CN117448375A - Double-fluorescent protein co-expression system for lepidopteran insect cells and application thereof - Google Patents
Double-fluorescent protein co-expression system for lepidopteran insect cells and application thereof Download PDFInfo
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Abstract
The invention discloses a double fluorescent protein co-expression system for lepidopteran insect cells and application thereof. The double fluorescent protein co-expression system comprises a recombinant vector which is inserted into a pIZ-V5-His vector of an EGFP gene fragment or an mCherry gene fragment respectively, wherein the nucleotide sequence of the EGFP gene fragment is shown as SEQ ID NO.1, and the nucleotide sequence of the mCherry gene fragment is shown as SEQ ID NO. 2. According to the double fluorescent protein co-expression system for lepidopteran insect cells, EGFP and mCherry genes are respectively inserted into a pIZ-V5-His carrier, and the EGFP and mCherry designed are inserted into the forward positions (HindIII and BamHI sites) of enzyme cutting sites, so that the utilization rate of enzyme cutting sites of a carrier skeleton is improved to the greatest extent while fluorescent protein fusion is realized; the recombinant expression vector can be used for promoting expression of green or red double-fluorescence fusion proteins in silkworm BmN cells, can be used for functional research such as protein detection and interaction, realizes visualization of protein expression and localization to be detected, and can be used for detecting protein expression and interaction by using antibodies of EGFP and mCherry proteins.
Description
Technical Field
The invention relates to a double-fluorescent protein co-expression system for lepidopteran insect cells and application thereof, belonging to the field of genetic engineering.
Background
Compared with the model organisms such as mice, drosophila and the like, commercial antibodies of silkworm proteins are few, and the customization of protein antibodies is long in time and high in price, and has a certain obstacle to the interaction experiments of proteins such as Co-IP, pull-down and the like. Therefore, the development of a high-efficiency and rapid silkworm fusion protein expression system is important. Moreover, the silkworm cells have poor adherence, the growth speed is low, and the silkworm cells are not easy to treat frequently, so that the development of efficient and directly visible protein expression vectors is very necessary for scientific research on cell level.
pIZ-V5-His vectors contain the OpIE2 promoter for constitutive expression and are commonly used insect cell expression vectors. The vector C-terminal V5 epitope and polyhistidine (6 XHis) sequence can be detected by using a V5 antibody and rapidly purified by using nickel chelating resin, and is an ideal tag fusion plasmid, but the vector has no fluorescent label. Furthermore, the Zeocin resistance gene of the vector can be used for rapidly selecting a stably transfected cell line, but silkworm BmN cells are sensitive to Zeocin, so that the stably transfected cell line is difficult to select, and high-efficiency expression is directly observed to be more effective for silkworm cells. The fluorescent expression vector used in the current silkworm BmN cells has great limitation. Three significant drawbacks are that one of them, such as pIHT/V5-His vector, expresses fluorescence, but the target protein is not fused with fluorescence; secondly, as in the existing some fluorescence expression vectors, the utilization efficiency of enzyme cutting sites is not fully considered; thirdly, there is no system capable of expressing dual fluorescence in the same cell, making protein expression both visible and detectable.
Disclosure of Invention
The invention aims to: a first object of the present invention is to provide a double fluorescent protein co-expression system for lepidopteran insect cells. The second object of the invention is to provide a construction method of the double fluorescent protein coexpression system for lepidopteran insect cells. The third object of the present invention is to provide the use of the above-mentioned double fluorescent protein coexpression system for lepidopteran insect cells. It is a fourth object of the present invention to provide a method for detecting whether two proteins interact.
The technical scheme is as follows: the invention relates to a double-fluorescent protein co-expression system for lepidopteran insect cells, which comprises a recombinant vector 1 and a recombinant vector 2, wherein the recombinant vector 1 is a pIZ-V5-His vector inserted with EGFP gene fragments, and the nucleotide sequence of the EGFP gene fragments is shown as SEQ ID NO. 1; the recombinant vector 2 is a pIZ-V5-His vector inserted into a mCherry gene fragment, and the nucleotide sequence of the mCherry gene fragment is shown as SEQ ID NO. 2.
The invention not only comprehensively utilizes the dual fluorescence characteristics of EGFP and mCherry, but also designs the site at the forefront end of the plasmid enzyme cutting site skeleton, has very high enzyme cutting site utilization rate, and has higher selectivity especially when the enzyme cutting site contains more internal enzyme cutting sites compared with the insertion sequence.
The invention discloses a construction method of a double fluorescent protein coexpression system for lepidopteran insect cells, which comprises the following steps:
(1) PCR amplification is carried out by taking pEGFP-N3 plasmid as a template to obtain EGFP gene fragments, wherein the nucleotide sequence of the EGFP gene fragments is shown as SEQ ID NO. 1; PCR amplification is carried out by taking pCMV-C-mCherry plasmid as a template to obtain mCherry gene fragments, and the nucleotide sequence of the mCherry gene fragments is shown as SEQ ID NO. 2;
(2) Double enzyme digestion is carried out on the EGFP gene fragment, the mCherry gene fragment and the pIZ-V5-His by using HindIII and BamHI respectively, and then the EGFP gene fragment and the mCherry gene fragment are respectively connected with pIZ-V5-His vectors, thus obtaining recombinant vectors pIZ-EGFP-V5-His and pIZ-mCherry-V5-His.
Further, the nucleotide sequences of the primer pair used for PCR amplification of EGFP gene fragment and mCherry gene fragment in the step (1) are shown in SEQ ID NO. 3-4.
The invention relates to an application of a double-fluorescent protein co-expression system for lepidopteran insect cells in detecting whether two proteins interact with each other.
Further, the protein is Co-IP or pull-down protein.
The invention discloses a method for detecting whether two proteins interact, which comprises the following steps: inserting gene fragments of two proteins to be detected into different recombinant vectors in the system respectively, then co-transfecting host cells in equal proportion, observing the fluorescent protein expression condition of transfected cells, and if red/green fluorescence can be observed, the system can be used for interaction detection of the two proteins to be detected; if only red fluorescence or green fluorescence is observed, the system is not applicable to interaction detection of two proteins to be detected.
Further, the host cell is a BmN cell.
Further, the proteins to be tested are the Bmp53 protein and the Mdm2-like protein.
The nucleotide sequence of the inserted gene fragment of the Bmp53 protein is shown as SEQ ID NO.9, and the nucleotide sequence of the inserted gene fragment of the Mdm2-like protein is shown as SEQ ID NO. 10.
Further, the nucleotide sequence of a primer pair used for amplifying the inserted gene fragment of the Bmp53 protein is shown as SEQ ID NO. 5-6; the nucleotide sequence of the primer pair used for amplifying the inserted gene fragment of the Mdm2-like protein is shown as SEQ ID NO. 7-8.
Further, the gene fragment insertion sites of the Bmp53 protein are BamHI and XhoI, and the gene fragment insertion sites of the Mdm2-like protein are EcoRI and XhoI.
The insertion sites of the gene fragment of the Bmp53 protein are selected as BamHI and XhoI, so that invalid amino acid sequences before fluorescent protein, bmp53 and C-terminal V5-His tag can be reduced to the greatest extent.
The beneficial effects are that: compared with the prior art, the invention has the following remarkable advantages: according to the double fluorescent protein co-expression system for lepidopteran insect cells, EGFP and mCherry genes are respectively inserted into a pIZ-V5-His carrier, and the EGFP and mCherry designed are inserted into the forward positions (HindIII and BamHI sites) of enzyme cutting sites, so that the utilization rate of enzyme cutting sites of a carrier skeleton is improved to the greatest extent while fluorescent protein fusion is realized; the recombinant expression vector can be used for promoting expression of green or red double-fluorescence fusion proteins in silkworm BmN cells, can be used for functional research such as protein detection and interaction, realizes visualization of protein expression and localization to be detected, and can be used for detecting protein expression and interaction by using EGFP and mCherry protein antibodies. The double fluorescent protein co-expression system is used for expressing silkworm apoptosis regulating protein Bmp53 and potential ubiquitination protein Mdm2-like, and successfully visualizes the expression, positioning and interaction of the two proteins.
Drawings
FIG. 1 is a vector map of pIZ-EGFP-V5-His and a fused silkworm gene (Bmp 53 as an example).
FIG. 2 is a vector map of pIZ-mCherry-V5-His and its fused silkworm gene (Mdm 2-like, for example).
FIG. 3 shows fluorescence and protein expression levels of the constructed vector.
FIG. 4 is a diagram of dual fluorescent expression and protein localization analysis of the constructed vector.
Detailed Description
The technical scheme of the invention is further described below with reference to the accompanying drawings.
Example 1 design and cloning of EGFP and mCherry sequences
EGFP fragment was obtained by PCR amplification from plasmid pEGFP-N3 (Biowind, # 34) stored in this experiment, mCherry fragment was obtained by PCR amplification from pCMV-C-mCherry plasmid (Beyotime, # D2628), and a part of the PCR product was recovered after gel running identification. Wherein EGFP and mCherry are identical at the first 21 and last 22 bases, so EGFP and mCherry amplification primers are identical and the DNA templates are different.
The amplification primers were as follows:
EM-F(HindIII):CCCAAGCTTATGGTGAGCAAGGGCGAGGA(SEQ ID NO.3)
EM-R(BamHI):CGCGGATCCCTTGTACAGCTCGTCCATGC (SEQ ID NO. 4) PCR amplification reaction conditions were as follows:
the EGFP sequence was amplified as follows (no termination code, SEQ ID NO. 1):
CCCAAGCTTATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGCCTAGGCGC
the mCherry sequence was amplified as follows (no stop codon, SEQ ID NO. 2):
CCCAAGCTTATGGTGAGCAAGGGCGAGGAGGATAACATGGCCATCATCAAGGAGTTCATGCGCTTCAAGGTGCACATGGAGGGCTCCGTGAACGGCCACGAGTTCGAGATCGAGGGCGAGGGCGAGGGCCGCCCCTACGAGGGCACCCAGACCGCCAAGCTGAAGGTGACCAAGGGTGGCCCCCTGCCCTTCGCCTGGGACATCCTGTCCCCTCAGTTCATGTACGGCTCCAAGGCCTACGTGAAGCACCCCGCCGACATCCCCGACTACTTGAAGCTGTCCTTCCCCGAGGGCTTCAAGTGGGAGCGCGTGATGAACTTCGAGGACGGCGGCGTGGTGACCGTGACCCAGGACTCCTCCCTGCAGGACGGCGAGTTCATCTACAAGGTGAAGCTGCGCGGCACCAACTTCCCCTCCGACGGCCCCGTAATGCAGAAGAAGACCATGGGCTGGGAGGCCTCCTCCGAGCGGATGTACCCCGAGGACGGCGCCCTGAAGGGCGAGATCAAGCAGAGGCTGAAGCTGAAGGACGGCGGCCACTACGACGCTGAGGTCAAGACCACCTACAAGGCCAAGAAGCCCGTGCAGCTGCCCGGCGCCTACAACGTCAACATCAAGTTGGACATCACCTCCCACAACGAGGACTACACCATCGTGGAACAGTACGAACGCGCCGAGGGCCGCCACTCCACCGGCGGCATGGACGAGCTGTACAAGCCTAGGCGC
the amplified sequences were identified by agarose gel electrophoresis and then purified using a PCR product purification kit (Shanghai Biotechnology Co.,B518141-0100) And (5) recycling.
EXAMPLE 2 construction of pIZ-EGFP-V5-His and pIZ-mCherry-V5-His vectors
The EGFP, mCherry fragment and pIZ-V5-His vector recovered in step 1 were digested simultaneously with HindIII (NEB, # 3104) and BamHI (NEB, # R3136), respectively, at 37℃for 1 hour, and then the products were recovered. The enzyme digestion system is as follows:
| DNA (plasmid or gene) | 2μg |
| BamHI-HF | 2μl |
| HindIII-HF | 2μl |
| rCutSmart TM Buffer | 5μl |
| ddH 2 O | up to 50μl |
The recovered EGFP and mCherry fragments are respectively connected with pIZ-V5-His vectors (Youbao biological Co., # VT 1178) and are transformed into DH5a competent cells, positive clone shaking bacteria are selected for double enzyme digestion detection after plating, and pIZ-EGFP-V5-His and pIZ-mCherry-V5-His vectors are respectively constructed. The connection system is as follows:
| pIZ-V5-His enzyme digestion and recovery | 1μL |
| EGFP or mCherry restriction recovery | 2∽4μl |
| Solution I | 5μl |
| ddH 2 O | up to 10μl |
EXAMPLE 3 construction of pIZ-EGFP-Bmp53-V5-His and pIZ-mCherry-Mdm2-V5-His vectors
The pIZ-EGFP-V5-His vector obtained in example 2 was inserted into silkworm apoptosis controlling protein Bmp53 sequence at BamHI and XhoI sites, and the pIZ-mCherry-V5-His vector was inserted into potential ubiquitinated protein Mdm2-like sequence at EcoRI and XhoI sites, so as to construct pIZ-EGFP-Bmp53-V5-His and pIZ-mCherry-Mdm2like-V5-His vectors, respectively. This step is particularly focused on frameshift mutations to prevent fusion with the C-terminal V5-His antibody, and is particularly focused on the specific cleavage site of the gene. The amplification template was saved for the present laboratory clone and submitted for published sequence Mdm2-like (GenBank: OP 966773.1)/Bmp 53 (GenBank: OP 966772.1).
The Bmp53 gene amplification primers were as follows:
p53-EF(BamHI)：AAGGATCCATGAAACACGAAATCATGAC(SEQ ID NO.5)；
p53-ER(XhoI(NS)):ATACTCGAGccTTCGCTGGCATGTTTCGT(SEQ ID NO.6)；
the Mdm2-like gene amplification primers were as follows:
mChery-M2-F:CCGGAATTCaATGAACACAACATTTTGT(SEQ ID NO.7)；
mChery-M2-R:ATACTCGAGaaTAGCACGACGGCGCGC(SEQ ID NO.8)；
the PCR amplification reaction conditions were as follows:
the Bmp53 gene amplification sequence is as follows (SEQ ID NO. 9):
AAGGATCCATGAAACACGAAATCATGACATCTCTTGAGGTTGCCACATGCGAAGATGACATAGTGAACATTGATTTGAACACTATACCGGATGAAGCATTGTTTCAAGGGGGGATACACGATCAAGTTGACATTGGAGTGCTAGATGACATTCCGTACATAATCAGTGATGTGTCTAACAATTCAAACAATTCCTTTCCTCTTGGACCCCGAGGACCGCCGCCTCGCAAAGACGATCCCGGGCAATACAACTTCAGCGTCGAAATCCACAGCAAGGATACACACAAGAAGAAGTTCTTGTTCTCGCACAAGCTGAACCGGATCTACGTGAACATGGAGACCGACTTCGCTGTCCAGTTCAACTGGGAGCTGGTGGACCTCGCCGTGACGCAGATGTACGTGCGCGCCACCGTCGTCTTCGAGGACGAGTCCCAGGCCGAGAAGAGAGTCGAGAGATGCATCCAGCACAAACTCTGCAGTTCCGATAAAGGCCAGGACCGCGTCGTGTCGGAGAACGTGCTGCGCTCGTCGCGGCCCCTGGGCACCAACGACGTGCAGTACTGCGGCCACCCGGACGACCCCGACTACTGGTACTCCGTGCTGGTGCAGCTGCCCAAGCCGGGCCGGGAGCCTTGCACGCACGCCTTCAAGTTCGTCTGCAAGAACTCCTGCAGCACCGGCATCAACCGCCGCTCCATAGCCGTCATCTTCACCCTGGAGAGCGCCTCCGGCTCGGTGCTGGGGCGGCAGACGGTGGGCGCGCGGGTGTGCTCGTGCACGGCGCGCGACATGTGCAAGGACGAGGAGGCGGAGGGCGCGCGCGCGGCCAGGAAGCGGCCCCGGCCCGCGCAGTCGCGCCTCCTCAAGAAGATCAAGCTGGAGACCGTCGGCGACCTGCCCGTCGACGCCGAGACCCTCACCCTGCCGCCGCTGGAAATTATCGGAGCCAAAACGGTGAAGACCGGCCTGGAAGTGATGCTGCGCATGATGGAGCAGGCGGCCCACTTCCACAAGCACGACCAGCTCGCCGCCGACGGCTACCAGCGCCGCGTCGCCAGCCTCCGACAGTACATCGACACGCTCGACGCCAAACCGACGAAACATGCCAGCGAAggCTCGAGATA
the amplification sequence of the Mdm2-like gene is as follows (SEQ ID No. 10):
CCGGAATTCaATGAACACAACATTTTGTTCAGAAACTTCAGTAAGCCGACGATGTTCGACTGAGACAATCTACAGCATACAAGGAAAAGAAACAGATTTCGCGCGAGACACCAGCGACACAGATGCAAGTATATTCGATACAGACGCCGAGATCGAGTTCGAGCCGGCCTCTGACGAGGAAGAGGACCAGGCGCCGGTGGACGAGGACACGTCGGCGTCTTTCACTGATGGTGAAATAATAAAGACCAAAGTGCTAGAGGTGAGCGTGGGCGACGACGGCGACCTGCAGCTCGCGGACTCTGAGCAGACTGACTCGCAGGACAGCGACAGCGAGATCGACCTGTACGACTTCTGGCACTGCATCAAGTGCAGAGCCGAGAACAACGTTCCGTTTTATAGATATTGTCAGAAGTGCTTCCAGGTCCGCAAAAACTTCTTTCCTCCGCGGCCGAAACGTAAACGCAAACGCGGCACGACGGTCACCCCGGAGCTGGACGCGGTGCCCAGGACTCTGTCCCAGGACTCCGGCATCCAGTCCGTGCACAGTCAAGAACTGTTCAACAGCCAGGACGCGACCCACGGTGAGGGCACTTCGCGAGACGTTGACAGTAGCGCTGAGTTCACAGGTCGAGCGAGGAAGAGACGCGCCCGGTCGGTCGACGGCGGCTCCAAGCGAACGAAGCTCGGCTCCGCCTCCGGATCCGGATCCGACTCCGGCTCTGGAGGCAACTCGGAGTCCCTGACCAAGTCCGTCAGCGATCCGTCCGTGGCGATCGACGAGCCGCTGAAAATGACGAAAAAAGTAATATCGGATAGGATCAAACAAAAGCTAGACAAGGAGCTGTGCATCGTGTGCGACTCCGAACCCAAGACCGGCGTGTTCGTGCACGGGCACCTCGCGCACATCTGCTGCTGCTACAAGTGCGCCGTCAAGGTGTGGGCGCGCGCCCGCCGCTGCCCCGTCTGCAACCGCAGGGTCAGCAACGTGCTGCGCGCCGTCGTGCTAttCTCGAGATA
the amplified sequences were identified by agarose gel electrophoresis and then purified using a PCR product purification kit (Shanghai Biotechnology Co.,B518141-0100) And (5) recycling. The recovered Bmp53 sequence, pIZ-EGFP-V5-His, was digested with BamHI (NEB, # R3136) and XhoI (NEB, # R0146), respectively, in the following manner:
| DNA (plasmid or gene) | 2μg |
| BamHI-HF | 2μl |
| XhoI | 2μl |
| rCutSmart TM Buffer | 5μl |
| ddH 2 O | up to 50μl |
The recovered product, the Mdm2-like sequence, pIZ-mCherry-V5-His was digested with EcoRV (NEB, # R3195) and XhoI (NEB, # R0146), respectively, in the following manner:
the above digested products were subjected to a PCR product purification kit (Shanghai Ind Co., ltd.,B518141-0100) And (5) recycling. Bmp53 sequence is connected with pIZ-EGFP-V5-His, mdm2-like sequence is connected with pIZ-mCherry-V5-His, and is transformed into DH5a competent cells, positive clone shaking bacteria are picked by plating and double enzyme digestion detection is carried out, and pIZ-EGFP-Bmp53-V5-His and pIZ-mCherry-Mdm2-V5-His vectors are respectively constructed. The connection system is as follows:
example 4 transfection and detection of double fluorescent fusion expression vectors
The transfection procedure was performed according to Entranster-D4000 (Engreen) instructions. BmN cells were seeded 24h before transfection (3X 10 5 ) Cultures were performed in 6-well plates at 28 ℃. When the cell culture density reaches 60-80%, the cell can be transfected. The transfection reagent Entranster-D4000 stored at 4℃was removed and mixed with shaking. 100 μl of TC-100 free serum free medium was added to each of 2 sterilized 1.5ml centrifuge tubes, labeled tubes 1, 2, respectively. 10 μl of transfection reagent was added to tube 1 and mixed well, and pIZ-EGFP-V5-His was added to tube 2 and mixed well. Incubate at room temperature for 5min. Tubes 1, 2 were mixed and the mixture incubated at room temperature for 15min at 25 ℃. The BmN cell culture dish was taken out of the incubator, the above mixture was added to the dish, and immediately after gently mixing, the dish was placed in the incubator at 28℃for cultivation. After 6h incubation, the BmN cell culture dish was replaced with normal oneTC-100 medium (fetal bovine serum was mixed with TC100 medium at a 1:9 volume ratio).
Silkworm BmN cells were transfected by replacing tube 2 with pIZ-mCherry-V5-His, pIZ-EGFP-Bmp53-V5-His, pIZ-mCherry-Mdm2like-V5-His, pIZ-EGFP-Bmp53-V5-His+ pIZ-mCherry-Mdm2like-V5-His (co-transfected plasmid ratio 1:1) according to the procedure described above.
The expression of the fluorescent protein was observed after 72 hours in the transfected cells, and the results were: pIZ-EGFP-V5-His, pIZ-EGFP-Bmp53-V5-His transfected silkworm BmN cells, green fluorescence was observed under FITC microscope (FIG. 3A), and pIZ-EGFP-Bmp53-V5-His transfection was mainly expressed in the nucleus (FIG. 4C); pIZ-mCherry-V5-His, pIZ-mCherry-Mdm2like-V5-His transfected silkworm BmN cells, red fluorescence was observed under FITC microscope (FIG. 3B), and pIZ-mCherry-Mdm2like-V5-His transfection was expressed mainly in nuclei (FIG. 4D); pIZ-EGFP-Bmp53-V5-His and pIZ-mCherry-Mdm2like-V5-His mixed-transferred silkworm BmN cells are observed to be red/green fluorescent light under a FITC microscope (figure 4B), which shows that the modified fusion vector can realize double-fluorescent mixed expression of different proteins, and provides a new tool for directly observing the positioning of the proteins. FIG. 4A shows the BmN cells of the mixed silkworm of pIZ-EGFP-Bmp53-V5-His and pIZ-mCherry-Mdm2like-V5-His in a white light state.
The transfected cells were subjected to protein isolation using Nuclear and Cytoplasmid Extration Kit (well known as century Co., # CW 01995) kit for the isolation of protein from the cytoplasm of pIZ-EGFP-Bmp53-V5-His, pIZ-mCherry-Mdm2like-V5-His and the mixed cells of these, EGFP-Bmp53 was successfully detected by EGFP antibody (Norleote Co., # ATM 1006) and mCherry-Mdm2like fusion protein by mCherry antibody (Hua Anbio Co., # HA 500049) using the nucleoprotein as an immunoblotting. The applicability of the carrier in functional researches such as protein interaction is demonstrated.
Claims (10)
1. The double-fluorescent protein coexpression system for lepidopteran insect cells is characterized by comprising a recombinant vector 1 and a recombinant vector 2, wherein the recombinant vector 1 is a pIZ-V5-His vector inserted with EGFP gene fragments, and the nucleotide sequence of the EGFP gene fragments is shown as SEQ ID NO. 1; the recombinant vector 2 is a pIZ-V5-His vector inserted with a mCherry gene fragment, and the nucleotide sequence of the mCherry gene fragment is shown as SEQ ID NO. 2.
2. A method of constructing a dual fluorescent protein co-expression system for lepidopteran insect cells according to claim 1, comprising the steps of:
(1) PCR amplification is carried out by taking pEGFP-N3 plasmid as a template to obtain EGFP gene fragments, wherein the nucleotide sequence of the EGFP gene fragments is shown as SEQ ID NO. 1; PCR amplification is carried out by taking pCMV-C-mCherry plasmid as a template to obtain mCherry gene fragments, and the nucleotide sequence of the mCherry gene fragments is shown as SEQ ID NO. 2;
(2) Double enzyme digestion is carried out on the EGFP gene fragment, the mCherry gene fragment and the pIZ-V5-His by using HindIII and BamHI respectively, and then the EGFP gene fragment and the mCherry gene fragment are respectively connected with pIZ-V5-His vectors, thus obtaining recombinant vectors pIZ-EGFP-V5-His and pIZ-mCherry-V5-His.
3. The construction method according to claim 2, wherein the primer pair nucleotide sequences used in the PCR amplification of EGFP gene fragment and mCherry gene fragment in step (1) are shown in SEQ ID NO. 3-4.
4. Use of a dual fluorescent protein co-expression system for lepidopteran insect cells according to claim 1 for detecting whether two proteins interact.
5. A method of detecting whether two proteins interact, comprising the steps of: inserting gene fragments of two proteins to be detected into different recombinant vectors in the system of claim 1 respectively, then co-transfecting host cells by equal proportion mixing, observing the fluorescent protein expression condition of transfected cells, and if red/green fluorescence can be observed, the system can be used for interaction detection of the two proteins to be detected; if only red fluorescence or green fluorescence is observed, the system is not applicable to interaction detection of two proteins to be detected.
6. The method of claim 5, wherein the host cell is a BmN cell.
7. The method of claim 5, wherein the proteins to be tested are Bmp53 protein and Mdm2-like protein.
8. The method according to claim 7, wherein the nucleotide sequence of the inserted gene fragment of the Bmp53 protein is shown in SEQ ID NO.9, and the nucleotide sequence of the inserted gene fragment of the Mdm2-like protein is shown in SEQ ID NO. 10.
9. The method according to claim 8, wherein the nucleotide sequences of the primer pair used for amplifying the inserted gene fragment of the Bmp53 protein are shown in SEQ ID NO. 5-6; the nucleotide sequence of the primer pair used for amplifying the inserted gene fragment of the Mdm2-like protein is shown as SEQ ID NO. 7-8.
10. The method according to claim 7, wherein the gene fragment insertion sites of the Bmp53 protein are BamHI and XhoI, and the gene fragment insertion sites of the Mdm2-like protein are EcoRI and XhoI.
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