CN113201058A

CN113201058A - Green fluorescent protein Clover4, bioluminescence resonance energy transfer-based probe derived from green fluorescent protein Clover4 and application of bioluminescence resonance energy transfer-based probe

Info

Publication number: CN113201058A
Application number: CN202110489938.4A
Authority: CN
Inventors: 储军; 邓梦颖; 袁静; 金宗文
Original assignee: Shenzhen Institute of Advanced Technology of CAS
Current assignee: Shenzhen Institute of Advanced Technology of CAS
Priority date: 2021-05-06
Filing date: 2021-05-06
Publication date: 2021-08-03
Anticipated expiration: 2041-05-06
Also published as: CN113201058B

Abstract

The invention relates to a green fluorescent protein Clover4, a derived probe based on bioluminescence resonance energy transfer and application thereof. Specifically discloses a green fluorescent protein Clover4, wherein the amino acid sequence of the green fluorescent protein has 11 mutation sites compared with the amino acid sequence of Clover. The discloses a protein pair for bioluminescence resonance energy transfer detection and a probe based on bioluminescence resonance energy transfer, wherein the protein pair comprises the green fluorescent protein. The green fluorescent protein disclosed by the invention is used for a probe for bioluminescence resonance energy transfer, so that the detection limit is reduced, the detection sensitivity and the detection dynamic range are improved, an antibody can be detected in an antibody environment with lower concentration, and early diagnosis is facilitated.

Description

Green fluorescent protein Clover4, bioluminescence resonance energy transfer-based probe derived from green fluorescent protein Clover4 and application of bioluminescence resonance energy transfer-based probe

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a green fluorescent protein Clover4 and a BRET serum antibody detection system derived from the green fluorescent protein Clover 4.

Background

Bioluminescence resonance energy transfer technology

Bioluminescence has become one of the important means of living body optical imaging due to the characteristics of no need of optical excitation and low background. However, luciferase (which generates photons by catalyzing biochemical reactions of substrates) has not only low quantum yield but also a shorter wavelength of emitted photons, thus limiting its application in animals. The Bioluminescence Resonance Energy Transfer (BRET) technology based on luciferase and fluorescent protein solves the above problems well. Bioluminescence Resonance Energy Transfer (BRET) is used as an efficient optical molecular ruler, the technical principle of which is based on non-radiative energy transfer, the process of transferring the energy of the excited state of the luciferase donor to the excited state of the fluorescent protein acceptor by intermolecular electric dipole interaction (Xu Y, Piton D W, Johnson C H.A Biologics Response Energy Transfer (BRET) system: application to interaction of cyclic addition proteins, proceedings of the National Academy of Sciences,1999,96(1):151-156.) only when the emission spectrum of the energy donor luciferase overlaps with the excitation spectrum of the energy fluorescent acceptor (e.g., fluorescent protein), and the spatial distance between the donor and the acceptor is close (<10nm), the donor may catalyze the substrate to release a light signal, which is transferred (partially or completely) to the acceptor molecule, causing the acceptor molecule to generate a light signal. The method is a homogeneous detection technology realized in a single liquid phase, does not need the participation of any solid medium (magnetic beads, microspheres and the like), and has attracted wide attention of broad students in recent ten years. Because an exogenous excitation light source is not needed (background signal is low) and the operation is simple, the technology can realize rapid high-sensitivity detection and is widely used for biomedical basic research and high-throughput drug screening.

Serum antibody detection mNeon Green-LUMABS system

BRET detection for in vitro diagnostics has been around for several years, but research is very rare, focusing only on the detection of antibodies. In 2016, the smart phone real-time analysis BRET detection technology for the antibodies in plasma was realized for the first time in Maarten Merkx laboratory by modifying the existing antibody detection probe AbSense, and the detection system was named LUMABS (Arts R, H.I., Zijlema SE, Thijssen V, Belen SHE, Merkx M.Detection of antibodies in blood plasma using biologicalinecense sensor proteins and a smartphone analytical Chemistry 88(8), 4525-4532 (2016)). The system directionally introduces different help structural domains between luciferase Nluc and green fluorescent protein mNeonGreen, and constructs an intramolecular BRET probe. Under the condition of no antibody, the probe has higher BRET efficiency between Nluc and mNeonGreen, and emits green fluorescence; when antibodies are present, they bind specifically to the antigen tag in the LUMABS system, which keeps Nluc away from meneongreen, reducing BRET efficiency and giving blue fluorescence. When the BRET probe is applied to a detection system, the BRET probe can be designed aiming at the antibody in plasma, so that the high specificity of detection is ensured, and the possibility is provided for the future clinical application. Meanwhile, the probe does not need to be excited by exogenous light, and the light signal is derived from the chemical oxidation process of luciferase catalysis substrate luminescence, so that the background signal of the plasma is effectively reduced, and the high signal-to-noise ratio of the detection is ensured; meanwhile, besides no excitation light source is needed (the light path is simple in design), the BRET detection step is simple, the miniaturization and the portability of the detection instrument are possible, and the instantaneity of detection is guaranteed.

In the presence of antibodies that bind specifically to the antigen tag in the mNeon Green-LUMABS system, such that Nluc is far from mNeon Green, BRET efficiency falls to a low state, which is difficult to change due to the distance. However, in the absence of antibodies, the distance between Nluc and meneon green is small, and the BRET efficiency at this time is mainly determined by the pair of resonance energy transfer pairs Nluc and meneon green itself. When the BRET efficiency is high, the change in BRET (i.e., dynamic range) due to the presence or absence of an antibody is large, and the sensitivity (sensitivity) of detection is high. However, the biggest disadvantage of the meneon green-LUMABS system is that its BRET efficiency is not high without antibody, resulting in low fluctuation of BRET efficiency (small dynamic range) in practical antibody detection, which greatly limits the detection sensitivity of antibody in plasma, so that the technology is limited to scientific exploration and cannot be applied clinically.

The first wild-type green fluorescent protein (wtGFP) to be discovered was jellyfish green fluorescent protein (avGFP), consisting of 238 amino acids and having a molecular weight of about 27 kDa. Ser65-Tyr66-Gly67 in the fluorescent protein molecule spontaneously forms a luminescent group in the presence of oxygen, is positioned in the middle, is surrounded by 11 beta folds to form a barrel-shaped structure, and can emit weak green fluorescence under the excitation of ultraviolet light (Zimmer M.Green fluorescent proteins (GFP) applications, structure, and related photophysical viewer. chem Rev,2002,3: 759-782.). High-brightness green fluorescent protein Clover has been developed by site-directed modification of specific amino acids of the luminescent domain of jellyfish green fluorescent protein (Lam AJ, S. -P.F., Gong Y, Marshall JD, Cranfill PJ, Baird MA, McKeown MR, Wiedemann J, Davidson MW, Schnitzer MJ, Tsien RY, Lin MZ., Improving FRET dynamic range with bright green and red fluorescent proteins Nature Methods 9,1005 ion 1012 (2012)). In addition to the jellyfish green fluorescent protein, the green fluorescent protein mNeonGreen (Shanner NC, L.G., Chammas A, Ni Y, Cranfill PJ, Baird MA, Sell BR, Allen JR, Day RN, Israelsson M, Davidson MW, Wang J.A bright monomeric green fluorescent protein derivative from branched brain cell membrane method 10 (5)), 407-409 (2013.), which has a higher brightness than that of the Clover, is the current highest-brightness green fluorescent protein, but still fails to satisfy the requirement of high detection sensitivity. Therefore, further engineering of the fluorescent protein and design of new luciferase and fluorescent protein BRET pairs are urgently needed.

Disclosure of Invention

The invention aims to: provides a high-performance green fluorescent protein, which can provide higher brightness and better monomer property and pH stability. Furthermore, the luciferase gene and luciferase (Nluc) form a new bioluminescence resonance energy transfer system, and a Clover4-LUMABS series probe is further constructed, so that a BRET antibody instant detection system is developed. The instant detection system can have high BRET efficiency under the condition of no antibody, thereby having large dynamic range and detection sensitivity when actually detecting the antibody, and leading the technology to be truly clinically accessible.

One aspect of the present invention provides a green fluorescent protein Clover4, which has the following mutation sites compared with the amino acid sequence of Clover: S72A, Q80L, S86A, K101E, T153M, Q157A, R168Y, L178V, a206T, L221V, F223R.

In some embodiments of the invention, the amino acid sequence of the green fluorescent protein Clover4 is set forth in SEQ ID No: 1 is shown.

SEQ ID No：1

MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKFICTTGKLPVPWPTLVTTFGYGVACFARYPDHMKLHDFFKAAMPEGYVQERTISFEDDGTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYIMADKAKNGIKANFKIYHNVEDGSVQVADHYQQNTPIGDGPVLLPDNHYLSHQSTLSKDPNEKRDHMVLVERVTAAGITHGMDELYK。

In another aspect of the invention, there is provided a polynucleotide sequence encoding the green fluorescent protein Clover4 described above.

In some embodiments of the invention, the polynucleotide sequence has the sequence shown in SEQ ID No. 2.

SEQ ID No.2

ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTCCGCGGCGAGGGCGAGGGCGATGCCACCAACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCTTCGGCTACGGCGTGGCCTGCTTCGCCAGGTACCCCGACCACATGAAGCTGCACGACTTCTTCAAGGCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTCTTTCGAGGACGACGGTACCTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTTCAACAGCCACAACGTCTATATCATGGCCGACAAGGCCAAGAACGGCATCAAGGCTAACTTCAAGATCTACCACAACGTTGAGGACGGCAGCGTGCAGGTGGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCCATCAGTCCACCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGGTGGAGCGCGTGACCGCCGCCGGGATTACACATGGCATGGACGAGCTGTACAAG

In yet another aspect, the present invention provides a protein pair for bioluminescence resonance energy transfer detection, which comprises the green fluorescent protein Clover4 as a bioluminescence resonance energy transfer acceptor, and a bioluminescence resonance energy transfer donor.

In some embodiments of the invention, the bioluminescent resonance energy transfer donor is selected from luciferase.

In yet another aspect, the present invention provides a bioluminescence resonance energy transfer based probe comprising the above-described protein pair for bioluminescence resonance energy transfer detection.

In some embodiments of the invention, the pair of proteins for use in a bioluminescence resonance energy transfer assay comprises a bioluminescence resonance energy transfer acceptor as described above and a bioluminescence resonance energy transfer donor; the bioluminescent resonance energy transfer acceptor is the green fluorescent protein Clover 4.

In some specific embodiments of the invention, the bioluminescence resonance energy transfer based probe further comprises a fragment comprising an antigenic epitope and a helper domain.

In some embodiments of the invention, the epitope in the epitope-containing fragment is selected from an epitope fragment in a viral protein.

In some embodiments of the invention, the viral protein is dengue virus, avian influenza virus, aids virus, EB virus, hepatitis b virus or coronavirus.

In some embodiments of the invention, the dengue virus epitope fragment is represented by SEQ ID NO. 3.

In some embodiments of the invention, the epitope fragment of avian influenza virus is represented by SEQ ID No. 4.

In some embodiments of the present invention, the epitope fragment of HIV is represented by SEQ ID NO. 5.

In some embodiments of the invention, the epitope fragment of EB virus is shown as SEQ ID NO.6 or SEQ ID NO. 7.

In some embodiments of the invention, the epitope fragment of hepatitis B virus is represented by SEQ ID NO. 8.

In some embodiments of the invention, the epitope fragment of the novel coronavirus is represented by SEQ ID NO. 9.

In some embodiments of the invention, the helper domain is selected from the SH3/sp1 domain.

In some embodiments of the invention, the probe further comprises a fluorescent protein Clover4 selected from the group consisting of: a fluorescent protein Clover4 cyclized rearrangement (cp) derivative formed by connecting the original N-terminal and C-terminal and opening the peptide chain at other sites of the fluorescent protein Clover 4; and linking a helper domain and a fragment containing an epitope to the N-terminal and C-terminal of the opening site of the fluorescent protein Clover4 derivative. Other sites of opening are, for example, at any of positions 2-237 in the protein sequence, for example, at position 157 or 173.

In yet another aspect, the present invention provides the use of the above-described green fluorescent protein Clover4, or the cyclized rearranged derivative of fluorescent protein Clover4, of the present invention as a bioluminescence resonance energy transfer receptor in a bioluminescence resonance energy transfer assay;

the cyclized rearrangement derivative of the fluorescent protein Clover4 is: a cyclized rearrangement derivative formed by connecting the N terminal and the C terminal of the fluorescent protein Clover4 originally shown as SEQ ID NO.1 and opening a peptide chain at other sites.

In still another aspect, the present invention provides the use of the above-mentioned green fluorescent protein Clover4, or the cyclized and rearranged derivative of fluorescent protein Clover4 in the preparation of a probe or reagent for bioluminescence resonance energy transfer based assay;

In some embodiments of the invention, in the above uses, the green fluorescent protein Clover4 is used in conjunction with a bioluminescent resonance energy transfer donor, preferably, the bioluminescent resonance energy transfer donor is selected from the group consisting of luciferase.

In a further aspect, the present invention provides a kit comprising the above bioluminescent resonance energy transfer based probe.

In some embodiments of the invention, the kit is for serum detection.

In a further aspect, the invention provides the use of the probe based on bioluminescence resonance energy transfer in the preparation of a reagent for detecting specific antibodies in serum.

In some embodiments of the invention, the specific antibody is an antibody capable of binding to an epitope in the probe.

In some embodiments of the invention, the detection is a qualitative or quantitative detection.

In a further aspect, the present invention provides a method for detecting an antibody in a biological sample, the method comprising the step of detecting an analyte with the bioluminescence resonance energy transfer based probe as described above.

In some specific embodiments of the present invention, the detection method is a qualitative or quantitative detection of an antibody in an analyte.

In some specific embodiments of the present invention, the substance to be detected is a liquid sample, preferably a serum sample.

In some embodiments of the present invention, the detection method comprises contacting the test object with the probe, and observing the bioluminescence resonance energy transfer efficiency of the probe when the test object is contained or not contained in the detection system.

Advantageous effects

(1) According to the invention, a green fluorescent protein Clover4 is screened out through mutation by fluorescent protein engineering, the spectral property of the green fluorescent protein Clover4 is similar to that of the original protein Clover, the brightness is higher than that of Clover, and the monomer property and the pH stability are better than those of Clover;

(2) the BRET efficiency (hibRET) in serum was higher for the cloven epitope, the Clover4-LUMABS series probe than for the mNeon Green-LUMABS probe, when no antibody was added. Wherein the dose of Clover4-HIV and Clover4-DEN1 is 4-5 times higher than that of mNeonG-HIV and mNeonG-DEN 1.

(3) For four different epitopes, the dynamic range of the Clover4-LUMABS series probe was higher than that of the mNeon Green-LUMABS probe in antibody detection. The dynamic range of detection of the new crown antibody is more than 20 times of that of the mNeon Green probe.

(4) For four different epitopes, the Clover4-LUMABS series of probes had lower minimum detection limits on antibody detection than the mNeon Green-LUMABS probes. The antibody can be detected in the environment of lower concentration of the antibody, and early diagnosis is facilitated.

Drawings

FIG. 1 (a) is the sequence of fluorescent proteins Clover and Clover4, wherein the Clover4 mutation site is labeled; luminescent groups in black boxes; (b) is Clover crystal structure (PDB No. 5WJ 2); (c) is Clover4 excitation and emission spectrum.

FIG. 2 is a graph of HPLC results for Clover, mClover3, Clover4, and mEGFP at 100 μ M (a) and 5 μ M (b) concentrations. The later the peak is taken (i.e., the larger the volume at which the peak is taken) the more monomeric.

FIG. 3 is the pH stability results of Clover 4.

FIG. 4 is a schematic diagram of the structure of Clover4-LUMABS probe.

FIG. 5 shows the dynamic range of antibody detection by the LUMABS-HIV probe and the detection range of antibody concentration after incubation in serum for 30 minutes. (a) Emission spectra of mNeonG-LUMABS-HIV (left) and Clover4-LUMABS-HIV (right) probes; (b) comparing emission spectra of the LUMABS-HIV series probes in the absence of antibodies; (c) BRET efficiency of the probe in the presence or absence of 1nM anti-HIV-p17 antibody, respectively; (d) titration curves for mNeonG-LUMABS-HIV (left) and Clover4-LUMABS-HIV (right) probes against 0-100nM anti-HIV-p17 antibody. And (4) determining a linear calibration curve of the detection range. Error line: mean ± SD ═ 3. The significant difference is as follows: p is less than 0.001; p < 0.05.

Fig. 6 is the hiBRET of the LUMABS probe without incubation. Error line: mean ± SD ═ 3. The significant difference is as follows: p is less than 0.001; p ═ 0.358.

FIG. 7 is a graph of BRET efficiency of mNeong-LUMABS (a) and Clover4-LUMABS (b) series of probes with and without 1nM of different antibodies, respectively, after 30 minutes incubation in serum. Error line: mean ± SD ═ 3. The significant difference is as follows: p is less than 0.001; p < 0.05; and P is 0.823.

Detailed Description

In order to make the aforementioned objects, features and advantages of the present invention more comprehensible, specific embodiments thereof are described in detail below, but the present invention is not to be construed as being limited to the implementable range thereof.

Example 1 construction and photophysical Properties detection of the Green fluorescent protein Clover4

(1) The crystal structure analysis of green fluorescent protein Clover (figure 1b) is carried out, and the comparison with homologous sequence is carried out, the reasonable design and the site-directed mutation are carried out to the key site influencing the fluorescent protein spectrum and the amino acid interacting with the key site, then the mutant is expressed and screened on the constitutive expression vector pNCS, and the used expression strain is Stellar. In order to ensure the integrity of the library, 10 clones are set for each mutant, and finally, the fluorescence properties of the mutants are detected through visual discrimination and blue LED exciting light penetrating through an orange acrylic filter, and the monoclonal of the fluorescent protein expressing the spectrum blue shift is screened out. Non-conserved amino acid residues around the luminophore of the spectrally blue-shifted mutant fluorescent protein were then further site-directed mutated to stabilize the luminophore, and a high quantum yield (high brightness) monoclonal was selected, named as Clover4, which was mutated at 11 sites on the basis of Clover (S72A, Q80L, S86A, K101E, T153M, Q157A, R168Y, L178V, a206T, L221V, F223R), and the results of comparing the sequences of Clover and Clover4 are shown in fig. 1 a.

(2) Bacteria expressing Clover4 were lysed using B-PER II (purchased from Pierce), followed by protein purification using HisPur Cobalt Resin (purchased from Pierce), followed by Econo-Pac 10DG gravity flow chromatography (purchased from Bio-Rad, USA)And (4) desalting. After the above protein purification steps were completed, the single photon excitation and emission spectra of Clover4 were examined using Lambda35 UV/VIS and LS-55 fluorescence spectrometer (purchased from Perkin Elmer, Inc.). As shown in Table 1 and FIG. 1c, Clover4 has an excitation peak of 506nm and an emission peak of 516nm, similar to Clover. Its extinction coefficient at the peak was 115mM^-1cm^-1The quantum yield was 0.77 (see Table 1) and the molecular brightness was 89, which is higher than Clover.

(3) The purified Clover4 protein was concentrated to a high concentration of 100. mu.M and a low concentration of 5. mu.M, respectively, and subjected to chromatographic analysis using high performance liquid chromatography (Shimadzu LC20A) to detect the protein monosomy. As shown in FIG. 2, at both high and low concentrations, Clover4 was more monomeric than Clover and its other derivative, mClover3(mClover3 structure see 201910057635.8), which was closer to a single monomer.

(4) The purified Clover4 protein was concentrated to a high concentration of 5mg/mL and the readings of fluorescence from Clover4 excited by excitation light of the same wavelength in buffers of different pH values were examined using Lambda35 UV/VIS and LS-55 fluorescence spectrometer (purchased from Perkin Elmer Co.) and the pKa of the protein was calculated based on this. As shown in Table 1 and FIG. 3, Clover4 has a pKa of 5.6, which is lower than that of Clover, mClover3 and mNeonGreen, and has better pH stability than Clover.

TABLE 1 characterization of related Green fluorescent protein Properties

And (4) surface note: a luminance is calculated as the product of peak EC and QY

Example 2 use of Clover4 in the detection of Clover4-LUMABS System by BRET and serum antibodies (mNeonGreen in the Probe abbreviated as mNeong)

(1) Clover4-LUMABS-HIV probe

First, mNEnG-LUMABS-HIV and Clover4-LUMABS-HIV probes are respectively constructed, the structural schematic diagram of the probes is shown in figure 4, mNEnG-LUMABS-HIV is a probe which connects luciferase (Nluc) and mNeonGreen through a sequence containing HIV epitope, the probe also comprises SH3/sp1 structural domain, Clover4-LUMABS-HIV is a probe which connects luciferase (Nluc) and Clover4 through a sequence containing HIV epitope, and the probe also comprises SH3/sp1 structural domain. The epitopes are shown in Table 2.

The results of the tests, which were performed in bovine serum (FBS), are shown in Table 3, and show that the BRET efficiency (hibRET) of the Clover4-LUMABS-HIV probe under antibody-free conditions is 4.6 times higher than that of mNEN G-LUMABS-HIV (wherein Clover4-LUMABS-HIV is 14.00 and mNEN G-LUMABS-HIV is 3.07). In the detection of anti-HIV-p17 antibody, anti-HIV-p17 antibody and the probe were respectively incubated for 30 minutes, and then detection was performed to evaluate the dynamic range of the probe, and the Clover4 probe showed a very large dynamic range (dynamic range): 807.7 + -9.9%, is more than four times as much as mNeon Green. Dynamic Range equation (hiBRET)^b-loBRET)/loBRET, wherein loBRET is the BRET efficiency in the presence of an antibody. HiBRET^bBRET efficiency after 30 min incubation without antibody, loBRET after 30 min incubation with antibody.

At the same time, the Clover-LUMABS-HIV and mCloror 3-LUMABS-HIV probes of the same protein series with Clover4 are constructed, the hiBRET and the dynamic range of the probes are both smaller than those of the mNeonGreen probes, and the dynamic range of the Clover4 probe is more than 9 times of those of the mNeonGreen probes. It was shown that the probe of the present invention has higher sensitivity.

In addition, the detection range (detection range) and the minimum detection limit (limit of detection, LOD) of the four probe pairs were determined by titration of anti-HIV-p17 antibodies at different concentrations (0-100 nM). The detection range of the Clover4 probe was 3-120pM, and the LOD was 2.5pM calculated according to the 3. sigma. standard. The detection ranges of the mNeonGreen, Clover and mClover3 probes are 20-500pM, 25-500pM and 30-500pM respectively, and the LODs are 14pM, 24pM and 25pM respectively. Lower concentrations of antibody were shown to be detectable by Clover4-LUMABS-HIV compared to other probes, e.g., one tenth of the lowest detection limit of the mCloror 3-LUMABS-HIV probe.

To analyze the mechanism of high BRET efficiency of the Clover4 probe, the present inventors constructed two cyclo-rearranged fluorescent protein (cpFP) -based Clover4 derivatives probe cp157Clover4-LUMABS-HIV and cp173Clover4-LUMABS-HIV, whose hibRETs were 50% and 70% of the Clover4 probe, respectively. The cp157Clover4-LUMABS-HIV and cp173Clover4-LUMABS-HIV are peptide chains opened at 157 th and 173 th positions of the Clover4 fluorescent protein, respectively, and a helper domain and an epitope-containing fragment are linked at the open position. From the experimental results, the Clover4 derivative probes cp157Clover4-LUMABS-HIV and cp173Clover4-LUMABS-HIV have higher hiBRET than mNeong-LUMABS-HIV, Clover-LUMABS-HIV and mCLOver3-LUMABS-HIV, although lower hiBRET than Clover4 probes Clover 4-LUMABS-HIV. In other words, the Clover4 fluorescent protein obtained by mutation greatly improves the probe sensitivity in the bioluminescence resonance energy transfer technology probe. In the probe, the Clover4 fluorescent protein derivative obtained by opening peptide chains at different sites of Clover4 fluorescent protein still brings higher sensitivity and hiBRET and larger dynamic range for the bioluminescence resonance energy transfer technology probe than other fluorescent proteins.

In addition, a probe Clover4-LUMABS-HIV w/o SH3/sp1 with the SH3/sp1 domain removed is constructed, and the detection function is basically lost.

The LUMABS-HIV probe assay data are shown in Table 3 or FIG. 5.

(2) Clover4-LUMABS series probes with different antigen epitopes

To verify the adaptability of Clover4-LUMABS, Clover4-LUMABS series probes of different lengths and structures were constructed against different epitopes of different viruses, including Clover4-LUMABS-HA against avian influenza virus and Clover4-LUMABS-DEN1 against dengue virus, Clover4-LUMABS-EBNA1 and Clover4-LUMABS-VCA against nasopharyngeal carcinoma EB virus, Clover4-LUMABS-HBV against hepatitis B virus and Clover4-LUMABS-S1 RBD probes against neocoronaviruses. The viral and antigenic epitopes (epitopes) present in this section are shown in Table 2.

TABLE 2 epitope information on related antigens

Meanwhile, corresponding mNeon Green probes are also constructed in a one-to-one correspondence manner, namely, mNeon Green replaces the probes constructed by Clover 4. The results are shown in FIG. 6, where all Clover4 probes had higher hiBRET than the same epitope mNeon Green probe: 1) DEN1, Clover4 probe 5.5 times as much as mNeon Green probe (16.63 for Clover4 probe and 3.01 for mNeon Green probe); 2) HA, Clover4 probe was 2.2 times as much as meneon green probe (Clover4 probe 6.09, meneon green probe 2.80); 3) VCA, Clover4 probe was 3.3 times as much as meneon green probe (Clover4 probe 4.66, meneon green probe 1.40); 4) EBNA1, Clover4 probe was 4.2 times as high as mNeonGreen probe (9.30 for Clover4 probe and 2.20 for mNeonGreen probe); 5) HBV, the Clover4 probe was 1.3 times as much as the mNeon Green probe (Clover4 probe was 3.95, mNeon Green probe was 3.01); 6) S1-RBD, Clover4 probe was 1.9 times as large as mNeonGreen probe (5.60 for Clover4 probe and 2.88 for mNeonGreen probe).

The detection effect of the Clover4-LUMABS series probe on the antibodies of avian influenza, dengue fever, new corona and other viruses is detected. The results are shown in FIG. 7, with all Clover4 probes having a higher dynamic range than the same epitope mNeon Green probe: 1) DEN1, 5.7 times more of the Clover4 probe than the mNeon Green probe (498.7 + -24.4% for the Clover4 probe, 87.9 + -3.5% for the mNeon Green probe); 2) HA, Clover4 probe was 2.5 times as much as mNeon Green probe (247.8 + -1.7% for Clover4 probe, 99.7 + -1.6% for mNeon Green probe); 3) S1-RBD, Clover4 probe was 20-fold higher than mNeonGreen probe (Clover4 probe 105.9. + -. 8.4%, mNeonGreen probe 5.3. + -. 0.8%).

Different concentrations (0-100nM) of antibody were titrated against the three viruses to determine the detection range and the lowest detection limit. 1) The DEN1, Clover4 probes detected in the range of 8-200pM, LOD 7.7 pM. The detection range of the mNeonGreen probe is 25-1000pM respectively, and the LOD is 22.7 pM. (ii) a 2) The HA, Clover4 probes detected in the range of 8-200pM, LOD 7.5 pM. The detection range of the mNeonGreen probe is 300-500pM respectively, and the LOD is 25 pM. 3) The detection range of the S1-RBD, Clover4 probe is 20-200pM, and the LOD is 15.5 pM. The mNeon Green probe cannot be directly measured due to too high binding concentration. It was demonstrated that both the Clover4 series probes detected lower concentrations of antibody than the meneon green series probes.

All probe detection data are listed in table 3.

TABLE 3 LUMABS Probe assay data

And (4) surface note: HiBRET^aThe probe is not incubated; HiBRET^bProbe incubation for 30 minutes; LOD: the lowest detection limit.

SEQUENCE LISTING

<110> Shenzhen advanced technology research institute

<120> a green fluorescent protein Clover4 and derived bioluminescence resonance energy transfer-based probe and method

Applications of

<130> CP121010311C

<160> 9

<170> PatentIn version 3.3

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<213> Artificial sequence

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Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu

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Phe Gly Tyr Gly Val Ala Cys Phe Ala Arg Tyr Pro Asp His Met Lys

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Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly

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Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr

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<400> 2

atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 60

ggcgacgtaa acggccacaa gttcagcgtc cgcggcgagg gcgagggcga tgccaccaac 120

ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc 180

ctcgtgacca ccttcggcta cggcgtggcc tgcttcgcca ggtaccccga ccacatgaag 240

ctgcacgact tcttcaaggc cgccatgccc gaaggctacg tccaggagcg caccatctct 300

ttcgaggacg acggtaccta caagacccgc gccgaggtga agttcgaggg cgacaccctg 360

gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 420

aagctggagt acaacttcaa cagccacaac gtctatatca tggccgacaa ggccaagaac 480

ggcatcaagg ctaacttcaa gatctaccac aacgttgagg acggcagcgt gcaggtggcc 540

gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 600

tacctgagcc atcagtccac cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 660

ctggtggagc gcgtgaccgc cgccgggatt acacatggca tggacgagct gtacaag 717

<210> 3

<211> 8

<212> PRT

<213> Artificial sequence

<400> 3

Glu His Lys Tyr Ser Trp Lys Ser

1 5

<210> 4

<211> 9

<212> PRT

<213> Artificial sequence

<400> 4

Tyr Pro Tyr Asp Val Pro Asp Tyr Ala

1 5

<210> 5

<211> 12

<212> PRT

<213> Artificial sequence

<400> 5

Glu Leu Asp Arg Trp Glu Lys Ile Arg Leu Arg Pro

1 5 10

<210> 6

<211> 58

<212> PRT

<213> Artificial sequence

<400> 6

Ser Thr Ala Val Ala Gln Ser Ala Thr Pro Ser Val Ser Ser Ser Ile

1 5 10 15

Ser Ser Leu Arg Ala Ala Thr Ser Gly Ala Thr Ala Ala Ala Ser Ala

20 25 30

Ala Ala Ala Val Asp Thr Gly Ser Gly Gly Gly Gly Gln Pro His Asp

35 40 45

Thr Ala Pro Arg Gly Ala Arg Lys Lys Gln

50 55

<210> 7

<211> 59

<212> PRT

<213> Artificial sequence

<400> 7

Pro Pro Arg Arg Pro Pro Pro Gly Arg Arg Pro Phe Phe His Pro Val

1 5 10 15

Gly Glu Ala Asp Tyr Phe Glu Tyr His Gln Glu Gly Gly Pro Asp Gly

20 25 30

Glu Pro Asp Val Pro Pro Gly Ala Ile Glu Gln Gly Pro Ala Asp Asp

35 40 45

Pro Gly Glu Gly Pro Ser Thr Gly Pro Arg Gly

50 55

<210> 8

<211> 71

<212> PRT

<213> Artificial sequence

<400> 8

Asp Tyr Gln Gly Met Leu Pro Val Cys Pro Leu Leu Pro Gly Thr Thr

1 5 10 15

Thr Thr Ser Thr Gly Pro Cys Lys Thr Cys Thr Thr Pro Ala Ala Asn

20 25 30

Ile Ser Met Phe Pro Ser Cys Cys Cys Thr Lys Pro Ser Asp Gly Asn

35 40 45

Cys Thr Cys Ile Pro Ile Pro Ser Ser Trp Ala Phe Ala Arg Phe Leu

50 55 60

Trp Glu Trp Ala Ser Val Arg

65 70

<210> 9

<211> 200

<212> PRT

<213> Artificial sequence

<400> 9

Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg

1 5 10 15

Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val

20 25 30

Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys

35 40 45

Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn

50 55 60

Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile

65 70 75 80

Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro

85 90 95

Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp

100 105 110

Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys

115 120 125

Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln

130 135 140

Ala Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe

145 150 155 160

Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln

165 170 175

Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala

180 185 190

Thr Val Cys Gly Pro Lys Lys Ser

195 200

Claims

1. A green fluorescent protein Clover4, wherein the amino acid sequence of said green fluorescent protein has the following mutation sites compared to the amino acid sequence of Clover: S72A, Q80L, S86A, K101E, T153M, Q157A, R168Y, L178V, a206T, L221V, F223R;

preferably, the amino acid sequence of said green fluorescent protein Clover4 is as shown in SEQ ID No: 1 is shown.

2. A polynucleotide sequence encoding the green fluorescent protein Clover4 of claim 1.

3. A pair of proteins for use in bioluminescence resonance energy transfer assays comprising the green fluorescent protein Clover4 as a bioluminescence resonance energy transfer acceptor according to claim 1 or 2; the protein pair further comprises a resonance energy transfer donor;

preferably, the bioluminescence resonance energy transfer donor is selected from luciferase.

4. A bioluminescence resonance energy transfer based probe, wherein the bioluminescence resonance energy transfer based probe comprises the pair of proteins for resonance energy transfer detection of claim 3;

preferably, the bioluminescence resonance energy transfer based probe further comprises a fragment comprising an antigenic epitope and a helper domain;

more preferably, the helper domain is selected from the group consisting of the SH3/sp1 domain;

more preferably, the epitope in the epitope-containing fragment is selected from the group consisting of epitope fragments in viral proteins.

5. The probe of claim 4, wherein the fluorescent protein Clover4 is further selected from the group consisting of: connecting the N end and the C end of the fluorescent protein Clover4 originally shown as SEQ ID NO.1, and opening a peptide chain at other sites of the fluorescent protein Clover4 to form a fluorescent protein Clover4 cyclic rearrangement derivative; and linking a helper domain and a fragment containing an epitope to the N-terminal and C-terminal of the opening site of the fluorescent protein Clover4 derivative.

6. Use of the green fluorescent protein Clover4, or the cyclized rearranged derivative of fluorescent protein Clover4, of claim 1 as a resonance energy transfer donor or acceptor in a resonance energy transfer assay;

the fluorescent protein Clover4 cyclized rearrangement derivative is: a cyclized rearrangement derivative formed by connecting the N terminal and the C terminal of the fluorescent protein Clover4 originally shown as SEQ ID NO.1 and opening a peptide chain at other sites.

7. Use of the green fluorescent protein Clover4, or the cyclized rearranged derivative of fluorescent protein Clover4, of claim 1 in the preparation of a probe or reagent for resonance energy transfer based detection;

8. Use according to claim 6 or 7, wherein the green fluorescent protein Clover4 is used in combination with a bioluminescent resonance energy transfer donor, preferably wherein the bioluminescent resonance energy transfer donor is selected from the group consisting of luciferase.

9. A kit comprising the above-mentioned probe based on bioluminescence resonance energy transfer;

preferably, the kit is for serum detection.

10. Use of the bioluminescence resonance energy transfer based probe of claim 4 or 5 or the pair of proteins of claim 3 in the preparation of a reagent for the detection of specific antibodies in serum;

preferably, the detection is a qualitative or quantitative detection.