise2N and mTurquoise2C, are amplified by polymerase chain reaction by using cDNA of the mTurquoise2 protein as a template. (Note: C1/N1 indicates that the relative positions of the fluorescent protein and the fusion protein are different and that the primers used for amplification are different; wherein C1 indicates that the fluorescent protein is at the N-terminus and the fusion protein is at the C-terminus; and N1 is the opposite.)
1. Obtainment of mTurquoise2N-C1
With the primer sequences:
polymerase chain reaction was performed using cDNA of mTurquoise2 as a template.
The solution system of the reaction is as follows:
the PCR reaction steps are as follows:
obtaining a 465bp product, and sequencing to obtain a nucleotide sequence of the product which is shown as a sequence SEQ ID NO: 1, the product is named as mTurquoise2N-C1, the coded protein is mTurquoise2N-C1, and the amino acid sequence is SEQ ID NO: 11.
2. obtaining of mTurquoise2C-C1
With the primer sequences:
polymerase chain reaction was performed using cDNA of mTurquoise2 as a template.
The reaction system and the steps are the same as those of 1, and the mTurquoise2N-C1 is obtained, so that a 252bp product is obtained, and the nucleotide sequence of the product is shown as a sequence SEQ ID NO: 2, the product is named as mTurquoise2C-C1, the coded protein is mTurquoise2C, and the amino acid sequence is SEQ ID NO: 12.
3. obtaining of mTurquoise2N-N1
The primer sequences are utilized:
polymerase chain reaction was performed using cDNA of mTurquoise2 as a template.
The reaction system and the steps are the same as those of 1, and mTurquoise2N-C1, so that a 465bp product is obtained, and the nucleotide sequence of the product is shown as a sequence SEQ ID NO: 1, the product is named as mTurquoise2N-N1, the coded protein is mTurquoise2N, and the amino acid sequence is SEQ ID NO: 11.
4. obtaining of mTurquoise2C-N1
With the primer sequences:
polymerase chain reaction was performed using cDNA of mTurquoise2 as a template.
The reaction system and the steps are the same as those of 1, and the mTurquoise2N-C1 is obtained, so that a 252bp product is obtained, and the nucleotide sequence of the product is shown as a sequence SEQ ID NO: 2, the product is named as mTurquoise2C-N1, the coded protein is mTurquoise2C, and the amino acid sequence is SEQ ID NO: 12.
and (3) amplifying four DNA fragments EYFP-C1 and EYFP-C1, EYFP-N1 and EYFP-N1 which respectively express two protein fragments of EYFP and EYFP by using cDNA of the EYFP protein as a template through polymerase chain reaction.
5. Acquisition of EYFPN-C1
With the primer sequences:
and performing polymerase chain reaction by using cDNA of EYFP as a template.
The reaction system and the steps are the same as those of 1, and mTurquoise2N-C1, so that a 465bp product is obtained, and the nucleotide sequence of the product is shown as a sequence SEQ ID NO: 3, the product is named EYFPN-C1, the coded protein is EYFPN, and the amino acid sequence is SEQ ID NO: 13.
6. acquisition of EYFPC-C1
With the primer sequences:
and performing polymerase chain reaction by using cDNA of EYFP as a template.
The reaction system and the steps are the same as those of 1, and mTurquoise2N-C1, a 255bp product is obtained, and the nucleotide sequence of the product is shown as a sequence SEQ ID NO: 4, the product is named as EYFPC-C1, the coded protein is EYFPC, and the amino acid sequence is shown as SEQ ID NO: 14.
7. acquisition of EYFPN-N1
With the primer sequences:
and performing polymerase chain reaction by using cDNA of EYFP as a template.
The reaction system and the steps are the same as those of 1, and mTurquoise2N-C1, so that a 465bp product is obtained, and the nucleotide sequence of the product is shown as a sequence SEQ ID NO: 3, the product is named as EYFPN-N1, the coded protein is EYFPN, the amino acid sequence is shown as a sequence SEQ ID NO: 13.
8. acquisition of EYFPC-N1
With the primer sequences:
and performing polymerase chain reaction by using cDNA of EYFP as a template.
The reaction system and the steps are the same as those of 1, and mTurquoise2N-C1, a 255bp product is obtained, and the nucleotide sequence of the product is shown as a sequence SEQ ID NO: 4, the product is named as EYFPC-N1, the coded protein is EYFPC, and the amino acid sequence is shown as SEQ ID NO: 14.
secondly, the construction of the vector pcDNA3.1-mTurquoise2N-C, pcDNA3.1-mTurquoise2C-C, pcDNA3.1-EYFPN-C, pcDNA3.1-EYFPC-C, pcDNA3.1-mTurquoise2N-N, pcDNA3.1-mTurquoise2C-N, pcDNA3.1-EYFPN-N, pcDNA3.1-EYFPC-N
1. Construction of pcDNA3.1-mTurquoise2N-C, pcDNA3.1-mTurquoise2C-C, pcDNA3.1-EYFPN-C, and pcDNA3.1-EYFPC-C
Carrying out DNA single enzyme digestion reaction on the PCR products mTurquoise2N-C1, mTurquoise2C-C1, EYFPN-C1 or EYFP-C1 (the following reaction system is replaced by insert) obtained in the step one, wherein the used enzyme is KpnI, and the reaction system is as follows:
the reaction conditions were 37 ℃ incubation for 1 hour.
The pcDNA3.1MycHis-B vector (Thermo, Cat. No. V80020) was also digested with KpnI in the following manner:
the reaction conditions were 37 ℃ incubation for 1 hour.
After completion of the digestion of the vector, it was necessary to further digest it with alkaline phosphatase for 1 hour to remove the phosphate group at the 5' -end of the vector DNA. The reaction system is as follows:
the reaction conditions were 37 ℃ incubation for 1 hour.
The resulting enzyme-cleaved products were subjected to agarose gel electrophoresis, respectively. Agarose was purchased from Shanghai Biotech, Inc., and 1.0g of agarose was dissolved in 100ml of 1 XTAE buffer, heated to boiling by microwave, placed in a mold, cooled, and then solidified to form. 50 XTAE was purchased from Shanghai Producer, and diluted with ddH2O to give 1 XTAE buffer. Electrophoresis was performed in a1 × TAE buffer, with a set voltage of 120V and a current of 150-. The product strip is cut and recovered, and the recovery reaction uses a glue recovery kit of Shanghai biological engineering technology Limited. The recovered product is ligated in a ligation system. The connecting system is as follows:
the reaction conditions were 16 ℃ for 8 hours.
The resulting ligation product was transformed into TOP10 competent cells by adding 10. mu.l of the product to 50. mu.l of competent cells on ice and incubating for 30 min on ice, then for 1 min at 42 ℃ and then for 2 min on ice after removal. Then adding 1ml LB culture medium, mixing, shaking at 37 deg.C constant temperature shaking table at 250rpm for 1 hr to obtain product, coating on agarose gel plate containing ampicillin and nutrient components, and growing in 37 deg.C constant temperature oven for 12 hr to obtain 4 clones, clone 1, clone 2, clone 3 and clone 4.
6 clones of each clone were selected and cultured overnight in LB medium containing ampicillin, plasmids of clones 1-4 were extracted and sent to the organism for sequencing, and the results were as follows:
the positive plasmid 1 is a plasmid obtained by converting a sequence SEQ ID NO: 1 into the KpnI site of pcDNA3.1MycHis-B vector, named pcDNA3.1-mTurquoise 2N-C;
the positive plasmid 2 is a plasmid obtained by converting a sequence SEQ ID NO: 2a plasmid inserted between KpnI sites of the pcDNA3.1MycHis-B vector and named pcDNA3.1-mTurquoise 2C-C;
the positive plasmid 3 is a plasmid obtained by converting a sequence SEQ ID NO: 3 a plasmid obtained by inserting the plasmid into KpnI sites of the pcDNA3.1MycHis-B vector, and the plasmid is named pcDNA3.1-EYFPN-C;
the positive plasmid 4 is a plasmid obtained by converting a sequence SEQ ID NO: 4 inserting the plasmid obtained between KpnI sites of the pcDNA3.1MycHis-B vector, and the plasmid is named pcDNA3.1-EYFPC-C;
2. construction of pcDNA3.1-mTurquoise2N-N, pcDNA3.1-mTurquoise2C-N, pcDNA3.1-EYFPN-N, and pcDNA3.1-EYFPC-N
Carrying out single DNA enzyme digestion reaction on the PCR products mTurquoise2N-N1, mTurquoise2C-N1, EYFPN-N1 or EYFPC-N1 (the following reaction system is replaced by insert) obtained in the step one, wherein the used enzyme is XbaI, and the reaction system is as follows:
the reaction conditions were 37 ℃ incubation for 1 hour.
The pcDNA3.1MycHis-B vector (Thermo, Cat. No. V80020) was also digested with XbaI in the following manner:
the reaction conditions were 37 ℃ incubation for 1 hour.
After completion of the digestion of the vector, it was necessary to further digest it with alkaline phosphatase for 1 hour to remove the phosphate group at the 5' -end of the vector DNA. The reaction system is as follows:
the reaction conditions were 37 ℃ incubation for 1 hour.
The obtained enzyme digestion products were recovered and ligated (system as above), and the obtained ligation products were transformed into TOP10 competent cells to obtain 4 clones, clone 5, clone 6, clone 7 and clone 8, respectively.
6 clones of each clone were selected and cultured overnight in LB medium containing ampicillin, plasmids of clones 1-4 were extracted and sent to the organism for sequencing, and the results were as follows:
the positive plasmid 5 is a plasmid obtained by converting a sequence SEQ ID NO: 1 into the XbaI site of pcDNA3.1MycHis-B vector, named pcDNA3.1-mTurquoise 2N-N;
the positive plasmid 6 is a plasmid obtained by converting a sequence SEQ ID NO: 2 inserting into XbaI site of pcDNA3.1MycHis-B vector, named pcDNA3.1-mTurquoise 2C-N;
the positive plasmid 7 is obtained by inserting the sequence SEQ ID NO-3 in the sequence table into XbaI sites of the pcDNA3.1MycHis-B vector, and is named as pcDNA3.1-EYFPN-N;
the positive plasmid 8 is obtained by inserting the sequence SEQ ID NO-4 in the sequence table into XbaI sites of the pcDNA3.1MycHis-B vector, and is named as pcDNA3.1-EYFPC-N;
thirdly, obtaining CENP-TWSX compound protein CENPT-Bonsai/CENPW/CENPS/CENPX
PCR was carried out using cDNAs as templates of CENPT-Bonsai (CENPT-Bonsai is an amino acid sequence designed by artificial modification of the protein sequence of CENPT), CENPW, CENPS, CENPX (all of which can be artificially synthesized, genbank numbers are NM-025082, NM-001286524, NM-199294, NM-001271006, respectively), under the conditions and system as described in example 1, and primers as follows:
artificially synthesized cDNA of CENPT is used as a template, and the primers are as follows:
CENPT-Bonsai-F：
ACAGGATCCGGTGGATCTGGTGGACAGGCCAGTGGGCACTTG(SEQ ID NO：31)
CENPT-Bonsai-R：ACACTCGAGTTCTGGGCAGGGAAGACAGAG(SEQ ID NO：32)
using cDNA of CENPW as a template, and using primers as follows:
CENPW-F：
ACAGGATCCGGTGGATCTGGTGGAGCGCTGTCGACCATAGTC(SEQ ID NO：33)
CENPW-R：ACACTCGAGTTACCTCTGCTCTTCTTTAGAATTACC(SEQ ID NO：34)
using cDNA of CENPS as a template, and using primers as follows:
CENPS-F：
ACAGGATCCGGTGGATCTGGTGGAGCGGAGACCGAGGAGCAG(SEQ ID NO：35)
CENPS-R：ACACTCGAGTTATTCTCACTTTCCACCACTCCAGC(SEQ ID NO：36)
using cDNA of CENPX as a template, and using primers as follows:
CENPX-F：ACAAGATCTGGATCTGAGGGAGCAGGAGCTGGA(SEQ ID NO：37)
CENPX-R：ACACTCGAGTTGAAGTCCAGGAGCAGCTGCG(SEQ ID NO：38)
the PCR product size of CENPT-Bonsai is 462bp, and the nucleotide is shown as a sequence SEQ ID NO: 5, the amino acid sequence of which is the sequence SEQ ID NO: 15;
the PCR product of CENPW is 261bp, and the nucleotide is sequenced to obtain a nucleotide sequence shown as a sequence SEQ ID NO: 6, the amino acid sequence of which is the sequence SEQ ID NO: 16;
the PCR product of CENPS is 402bp in size, and the nucleotide is sequenced to obtain a nucleotide sequence SEQ ID NO: 7, the amino acid sequence of which is the sequence SEQ ID NO: 17;
the PCR product of CENPX has the size of 240bp, and the nucleotide is a sequence SEQ ID NO: 8, the amino acid sequence of which is the sequence SEQ ID NO: 18.
the obtained PCR products are respectively subjected to agarose gel electrophoresis and gel recovery reaction, and the method is the same as the above.
The recovered product was digested with the DNA restriction enzyme BamHI/XhoI, and the constructed plasmid vectors pcDNA3.1-mTurquoise2N-C, pcDNA3.1-mTurquoise2C-C, pcDNA3.1-EYFPN-C and pcDNA3.1-EYFPC-C were digested with the same enzymes. And (3) carrying out agarose gel electrophoresis and gel recovery reaction on the enzyme digestion product to obtain corresponding insert and a carrier band. The ligation reaction was performed in the following pairing manner:
pcDNA3.1-mTurquoise2N-C：：CENPT-Bonsai(a)
pcDNA3.1-mTurquoise2C-C：：CENPW(b)
pcDNA3.1-EYFPN-C：：CENPS(c)
pcDNA3.1-EYFPC-C：：CENPX(d)
the ligation product was transformed into competent cells of TOP10 strain and selected for monoclonal amplification, resulting in 4 clones in total, clones 1) -4). The plasmids of the clones 1) to 4) are respectively extracted and sent to Shanghai's company for sequencing, and the positive plasmids with correct sequencing are as follows:
(a) the sequence of SEQ ID NO: 5 into the BamHI/XhoI cleavage site of pcDNA3.1-mTurquoise2N-C, named pcDNA3.1-mTurquoise 2N-CENPT-Bonsai;
(b) the sequence of SEQ ID NO: 6 is inserted into the BamHI/XhoI enzyme cutting sites of pcDNA3.1-mTurquoise2C-C to obtain a recombinant plasmid which is named pcDNA3.1-mTurquoise 2C-CENPW;
(c) the sequence of SEQ ID NO: 7 is inserted into BamHI/XhoI enzyme cutting sites of pcDNA3.1-YFPN-C to obtain a recombinant plasmid which is named pcDNA3.1-YFPN-CENPS;
(d) the sequence of SEQ ID NO: 8 is inserted into BamHI/XhoI enzyme cutting sites of pcDNA3.1-YFP-C to obtain a recombinant plasmid which is named pcDNA3.1-YFP-CENPX;
transfection, expression and detection of four recombinant proteins of CENP-TWSX compound HeLa cells were transfected with the four plasmids a) to d) described above. The transfection method comprises the following steps: HeLa cells (purchased from ATCC in USA) were plated on 35mm glass-bottom plates (Fisher Scientific, MatTek/P35G-1.5-20-C), and 2ml of DMEM medium (Invitrogen #31600-034) containing 10% FBS (Atlanta # S11550) was added to each well and cultured in a 37-degree constant temperature incubator containing 8% CO 2. After 24 hours, the medium was changed to 400. mu.l Opti-MEM (Invitrogen # 11058-. Another two sterile 1.5ml centrifuge tubes were added to 100. mu.l of Opti-MEM medium, respectively. One of the tubes was added with 4. mu.l of Lipofectamine 3000 transfection reagent (Invitrogen #11668-019), and the other tube was added with recombinant plasmids (1. mu.g each of the above 4 plasmids, and 0.5. mu.g of H2B-mCherry plasmid was added). Mixing, and standing for 5 min. Then the solutions in the two centrifuge tubes were mixed, mixed well, centrifuged and left to stand for 20 minutes. Then, the mixture was dropped into a 35mm glass-bottomed culture dish containing 2ml of the medium, and after culturing for 4 hours in an incubator, the medium was replaced with DMEM complete medium containing 10% FBS.
After transfection, HeLa cells were individually synchronized by adding 2mM Thymidine (Sigma-Aldrich # T1895) to the medium, releasing the cells after 16 hours by replacing with DMEM complete medium containing 10% FBS and 1% Glutamine, and after 7.5 hours, obtaining cells synchronized to the late G2 stage by replacing the medium with CO preheated at 37 ℃ and containing 10% FBS and 1% Glutamine 2 An Independent Medium (Thermo, #18045088) was used to observe a sample of live cells using a Delta Vision microscope using a 63 Xoil lens. Shoot 0.5 μm × 3 layers, channel: CFP/CFP; CFP/YFP; YFP/YFP; TRITC, frame/10 min.
The specific results are shown in fig. 1:
wherein, 1A is a), b), c), d) four kinds of plasmids expressed fusion protein mTurquoise2N-CENPT-Bonsai, mTurquoise2N-CENPW, EYFPN-CENPS and EYFPC-CENPX structural schematic diagram;
1B, transfection expressing two pairs of fusion proteins mTurquoise2N-CENPT-Bonsai and mTurquoise2C-CENPW, EYFPN-CENPS and EYFPC-CENPX in HeLa cells, respectively. After 24 hours of transfection, immunofluorescent staining was performed, both ACA and DNA were stained, and finally the images were collected under a DeltaVision fluorescence microscope after mounting. The green channel is the fluorescent signal after CENPT-Bonsai/CENPW, CENPS/CENPX complementation, respectively, and it can be seen that two pairs of fluorescent proteins can be complemented and correctly positioned in the centromere/kinetochore region.
1C, an expression plasmid expressing the fusion protein as shown in FIG. 1A and a mCherry-H2B expression plasmid (indicating chromatin/chromosomes) were co-transfected into HeLa cells. Viable cells were imaged 24 hours after transfection under a DeltaVision fluorescence microscope. 0.5 μm × 3 layers were photographed using Olympus 63 × oil lens, and the following channels were photographed, respectively: CFP/CFP; CFP/YFP; YFP/YFP; TRITC, frame/10 min. The first behavior in this figure is chromatin signal/mCherry-H2B, the second behavior is CFP/CFP signal, the third behavior is YFP/YFP signal, and the fourth behavior is FRET ratio characterized by cold and warm colors. This figure shows that 5 plasmids of our transfection were expressed in the cells, mTurquoise2N-CENPT-Bonsai which after complementation with mTurquoise2C-CENPW yielded a fluorescent signal for mTurquoise2 and could be positively localized to the centromere, EYFPN-CENPS which after complementation with EYFPC-CENPX yielded an EYFP signal and could be positively localized to the centromere. The fourth row shows a warm FRET ratio, indicating the presence of a FRET signal.
1D, statistical plots of the calculated FRET ratios were collected at different time points. The figure shows the mean value of FRET for each spot (centromere) after statistics, indicating that FRET data is authentic.
Example 2 analysis of the interaction of four proteins CENP-T/W and Spc24/Spc25 Using four-molecule FRET
Construction of Spc24 and Spc25 fusion proteins
1. Acquisition of Spc25/Spc 24-expressing DNA
PCR was carried out using Spc24/Spc25 (amino acid sequence of Spc24 is SEQ ID NO: 19, nucleotide sequence is SEQ ID NO: 9, amino acid sequence of Spc25 is SEQ ID NO: 20, nucleotide sequence is SEQ ID NO: 10; both of which can be artificially synthesized, and genbank numbers are NM-182513.3 and NM-020675.4, respectively) cDNAs as templates, under the conditions and in the system as described in example 1, with primers as follows:
the cDNA of Spc24 was used as template, and the primers used for Spc24 were:
Spc24-F：ACAGGATCCGCCACCATGGCCGCCTTCCGCGAC(SEQ ID NO：39)
Spc24-R：ACAGAATTCAACCACTCGGTGTCCACCAG(SEQ ID NO：40)
the cDNA of Spc25 was used as template, and the primers used for Spc25 were:
Spc25-F：
ACAGGATCCGCCACCATGGTAGAGGACGAACTGG(SEQ ID NO：41)
Spc25-R：
ACAGAATTCAAATTATAAACCGTGGCAGTAAAAGC(SEQ ID NO：42)
the PCR product of Spc24 is 591bp in size, and the nucleotide is sequenced to obtain the nucleotide sequence shown as SEQ ID NO: 9, the amino acid sequence of which is the sequence SEQ ID NO: 19;
the size of the PCR product of Spc25 is 672bp, and the nucleotide is sequenced to obtain a nucleotide sequence shown as SEQ ID NO: 10, the amino acid sequence of which is the sequence SEQ ID NO: 20.
the obtained PCR products are respectively subjected to agarose gel electrophoresis and gel recovery reaction, and the method is the same as the above.
The recovered product is digested with BamHI/EcoRI enzyme, and the constructed plasmid vectors pcDNA3.1-YFPN-N and pcDNA3.1-YFPC-N are digested simultaneously. And (3) carrying out agarose gel electrophoresis and gel recovery reaction on the enzyme digestion product to obtain corresponding insert and a carrier band. The ligation reaction was performed in the following pairing manner:
pcDNA3.1-YFPN-N：：Spc24(e)
pcDNA3.1-YFPC-N：：Spc25(f)
the ligation product was transformed into competent cells of TOP10 strain and selected for monoclonal amplification, resulting in 2 clones in total, clones 5) -6). The plasmids of clones 5) to 6) were extracted and digested with BamHI/EcoRI to obtain fragments of about 600bp and about 700bp, respectively, and the plasmids from which the digestion products were obtained were designated as positive plasmids e) to f).
The positive plasmids e) to f) were sequenced separately, and the results were as follows:
the positive plasmid e) is a plasmid obtained by converting a sequence SEQ ID NO: 9 is inserted into the BamHI/EcoRI enzyme cutting sites of pcDNA3.1-YFPN-N to obtain a carrier which is named pcDNA3.1-YFPN-Spc 24;
the positive plasmid f) is a plasmid obtained by converting a sequence SEQ ID NO: 10 is inserted into the BamHI/EcoRI enzyme cutting site of pcDNA3.1-YFPC-N to obtain a carrier which is named pcDNA3.1-YFPC-Spc 25;
the sequencing reaction was performed by Shanghai Biotech.
Secondly, detecting the expression and interaction of the CENPT-Bonsai, CENPW, Spc24 and Spc25 proteins in cells
1. HeLa cells were transfected with the four plasmids a), b), e) and f) described above. Transfection method as in example 1).
After transfection, HeLa cells were individually synchronized by adding 2mM Thymidine (Sigma-Aldrich # T1895) to the medium, releasing the cells after 16 hours by replacing with DMEM complete medium containing 10% FBS and 1% Glutamine, and after 7.5 hours, obtaining cells synchronized to the late G2 stage by replacing the medium with CO preheated at 37 ℃ and containing 10% FBS and 1% Glutamine 2 An Independent Medium (Thermo, #18045088) was used to observe a sample of live cells using a Delta Vision microscope using a 63 Xoil lens. Shoot 0.5 μm × 3 layers, channel: CFP/CFP; CFP/YFP; YFP/YFP; TRITC, frame/10 min.
The specific results are shown in fig. 2:
2A is a), b), e), f) four kinds of plasmids expressed fusion protein mTurquoise2N-CENPT-Bonsai, mTurquoise2N-CENPW, Spc24-EYFPN and Spc25-EYFPC structural schematic diagram;
2B is a pair of fusion proteins for expressing Spc24-EYFPN and Spc25-EYFPC in HeLa cells by transfection. After 24 hours of transfection, immunofluorescent staining was performed, both ACA and DNA were stained, and finally the images were collected under a DeltaVision fluorescence microscope after mounting. The green channel is the fluorescent signal after the complementation of Spc24/Spc25, and thus, two pairs of fluorescent proteins can be complemented and correctly positioned in the kinetochore region.
2C, co-transfection of an expression plasmid expressing the fusion protein as shown in figure 2A and a mCherry-H2B expression plasmid (indicating chromatin/chromosome) into HeLa cells. Viable cells were imaged 24 hours after transfection under a DeltaVision fluorescence microscope. 0.5 μm × 3 layers were photographed using Olympus 63 × oil lens, and the following channels were photographed, respectively: CFP/CFP; CFP/YFP; YFP/YFP; TRITC, frame/10 min. The first behavior in this figure is chromatin signal/mCherry-H2B, the second behavior is CFP/CFP signal, the third behavior is YFP/YFP signal, and the fourth behavior is FRET ratio characterized by cold and warm colors. This figure shows that 5 plasmids of our transfection were expressed in the cells, that mTurquoise2N-CENPT-Bonsai after complementation with mTurquoise2C-CENPW gave a fluorescent signal for mTurquoise2 and could be positively located in the centromere, and that SPC24-EYFP after complementation with SPC25-EYFP gave a EYFP signal and could be positively located in the centromere. The fourth row shows a warm FRET ratio, indicating the presence of a FRET signal.
And 2D, collecting statistical graphs of the calculated FRET ratio values at different time points, wherein the graphs show the average value of the FRET of each point (centromere) obtained after statistics, and the FRET data are proved to be credible.
The above-mentioned embodiments are intended to illustrate the objects, technical solutions and advantages of the present invention in further detail, and it should be understood that the above-mentioned embodiments are only exemplary embodiments of the present invention and are not intended to limit the present invention, and any modifications, equivalents, improvements and the like made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A recombinant vector combination comprises a first recombinant vector, a second recombinant vector, a third recombinant vector and a fourth recombinant vector,
wherein, the first recombinant vector contains a coding gene of a first segment of the first fluorescent protein, the second recombinant vector contains a coding gene of a second segment of the first fluorescent protein, and the first segment and the second segment of the first fluorescent protein form the first fluorescent protein;
the third recombinant vector contains a coding gene of a first fragment of the second fluorescent protein, the fourth recombinant vector contains a coding gene of a second fragment of the second fluorescent protein, and the first fragment and the second fragment of the second fluorescent protein form the second fluorescent protein;
wherein the first fluorescent protein and the second fluorescent protein are capable of generating a fluorescence resonance energy transfer signal.
2. The recombinant vector combination according to claim 1, wherein the first fluorescent protein is a cyan fluorescent protein and the second fluorescent protein is a yellow fluorescent protein.
3. The recombinant vector combination according to claim 1, wherein the first fluorescent protein is Enhanced Cyan Fluorescent Protein (ECFP), mTurquoise2, or mCErulean, and the second fluorescent protein is Enhanced Yellow Fluorescent Protein (EYFP) or Venus.
4. The recombinant vector combination according to claim 1, wherein the first and second recombinant vectors are obtained by inserting the first and second fragments of the first fluorescent protein into the same sites of the vector, respectively, and the third and fourth recombinant vectors are obtained by inserting the first and second fragments of the second fluorescent protein into the same sites of the vector, respectively.
5. The recombinant vector combination according to claim 1, wherein the first fluorescent protein is mTurquoise2, the first fragment of which is SEQ ID NO: 11, and the second fragment is the amino acid sequence shown in SEQ ID NO: 12.
6. The recombinant vector combination according to claim 5, wherein the genes encoding the first and second fragments of the first fluorescent protein are inserted into the pcDNA3.1/myc-His-B vector, respectively, preferably the genes encoding the first and second fragments of the first fluorescent protein are inserted into the KpnI site or XbaI site of the pcDNA3.1/myc-His-B vector, respectively.
7. The recombinant vector combination according to claim 1, wherein the second fluorescent protein is EYFP, the first fragment of which is SEQ ID NO: 13, and the second fragment is the amino acid sequence shown in SEQ ID NO: 14, or a pharmaceutically acceptable salt thereof.
8. The recombinant vector combination according to claim 7, wherein the genes encoding the first and second fragments of the second fluorescent protein are inserted into the pcDNA3.1/myc-His-B vector, respectively, preferably the genes encoding the first and second fragments of the second fluorescent protein are inserted into the Kpn I site or Xba I site of the pcDNA3.1/myc-His-B vector, respectively.
9. A method for detecting in real time the interaction of four test proteins using a combination of recombinant vectors according to any one of claims 1 to 8, comprising:
respectively inserting the coding genes of the four proteins to be detected into a first recombinant vector, a second recombinant vector, a third recombinant vector and a fourth recombinant vector to obtain recombinant vectors for respectively expressing the four fusion proteins;
transferring the recombinant vector expressing the four fusion proteins into the same host cell;
and detecting the fluorescence signal of the host cell, and judging whether the four proteins to be detected interact with each other.
10. The method of claim 9, wherein if a FRET signal is generated in the host cell, there are interactions between the four test proteins and the spatial distance of the interactions is less than 10 nanometers;
if no FRET signal is produced in the host cell, there is no interaction or the spatial distance between the four test proteins is greater than 10 nanometers.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103290091A (en) * | 2012-03-22 | 2013-09-11 | 华中科技大学 | Protein interaction detection method with low false positive rate |
| CN104991072A (en) * | 2015-06-16 | 2015-10-21 | 西北农林科技大学 | Manufacturing method and application of insect in-vitro protein interaction detecting system |
| US20210388411A1 (en) * | 2018-07-27 | 2021-12-16 | Feed Research Institute, Chinese Academy Of Agricultural Sciences | Recombinant expression vector applicable to rapid screening for recombinant strain and application |
-
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- 2021-02-02 CN CN202110144958.8A patent/CN114836470A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103290091A (en) * | 2012-03-22 | 2013-09-11 | 华中科技大学 | Protein interaction detection method with low false positive rate |
| CN104991072A (en) * | 2015-06-16 | 2015-10-21 | 西北农林科技大学 | Manufacturing method and application of insect in-vitro protein interaction detecting system |
| US20210388411A1 (en) * | 2018-07-27 | 2021-12-16 | Feed Research Institute, Chinese Academy Of Agricultural Sciences | Recombinant expression vector applicable to rapid screening for recombinant strain and application |
Non-Patent Citations (6)
| Title |
|---|
| Y. JOHN SHYU等: ""Visualization of AP-1–NF-κB ternary complexes in living cells by using a BiFC-based FRET"", 《PROC NATL ACAD SCI U S A》, vol. 105, no. 1, pages 288 - 152 * |
| YUTAKA KODAMA等: ""Bimolecular fluorescence complementation (BiFC):A 5-year update and future perspectives"", 《BIOTECHNIQUES》, vol. 53, no. 5, pages 288 * |
| 刘爱平等: "《细胞生物学荧光技术原理与应用》", 28 February 2007, 中国科学技术大学出版社, pages: 238 * |
| 姚亭秀;: "3种探究蛋白质互作实验技术简介", 生物学通报, no. 05 * |
| 沈珂;吴群英;蒋晓山;: "双分子荧光互补技术在蛋白质相互作用研究中的应用", 中国医学创新, no. 12, pages 132 - 135 * |
| 金艳燕;邓君;金良韵;肖忠新;徐志卿;张进禄;赵君朋;薛冰;: "荧光共振能量转移技术阳性参照质粒的构建及应用", 山西医科大学学报, no. 11 * |
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