Bimolecular fluorescence complementation system based on red fluorescent protein mScarlet3 and its construction method and application

Abstract

The present application discloses a bimolecular fluorescence complementation system based on the red fluorescent protein mScarlet3, its construction method, and its application in the imaging of protein-protein interactions. The red fluorescent protein mScarlet3 is a protein composed of 229 amino acids. The present invention applies the mScarlet3 fluorescent protein to bimolecular fluorescence complementation, and selects the fluorescent protein splitting sites 154 / 155 and 169 / 170 with strong signals and extremely low background signals. Compared with the previous bimolecular fluorescence complementation system based on red fluorescent protein, the red fluorescent protein used in this system has the characteristics of low dimerization degree, high intrinsic brightness, and short maturation time. Therefore, the system developed by it has the characteristics of low background, high signal-to-noise ratio, and short observation time. Based on these advantages, this system can be used to further screen other protein-protein interactions.

Description

Technical Field

[0001] The present application belongs to the technical field of fluorescence imaging of protein interactions in living cells, and specifically relates to the development and application of a bimolecular fluorescence complementation system. Background Art

[0002] Protein-protein interactions and protein-nucleic acid interactions underlie many intracellular biological processes. Studying these interactions between biomacromolecules not only helps us understand cellular physiological mechanisms at the molecular level, but also serves as an effective means of discovering many new drug targets. Over the past few decades, numerous techniques have been developed to study protein-protein interactions, such as Fluorescence Resonance Energy Transfer (FRET), co-immunoprecipitation (Co-Immunoprecipitation), and Yeast Two-Hybrid Assay (Yeast Two-Hybrid Assay). While each has its own advantages, they also have limitations. Among these techniques, bimolecular fluorescence complementation (BiFC) has gained favor among researchers as a method that allows for direct in situ observation of protein-protein interactions under physiological conditions.

[0003] Fluorescent proteins such as green fluorescent protein (GFP) consist of a barrel-shaped structure composed of β-pleated sheets and a chromophore composed of several amino acids at the center of the barrel. Bimolecular fluorescence complementation involves the splitting and reassembling of fluorescent proteins: the fluorescent protein is separated into its N-terminus and C-terminus, which are then fused to two test proteins. If the two test proteins do interact, the covalently linked N- and C-termini of the fluorescent protein will reassemble into a complete fluorescent protein structure due to their spatial proximity and emit fluorescence. The advantages of bimolecular fluorescence complementation include its ease of operation, high sensitivity, and ability to detect weak and transient interactions. Over the past decade, an increasing number of fluorescent proteins have been used in bimolecular fluorescence complementation, which has not only enriched the spectral coverage of the technology but also improved the system's performance.

[0004] Among red fluorescent proteins, bimolecular fluorescence complementation systems based on mRFP1-Q66T, mCherry, and mScarlet I have been developed. However, due to the performance defects of each of these fluorescent proteins, the bimolecular fluorescence complementation systems developed therefrom may also show deficiencies in some aspects. For example, earlier developed RFPs such as mRFP1-Q66T and mCherry have problems such as low quantum yield, photochromism or residual dimerization tendency, which may reduce the signal-to-noise ratio of detection to a certain extent. The first-generation proteins in the mScarlet series have high intrinsic brightness, no photochromism, and are completely monomeric RFPs, but they also have the problem of relatively slow maturation time, which may cause the subcellular localization of fluorescence observed in bimolecular fluorescence complementation to be inconsistent with the actual protein interaction. mScarlet I, a variant of the first-generation mScarlet, has been greatly improved in maturation time and degree of maturation, but its intrinsic brightness is lower than that of the first-generation mScarlet protein. Therefore, this application is proposed to address the problems of false positives, weak brightness, slow maturation time, etc. in existing bimolecular fluorescence complementation systems. Summary of the Invention

[0005] The first purpose of the present application is to provide a bimolecular fluorescence complementation system based on the red fluorescent protein mScarlet3 to overcome the problems of false positive, weak brightness and slow maturation time existing in the prior art.

[0006] To achieve the aforementioned invention objectives, the technical solutions adopted in this application include:

[0007] A bimolecular fluorescence complementation system based on red fluorescent protein mScarlet3 comprises a first vector and a second vector. The red fluorescent protein mScarlet3 is a protein composed of 229 amino acids, and the amino acid sequence of the red fluorescent protein mScarlet3 is shown in SEQ ID NO.1. The first vector comprises a gene sequence for expressing a first protein fragment, and the second vector comprises a gene sequence for expressing a second protein fragment. When the first and second protein fragments are brought into proximity, they can reconstruct to form the red fluorescent protein mScarlet3. The first protein fragment is a protein fragment composed of amino acids from positions 1 to n of the red fluorescent protein mScarlet3, and the second protein fragment is a protein fragment composed of amino acids from positions (n+1) to 229 of the red fluorescent protein mScarlet3. n is 154 or 169.

[0008] In some embodiments, when n is 154, the first protein fragment is the mSc154N protein fragment, and the second protein fragment is the mSc155C protein fragment; the gene sequence for expressing mSc154N comprises the gene sequence SEQ ID NO.3 and the gene sequence for expressing the mSc155C protein fragment comprises the gene sequence SEQ ID NO.4.

[0009] In other embodiments, when n is 169, the first protein fragment is the mSc169N protein fragment, and the second protein fragment is the mSc170C protein fragment; the gene sequence for expressing the mSc169N protein fragment includes the gene sequence SEQ ID NO.5 and the gene sequence for expressing the mSc170C protein fragment includes the gene sequence SEQ ID NO.6.

[0010] Furthermore, the first vector further comprises a first gene sequence, the first gene sequence being used to express a first test protein, and the first vector being used to express a first fusion protein, which is a fusion protein of the first test protein and a first protein fragment. The second vector further comprises a second gene sequence, the second gene sequence being used to express a second test protein, and the second vector being used to express a second fusion protein, which is a fusion protein of the second protein and a second protein fragment.

[0011] The second object of the present application is to provide a method for constructing a bimolecular fluorescence complementation system based on the red fluorescent protein mScarlet3, comprising the following steps:

[0012] S1: The gene sequence of the red fluorescent protein mScarlet3 is cloned into a first eukaryotic expression vector to synthesize a gene vector containing mScarlet3;

[0013] S2: Using the gene vector containing mScarlet3 as a template, the gene sequence for expressing the first protein fragment and the gene sequence for expressing the second protein fragment are obtained by polymerase chain reaction;

[0014] S3: Using a recombinase, the gene sequence for expressing the first protein fragment is inserted into the second eukaryotic expression vector to form a first vector; using a recombinase, the gene sequence for expressing the second protein fragment is inserted into the third eukaryotic expression vector to form a second vector;

[0015] The red fluorescent protein mScarlet3 is composed of 229 amino acids, its amino acid sequence is shown in SEQ ID NO1, and its gene sequence is shown in SEQ ID NO2; when the first protein fragment and the second protein fragment are close to each other, they can be reconstructed to form the red fluorescent protein mScarlet3; wherein: the first protein fragment is a protein fragment composed of amino acids 1 to n of the red fluorescent protein mScarlet3, and the second protein fragment is a protein fragment composed of amino acids (n+1) to 229 of the red fluorescent protein mScarlet3; n is 154 or 169.

[0016] Furthermore, the first eukaryotic expression vector includes one of pcDNA3.1(+) vector, pUC57, and pUC19; the second eukaryotic expression vector includes one of pcDNA3.1(+) vector, pUC57, and pUC19; and the third eukaryotic expression vector includes one of pcDNA3.1(+) vector, pUC57, and pUC19.

[0017] In some embodiments, the first eukaryotic expression vector is a pcDNA3.1(+) vector, the gene vector containing mScarlet3 is pcDNA3.1-mScarlet3; the gene sequence for expressing mSc154N is the gene sequence SEQ ID NO. 3, and the gene sequence for expressing the mSc155C protein fragment is the gene sequence SEQ ID NO. 4; in step S2:

[0018] When the gene sequence SEQ ID NO.3 was obtained by polymerase chain reaction using pcDNA3.1-mScarlet3 as a template,

[0019] The upstream primers are:

[0020] 5'-ACCCAAGCTGGCTAGCCACCATGGATAGCACCGAGGCAGT-3'

[0021] The downstream primers are:

[0022] 5'-ACGGGCCCTCTAGACTCGAGTCAGTCCTCGGGGTACAACC-3';

[0023] When the gene sequence SEQ ID NO.4 was obtained by polymerase chain reaction using pcDNA3.1-mScarlet3 as a template,

[0024] The upstream primers are:

[0025] 5'-CCAAGCTGGCTAGCCACCATGGTCGTGCTGAAGGGCG-3'

[0026] The downstream primers are:

[0027] 5'-GGCCCTCTAGACTCGAGTCAGGAGCCACCGGAGCCGCCGG-3'.

[0028] In other embodiments, the first eukaryotic expression vector is a pcDNA3.1(+) vector, the gene vector containing mScarlet3 is pcDNA3.1-mScarlet3; the gene sequence for expressing mSc169N is the gene sequence SEQ ID NO. 5, and the gene sequence for expressing the mSc170C protein fragment is the gene sequence SEQ ID NO. 6; in step S2:

[0029] When the gene sequence SEQ ID NO.5 was obtained by polymerase chain reaction using pcDNA3.1-mScarlet3 as a template,

[0030] The upstream primers are:

[0031] 5'-CCAAGCTGGCTAGCCACCatggatagcaccgaggcagt-3'

[0032] The downstream primers are:

[0033] 5'-ACGGGCCCTCTAGACTCGAGTCAgtccttcaggcgcag-3'

[0034] When the gene sequence SEQ ID NO.6 was obtained by polymerase chain reaction using pcDNA3.1-mScarlet3 as a template,

[0035] The upstream primers are:

[0036] 5'-CAAGCTGGCTAGCCACCatgggcggccgctacctg-3';

[0037] The downstream primers are:

[0038] 5'-CCTCTAGACTCGAGTCAggagccaccggagccgccg-3'.

[0039] The third object of the present invention is to provide the application of the bimolecular fluorescence complementation system based on red fluorescent protein mScarlet3 in the imaging of protein-protein interactions in the first object.

[0040] Further, the following steps are included:

[0041] Step A1: cloning the gene sequences of the first test protein and the second test protein into the first vector and the second vector, respectively, to obtain the first test protein-first vector plasmid and the second test protein-second vector plasmid;

[0042] Step A2: Using a transfection reagent, the constructed test protein-first vector plasmid and the test protein-second vector plasmid are co-transfected into the cells to be tested;

[0043] Step A3: After culturing for a period of time, perform fluorescence microscopy imaging.

[0044] Compared to existing technologies, this invention applies the mScarlet3 fluorescent protein to bimolecular fluorescence complementation, selecting a fluorescent protein splitting site with a strong signal and extremely low background signal. Compared to previous bimolecular fluorescence complementation systems based on red fluorescent protein, the red fluorescent protein used in this system has a low degree of dimerization, high intrinsic brightness, and a short maturation time. As a result, the system developed with this method has low background, high signal-to-noise ratio, and short observation time. Based on these advantages, this system can be applied to further screen for other protein-protein interactions. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 pcDNA3.1-mScarlet3 plasmid map.

[0046] Figure 2 Plasmid maps of the first vector and the second vector.

[0047] Figure 3 Plasmid maps of EGFP-mSc154N, mSc155C-EGFP, EGFP-mSc169N, and mSc170C-EGFP.

[0048] Figure 4 (A): Fluorescence emitted by cells 16 hours after co-transfection of EGFP-mSc154N and mSc155C-EGFP; (B): Fluorescence emitted by cells 16 hours after co-transfection of EGFP-mSc169N and mSc170C-EGFP.

[0049] Figure 5bJun-mSc154N, mSc155C-bFos, mSc155C-bFosdZIP plasmid maps.

[0050] Figure 6 (A): Fluorescence of cells 16 hours after co-transfection of bJun-mSc154N and mSc155C-bFos; (B): Fluorescence of cells 16 hours after co-transfection of bJun-mSc154N and mSc155C-bFosdZIP.

[0051] Figure 7. Plasmid maps of ORC1-mSc169N and mSc170C-HMGA1a.

[0052] Figure 8 Fluorescence of cells 16 hours after co-transfection of ORC1-mSc169N and mSc170C-HMGA1a. DETAILED DESCRIPTION

[0053] The present application is further described below with reference to the embodiments, but is not limited thereto.

[0054] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources. The terms "first" and "second" in this application are used to distinguish different objects rather than to describe a specific order.

[0055] This application provides a bimolecular fluorescence complementation system based on the red fluorescent protein mScarlet3, comprising a first vector and a second vector. The red fluorescent protein mScarlet3 is a protein composed of 229 amino acids, and the amino acid sequence of the red fluorescent protein mScarlet3 is shown in SEQ ID NO. 1. The first vector contains a gene sequence for expressing a first protein fragment, and the second vector contains a gene sequence for expressing a second protein fragment. When the first and second protein fragments are brought into proximity, they reconstitute to form the red fluorescent protein mScarlet3.

[0056] This application discloses two sets of bimolecular fluorescence complementation systems based on the red fluorescent protein mScarlet3. The following introduces the two sets of bimolecular fluorescence complementation systems and their construction methods and applications.

[0057] Example 1:

[0058] The first set of bimolecular fluorescence complementation system consists of a first vector containing the sequence SEQ ID NO.3 ( Figure 2A ) and a second vector comprising the sequence SEQ ID NO4 ( Figure 2B ). Specifically, the first protein fragment is composed of amino acids 1 to 154 of the red fluorescent protein mScarlet3, named mSc154N; the second protein fragment is composed of amino acids 155 to 229 of the red fluorescent protein mScarlet3, named mSc155C.

[0059] The construction method of the first bimolecular fluorescence complementation system is as follows:

[0060] Step S1: Synthesize a gene vector containing mScarlet3. The gene sequence of mScarlet3 is shown in SEQ ID NO.2. The gene sequence of mScarlet3 is cloned into the pcDNA3.1(+) vector and named pcDNA3.1-mScarlet3. The map of the synthesized vector is shown in Figure 1 shown.

[0061] Step S2: PCR was performed using pcDNA3.1-mScarlet3 as a template to amplify the sequence SEQ ID NO. 3. Those skilled in the art will appreciate that appropriate upstream and downstream primers can be designed based on the actual situation. It should be noted that when designing primers, homologous sequences that can be recognized by the recombinase in step S3 need to be added to the primers. In this example, the upstream primer used to amplify the sequence SEQ ID NO. 3 is:

[0062] 5'-ACCCAAGCTGGCTAGCCACCATGGATAGCACCGAGGCAGT-3'

[0063] The downstream primers are:

[0064] 5'-ACGGGCCCTCTAGACTCGAGTCAGTCCTCGGGGTACAACC-3'

[0065] Step S3: Use recombinase to insert the sequence SEQ ID NO.3 for expressing mSc154N into the pcDNA3.1 vector to form the first vector containing the sequence SEQ ID NO.3. The plasmid map is shown in FIG. Figure 2A shown.

[0066] Those skilled in the art will appreciate that the recombinase is not limited to a specific form, such as separate recombinase and buffer or a premixed solution. In this embodiment, the "Novorec Plus One Step Cloning Kit" was used. Alternatively, the "Vazyme ClonExpress Ultra One Step Cloning Kit" and the "pEASY-Basic Seamless Cloning and Assembly Kit" can also achieve the functions of this embodiment.

[0067] Those skilled in the art will also appreciate that pcDNA3.1 vector fragments can be obtained by a variety of methods. In this example, pcDNA3.1-mScarlet3 was used as a template to amplify and recover pcDNA3.1 vector fragments. The upstream primers used were:

[0068] 5'-CTCGAGTCTAGAGGGCCCGTTTAAACCCGC-3'

[0069] The downstream primers are:

[0070] 5'-CATGGTGGCTAGCCAGCTTGGG-3'

[0071] Similarly, when using pcDNA3.1-mScarlet3 as a template for PCR amplification of sequence SEQ ID NO. 4, appropriate upstream and downstream primers can be designed based on the actual situation. It should be noted that when designing primers, it is necessary to add homologous sequences that can be recognized by the recombinase to the primers. In this example, the upstream primer used to amplify sequence SEQ ID NO. 4 is:

[0072] 5'-CCAAGCTGGCTAGCCACCATGGTCGTGCTGAAGGGCG-3'

[0073] The downstream primers are:

[0074] 5'-GGCCCTCTAGACTCGAGTCAGGAGCCACCGGAGCCGCCGG-3'

[0075] The upstream primers used to amplify the pcDNA3.1 vector fragment using pcDNA3.1-mScarlet3 as a template are:

[0076] 5'-CTCGAGTCTAGAGGGCCCGTTTAAACCCGC-3'

[0077] The downstream primers are:

[0078] 5'-CATGGTGGCTAGCCAGCTTGGG-3'

[0079] The above primers were used to amplify and recover the pcDNA3.1 vector fragment. Then, the sequence SEQ ID NO.4 for expressing mSc154N was inserted into the pcDNA3.1 vector using a recombinase to form a second vector containing the sequence SEQ ID NO.4. The plasmid map is shown in FIG. Figure 2B shown.

[0080] The first vector and the second vector constructed above were transformed into Escherichia coli respectively. After the sequences were verified to be correct by sequencing, the endotoxin-free plasmid was extracted using an endotoxin-free plasmid mini-midiprep kit.

[0081] Application of the first bimolecular fluorescence complementation system:

[0082] Step A1: The gene sequences of the first test protein and the second test protein are cloned into the first vector and the second vector, respectively, to obtain the first test protein-first vector plasmid and the second test protein-second vector plasmid.

[0083] Specifically include:

[0084] After constructing a first vector containing the sequence of SEQ ID NO. 3 and a second vector containing the sequence of SEQ ID NO. 4, PCR is used to amplify the DNA sequences of the first and second test proteins from existing plasmids or cDNA libraries. Those skilled in the art will appreciate that the orientation of the first and second test proteins relative to mSc154N or mSc155C can be adjusted based on actual conditions.

[0085] The present application does not limit the first test protein and the second test protein. In some embodiments, the first test protein and the second test protein can be known interacting proteins, such as the first test protein is FKBP protein and the second test protein is FRB protein; or the first test protein is Bak protein and the second test protein is Bcl-XL protein; or the first test protein is bJun protein and the second test protein is bFos protein, etc. The system of the present application is used to determine whether a specific protein-protein interaction exists in the cell. In other embodiments, the first test protein and the second test protein can be proteins whose interaction is unknown, and the system provided by the present application can be used to determine whether the two proteins under study have an interaction.

[0086] In this embodiment, the enhanced green fluorescent protein (Enhanced Green Fluorescent Protein) that can form a homodimer is taken as an example, hereinafter referred to as EGFP. That is to say, in this embodiment, the first protein to be tested and the second protein to be tested are both EGFP. Using the pX458 plasmid as a template, primers for amplifying the EGFP fragment are designed and synthesized. It should be noted that when designing primers, it is necessary to add a linker sequence between EGFP and the mSc154N protein fragment or between EGFP and the mSc155C protein fragment, and it is necessary to add a homologous sequence that can be recognized by the recombinase to the primer. The linker amino acid sequence of EGFP connected to the mSc154N protein fragment is such as the sequence SEQ ID NO.7, and the gene sequence is such as the sequence SEQ ID NO.8.

[0087] When EGFP is linked to the N-terminus of the mSc154N protein fragment, the upstream primer used to amplify EGFP is:

[0088] 5'-CCCAAGCTGGCTAGCCACCATGGTGAGCAAGGGCGAGGAG-3'

[0089] The downstream primers are:

[0090] 5'-ACTGCCTCCACCTCCGCTACCTCCACCGCCCTTGTACAGCTCGTCCATGCCG-3'

[0091] When amplifying the first vector sequence using the first vector containing the sequence SEQ ID NO. 3 as a template, the upstream primer is:

[0092] 5'-TAGCGGAGGTGGAGGCAGTATGGATAGCACCGAGGCAG-3'

[0093] The downstream primers are:

[0094] 5'-CATGGTGGCTAGCCAGCTTGGG-3'

[0095] The above primers were used to amplify and recover the first vector fragment, and then the EGFP was inserted into the first vector containing the sequence SEQ ID NO.3 using a recombinase to form pcDNA3.1-EGFP-154N. The plasmid map is shown in FIG. Figure 3A shown.

[0096] The amino acid sequence of the linker connecting EGFP and the mSc155C protein fragment is shown in SEQ ID NO.9, and the gene sequence is shown in SEQ ID NO.10.

[0097] When EGFP is linked to the C-terminus of the mSc155C protein fragment, the upstream primer used to amplify EGFP is:

[0098] 5'-GGCTCTGGCTCAGGCAGTATGGTGAGCAAGGGCGAGGAG-3'

[0099] The downstream primers are:

[0100] 5'-TTTAAACGGGCCCTCTAGACTCGAGTCACTTGTACAGCTCGTCC-3'

[0101] When amplifying the second vector sequence using the second vector containing the sequence SEQ ID NO. 4 as a template, the upstream primer is:

[0102] 5'-GGCTCTGGCTCAGGCAGTATGGTGAGCAAGGGCGAGGAG-3'

[0103] The downstream primers are:

[0104] 5'-CATGGTGGCTAGCCAGCTTGGG-3'

[0105] The second vector fragment was amplified and recovered using the above primers, and then EGFP was inserted into the second vector containing the sequence SEQ ID NO.4 using recombinase to form pcDNA3.1-155C-EGFP. The plasmid map is shown in FIG. Figure 3B shown.

[0106] Step A2: The pcDNA3.1-EGFP-154N and pcDNA3.1-155C-EGFP constructed above were transformed into Escherichia coli. After the sequences were verified by sequencing, the endotoxin-free plasmid was extracted using an endotoxin-free plasmid mini-midiprep kit.

[0107] Lipofectamine 3000 was used to transfect the two plasmids into the cells to be tested at a ratio of 1:1.

[0108] Step A3: After 12 to 24 hours, observe the cells under a fluorescence microscope. Figure 4 As shown in A (the cells to be tested are HEK293T cells), the transfected cells exhibit bright red fluorescence.

[0109] In another embodiment, bJun protein is used as the first protein to be detected, and bFos protein is used as the second protein to be detected.

[0110] 1. pcDNA3.1(+)-lifeact-mScarlet3 was synthesized by Sangon Biotechnology Co., Ltd.

[0111] bJun and bFos sequences were obtained by PCR from existing plasmids in the laboratory. It should be noted that when designing primers, a linker sequence needs to be added between bJun or bFos and mSc154N or mSc155C, and a homologous sequence that can be recognized by the recombinase needs to be added to the primers.

[0112] Using the first vector or the second vector as a template, the first vector sequence or the second vector sequence was obtained by PCR amplification and recovery. Using the Novorec PCR One-Step Directional Cloning Kit, bJun was inserted into the first vector to obtain the plasmid bJun-mSc154N. Figure 5A shown.

[0113] The same method was used to clone bFos into the second vector to obtain plasmid bFos-mSc155C, as shown in Figure 5B shown.

[0114] Using bFos-mSc155C as a template, a bFos lacking the ZIP domain that interacts with bJun was obtained by PCR and named bFosdZIP. It was cloned together with mSc155C into the pcDNA3.1 vector to obtain the plasmid bFosdZIP-mSc155C. Figure 5C shown.

[0115] 2. Transient Transfection of HEK293T Cells

[0116] The human renal epithelial cell line HEK293T was maintained by the inventor's laboratory.

[0117] bJun-mSc154N and bFos-mSc155C, bJun-mSc154N and bFosdZIP-mSc155C (as a negative control) were co-transfected into HEK293T cells at a 1:1 ratio using Lipofectamine 3000 liposome transfection reagent.

[0118] 16 hours after transfection, cells were observed using a fluorescence microscope and the fluorescence signals were recorded.

[0119] 3. Observe the results

[0120] like Figure 6 As shown in A, cells transfected with bJun-mSc154N and bFos-mSc155C emit bright red fluorescence. Figure 6 As shown in B, the fluorescence brightness of the negative control, i.e., cells transfected with bJun-mSc154N and bFosdZIP-mSc155C, was much lower than that of cells transfected with bJun-mSc154N and bFos-mSc155C, indicating that there is an interaction between bJun and bFos, and that this interaction is dependent on the ZIP sequence in bFos.

[0121] Example 2:

[0122] The second set of bimolecular fluorescence complementation system consists of a first vector containing the sequence SEQ ID NO.5 ( Figure 2C ) and a second vector comprising the sequence SEQ ID NO.6 ( Figure 2D ). Specifically, the first protein fragment is composed of amino acids 1 to 169 of the red fluorescent protein mScarlet3, named mSc169N; the second protein fragment is composed of amino acids 170 to 229 of the red fluorescent protein mScarlet3, named mSc170C.

[0123] The construction method of the second bimolecular fluorescence complementation system is as follows:

[0124] Step S1: As in Example 1, a gene vector containing mScarlet3 was synthesized. The gene sequence of mScarlet3 is shown in SEQ ID NO. 2. The gene sequence of mScarlet3 was cloned into the pcDNA3.1(+) vector and named pcDNA3.1-mScarlet3. The synthesized vector map is shown in FIG. Figure 1 shown.

[0125] Step S2: PCR was performed using pcDNA3.1-mScarlet3 as a template to amplify the sequence SEQ ID NO. 5. Those skilled in the art will appreciate that appropriate upstream and downstream primers can be designed based on the actual situation. It should be noted that when designing primers, homologous sequences that can be recognized by the recombinase in step S3 need to be added to the primers. In this example, the upstream primer used to amplify the sequence SEQ ID NO. 5 is:

[0126] 5'-CCAAGCTGGCTAGCCACCATGGATAGCACCGAGGCAGT-3'

[0127] The downstream primers are:

[0128] 5'-ACGGGCCCTCTAGACTCGAGTCAGTCCTTCAGGCGCAG-3'

[0129] Step S3: Use recombinase to insert the sequence SEQ ID NO.5 for expressing mSc169N into the pcDNA3.1 vector to form the first vector containing the sequence SEQ ID NO.5. The plasmid map is shown in FIG. Figure 2C shown.

[0130] Those skilled in the art will appreciate that the recombinase is not limited to a specific form, such as separate recombinase and buffer or a premixed solution. In this embodiment, the "Novorec Plus One Step Cloning Kit" was used. Alternatively, the "Vazyme ClonExpress Ultra One Step Cloning Kit" and the "pEASY-Basic Seamless Cloning and Assembly Kit" can also achieve the functions of this embodiment.

[0131] Those skilled in the art will also appreciate that pcDNA3.1 vector fragments can be obtained by a variety of methods. In this example, pcDNA3.1-mScarlet3 was used as a template to amplify and recover pcDNA3.1 vector fragments. The upstream primers used were:

[0132] 5'-CTCGAGTCTAGAGGGCCCGTTTAAACCCGC-3'

[0133] The downstream primers are:

[0134] 5'-CATGGTGGCTAGCCAGCTTGGG-3'

[0135] Similarly, when using pcDNA3.1-mScarlet3 as a template for PCR amplification of sequence SEQ ID NO. 6, appropriate upstream and downstream primers can be designed based on the actual situation. It should be noted that when designing primers, it is necessary to add homologous sequences that can be recognized by the recombinase to the primers. In this example, the upstream primer used to amplify sequence SEQ ID NO. 6 is:

[0136] 5'-CAAGCTGGCTAGCCACCATGGGCGGCCCGCTACCTG-3'

[0137] The downstream primers are:

[0138] 5'-CCTCTAGACTCGAGTCAGGAGCCACCGGAGCCGCCG-3'

[0139] The upstream primers used to amplify the pcDNA3.1 vector fragment using pcDNA3.1-mScarlet3 as a template are:

[0140] 5'-CTCGAGTCTAGAGGGCCCGTTTAAACCCGC-3'

[0141] The downstream primers are:

[0142] 5'-catGGTGGCTAGCCAGCTTGGG-3'

[0143] The above primers were used to amplify and recover the pcDNA3.1 vector fragment. Then, the sequence SEQ ID NO.6 for expressing mSc170C was inserted into the pcDNA3.1 vector using a recombinase to form a second vector containing the sequence SEQ ID NO.6. The plasmid map is shown in FIG. Figure 2D shown.

[0144] The first vector and the second vector constructed above were transformed into Escherichia coli respectively. After the sequences were verified to be correct by sequencing, the endotoxin-free plasmid was extracted using an endotoxin-free plasmid mini-midiprep kit.

[0145] Application of the second set of bimolecular fluorescence complementation system:

[0146] Step A1: The gene sequences of the first test protein and the second test protein are cloned into the first vector and the second vector, respectively, to obtain the first test protein-first vector plasmid and the second test protein-second vector plasmid.

[0147] Specifically include:

[0148] After constructing a first vector containing the sequence of SEQ ID NO. 5 and a second vector containing the sequence of SEQ ID NO. 6, DNA sequences of the two test proteins are amplified using PCR from existing plasmids or cDNA libraries. Those skilled in the art will appreciate that the orientation of the first and second test proteins relative to mSc154N or mSc155C can be adjusted based on actual conditions.

[0149] In this example, EGFP, which can form homodimers, is used as an example. That is, in this example, both the first test protein and the second test protein are EGFP. Primers for amplifying the EGFP fragment are designed and synthesized using the pX458 plasmid as a template. It should be noted that when designing primers, a linker sequence needs to be added between EGFP and mSc169N or mSc170C, and a homologous sequence that can be recognized by the recombinase needs to be added to the primer. The amino acid sequence of the linker connecting EGFP and mSc169N is shown in SEQ ID NO.7, and the gene sequence is shown in SEQ ID NO.8.

[0150] When EGFP was linked to the N-terminus of mSc169N, the upstream primer used to amplify EGFP was:

[0151] 5'-CCCAAGCTGGCTAGCCACCatggtgagcaagggcgaggag-3'

[0152] The downstream primers are:

[0153] 5'-actgcctccacctccgctacctccaccgcccttgtacagctcgtccatgccg-3'

[0154] When amplifying the vector sequence using the first vector containing the sequence SEQ ID NO.5 as a template, the upstream primer is:

[0155] 5'-tagcggaggtggaggcagtatggatagcaccgaggcag-3'

[0156] The downstream primers are:

[0157] 5'-CATGGTGGCTAGCCAGCTTGGG-3'

[0158] The above primers were used to amplify and recover the vector fragment, and then the EGFP was inserted into the first vector containing the sequence SEQ ID NO.5 using recombinase to form pcDNA3.1-EGFP-169N. The plasmid map is shown in FIG. Figure 3C shown.

[0159] The amino acid sequence of the linker connecting EGFP and mSc170C is shown in SEQ ID NO.9, and the gene sequence is shown in SEQ ID NO.10.

[0160] When EGFP is linked to the C-terminus of mSc170C, the upstream primer used to amplify EGFP is:

[0161] 5'-GGCTCTGGCTCAGGCAGTATGGTGAGCAAGGGCGAGGAG-3'

[0162] The downstream primers are:

[0163] 5'-TTTAAACGGGCCCTCTAGACTCGAGTCACTTGTACAGCTCGTCC-3'

[0164] When amplifying the vector sequence using the second vector containing the sequence SEQ ID NO.6 as a template, the upstream primer is:

[0165] 5'-CTCGAGTCTAGAGGGCCCGTTTAAACCCGC-3'

[0166] The downstream primers are:

[0167] 5'-ACTGCCTGAGCCAGAGCCGGAGCCACCGGAGCCGCCGG-3'

[0168] The above primers were used to amplify and recover the vector fragment, and then the EGFP was inserted into the second vector containing the sequence SEQ ID NO.6 using recombinase to form pcDNA3.1-170C-EGFP. The plasmid map is shown in Figure 3D shown.

[0169] The pcDNA3.1-EGFP-169N and pcDNA3.1-170C-EGFP constructed above were transformed into Escherichia coli. After the sequences were verified by sequencing, the endotoxin-free plasmids were extracted using an endotoxin-free plasmid mini-midiprep kit.

[0170] Lipofectamine 3000 was used to transfect the two plasmids into the cells to be tested at a ratio of 1:1.

[0171] After 12 to 24 hours, observe the cells under a fluorescence microscope. Figure 4 As shown in B, the transfected cells exhibited bright red fluorescence.

[0172] Application of the second set of bimolecular fluorescence complementation system:

[0173] Taking the previously reported interacting proteins ORC1 and HMGA1a as an example, ORC1 protein is used as the first protein to be tested, and HMGA1a protein is used as the second protein to be tested.

[0174] 1. pcDNA3.1(+)-lifeact-mScarlet3 was synthesized by Sangon Biotechnology Co., Ltd.

[0175] 1.1 Using qPCR human reference total RNA (Takara) or mRNA extracted from cells as a template, synthesize cDNA using the ReverTra Ace reverse transcription kit.

[0176] 1.2 The ORC1 and HMGA1a gene sequences were obtained by PCR using the synthesized cDNA as a template. It should be noted that when designing primers, linker sequences need to be added between ORC1 and mSc169N and between HMGA1a and mSc170C, and homologous sequences that can be recognized by the recombinase need to be added to the primers.

[0177] 1.3 Using the first vector or the second vector as a template, the first vector or the second vector sequence is obtained by PCR amplification and recovery.

[0178] 1.4 Use the Novorec PCR one-step directional cloning kit to insert ORC1 into the first vector to obtain plasmid ORC1-mSc169N, as shown in Figure 7A shown.

[0179] 1.5 Use the same method to clone HMGA1a into the second vector to obtain plasmid mSc170C-HMGA1a, as shown in Figure 7B shown.

[0180] 2. Transient Transfection of Hela Cells

[0181] Hela cells were preserved by the inventor's laboratory.

[0182] 2.1 ORC1-mSc154N and mSc170C-HMGA1a were co-transfected into Hela cells at a 1:1 ratio using Lipofectamine 3000 liposome transfection reagent.

[0183] 2.2 16 hours after transfection, observe the cells using a fluorescence microscope and record the fluorescence signal.

[0184] 3. Observe the results

[0185] like Figure 8 As shown, cells transfected with ORC1-mSc169N and mSc170C-HMGA1a emitted bright red fluorescence, indicating the interaction between ORC1 and HMGA1a.

[0186] SEQ ID NO.1

[0187] MDSTEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTAKLRVTKGGPLPFSWDI

[0188] LSPQFMYGSRAFTKHPADIPDYWKQSFPEGFKWERVMNFEDGGAVSVAQDTSLEDGTLIY

[0189] KVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDVVLKGDIKMALRLKDGGRYLADFKT

[0190] TYRAKKPVQMPGAFNIDRKLDITSHNEDYTVVEQYERSVARHSTGGSGGSSEQ ID NO.2

[0191] ATGGATAGCACCGAGGCAGTGATCAAGGAGTTCATGCGGTTCAAGGTGCACATGGAGG

[0192] GCTCCATGAACGGCCACGAGTTCGAGATCGAGGGCGAGGGCGAGGGCCGCCCCTACG

[0193] AGGGCACCCAGACCGCCAAGCTGAGGGTGACCAAGGGTGGCCCCCTGCCCTTCTCCTG

[0194] GGACATCCTGTCCCCTCAGTTCATGTACGGCTCCAGGGCCTTCACGAAGCACCCCGCCG

[0195] ACATCCCCGACTACTGGAAGCAGTCCTTCCCCGAGGGCTTCAAGTGGGAGCGCGTGAT

[0196] GAACTTCGAGGACGGCGGCGCCGTGTCCGTGGCCCAGGACACCTCCCTGGAGGACGGC

[0197] ACCCTGATCTACAAGGTGAAGCTCCGCGGCACCAACTTCCCTCCTGACGGCCCCGTAAT

[0198] GCAGAAGAAGACAATGGGCTGGGAAGCATCCACCGAGCGGTTGTACCCCGAGGACGT

[0199] CGTGCTGAAGGGCGACATTAAGATGGCCCTGCGCCTGAAGGACGGCGGCCGCTACCTG

[0200] GCGGACTTCAAGACCACCTACAGGGCCAAGAAGCCCGTGCAGATGCCCGGCGCCTTCA

[0201] ACATCGACCGCAAGTTGGACATCACATCCCACAACGAGGACTACACCGTGGTGGAACA

[0202] GTACGAACGCTCCGTGGCCCGCCACTCCACCGGCGGCTCCGGTGGCTCCSEQ ID NO.3

[0203] ATGGATAGCACCGAGGCAGTGATCAAGGAGTTCATGCGGTTCAAGGTGCACATGGAGG

[0204] GCTCCATGAACGGCCACGAGTTCGAGATCGAGGGCGAGGGCGAGGGCCGCCCCTACG

[0205] AGGGCACCCAGACCGCCAAGCTGAGGGTGACCAAGGGTGGCCCCCTGCCCTTCTCCTG

[0206] GGACATCCTGTCCCCTCAGTTCATGTACGGCTCCAGGGCCTTCACGAAGCACCCCGCCG

[0207] ACATCCCCGACTACTGGAAGCAGTCCTTCCCCGAGGGCTTCAAGTGGGAGCGCGTGAT

[0208] GAACTTCGAGGACGGCGGCGCCGTGTCCGTGGCCCAGGACACCTCCCTGGAGGACGGC

[0209] ACCCTGATCTACAAGGTGAAGCTCCGCGGCACCAACTTCCCTCCTGACGGCCCCGTAAT

[0210] GCAGAAGAAGACAATGGGCTGGGAAGCATCCACCGAGCGGTTGTACCCCGAGGACSEQ ID NO.4

[0211] ATGGTCGTGCTGAAGGGCGACATTAAGATGGCCCTGCGCCTGAAGGACGGCGGCCGCT

[0212] ACCTGGCGGACTTCAAGACCACCTACAGGGCCAAGAAGCCCGTGCAGATGCCCGGCGC

[0213] CTTCAACATCGACCGCAAGTTGGACATCACATCCCACAACGAGGACTACACCGTGGTG

[0214] GAACAGTACGAACGCTCCGTGGCCCGCCACTCCACCGGCGGCTCCGGTGGCTCCSEQ ID NO.5

[0215] ATGGATAGCACCGAGGCAGTGATCAAGGAGTTCATGCGGTTCAAGGTGCACATGGAGG

[0216] GCTCCATGAACGGCCACGAGTTCGAGATCGAGGGCGAGGGCGAGGGCCGCCCCTACG

[0217] AGGGCACCCAGACCGCCAAGCTGAGGGTGACCAAGGGTGGCCCCCTGCCCTTCTCCTG

[0218] GGACATCCTGTCCCCTCAGTTCATGTACGGCTCCAGGGCCTTCACGAAGCACCCCGCCG

[0219] ACATCCCCGACTACTGGAAGCAGTCCTTCCCCGAGGGCTTCAAGTGGGAGCGCGTGAT

[0220] GAACTTCGAGGACGGCGGCGCCGTGTCCGTGGCCCAGGACACCTCCCTGGAGGACGGC

[0221] ACCCTGATCTACAAGGTGAAGCTCCGCGGCACCAACTTCCCTCCTGACGGCCCCGTAAT

[0222] GCAGAAGAAGACAATGGGCTGGGAAGCATCCACCGAGCGGTTGTACCCCGAGGACGT

[0223] CGTGCTGAAGGGCGACATTAAGATGGCCCTGCGCCTGAAGGACSEQ ID NO.6

[0224] ATGGGCGGCCGCTACCTGGCGGACTTCAAGACCACCTACAGGGCCAAGAAGCCCGTGC

[0225] AGATGCCCGGCGCCTTCAACATCGACCGCAAGTTGGACATCACATCCCACAACGAGGA

[0226] CTACACCGTGGTGGAACAGTACGAACGCTCCGTGGCCCGCCACTCCACCGGCGGCTCC

[0227] GGTGGCTCC

[0228] SEQ ID NO.7

[0229] GGGGSGGGGS

[0230] SEQ ID NO.8

[0231] GGCGGTGGAGGTAGCGGAGGTGGAGGCAGTSEQ ID NO.9GSGSGSSEQ IDNO.10GGCTCTGGCTCAGGCAGT

Claims

1. A bimolecular fluorescence complementation system based on the red fluorescent protein mScarlet3, characterized by: The invention comprises a first vector and a second vector; the red fluorescent protein mScarlet3 is a protein composed of 229 amino acids, and the amino acid sequence of the red fluorescent protein mScarlet3 is shown in SEQ ID NO.1; the first vector is a vector containing a gene sequence for expressing a first protein fragment, and the second vector is a vector containing a gene sequence for expressing a second protein fragment. When the first protein fragment and the second protein fragment are close to each other, they can be reconstructed to form the red fluorescent protein mScarlet3; wherein: the first protein fragment is a protein fragment composed of amino acids at positions 1 to n of the red fluorescent protein mScarlet3, and the second protein fragment is a protein fragment composed of amino acids at positions (n+1) to 229 of the red fluorescent protein mScarlet3; and n is 154 or 169.

2. The bimolecular fluorescence complementation system based on red fluorescent protein mScarlet3 according to claim 1, characterized in that: When n is 154, the first protein fragment is the mSc154N protein fragment, and the second protein fragment is the mSc155C protein fragment; the gene sequence for expressing mSc154N contains the gene sequence SEQ ID NO.3 and the gene sequence for expressing the mSc155C protein fragment contains the gene sequence SEQ ID NO.4; when n is 169, the first protein fragment is the mSc169N protein fragment, and the second protein fragment is the mSc170C protein fragment; the gene sequence for expressing the mSc169N protein fragment contains the gene sequence SEQ ID NO.5 and the gene sequence for expressing the mSc170C protein fragment contains the gene sequence SEQ ID NO.

6.

3. The bimolecular fluorescence complementation system based on red fluorescent protein mScarlet3 according to claim 1, characterized in that: The first vector further includes a first gene sequence, the first gene sequence is used to express a first protein to be tested, the first vector is used to express a first fusion protein, and the first fusion protein is a fusion protein of the first protein to be tested and the first protein fragment.

4. The bimolecular fluorescence complementation system based on red fluorescent protein mScarlet3 according to claim 1, characterized in that: The second vector further includes a second gene sequence, the second gene sequence is used to express a second protein to be tested, the second vector is used to express a second fusion protein, and the second fusion protein is a fusion protein of the second protein and the second protein fragment.

5. A method for constructing a bimolecular fluorescence complementation system based on red fluorescent protein mScarlet3, characterized in that: The steps include: S1: The gene sequence of the red fluorescent protein mScarlet3 is cloned into a first eukaryotic expression vector to synthesize a gene vector containing mScarlet3; S2: using the gene vector containing mScarlet3 as a template, obtaining a gene sequence for expressing the first protein fragment and a gene sequence for expressing the second protein fragment by polymerase chain reaction; S3: Using a recombinase, the gene sequence for expressing the first protein fragment is inserted into the second eukaryotic expression vector to form a first vector; using a recombinase, the gene sequence for expressing the second protein fragment is inserted into the third eukaryotic expression vector to form a second vector; The red fluorescent protein mScarlet3 is composed of 229 amino acids, its amino acid sequence is shown in SEQ ID NO1, and its gene sequence is shown in SEQ ID NO2; when the first protein fragment and the second protein fragment are close to each other, they can be reconstructed to form the red fluorescent protein mScarlet3; wherein: the first protein fragment is a protein fragment composed of amino acids 1 to n of the red fluorescent protein mScarlet3, and the second protein fragment is a protein fragment composed of amino acids (n+1) to 229 of the red fluorescent protein mScarlet3; n is 154 or 169.

6. The method for constructing a bimolecular fluorescence complementation system based on red fluorescent protein mScarlet3 according to claim 5, characterized in that: The first eukaryotic expression vector includes one of pcDNA3.1(+) vector, pUC57, and pUC19; the second eukaryotic expression vector includes one of pcDNA3.1(+) vector, pUC57, and pUC19; and the third eukaryotic expression vector includes one of pcDNA3.1(+) vector, pUC57, and pUC19.

7. The method for constructing a bimolecular fluorescence complementation system based on red fluorescent protein mScarlet3 according to claim 5, characterized in that: The first eukaryotic expression vector is a pcDNA3.1(+) vector, and the gene vector containing mScarlet3 is pcDNA3.1-mScarlet3; the gene sequence for expressing mSc154N is the gene sequence SEQ ID NO.3, and the gene sequence for expressing the mSc155C protein fragment is the gene sequence SEQ ID NO.4; in step S2: When the gene sequence SEQ ID NO.3 was obtained by polymerase chain reaction using pcDNA3.1-mScarlet3 as a template, The upstream primers are: 5'-ACCCAAGCTGGCTAGCCACCATGGATAGCACCGAGGCAGT-3' The downstream primers are: 5'-ACGGGCCCTCTAGACTCGAGTCAGTCCTCGGGGTACAACC-3'; When the gene sequence SEQ ID NO.4 was obtained by polymerase chain reaction using pcDNA3.1-mScarlet3 as a template, The upstream primers are: 5'-CCAAGCTGGCTAGCCACCATGGTCGTGCTGAAGGGCG-3' The downstream primers are: 5'-GGCCCTCTAGACTCGAGTCAGGAGCCACCGGAGCCGCCGG-3'.

8. The method for constructing a bimolecular fluorescence complementation system based on red fluorescent protein mScarlet3 according to claim 5, characterized in that: The first eukaryotic expression vector is a pcDNA3.1(+) vector, and the gene vector containing mScarlet3 is pcDNA3.1-mScarlet3; the gene sequence for expressing mSc169N is the gene sequence SEQ ID NO.5, and the gene sequence for expressing the mSc170C protein fragment is the gene sequence SEQ ID NO.6; in step S2: When the gene sequence SEQ ID NO.5 was obtained by polymerase chain reaction using pcDNA3.1-mScarlet3 as a template, The upstream primers are: 5'-CCAAGCTGGCTAGCCACCatggatagcaccgaggcagt-3' The downstream primers are: 5'-ACGGGCCCTCTAGACTCGAGTCAgtccttcaggcgcag-3' When the gene sequence SEQ ID NO.6 was obtained by polymerase chain reaction using pcDNA3.1-mScarlet3 as a template, The upstream primers are: 5'-CAAGCTGGCTAGCCACCatgggcggccgctacctg-3'; The downstream primers are: 5'-CCTCTAGACTCGAGTCAggagccaccggagccgccg-3'.

9. An application of the bimolecular fluorescence complementation system based on the red fluorescent protein mScarlet3 according to any one of claims 1 to 4 in imaging protein-protein interactions.

10. The use according to claim 9, characterized in that The steps include: Step A1: cloning the gene sequences of the first test protein and the second test protein into the first vector and the second vector, respectively, to obtain the first test protein-first vector plasmid and the second test protein-second vector plasmid; Step A2: Using a transfection reagent, the constructed test protein-first vector plasmid and the test protein-second vector plasmid are co-transfected into the cells to be tested; Step A3: After culturing for a period of time, perform fluorescence microscopy imaging.

Images