WO2022232983A1 - 一种绿色荧光蛋白Clover4及其衍生的基于生物发光共振能量转移的探针和应用 - Google Patents

一种绿色荧光蛋白Clover4及其衍生的基于生物发光共振能量转移的探针和应用 Download PDF

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WO2022232983A1
WO2022232983A1 PCT/CN2021/091875 CN2021091875W WO2022232983A1 WO 2022232983 A1 WO2022232983 A1 WO 2022232983A1 CN 2021091875 W CN2021091875 W CN 2021091875W WO 2022232983 A1 WO2022232983 A1 WO 2022232983A1
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clover4
fluorescent protein
energy transfer
resonance energy
probe
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French (fr)
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储军
邓梦颖
袁静
金宗文
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深圳先进技术研究院
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/576—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

Definitions

  • the invention belongs to the field of biotechnology, and in particular relates to a green fluorescent protein Clover4 and a BRET serum antibody detection system derived therefrom.
  • luciferase which generates photons by catalyzing a biochemical reaction of a substrate
  • BRET Bioluminescence resonance energy transfer
  • Bioluminescence resonance energy transfer as an efficient optical "molecular ruler", its technical principle is based on non-radiative energy transfer, through the electric dipole interaction between molecules, the luciferase donor is excited The process of state energy transfer to the excited state of fluorescent protein acceptor (Xu Y, Piston D W, Johnson C H.
  • a bioluminescence resonance energy transfer (BRET) system application to interacting circadian clock proteins.
  • the probe In the absence of antibody, the probe has high BRET efficiency between Nluc and mNeonGreen, and emits green fluorescence; when there is antibody, the antibody specifically binds to the antigen tag in the LUMABS system, making Nluc and mNeonGreen far away, and the BRET efficiency is reduced. Fluoresces blue.
  • the detection BRET probe can be designed to target antibodies in plasma, which ensures the high specificity of detection and provides the possibility for future clinical applications.
  • the light signal originates from the chemical oxidation process of luciferase-catalyzed substrate luminescence, which effectively reduces the background signal of plasma and ensures a high signal-to-noise ratio of detection; at the same time, in addition to the need for no excitation light source (The optical path design is simple), and the BRET detection steps are simple, making it possible to miniaturize and portable the detection instrument, and ensure the immediacy of detection.
  • the antibody In the presence of the antibody, the antibody specifically binds to the antigen tag in the mNeonGreen-LUMABS system, making Nluc and mNeonGreen far away, and the BRET efficiency will drop to a very low state at this time. This low BRET due to distance is difficult to change. .
  • the distance between Nluc and mNeonGreen is small, and the BRET efficiency at this time is mainly determined by the resonance energy transfer pair of Nluc and mNeonGreen. If the BRET efficiency at this time is high, the BRET change (that is, the dynamic range) caused by the presence or absence of the antibody will be large, and the detection sensitivity will be high.
  • the biggest disadvantage of the mNeonGreen-LUMABS system is that its BRET efficiency in the absence of antibodies is not high, resulting in the fact that the BRET efficiency fluctuates only in a low range (small dynamic range) in the actual detection of antibodies, which greatly limits the The detection sensitivity of antibodies in plasma makes this technology limited to scientific exploration and cannot be used clinically.
  • wild-type green fluorescent protein is jellyfish green fluorescent protein (avGFP), which consists of 238 amino acids and has a molecular weight of about 27kDa. Ser65-Tyr66-Gly67 in the fluorescent protein molecule will spontaneously form a light-emitting group in the presence of oxygen, located in the middle, surrounded by 11 ⁇ -sheets to form a barrel structure, and can emit weak green fluorescence under ultraviolet light excitation ( Zimmer M. Green fluorescent protein (GFP) applications, structure, and related photophysical behavior. Chem Rev, 2002, 3:759-782.).
  • avGFP jellyfish green fluorescent protein
  • the high-brightness green fluorescent protein Clover was developed after the specific amino acid modification of the luminescent domain of the jellyfish green fluorescent protein (Lam AJ, S.-P.F., Gong Y, Marshall JD, Cranfill PJ, Baird MA, McKeown MR, Wiedenmann J, Davidson MW, Schnitzer MJ, Tsien RY, Lin MZ.. Improving FRET dynamic range with bright green and red fluorescent proteins. Nature Methods 9, 1005–1012 (2012).).
  • green fluorescent protein mNeonGreen derived from amphioxus (Shaner 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 derived from Branchiostoma lanceolatum. Nat Methods 10(5), 407-409(2013).)
  • its brightness is a little higher than Clover, both are green fluorescent proteins with the highest brightness at present, but still can not meet the high brightness The need for detection sensitivity. Therefore, it is urgent to further engineer fluorescent proteins and design new luciferase and fluorescent protein BRET pairs.
  • the purpose of the present invention is to provide a high-performance green fluorescent protein, which can provide higher brightness, better monomericity and pH stability. Further, it is combined with luciferase (Nluc) to form a new bioluminescence resonance energy transfer system, and then the Clover4-LUMABS series of probes are constructed to develop a BRET antibody instant detection system.
  • the instant detection system can have high BRET efficiency in the absence of antibodies, so that it has a large dynamic range and detection sensitivity when actually detecting antibodies, so that this technology can really enter the clinic.
  • One aspect of the present invention provides a green fluorescent protein Clover4.
  • the amino acid sequence of the green fluorescent protein has the following mutation sites: S72A, Q80L, S86A, K101E, T153M, Q157A, R168Y, L178V , A206T, L221V, F223R.
  • amino acid sequence of the green fluorescent protein Clover4 is shown in SEQ ID No: 1.
  • Another aspect of the present invention provides a polynucleotide sequence encoding the above-mentioned green fluorescent protein Clover4.
  • polynucleotide sequence has the sequence shown in SEQ ID No. 2.
  • Another aspect of the present invention provides a protein pair that can be used for bioluminescence resonance energy transfer detection, comprising the above-mentioned green fluorescent protein Clover4 as a bioluminescence resonance energy transfer acceptor, and a bioluminescence resonance energy transfer donor.
  • the bioluminescence resonance energy transfer donor is selected from luciferase.
  • bioluminescence resonance energy transfer-based probe the bioluminescence resonance energy transfer-based probe comprising the above-mentioned protein pair for bioluminescence resonance energy transfer detection.
  • the protein pair for bioluminescence resonance energy transfer detection includes the above-mentioned bioluminescence resonance energy transfer acceptor and bioluminescence resonance energy transfer donor; the bioluminescence resonance energy transfer acceptor is The above-mentioned green fluorescent protein Clover4 of the present invention.
  • the bioluminescence resonance energy transfer-based probe further includes a fragment comprising an epitope and a helper domain.
  • the epitope in the fragment comprising the epitope is selected from an epitope fragment in a viral protein.
  • the viral protein is dengue virus, avian influenza virus, HIV, Epstein-Barr virus, hepatitis B virus or coronavirus.
  • the antigenic epitope fragment of dengue virus is shown in SEQ ID NO.3.
  • the antigenic epitope fragment of avian influenza virus is shown in SEQ ID NO.4.
  • the antigenic epitope fragment of HIV is shown in SEQ ID NO.5.
  • the antigenic epitope fragment of Epstein-Barr virus is shown in SEQ ID NO.6 or SEQ ID NO.7.
  • the antigenic epitope fragment of hepatitis B virus is shown in SEQ ID NO.8.
  • the antigenic epitope fragment of the novel coronavirus is shown in SEQ ID NO.9.
  • the helper domain is selected from SH3/sp1 domains.
  • the fluorescent protein Clover4 is also selected from: connecting the N-terminus and C-terminus of the fluorescent protein Clover4 as originally shown in SEQ ID NO.
  • the fluorescent protein Clover4 cyclization rearrangement (cp) derivative formed by opening the peptide chain at other sites of the protein Clover4; and linking the helper domain and containing the antigenic epitope to the N-terminal and C-terminal of the open site of the fluorescent protein Clover4 derivative fragment.
  • Other sites that are opened are, for example, any of positions 2-237 in the protein sequence, such as positions 157 or 173.
  • Another aspect of the present invention provides the use of the above-mentioned green fluorescent protein Clover4 of the present invention, or a cyclization rearrangement derivative of the fluorescent protein Clover4 as a bioluminescence resonance energy transfer acceptor in bioluminescence resonance energy transfer detection;
  • the cyclization rearrangement derivative of the fluorescent protein Clover4 is: the cyclization rearrangement formed by connecting the N-terminus and C-terminus of the fluorescent protein Clover4 as originally shown in SEQ ID NO.1, and opening the peptide chain at other sites derivative.
  • Another aspect of the present invention provides the use of the above-mentioned green fluorescent protein Clover4 of the present invention, or a cyclization rearrangement derivative of the fluorescent protein Clover4 in the preparation of a probe or reagent based on bioluminescence resonance energy transfer detection;
  • the cyclization rearrangement derivative of the fluorescent protein Clover4 is: the cyclization rearrangement formed by connecting the N-terminus and C-terminus of the fluorescent protein Clover4 as originally shown in SEQ ID NO.1, and opening the peptide chain at other sites derivative.
  • the green fluorescent protein Clover4 is used in combination with a bioluminescence resonance energy transfer donor, preferably, the bioluminescence resonance energy transfer donor is selected from luciferase.
  • Another aspect of the present invention provides a kit comprising the above-mentioned bioluminescence resonance energy transfer-based probe.
  • the kit is used for serum detection.
  • Another aspect of the present invention provides the use of the above-mentioned bioluminescence resonance energy transfer-based probe in preparing a reagent for detecting specific antibodies in serum.
  • the specific antibody is an antibody capable of binding to an epitope in the probe.
  • the detection is qualitative detection or quantitative detection.
  • Yet another aspect of the present invention provides a method for detecting antibodies in a biological sample, the method comprising the step of detecting an object to be detected with the above-mentioned bioluminescence resonance energy transfer-based probe.
  • the detection method is qualitative detection or quantitative detection of the antibody in the analyte.
  • the substance to be detected is a liquid sample, preferably a serum sample.
  • the detection method includes contacting the analyte with the probe, and respectively observing the detection system containing the analyte or not containing the analyte. Bioluminescence resonance energy transfer efficiency.
  • the present invention has screened out a green fluorescent protein Clover4 by mutation through fluorescent protein engineering, and its spectral properties are similar to its starting protein Clover, its brightness is higher than that of Clover, and its monomericity and pH stability are better than that of Clover;
  • Clover4-LUMABS series probes in serum was higher than that of mNeonGreen-LUMABS probes when no antibody was added.
  • Clover4-HIV and Clover4-DEN1 are 4-5 times higher than mNeonG-HIV and mNeonG-DEN1.
  • the dynamic range of Clover4-LUMABS series probes in antibody detection is higher than that of mNeonGreen-LUMABS probes.
  • the dynamic range of detection of new crown antibodies is as high as 20 times that of mNeonGreen probe.
  • Clover4-LUMABS series probes in antibody detection was lower than that of mNeonGreen-LUMABS probe.
  • Antibodies can be detected at a lower concentration of antibodies, which is more conducive to early diagnosis.
  • FIG. 1 (a) is the sequence of the fluorescent proteins Clover and Clover4, in which the Clover4 mutation site is marked; the black box is the light-emitting group; (b) is the crystal structure of Clover (PDB No. 5WJ2); (c) is Clover4 Excitation and emission spectra.
  • Figure 2 is a graph of the HPLC results of Clover, mClover3, Clover4 and mEGFP at 100 ⁇ M (a) and 5 ⁇ M (b) concentrations. The later the peak appears (that is, the larger the volume when the peak appears), the stronger the monomer is.
  • FIG. 3 shows the pH stability results of Clover4.
  • Figure 4 is a schematic diagram of the structure of the Clover4-LUMABS probe.
  • Figure 5 shows the dynamic range and antibody concentration detection range of the LUMABS-HIV probe after incubation in serum for 30 minutes.
  • Clover4 site-directed mutagenesis of the non-conserved amino acid residues around the light-emitting group of the mutant fluorescent protein with spectral blue shift was performed to stabilize the light-emitting group, and a single clone with high quantum yield (high brightness) was screened, named Clover4, which is in On the basis of Clover, 11 sites were mutated (S72A, Q80L, S86A, K101E, T153M, Q157A, R168Y, L178V, A206T, L221V, F223R). The sequence comparison results of Clover and Clover4 are shown in Figure 1a.
  • Clover4 Concentrate the purified Clover4 protein to a high concentration of 100 ⁇ M and a low concentration of 5 ⁇ M, respectively, and perform chromatographic analysis with a high performance liquid chromatograph (Shimadzu LC20A) to detect the monomericity of the protein.
  • a high performance liquid chromatograph Shiadzu LC20A
  • Clover4 has better monomericity than Clover and its other derivative mClover3 (see 201910057635.8 for the structure of mClover3) at both high and low concentrations, and its state is closer to a monomer.
  • Clover4 Concentrate the purified Clover4 protein to a high concentration of 5 mg/mL, and use a Lambda35 UV/VIS and LS-55 fluorescence spectrometer (purchased from Perkin Elmer) to detect Clover4 in buffers with different pH values, the same wavelength The fluorescence readings emitted by the excitation light, and the pKa value of the protein is calculated accordingly. As shown in Table 1 and Figure 3, the pKa value of Clover4 is 5.6, which is a little lower than that of Clover, mClover3 and mNeonGreen, and the pH stability is better than that of Clover.
  • mNenG-LUMABS-HIV and Clover4-LUMABS-HIV probes were constructed respectively.
  • the schematic diagram of the probe structure is shown in Figure 4.
  • mNenG-LUMABS-HIV is a sequence containing HIV epitopes that connect luciferase (Nluc) and The probe of mNeonGreen also includes SH3/sp1 domain
  • Clover4-LUMABS-HIV is a probe that connects luciferase (Nluc) and Clover4 through a sequence containing HIV epitope, and the probe also includes SH3 /sp1 domain.
  • the antigenic epitopes are shown in Table 2.
  • the experimental results show that the BRET efficiency (hiBRET) of the Clover4-LUMABS-HIV probe under antibody-free conditions is 4.6 times that of mNenG-LUMABS-HIV (wherein 14.00 for Clover4-LUMABS-HIV and 3.07 for mNenG-LUMABS-HIV).
  • the anti-HIV-p17 antibody was incubated with the above probe for 30 minutes, and then the detection was used to evaluate the dynamic range of the probe.
  • the Clover4 probe showed that Great dynamic range: 807.7 ⁇ 9.9%, more than four times that of mNeonGreen.
  • Dynamic range calculation formula (hiBRET b -loBRET)/loBRET, where loBRET is the BRET efficiency in the presence of antibody.
  • hiBRET b is the BRET efficiency after 30 minutes of incubation without antibody
  • loBRET is the BRET efficiency after 30 minutes of incubation with the addition of antibody.
  • Clover-LUMABS-HIV and mClover3-LUMABS-HIV probes with the same protein series as Clover4 were constructed. Their hiBRET and dynamic range were smaller than those of mNeonGreen probe, and the dynamic range of Clover4 probe was more than 9 times that of them. It shows that the probe of the present invention has higher sensitivity.
  • the detection range and the limit of detection (LOD) of the above four probe pairs were determined by titration of different concentrations (0-100nM) of anti-HIV-p17 antibodies.
  • the detection range of the Clover4 probe was 3-120 pM, and the LOD was calculated to be 2.5 pM according to the 3 ⁇ criterion.
  • the detection ranges of mNeonGreen, Clover and mClover3 probes were 20-500pM, 25-500pM and 30-500pM, respectively, with LODs of 14pM, 24pM and 25pM, respectively. It was demonstrated that Clover4-LUMABS-HIV can detect antibodies at lower concentrations than other probes, eg, one-tenth the minimum detection limit of the mClover3-LUMABS-HIV probe.
  • Clover4 derivative probes cp157Clover4-LUMABS-HIV and cp173Clover4-LUMABS-HIV based on cyclized rearranged fluorescent protein (cpFP), their hiBRET respectively. 50% and 70% of the Clover4 probe.
  • the cp157Clover4-LUMABS-HIV and cp173Clover4-LUMABS-HIV open the peptide chain at positions 157 and 173 of the Clover4 fluorescent protein, respectively, and link a helper domain and a fragment containing an antigenic epitope at the open position.
  • Clover4 derivative probes cp157Clover4-LUMABS-HIV and cp173Clover4-LUMABS-HIV have lower hiBRET than the Clover4 probe Clover4-LUMABS-HIV, their hiBRET is higher than that of mNeonG-LUMABS-HIV, Clover- LUMABS-HIV, mClover3-LUMABS-HIV.
  • the Clover4 fluorescent protein obtained by the mutation of the present invention greatly improves the sensitivity of the probe in the bioluminescence resonance energy transfer technology probe.
  • Clover4 fluorescent protein derivatives obtained by opening the peptide chain at different sites of Clover4 fluorescent protein still bring bioluminescence resonance energy transfer technology probes with higher sensitivity and hiBRET than other fluorescent proteins, and larger dynamic range.
  • Clover4-LUMABS-HA for avian influenza virus
  • Clover4-LUMABS for dengue virus
  • LUMABS-DEN1 Clover4-LUMABS-EBNA1
  • Clover4-LUMABS-VCA for nasopharyngeal carcinoma Epstein-Barr virus
  • Clover4-LUMABS-HBV for hepatitis B virus
  • Clover4-LUMABS-S1RBD probe for new coronavirus. All viruses and epitopes in this part are shown in Table 2.
  • Clover4-LUMABS series probes Detect the detection effect of Clover4-LUMABS series probes on antibodies against viruses such as avian influenza, dengue fever and new crown.
  • the results are shown in Figure 7.
  • the dynamic range of all Clover4 probes is higher than that of mNeonGreen probes with the same epitope: 1) DEN1, Clover4 probes are 5.7 times that of mNeonGreen probes (Clover4 probes are 498.7 ⁇ 24.4%, mNeonGreen probe was 87.9 ⁇ 3.5%); 2) HA, Clover4 probe was 2.5 times more than mNeonGreen probe (Clover4 probe was 247.8 ⁇ 1.7%, mNeonGreen probe was 99.7 ⁇ 1.6%); 3) S1-RBD, Clover4 The probe was 20 times more powerful than the mNeonGreen probe (105.9 ⁇ 8.4% for the Clover4 probe and 5.3 ⁇ 0.8% for the mNeonGreen probe).
  • Antibodies to these three viruses were titrated at different concentrations (0-100 nM) to determine the detection range and the minimum detection limit.
  • the detection range of DEN1, Clover4 probe is 8-200pM, and the LOD is 7.7pM.
  • the detection range of the mNeonGreen probe was 25-1000pM, respectively, and the LOD was 22.7pM.
  • the detection range of HA, Clover4 probe is 8-200pM, and the LOD is 7.5pM.
  • the detection range of mNeonGreen probes was 300-500pM, respectively, and the LOD was 25pM.
  • the detection range of S1-RBD, Clover4 probe is 20-200pM, and the LOD is 15.5pM.
  • the mNeonGreen probe cannot be directly measured because the binding concentration is too high. It is proved that the Clover4 series probes can detect lower concentrations of antibodies than the mNeonGreen series probes.
  • hiBRET a probe not incubated
  • hiBRET b probe incubated for 30 minutes
  • LOD minimum detection limit

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Abstract

一种绿色荧光蛋白Clover4及其衍生的基于生物发光共振能量转移的探针和应用。绿色荧光蛋白Clover4的氨基酸序列与Clover的氨基酸序列相比，具有11个突变位点。包含绿色荧光蛋白Clover4的用于生物发光共振能量转移检测的蛋白对以及基于生物发光共振能量转移的探针。绿色荧光蛋白Clover4用于生物发光共振能量转移的探针后降低了检测限，提高了检测灵敏度和检测动态范围，可以在更低浓度抗体环境下检出抗体，更利于早期诊断。

Description

一种绿色荧光蛋白Clover4及其衍生的基于生物发光共振能量转移的探针和应用 技术领域
本发明属于生物技术领域，具体涉及一种绿色荧光蛋白Clover4及其衍生的BRET血清抗体检测系统。
背景技术
生物发光共振能量转移技术
生物发光由于其不需要光激发，背景低特点，已成为活体光学成像的重要手段之一。然而荧光素酶(通过催化底物的生化反应而产生光子)不仅量子产率低，而且发射光子的波长较短，因此限制其在动物体内的应用。基于荧光素酶和荧光蛋白的生物发光共振能量转移(BRET)技术则很好的解决了上述问题。生物发光共振能量转移(BRET)作为一种高效的光学“分子尺”，其技术原理是建立在非辐射能量转移的基础上，通过分子间的电偶极相互作用，将荧光素酶供体激发态能量转移到荧光蛋白受体激发态的过程(Xu Y,Piston D W,Johnson C H.A bioluminescence resonance energy transfer(BRET)system:application to interacting circadian clock proteins.Proceedings of the National Academy of Sciences,1999,96(1):151-156.)只有当能量供体荧光素酶的发射谱与能量荧光受体(如荧光蛋白)的激发谱相重叠，且供体和受体之间的空间距离接近(<10nm)时，供体可催化底物释放光信号，光信号能量(部分或全部)转移给受体分子，而使受体分子产生光信号。这是单一液相内实现的均相检测技术，无需任何固体介质(磁珠、微球等)的参与，近十几年受到了广大学者们的广泛关注。由于无需外源激发光源(背景信号低)和操作简单，该技术可以实现快速的高灵敏度检测，已被广泛用于生物医学基础研究和高通量药物筛选。
血清抗体检测mNeonGreen-LUMABS系统
体外诊断的BRET检测几年来逐渐兴起，但研究非常少，仅集中在抗体的检测。2016年，Maarten Merkx实验室通过对已有抗体检测探针AbSense的改造，首次实现了用于血浆内抗体的智能手机即时分析BRET检测技术，并将该检测系统命名为LUMABS(Arts R,H.I.,Zijlema SE,Thijssen V,Beelen SHE,Merkx M.Detection of antibodies in blood plasma using bioluminescent sensor proteins and a smartphone.Analytical Chemistry 88(8),4525–4532(2016).)。该系统在荧光素酶Nluc和绿色荧光蛋白mNeonGreen之间定向引入不同的帮助结构域，构建成了一个分子内BRET探针。该探针在无抗体情况下，Nluc与mNeonGreen之间存在较高的BRET效率，发出绿色荧光；当存在抗体时，抗体与LUMABS系统中抗原标签 特异结合，使得Nluc与mNeonGreen远离，BRET效率降低，发出蓝色荧光。当应用于检测体系时，检测BRET探针可针对血浆内抗体靶向设计，保证了检测的高特异，为未来临床应用提供了可能性。同时由于探针无需外源光激发，光信号源于荧光素酶催化底物发光的化学氧化过程，有效降低血浆的背景信号，保证了检测的高信噪比；与此同时，除了无需激发光源(光路设计简单)，BRET检测步骤简单，使得检测仪器小型化、便携化成为可能，保证了检测的即时性。
在抗体存在的情况下，抗体与mNeonGreen-LUMABS系统中抗原标签特异结合，使得Nluc与mNeonGreen远离，BRET效率此时会降到很低的状态，这种由于距离导致的低BRET是很难改变的。但是在无抗体情况下，Nluc与mNeonGreen之间的距离小，此时的BRET效率主要由Nluc和mNeonGreen这一对共振能量转移对本身决定。此时的BRET效率如果高，那么抗体存在与否导致的BRET变化(也就是动态范围(dynamic range))就大，检测灵敏度(sensitivity)就高。然而mNeonGreen-LUMABS系统最大的缺点就在于，其在无抗体情况下的BRET效率并不高，导致在实际检测抗体时BRET效率仅在较低的区间波动(动态范围小)，这极大限制了血浆中抗体的检测灵敏度，使得这一技术仅限于科学探索，无法进行临床应用。
最早被发现的野生型绿色荧光蛋白(wtGFP)是水母绿色荧光蛋白(avGFP)，由238个氨基酸组成，分子量约27kDa。荧光蛋白分子中的Ser65-Tyr66-Gly67在氧气存在的情况下会自发形成发光基团，位于中间，周围由11个β折叠包围形成桶状结构，在紫外光激发下能够发出微弱的绿色荧光(Zimmer M.Green fluorescent protein(GFP)applications,structure,and related photophysical behavior.Chem Rev,2002,3:759-782.)。经过对水母绿色荧光蛋白发光结构域的特定氨基酸定点改造，高亮度的绿色荧光蛋白Clover被研发出来(Lam AJ,S.-P.F.,Gong Y,Marshall JD,Cranfill PJ,Baird MA,McKeown MR,Wiedenmann J,Davidson MW,Schnitzer MJ,Tsien RY,Lin MZ..Improving FRET dynamic range with bright green and red fluorescent proteins.Nature Methods 9,1005–1012(2012).)。除水母绿色荧光蛋白之外，来源于文昌鱼的绿色荧光蛋白mNeonGreen(Shaner 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 derived from Branchiostoma lanceolatum.Nat Methods 10(5),407-409(2013).)，其亮度比Clover高一点，均为目前亮度最高的绿色荧光蛋白，但仍无法满足高检测灵敏度的需要。因此，亟需进一步对荧光蛋白进行改造，并设计新的荧光素酶和荧光蛋白BRET对。
发明内容
本发明的目的在于：提供一种高性能绿色荧光蛋白，能够提供更高的亮度和更好的单体 性和pH稳定性。进一步地，将其与荧光素酶(Nluc)构成新的生物发光共振能量转移系统，进而构建的Clover4-LUMABS系列探针，从而开发BRET抗体即时检测系统。所述即时检测系统能够在无抗体情况下具有很高的BRET效率，从而在实际检测抗体时具有很大的动态范围和检测灵敏度，使得这一技术真正可以走入临床。
本发明一个方面提供了一种绿色荧光蛋白Clover4，所述绿色荧光蛋白的氨基酸序列与Clover的氨基酸序列相比，具有以下突变位点：S72A、Q80L、S86A、K101E、T153M、Q157A、R168Y、L178V、A206T、L221V、F223R。
在本发明的一些具体实施方案中，所述绿色荧光蛋白Clover4的氨基酸序列如SEQ ID No：1所示。
SEQ ID No：1
Figure PCTCN2021091875-appb-000001
本发明另一个方面提供了编码上述的绿色荧光蛋白Clover4的多核苷酸序列。
在本发明的一些具体实施方案中，所述多核苷酸序列具有如SEQ ID No.2所示序列。
SEQ ID No.2
Figure PCTCN2021091875-appb-000002
本发明再一个方面提供了一种能用于生物发光共振能量转移检测的蛋白对，其包括上述 的作为生物发光共振能量转移受体的绿色荧光蛋白Clover4，以及生物发光共振能量转移供体。
在本发明的一些具体实施方案中，生物发光共振能量转移供体选自荧光素酶。
本发明再一个方面提供了一种基于生物发光共振能量转移的探针，所述基于生物发光共振能量转移的探针包括上述用于生物发光共振能量转移检测的蛋白对。
在本发明的一些具体实施方案中，所述用于生物发光共振能量转移检测的蛋白对包括上述的生物发光共振能量转移受体以及生物发光共振能量转移供体；生物发光共振能量转移受体为本发明上述绿色荧光蛋白Clover4。
在本发明的一些具体的实施方案中，所述基于生物发光共振能量转移的探针还包括包含抗原表位的片段以及帮助结构域。
在本发明的一些具体实施方案中，所述包含抗原表位的片段中的抗原表位选自病毒蛋白中的抗原表位片段。
在本发明的一些具体实施方案中，所述的病毒蛋白为登革病毒、禽流感病毒、艾滋病毒、EB病毒、乙肝病毒或冠状病毒。
在本发明的一些具体实施方案中，登革病毒的抗原表位片段如SEQ ID NO.3所示。
在本发明的一些具体实施方案中，禽流感病毒的抗原表位片段如SEQ ID NO.4所示。
在本发明的一些具体实施方案中，艾滋病毒的抗原表位片段如SEQ ID NO.5所示。
在本发明的一些具体实施方案中，EB病毒的抗原表位片段如SEQ ID NO.6或SEQ ID NO.7所示。
在本发明的一些具体实施方案中，乙肝病毒的抗原表位片段如SEQ ID NO.8所示。
在本发明的一些具体实施方案中，新型冠状病毒的抗原表位片段如SEQ ID NO.9所示。
在本发明的一些具体实施方案中，所述的帮助结构域选自SH3/sp1结构域。
在本发明的一些具体的实施方案中，所述的探针中，其中的荧光蛋白Clover4还选自：连接原始如SEQ ID NO.1所示的荧光蛋白Clover4的N端和C端，在荧光蛋白Clover4的其他位点打开肽链所形成的荧光蛋白Clover4环化重排(cp)衍生物；并在荧光蛋白Clover4衍生物打开位点的N端和C端连接帮助结构域以及包含抗原表位的片段。打开的其他位点例如，在蛋白序列中第2-237位中的任意一个位置，例如第157位或第173位。
本发明再一个方面提供了本发明上述的绿色荧光蛋白Clover4，或荧光蛋白Clover4的环化重排衍生物作为生物发光共振能量转移检测中的生物发光共振能量转移受体的用途；
所述荧光蛋白Clover4的环化重排衍生物为：连接原始如SEQ ID NO.1所示的荧光蛋白Clover4的N端和C端，并在其他位点打开肽链所形成的环化重排衍生物。
本发明再一个方面提供了本发明上述的绿色荧光蛋白Clover4，或荧光蛋白Clover4的环化重排衍生物在制备基于生物发光共振能量转移检测的探针或试剂中的用途；
所述荧光蛋白Clover4的环化重排衍生物为：连接原始如SEQ ID NO.1所示的荧光蛋白Clover4的N端和C端，并在其他位点打开肽链所形成的环化重排衍生物。
在本发明的一些具体实施方案中，在上述用途中，所述绿色荧光蛋白Clover4与生物发光共振能量转移供体配合使用，优选地，生物发光共振能量转移供体选自荧光素酶。
本发明再一个方面提供了一种试剂盒，所述试剂盒中包含上述基于生物发光共振能量转移的探针。
在本发明的一些具体实施方案中，所述试剂盒用于血清检测用。
本发明再一个方面提供了上述基于生物发光共振能量转移的探针在制备检测血清中特定抗体的试剂中的用途。
在本发明的一些具体实施方案中，所述特定抗体为能够与探针中的抗原表位相结合的抗体。
在本发明的一些具体实施方案中，所述的检测为定性检测或定量检测。
本发明再一个方面提供了一种检测生物样品中抗体的方法，所述方法包括以上述基于生物发光共振能量转移的探针对待检测物进行检测的步骤。
在本发明的一些具体的实施方案中，所述的检测方法为对待测物中抗体进行定性检测或定量检测。
在本发明的一些具体的实施方案中，所述的待检测物为液体样品，优选为血清样本。
在本发明的一些具体的实施方案中，所述检测方法包括将待测物与所述探针进行接触，并分别观察检测体系中含有待测物或不含待测物时所述探针的生物发光共振能量转移效率。
有益效果
(1)本发明通过荧光蛋白工程，突变筛选出了一种绿色荧光蛋白Clover4，其光谱性质与其出发蛋白Clover相似，亮度比Clover高，单体性及pH稳定性比Clover好；
(2)对于七个不同的抗原表位，在没有添加抗体时，Clover4-LUMABS系列探针在血清中的BRET效率(hiBRET)均高于mNeonGreen-LUMABS探针。其中Clover4-HIV、Clover4-DEN1更是高达mNeonG-HIV、mNeonG-DEN1的4-5倍。
(3)对于四个不同的抗原表位，Clover4-LUMABS系列探针在抗体检测时的动态范围均 高于mNeonGreen-LUMABS探针。其中对新冠抗体的检测动态范围更是高达mNeonGreen探针的20倍。
(4)对于四个不同的抗原表位，Clover4-LUMABS系列探针在抗体检测时的最低检测限均低于mNeonGreen-LUMABS探针。可以在更低浓度抗体环境下检出抗体，更利于早期诊断。
附图说明
图1中(a)为荧光蛋白Clover和Clover4的序列，其中标记了Clover4突变位点；黑色方框中为发光基团；(b)为Clover晶体结构(PDB编号5WJ2)；(c)为Clover4激发和发射光谱。
图2为Clover、mClover3、Clover4以及mEGFP在100μM(a)和5μM(b)浓度下的HPLC结果图。出峰越晚(即出峰时体积越大)单体性越强。
图3为Clover4的pH稳定性结果。
图4为Clover4-LUMABS探针结构示意图。
图5为在血清中孵育30分钟后LUMABS-HIV探针检测抗体的动态范围和抗体浓度检测范围。(a)mNeonG-LUMABS-HIV(左)和Clover4-LUMABS-HIV(右)探针的发射光谱图；(b)LUMABS-HIV系列探针在无抗体时的发射光谱比较；(c)探针分别在有或无1nM anti-HIV-p17抗体时的BRET效率；(d)mNeonG-LUMABS-HIV(左)和Clover4-LUMABS-HIV(右)探针对0-100nM anti-HIV-p17抗体的滴定曲线。嵌套表:确定检测范围的线性校准曲线。误差线：mean±SD＝3。显著性差异：*，P＜0.001；**，P＜0.05。
图6为未经孵育时LUMABS探针的hiBRET。误差线：mean±SD＝3。显著性差异：*，P＜0.001；**，P＝0.358。
图7为在血清中孵育30分钟后mNeonG-LUMABS(a)和Clover4-LUMABS(b)系列探针分别在有或无1nM不同抗体时的BRET效率。误差线：mean±SD＝3。显著性差异：*，P＜0.001；***，P＜0.05；****，P＝0.823。
具体实施方式
为了使本发明的上述目的、特征和优点能够更加明显易懂，下面对本发明的具体实施方式做详细的说明，但不能理解为对本发明的可实施范围的限定。
实施例1绿色荧光蛋白Clover4的构建和光物理性质检测
(1)对绿色荧光蛋白Clover进行晶体结构分析(图1b)，并与同源序列比对，对影响荧光蛋白光谱的关键位点及与其相互作用的氨基酸进行合理设计和定点突变，然后在组成型表达载体pNCS上对突变体进行表达和筛选，所用表达菌株为Stellar。为确保文库的完整性，每个突变体设置10个克隆，最后通过肉眼分辨以及蓝色LED激发光透过橙色丙烯酸滤波器检测突变体的荧光性质，筛选出表达光谱蓝移的荧光蛋白的单克隆。随后进一步对光谱蓝移的突变体荧光蛋白发光基团周围的非保守氨基酸残基进行定点突变，稳定发光基团，筛选出高量子产率(高亮度)的单克隆，命名为Clover4，其在Clover的基础上突变了11个位点(S72A,Q80L,S86A,K101E,T153M,Q157A,R168Y,L178V,A206T,L221V,F223R)，Clover与Clover4序列对比结果见图1a。
(2)使用B-PER II(购买自皮尔斯公司)对表达Clover4的细菌进行裂解，然后采用HisPur Cobalt Resin(购买自皮尔斯公司)纯化蛋白，紧接着通过Econo-Pac 10DG重力流色谱柱(购买自美国Bio-Rad公司)去盐。完成以上蛋白纯化步骤后，使用Lambda35 UV/VIS and LS-55荧光光谱仪(购买自Perkin Elmer公司)检测Clover4的单光子激发光谱和发射光谱。如表1和图1c所示，Clover4的激发峰为506nm，发射峰为516nm，与Clover类似。其在峰值处的消光系数为115mM -1cm -1，量子产率为0.77(见表1)，分子亮度为89，比Clover高。
(3)将纯化的Clover4蛋白分别浓缩至100μM的高浓度和5μM的低浓度，并用高效液相色谱仪(岛津LC20A)进行层析分析，检测蛋白的单体性。如附图2所示，Clover4在高浓度和低浓度的情况下单体性均比Clover及其另外一个衍生物mClover3(mClover3结构见201910057635.8)更好，其状态更接近一个单体。
(4)将纯化的Clover4蛋白浓缩至5mg/mL的高浓度，并使用Lambda35 UV/VIS and LS-55荧光光谱仪(购买自Perkin Elmer公司)检测Clover4在不同pH值的缓冲液中，被同一波长的激发光激发所发出的荧光读值，并据此计算该蛋白的pKa值。如表1和图3所示，Clover4的pKa值为5.6，比Clover、mClover3和mNeonGreen都更低一点，pH稳定性比Clover好。
表1相关绿色荧光蛋白性能表征
Figure PCTCN2021091875-appb-000003
Figure PCTCN2021091875-appb-000004
表注：a亮度按峰值EC与QY的乘积计算
实施例2 Clover4在BRET及血清抗体检测Clover4-LUMABS系统中的应用(在探针中mNeonGreen简写为mNeonG)
(1)Clover4-LUMABS-HIV探针
首先分别构建了mNenG-LUMABS-HIV和Clover4-LUMABS-HIV探针，探针结构示意图如图4所示，mNenG-LUMABS-HIV为通过包含HIV抗原表位的序列连接荧光素酶(Nluc)和mNeonGreen的探针，探针中还包括SH3/sp1结构域，而Clover4-LUMABS-HIV为通过包含HIV抗原表位的序列连接荧光素酶(Nluc)和Clover4的探针，探针中还包括SH3/sp1结构域。抗原表位见表2。
通过在小牛血清(FBS)中进行测试，检测结果见表3，实验结果显示Clover4-LUMABS-HIV探针在无抗体条件下的BRET效率(hiBRET)为mNenG-LUMABS-HIV的4.6倍(其中Clover4-LUMABS-HIV为14.00，mNenG-LUMABS-HIV为3.07)。在anti-HIV-p17抗体的检测中，将anti-HIV-p17抗体与上述探针分别进行孵育，孵育时间为30分钟，然后进行检测用于评估探针的动态范围，Clover4探针显示出了极大的动态范围(dynamic range)：807.7±9.9％，是mNeonGreen的四倍多。动态范围计算公式(hiBRET b-loBRET)/loBRET，其中loBRET为抗体存在时的BRET效率。hiBRET b为无抗体时孵育30分钟后的BRET效率，loBRET为加入抗体后孵育30分钟后的BRET效率。
同时构建了与Clover4同一蛋白系列的Clover-LUMABS-HIV和mClover3-LUMABS-HIV探针，其hiBRET和动态范围均比mNeonGreen探针小，Clover4探针的动态范围是它们的9倍多。说明本发明的探针具有更高的灵敏度。
此外，通过对不同浓度(0-100nM)anti-HIV-p17抗体的滴定，确定了上述四个探针对的检测范围(detection range)和最低检测限(limit of detection，LOD)。Clover4探针的检测范围为3-120pM，LOD根据3σ标准计算为2.5pM。mNeonGreen、Clover和mClover3探针的检测范围分别为20-500pM、25-500pM和30-500pM，LOD分别为14pM、24pM和25pM。证明Clover4-LUMABS-HIV与其它探针相比可以检测到浓度更低的抗体，例如是 mClover3-LUMABS-HIV探针最低检测限的十分之一。
为分析Clover4探针高BRET效率的机理，本发明人构建了两个基于环化重排荧光蛋白(cpFP)的Clover4衍生物探针cp157Clover4-LUMABS-HIV和cp173Clover4-LUMABS-HIV，它们的hiBRET分别为Clover4探针的50％和70％。所述cp157Clover4-LUMABS-HIV和cp173Clover4-LUMABS-HIV分别为在Clover4荧光蛋白第157位和第173位打开肽链，并在打开位置连接帮助结构域和包含抗原表位的片段。从实验结果来看，Clover4衍生物探针cp157Clover4-LUMABS-HIV和cp173Clover4-LUMABS-HIV虽然比Clover4探针Clover4-LUMABS-HIV的hiBRET低，但是其hiBRET要高于mNeonG-LUMABS-HIV、Clover-LUMABS-HIV、mClover3-LUMABS-HIV。换言之，本发明突变获得的Clover4荧光蛋白在生物发光共振能量转移技术探针中极大的提高了探针灵敏度。在探针中，Clover4荧光蛋白不同位点打开肽链获得的Clover4荧光蛋白衍生物依然为生物发光共振能量转移技术探针带来了超过其他荧光蛋白的、更高的灵敏度和hiBRET，以及更大的动态范围。
此外，还构建了去除掉SH3/sp1结构域的探针Clover4-LUMABS-HIV w/o SH3/sp1，其检测功能基本丧失。
上述LUMABS-HIV探针检测数据均见表3或图5。
(2)不同抗原表位的Clover4-LUMABS系列探针
为了验证Clover4-LUMABS的适应性，针对不同病毒的不同抗原表位，构建了不同长度和结构的Clover4-LUMABS系列探针，包括针对禽流感病毒的Clover4-LUMABS-HA和登革病毒的Clover4-LUMABS-DEN1，针对鼻咽癌EB病毒的Clover4-LUMABS-EBNA1和Clover4-LUMABS-VCA，针对乙肝病毒的Clover4-LUMABS-HBV以及针对新冠病毒的Clover4-LUMABS-S1RBD探针。本部分所有的病毒及抗原表位(epitope)见表2。
表2相关抗原表位信息
Figure PCTCN2021091875-appb-000005
Figure PCTCN2021091875-appb-000006
同时，还一一对应地构建了相应的mNeonGreen探针，即以mNeonGreen替换Clover4构建的探针。结果见附图6，所有的Clover4探针的hiBRET都高于相同抗原表位的mNeonGreen探针：1)DEN1，Clover4探针是mNeonGreen探针的5.5倍(Clover4探针为16.63，mNeonGreen探针为3.01)；2)HA，Clover4探针是mNeonGreen探针的2.2倍(Clover4探针为6.09，mNeonGreen探针为2.80)；3)VCA，Clover4探针是mNeonGreen探针的3.3倍(Clover4探针为4.66，mNeonGreen探针为1.40)；4)EBNA1，Clover4探针是mNeonGreen探针的4.2倍(Clover4探针为9.30，mNeonGreen探针为2.20)；5)HBV，Clover4探针是mNeonGreen探针的1.3倍(Clover4探针为3.95，mNeonGreen探针为3.01)；6)S1-RBD,Clover4探针是mNeonGreen探针的1.9倍(Clover4探针为5.60，mNeonGreen探针为2.88)。
检测Clover4-LUMABS系列探针对禽流感、登革热和新冠等病毒的抗体的检测效果。结果见图7，所有的Clover4探针的动态范围都高于相同抗原表位的mNeonGreen探针：1)DEN1，Clover4探针是mNeonGreen探针的5.7倍(Clover4探针为498.7±24.4％，mNeonGreen探针为87.9±3.5％)；2)HA，Clover4探针是mNeonGreen探针的2.5倍(Clover4探针为247.8±1.7％，mNeonGreen探针为99.7±1.6％)；3)S1-RBD,Clover4探针是mNeonGreen探针的20倍(Clover4探针为105.9±8.4％，mNeonGreen探针为5.3±0.8％)。
对这三种病毒的抗体进行了不同浓度(0-100nM)的滴定，以确定检测范围和最低检测限。1)DEN1，Clover4探针的检测范围为8-200pM，LOD为7.7pM。mNeonGreen探针的检 测范围分别为25-1000pM，LOD为22.7pM。；2)HA，Clover4探针的检测范围为8-200pM，LOD为7.5pM。mNeonGreen探针的检测范围分别为300-500pM，LOD为25pM。3)S1-RBD,Clover4探针的检测范围为20-200pM，LOD为15.5pM。mNeonGreen探针因为结合浓度太高直接无法测算。证明Clover4系列探针均比mNeonGreen系列探针可以检测到浓度更低的抗体。
所有探针检测数据均在表3中列出。
表3 LUMABS探针检测数据
Figure PCTCN2021091875-appb-000007
表注：hiBRET a:探针未经孵育；hiBRET b:探针孵育30分钟；LOD：最低检测限。

Claims (15)

  1. 一种绿色荧光蛋白Clover4，其特征在于，所述绿色荧光蛋白的氨基酸序列与Clover的氨基酸序列相比，具有以下突变位点：S72A、Q80L、S86A、K101E、T153M、Q157A、R168Y、L178V、A206T、L221V、F223R；
    优选地，所述绿色荧光蛋白Clover4的氨基酸序列如SEQ ID No：1所示。
  2. 一种多核苷酸序列，其特征在于，所述的多核苷酸序列编码权利要求1所述的绿色荧光蛋白Clover4。
  3. 一种用于生物发光共振能量转移检测的蛋白对，其特征在于，其包括权利要求1或2所述的作为生物发光共振能量转移受体绿色荧光蛋白Clover4；蛋白对还包括生物发光共振能量转移供体；
    优选地，所述生物发光共振能量转移供体选自荧光素酶。
  4. 一种基于生物发光共振能量转移的探针，其特征在于，所述基于生物发光共振能量转移的探针包括权利要求3所述的用于共振能量转移检测的蛋白对。
  5. 根据权利要求4所述的探针，其特征在于，其中的荧光蛋白Clover4还选自：连接原始如SEQ ID NO.1所示的荧光蛋白Clover4的N端和C端，在荧光蛋白Clover4的其他位点打开肽链所形成的荧光蛋白Clover4环化重排衍生物；并在荧光蛋白Clover4衍生物打开位点的N端和C端连接帮助结构域以及包含抗原表位的片段。
  6. 根据权利要求4所述的探针，其特征在于，所述基于生物发光共振能量转移的探针还包括包含抗原表位的片段以及帮助结构域；
    优选地，所述的帮助结构域选自SH3/sp1结构域。
  7. 根据权利要求4所述的探针，其特征在于，所述包含抗原表位的片段中的抗原表位选自病毒蛋白中的抗原表位片段。
  8. 权利要求1所述绿色荧光蛋白Clover4，或荧光蛋白Clover4的环化重排衍生物作为共振能量转移检测中共振能量转移供体或受体中的用途；
    所述荧光蛋白Clover4环化重排衍生物为：连接原始如SEQ ID NO.1所示的荧光蛋白 Clover4的N端和C端，并在其他位点打开肽链所形成的环化重排衍生物。
  9. 权利要求1所述的绿色荧光蛋白Clover4，或荧光蛋白Clover4的环化重排衍生物在制备基于共振能量转移检测的探针或试剂中的用途；
    所述荧光蛋白Clover4环化重排衍生物为：连接原始如SEQ ID NO.1所示的荧光蛋白Clover4的N端和C端，并在其他位点打开肽链所形成的环化重排衍生物。
  10. 根据权利要求6或7所述的用途，其特征在于，所述绿色荧光蛋白Clover4与生物发光共振能量转移供体配合使用，优选地，生物发光共振能量转移供体选自荧光素酶。
  11. 一种试剂盒，其特征在于，所述试剂盒中包含上述基于生物发光共振能量转移的探针；
    优选地，所述试剂盒用于血清检测用。
  12. 权利要求4或5所述的基于生物发光共振能量转移的探针或权利要求3所述的蛋白对在制备检测血清中特定抗体的试剂中的用途；
    优选地，所述的检测为定性检测或定量检测。
  13. 一种检测生物样品中抗体的方法，其特征在于，所述方法包括以权利要求4-7任一项所述的基于生物发光共振能量转移的探针对待检测物进行检测的步骤；
    优选地，所述的方法为对待测物中抗体进行定性检测或定量检测。
  14. 根据权利要求3所述的方法，其特征在于，所述的待检测物为液体样品，优选为血清样本。
  15. 根据权利要求3所述的方法，其特征在于，所述检测方法包括将待测物与所述探针接触，并分别观察检测体系中含有待测物或不含待测物时所述探针的生物发光共振能量转移效率。
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