WO2016127302A1 - 检测细胞外基质mmp13的bret探针、基因、表达载体和构建方法 - Google Patents

检测细胞外基质mmp13的bret探针、基因、表达载体和构建方法 Download PDF

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WO2016127302A1
WO2016127302A1 PCT/CN2015/072618 CN2015072618W WO2016127302A1 WO 2016127302 A1 WO2016127302 A1 WO 2016127302A1 CN 2015072618 W CN2015072618 W CN 2015072618W WO 2016127302 A1 WO2016127302 A1 WO 2016127302A1
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gene
bret
seq
rluc8
encoding
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PCT/CN2015/072618
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French (fr)
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王大平
梁宇杰
段莉
李子刚
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深圳市第二人民医院
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    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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  • the invention belongs to the technical field of bioengineering, and particularly relates to a BRET probe, a gene, an expression vector and a construction method for detecting an extracellular matrix MMP13.
  • Matrix metalloproteinases are a group of Zn2+-dependent extracellular enzymes that are widely present in connective tissues and play an important role in the degradation of extracellular matrix.
  • MMP13 also known as collagenase-3
  • MMPs matrix metalloproteinase family
  • MMP13 is mainly produced by synovial cells, chondrocytes, neutrophils, etc., mainly by destroying the peptide chain between type H collagen to achieve the purpose of lysing cartilage. Therefore, an increase in the expression level of MMP13 in cartilage leads to osteoarthritis ( OA) One of the main factors of cartilage degeneration. Furthermore, after repeated verification, the MMP13 protein in osteoarthritis patients is ten times higher than that of normal people, and the expression level gradually increases with the progress of osteoarthritis. MMP13 gradually becomes a physiological indicator for the diagnosis and treatment of OA. And may provide a new target for the treatment of OA.
  • the detection method of the existing MMP13 the most commonly used is the double antibody sandwich assay, which is limited by the specific binding of the antibody and the limitation of the detection sensitivity, and the operation steps are cumbersome, and the content of the active MMP13 cannot be detected; In the process of the process, the cells need to be damaged, and it is not suitable for real-time dynamic monitoring of the protease in the living body.
  • the purpose of the embodiments of the present invention is to overcome the above-mentioned deficiencies of the prior art, and to provide a BRET probe, a gene, and an expression carrier for detecting the extracellular matrix MMP13 which can be expressed on the surface of a eukaryotic cell membrane. Body and construction method.
  • a BRET probe for detecting extracellular matrix MMP13 comprising a BRET bioluminescent donor and a BRET acceptor fluorescent protein, a polypeptide substrate specifically degraded by MMP13 between the BRET bioluminescent donor and the BRET acceptor fluorescent protein connection;
  • the BRET bioluminescent donor is a Rluc8 protein having the amino acid sequence of SEQ. ID. No. 1 of the Sequence Listing
  • the BRET acceptor fluorescent protein is a yellow fluorescent protein.
  • the above probe constructed by the present invention, the probe itself and the acceptor fluorescent protein are linked to each other by a MMP13-specific recognition degradation polypeptide substrate to generate BRET, and when the polypeptide substrate is specifically recognized by MMP13, BRET When the phenomenon disappears, only the emission signal emitted by the donor protein substrate can be detected to produce a fluorescent signal change, thereby achieving the detection purpose; and the C-terminus of the Rluc8 protein is fused to the transmembrane region containing the platelet-derived growth factor receptor (PDGFR), It can be anchored to the surface of the cell membrane, allowing for in vivo detection without damaging the cells, while also providing greater sensitivity.
  • PDGFR platelet-derived growth factor receptor
  • the present invention further provides a gene encoding the BRET probe for detecting the extracellular matrix MMP13, and an expression vector for expressing the gene in eukaryotic cells;
  • the coding gene comprises a BRET bioluminescence donor-encoding gene, a MMP13-specific recognition degradation polypeptide-encoding gene and a BRET-receptor fluorescent protein-encoding gene; the BRET bioluminescence donor-encoding gene has a sequence listing SEQ.
  • the present invention further provides a method for constructing the above expression vector, comprising the following steps:
  • a polypeptide substrate-encoding gene having the nucleotide sequence of SEQ. ID. No. 4 of the Sequence Listing is inserted between the Rluc8 protein-encoding gene and the coding gene of the yellow fluorescent protein in the fusion expression plasmid.
  • FIG. 1 is a schematic diagram of a BRET probe for detecting MMP13 of an extracellular matrix according to an embodiment of the present invention
  • FIG. 2 is a schematic diagram showing the degradation of BRET of a polypeptide substrate in the BRET probe of FIG. 1 by MMP13 specific recognition;
  • Figure 3 is a plasmid map of the plasmid pDisplay clone Rluc8 protein encoding gene according to an embodiment of the present invention
  • Figure 4 is a plasmid map of the plasmid of Figure 3 after further cloning the yellow fluorescent protein encoding gene;
  • Figure 5 is a plasmid map of the plasmid of Figure 4 after further insertion of the polypeptide-encoding gene;
  • Figure 6 is a fluorescence contrast diagram of the cell membrane surface under the confocal microscope, the BRET probe alone and the BRET probe binding expression on the cell membrane;
  • Figure 7 is a comparison of fluorescence results of chondrocytes after treatment with different MMP13 concentrations after transfection of BRET probes
  • Figure 8 is a graph showing changes in the shift in fluorescence intensity produced by changes in fluorescence shift in MMP13 activity using a BACS probe using a FACS flow cytometer.
  • the present invention provides a BRET probe for detecting MMP13 of extracellular matrix, the schematic diagram of which can be seen in FIG. 1 ; including BRET bioluminescent donor 1 and BRET receptor fluorescent protein 2, and BRET bioluminescent donor 1 and BRET receptor fluorescent protein 2 through MMP13 specificity Identifying the degraded polypeptide substrate 3 linkage;
  • the BRET bioluminescent donor is a Rluc8 protein having the amino acid sequence of SEQ. ID. No. 1 of the Sequence Listing;
  • the BRET acceptor fluorescent protein is a yellow fluorescent protein.
  • the above probe constructed by the present invention is based on a bioluminescence resonance energy transfer (BRET) mechanism, and BRET itself is a transfer process of non-radiative energy from a donor to a receptor; BRET needs to consist of two fusion proteins, one Fusion is the energy-providing luciferase, another fusion energy-receiving receptor fluorescent protein, luciferase can be activated by coelenterazine with hydrophobic and membrane permeability; when two fusion proteins interact, and When the distance is less than 10 nm, the donor will transfer its own energy to the receptor; if the two integrins are separated by shear to a distance exceeding 10 nm, as shown in Figure 2, the BRET phenomenon disappears from each other. When it happens, only the emitted light from the donor protein substrate can be detected.
  • BRET bioluminescence resonance energy transfer
  • the modified Rluc8 protein (renilla luciferase mutant) is used as a donor, and the yellow fluorescent protein is used as a receptor to make the emission spectrum of the donor luciferase and The overlap between the excitation spectra of the receptor protein enhances the BRET signal intensity; and the MMP13 specifically recognizes the degradation polypeptide substrate to link the donor and the receptor to produce BRET; and when the polypeptide substrate is specifically recognized by MMP13, The disappearance of BRET produces a change in fluorescence, and the expression level of MMP13 can be calculated based on the change in fluorescence.
  • the modified Rluc8 protein renilla luciferase mutant
  • the C-terminus of the above Rluc8 protein used in the present invention is fused with a platelet-derived growth factor receptor (PDGFR).
  • PDGFR platelet-derived growth factor receptor
  • the above BRET probe constructed in the present invention is different from FRET, and the donor does not require activation of exogenous light, and can be activated by directly adding a substrate, thereby avoiding problems of photobleaching and autofluorescence of the donor. Background subtraction of BRET is not required in the assay; it has higher sensitivity and a very low endogenous background.
  • the MMP13 in the present application specifically recognizes a degradation polypeptide substrate, and based on the requirement that the distance between the donor and the receptor is less than 10 nm, the oligonucleotide having a small length is usually used.
  • polypeptide substrate The amino acid sequence is GPLGMRGL, which has a residue length of 8 amino acids; in use, it has higher protection than the other types of degradable oligopeptides in the sensitivity of specific recognition and the degradability of degradation by MMP13. .
  • the yellow fluorescent protein of the acceptor selects one of the three fluorescent proteins YFP, cpYFP, and cpVenus. Since the linkage between the donor, the receptor and the linked substrate polypeptide in the probe can be carried out in an N-terminal, C-terminal orientation, wherein the C-terminus of the Rluc8 protein is fused to contain a platelet-derived growth factor receptor ( The transmembrane region of PDGFR) is used to anchor the cell membrane and thus can proceed from the N-terminus of the Rluc8 protein in the sequence of engagement with the C-terminus of the amino acid sequence of the polypeptide substrate.
  • PDGFR platelet-derived growth factor receptor
  • the above BRET probe of the present invention constructs a BRET probe by displaying luciferase BRET on the surface of a eukaryotic cell as a BRET signal for detecting a protease, and the probe can detect the activity of matrix metalloproteinase-13 at a cellular level, thereby improving detection.
  • the accuracy of proteinase in extracellular matrix as an ideal system for detecting matrix metalloproteinase-13 in eukaryotic extracellular matrix, can lay the foundation for the biological function of MMP13.
  • the BRET probe based on the above configuration has its own composition as a protein or a peptide, thereby realizing the expression and preparation of the protein. Therefore, the present invention also provides the coding gene of the above BRET probe as a whole, and the coding gene is based on the peptide.
  • the composition may comprise three parts and is spliced according to the order of composition: the BRET bioluminescence donor-encoding gene, the MMP13-specific recognition-degraded polypeptide substrate-encoding gene and the BRET-receptor fluorescent protein-encoding gene.
  • the coding gene thereof is a Rluc8 protein-encoding gene having the nucleotide sequence of SEQ. ID. No. 3 of the Sequence Listing.
  • the coding gene thereof has the base sequence of SEQ. ID. No. 4 of the Sequence Listing.
  • its receptor protein yellow fluorescent protein can There are three kinds, respectively corresponding to the YFP gene having the base sequence of SEQ.ID.No.5 of the sequence listing; or the cpYFP gene having the base sequence of SEQ.ID.No.6 of the sequence listing; or having the sequence listing SEQ.ID.No .7 base sequence of cpVensu gene.
  • the present invention further provides a fusion expression plasmid plasmid or expression vector based on the above protein BRET probe, and the coding gene of the probe is used for a plasmid or the like.
  • the vector is recombined to make it a recombinant expression vector.
  • the vector of the present invention is carried out using the pDisplay plasmid; the method for constructing the expression vector can be as follows, including the following steps:
  • the gene encoding the yellow fluorescent protein of the receptor protein is cloned into the multiple cloning site (MSC) of the expression plasmid pDisplay-Rluc8-MCS obtained in step S10, and ligated to the end of the Rluc8 protein-encoding gene to form luciferase and fluorescence.
  • MSC multiple cloning site
  • the fusion expression plasmid obtained by the invention can express the fluorescent protein by itself in eukaryotic cells, and the product after expression is a BRET probe, which can be directly detected; for example, in vitro detection of chondrocyte SW1353, the above can be The constructed fusion expression plasmid was transfected into chondrocyte SW1353; then cultured, and the fluorescent protein was expressed in the chondrocyte SW1353, and the relative fluorescence intensity of the three BRET fusions was detected by flow cytometry or fluorescence microplate reader.
  • the eukaryotic plasmid pDisplay is used as an expression vector, and other expression vector plasmids capable of good transfection and expression in bone cells can be used in the practice.
  • the above plasmid pDisplay is used in the present invention, and the multiple sites of pDisplay have BglII, PstI, HindIII, SalI multiple cleavage sites; in the above donor protein gene, receptor protein gene and polypeptide bottom
  • the expression sequence of the three-part insertion can be carried out by using these four cleavage sites of BglII, PstI, HindIII and SalI, respectively, to achieve accurate localization and convergence.
  • the pDisplay vector is double-digested (BgIII+PstI), and the linearized carrier is recovered after agarose gel electrophoresis;
  • the gene sequence of Rluc8 was amplified by PCR using the Rluc8 protein encoding gene having the base sequence of SEQ. ID. No. 3 as a template, and the gene amplification primer of Rluc8 was as follows:
  • TCCCGCGGCCCAAGCTTAAAACTGCAGT in the above reverse primer represents MCS (multiple cloning site) and includes three restriction sites (PstI + HindIII + SalI).
  • the corresponding enzyme cleavage sites can be introduced into the PCR, and then the PCR product is double-digested (BgIII+PstI), and finally ligated with the cleaved linearized vector, and the correctness of the sequence is confirmed by sequencing, and finally the sequencing is correct.
  • the Rluc8 expression plasmid pDisplay-Rluc8-MCS having the base sequence of SEQ. ID. No. 17 of the Sequence Listing was subjected to the next step.
  • step S20 after step S10, screening the recombinant pDisplay-Rluc8-MCS plasmid, performing amplification, large-scale plasmid extraction, double digestion (HindIII and SalI), and linearizing the vector after double-cutting by gel recovery;
  • the yellow fluorescent protein-encoding gene is amplified, and it is necessary to specifically design primers in the amplification to introduce a specific restriction site; therefore, primers are designed according to the type of yellow fluorescent protein as follows:
  • cpYFP is used as a yellow fluorescent protein
  • the base sequence of the plasmid constructed in this step can be verified by sequencing, and the Rluc8-cpYFP fragment in the plasmid is positive after recombination.
  • the sequence results can be found in the Sequence Listing SEQ. ID. No. 18.
  • step S30 after step S20, screening for a successful fusion expression plasmid, and then further recombining the coding gene of the artificially synthesized polypeptide substrate with the fusion expression plasmid;
  • sequence information of the expressed sequence gene in which the polypeptide substrate is used is as follows:
  • the above primers encoding the substrate polypeptide "GPLGMRGL" sequence of MMP13 were digested with PstI and HindIII after primer annealing pairing, and directly recovered by agarose gel electrophoresis with PstI and HindIII double digestion.
  • pDisplay-Rluc8-YFP, pDisplay-Rluc8-cpYFP or pDisplay-Rluc8-cpVenus for connection.
  • the above recombinant can finally obtain three recombinant vectors, that is, a fusion expression plasmid for obtaining a BRET probe, which can be expressed as: pDisplay-Rluc8-GPLGMRGL-YFP, pDisplay-Rluc8, respectively.
  • a fusion expression plasmid for obtaining a BRET probe which can be expressed as: pDisplay-Rluc8-GPLGMRGL-YFP, pDisplay-Rluc8, respectively.
  • -GPLGMRGL-cpYFP pDisplay-Rluc8-GPLGMRGL-cpVenus
  • the results of the recombinant plasmid are accurate and can be verified by sequencing.
  • pDisplay-Rluc8-GPLGMRGL-cpYFP was used.
  • the fusion expression plasmid of the BRET probe obtained in step S30 is transfected into the cell line of chondrocyte SW1353 by lipofection; the transfection step can be carried out according to the instructions of the lipofection reagent kit.
  • the staining reagent was used to transfect mammalian SW1353 chondrocytes, and one day before transfection, the density of each well of the 6-well plate reached 2*10 5 cells. After transfection, the above constructed expression plasmid itself has the ability to express in eukaryotic cells to generate BRET probes by itself.
  • the transfected cells were seeded in a 96-well plate, and a final concentration of 5 ⁇ M coelenterazine was added to each well. Approximately 100,000 cells per transfection were analyzed using a multi-function microplate reader fluorescence module, and a comprehensive reading of 10 min was used. Collect light through filters of two different wavelengths (475 nm, 525 nm, respectively).
  • the transfected cells were inoculated into the chamber slides, and a final concentration of 5 ⁇ M coelenterazine was added to each chamber.
  • the bioluminescent donor sea luciferase expressed by the probe utilized O2 and coelenterium. Fluorescein catalyzes the luminescence reaction, produces emitted light, transmits it to the yellow fluorescent protein to produce emitted light, and then uses a laser co-aggregation microscope to detect the relative fluorescence intensity of the cells.
  • the above probes can also be used to transfect the chondrocytes treated with different MMP13 concentrations, and then the relative intensity of BRET fluorescence is observed.
  • the results are shown in Fig. 7. From Fig. 7, it can be seen that as the concentration of MMP13 increases, the relative intensity of BRET fluorescence is attenuated.
  • the FACS flow cytometry test can also detect the activity of MMP13, the number of cells tested by cytometry and the amount of MMP13 added, and the change of fluorescence intensity caused by the change of BRET fluorescence shift.
  • the curve drawn is shown in Fig. 8. Show. It can also be seen from the curve that as MMP13 is added, the BRET fluorescence shift changes and the fluorescence intensity decreases.

Abstract

本发明提供了一种检测细胞外基质MMP13的BRET探针，包括BRET生物发光供体和BRET受体荧光蛋白，以及连接BRET生物发光供体和BRET受体荧光蛋白的MMP13特异性识别降解的多肽底物；BRET生物发光供体为具有序列表SEQ.ID.No.1的氨基酸序列的海肾的荧光素酶（Rluc8），BRET受体荧光蛋白为黄色荧光蛋白。

Description

检测细胞外基质MMP13的BRET探针、基因、表达载体和构建方法 技术领域
本发明属于生物工程技术领域，具体涉及一种检测细胞外基质MMP13的BRET探针、基因、表达载体和构建方法。
背景技术
基质金属蛋白酶(Matrix metallo proteinases，MMPs)是一组依赖Zn2+依赖的细胞外酶，广泛存在于各结缔组织中，在细胞外基质的降解中起重要作用。其中，MMP13又称为胶原酶-3，它是基质金属蛋白酶(MMPs)家族中重要的降解酶，能够特异性地降解胶原分子中三维螺旋结构的酶，其对于H型胶原的降解效率是所有基质蛋白酶中最高的。
MMP13主要由滑膜细胞、软骨细胞、中性粒细胞等产生，主要通过破坏H型胶原之间的肽链而达到裂解软骨的目的，因此在软骨中MMP13表达水平升高是导致骨关节炎(OA)节软骨退变的主要因素之一。并且进一步在反复的验证之后，骨关节炎患者中MMP13蛋白比正常人高十倍，且表达量随着骨性关节炎的进展而逐渐增加，MMP13逐渐成为OA诊断和治疗的一项生理性指标，并且可能为OA的治疗提供一个新的靶点。
而现有的MMP13的检测方法，最常用的是双抗体夹心法测定，受限于抗体的特异结合性以及检测灵敏度的限制，且操作步骤过程繁琐，并且无法检测到活性MMP13的含量；并且检测的过程中都需要损伤细胞，不适用于活体的蛋白酶的实时动态监测。
技术问题
本发明实施例的目的在于克服现有技术的上述不足，提供一种能够在真核细胞膜表面表达的用于检测细胞外基质MMP13的BRET探针、基因、表达载 体和构建方法。
发明内容
为了实现上述发明目的，本发明实施例的技术方案如下：
一种检测细胞外基质MMP13的BRET探针，包括BRET生物发光供体和BRET受体荧光蛋白，所述BRET生物发光供体和BRET受体荧光蛋白之间通过MMP13特异性识别降解的多肽底物连接；
其中，所述BRET生物发光供体为具有序列表SEQ.ID.No.1的氨基酸序列的Rluc8蛋白，所述BRET受体荧光蛋白为黄色荧光蛋白。
本发明构建的上述探针，探针自身的供体和受体荧光蛋白之间通过一可被MMP13特异性识别降解多肽底物连接产生BRET，当多肽底物被MMP13特异性识别降解之后，BRET现象消失，只能检测到供体蛋白底物发出的发射光产生荧光信号变化，从而实现检测目的；并且Rluc8蛋白的C端融合了含有血小板源生长因子受体(PDGFR)的跨膜区域，因此能锚定在细胞膜的表面，从而可以在不损伤细胞的情况下进行活体检测，同时还具有更高的灵敏度。
本发明进一步还提出编码上述检测细胞外基质MMP13的BRET探针的基因，以及该编码基因在真核细胞内进行表达的表达载体；其中，
编码基因包括顺次连接的BRET生物发光供体编码基因、MMP13特异性识别降解的多肽底物编码基因和BRET受体荧光蛋白编码基因；所述BRET生物发光供体编码基因为具有序列表SEQ.ID.No.3碱基序列的Rluc8蛋白编码基因。
针对本发明中真核表达载体的实施，本发明进一步还提出上述表达载体的构建方法，包括如下步骤：
将具有序列表SEQ.ID.No.3的碱基序列的Rluc8蛋白编码基因克隆至质粒pDisplay的多克隆位点，得到Rluc8表达质粒；
将黄色荧光蛋白的编码基因克隆至所述Rluc8表达质粒中与Rluc8蛋白编码基因末端衔接，形成荧光融合体表达质粒；
将具有序列表SEQ.ID.No.4碱基序列的多肽底物编码基因插入至所述融合体表达质粒中Rluc8蛋白编码基因和黄色荧光蛋白的编码基因之间。
附图说明
下面将结合附图及实施例对本发明作进一步说明，附图中：
图1为本发明实施例用于检测细胞外基质的MMP13的BRET探针示意图；
图2为图1的BRET探针中多肽底物被MMP13特异性识别降解BRET小时示意图；
图3为本发明实施例质粒pDisplay克隆Rluc8蛋白编码基因后的质粒图谱；
图4为图3中质粒进一步克隆黄色荧光蛋白编码基因后的质粒图谱；
图5为图4中质粒进一步插入多肽底物编码基因后的质粒图谱；
图6为激光共聚焦显微镜下的细胞膜表面、单独的BRET探针和BRET探针结合表达定位于细胞膜上的荧光对照图；
图7为BRET探针转染后，加入不同MMP13浓度处理后软骨细胞后荧光结果对照图；
图8为采用FACS流式细胞仪用BRET探针检测MMP13活性中荧光位移发生改变产生的荧光强度发生的位移改变。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白，以下结合附图及实施例，对本发明进行进一步详细说明。应当理解，此处所描述的具体实施例仅用以解释本发明，并不用于限定本发明。
本发明实施例提供一种用于检测细胞外基质的MMP13的BRET探针，其示意图可以参见图1所示；包括BRET生物发光供体1和BRET受体荧光蛋白2，且BRET生物发光供体1和BRET受体荧光蛋白2之间通过MMP13特异性 识别降解多肽底物3连接；
其中BRET生物发光供体为具有序列表SEQ.ID.No.1的氨基酸序列的Rluc8蛋白；BRET受体荧光蛋白为黄色荧光蛋白。
本发明构建的上述探针，基于生物发光共振能量转移(BRET)机理进行，BRET本身是一种非辐射的能量从供体传递到受体的转移过程；BRET需要由两个融合蛋白组成，一个融合是提供能量的荧光素酶，另外一个融合能量接受能量的受体荧光蛋白，荧光素酶可以由具有疏水性和膜渗透性的腔肠素来激活产生；当两个融合蛋白发生相互作用，并且距离小于10nm，供体便会将自身发出能量转移到受体；如果这两种整合蛋白被剪切分离至距离超过上述10nm，如图2所示，则相互之间的BRET现象消失，不会发生作用，就仅仅只能检测到供体蛋白底物发出的发射光。
在本发明中，利用BRET的构建探针，将改造后的Rluc8蛋白(海肾荧光素酶突变体)作为供体，同时以黄色荧光蛋白作为受体，使供体荧光素酶的发射光谱和受体蛋白激发光谱间的重叠，增强BRET信号强度；同时采用MMP13特异性识别降解多肽底物将供体和受体连接，使其产生BRET；并且当多肽底物被MMP13特异性识别降解后，BRET消失产生荧光变化，根据荧光的变化情况即可计算得到MMP13的表达水平。
同时在上述采用真核细胞表面展示荧光素酶BRET作为检测蛋白酶所引起BRET信号构建的BRET探针，本发明中采用的上述Rluc8蛋白的C端融合了含有血小板源生长因子受体(PDGFR)的跨膜区域，因此它真核表达的蛋白能锚定在细胞膜的表面，从而可以在不损伤细胞的情况下进行活体的检测。
并且本发明中构建的上述BRET探针和FRET不同，其供体不需要外源光的活化，直接加入底物便可以激活，这样可以避免了供体的光漂白和细胞自发荧光的问题，因此在测定中不需要进行BRET的背景扣除；相比具有更高的灵敏度以及极低的内源背景。
进一步在上述实施方式中，本申请中的MMP13特异性识别降解多肽底物，基于上述供体和受体距离小于10nm的要求，因此通常采用长度较小的寡 肽链，并且基于特异针对MMP13的作用的识别降解序列位点进行设计，多肽底物采用具有序列表SEQ.ID.No.2氨基酸序列的多肽序列，从序列表中可以看出，多肽底物的氨基酸序列为GPLGMRGL，具有8个氨基酸的残基长度；使用中，相比其他的类型可降解寡肽在特异性的识别的灵敏度和被MMP13降解的可降解性上，能有较高的保障。
同时进一步，基于供体和受体发出的荧光波长的重叠范围，受体的黄色荧光蛋白选择选择黄色荧光蛋白YFP、cpYFP、cpVenus这三种荧光蛋白中的一种。由于探针中的供体、受体和连接的底物多肽之间的连接，可以按照N端、C端衔接的方式进行，其中由于Rluc8蛋白的C端融合了含有血小板源生长因子受体(PDGFR)的跨膜区域用于锚定细胞膜，因此可以从Rluc8蛋白的N端开始与多肽底物氨基酸序列的C端衔接的顺序进行。
采用本发明的上述BRET探针通过真核细胞表面展示荧光素酶BRET作为检测蛋白酶所引起BRET信号构建BRET探针，该探针可以在细胞水平上检测基质金属蛋白酶-13的活性，提高了检测细胞外基质中蛋白质酶的准确性，作为检测真核细胞外基质中基质金属蛋白酶-13的较为理想系统，可以为MMP13的生物学功能研究奠定基础。
基于上述构造的BRET探针，其本身的各个组成均为蛋白质或者肽段，因此实现其蛋白的表达和制备，因此本发明还给出上述BRET探针整体的编码基因，编码基因根据其肽段的组成，可以包括三部分，并且按照组成的顺序进行拼接，三部分对应分别为：BRET生物发光供体编码基因、MMP13特异性识别降解的多肽底物编码基因和BRET受体荧光蛋白编码基因。
其中根据本发明中采用的上述供体Rluc8蛋白，其编码基因为具有序列表SEQ.ID.No.3碱基序列的Rluc8蛋白编码基因。
进一步对应可被MMP13特异性识别降解的多肽底物，其编码基因为具有序列表SEQ.ID.No.4碱基序列。
而其中，由于本身对应供体Rluc8蛋白，其受体蛋白的黄色荧光蛋白可以 有三种，分别对应为具有序列表SEQ.ID.No.5碱基序列的YFP基因；或具有序列表SEQ.ID.No.6碱基序列的cpYFP基因；或具有序列表SEQ.ID.No.7碱基序列的cpVensu基因。
为了使上述基因能够便于在真核细胞体内进行表达或者制备和研究，本发明进一步还提出一种基于上述蛋白BRET探针的融合表达质粒质粒或者表达载体，将上述探针的编码基因用质粒等载体进行重组，使其成为重组的表达载体。具体结合本发明中的上述序列，以及在真核细胞中能够进行良好的表达的情况，本发明中的载体采用pDisplay质粒进行；其表达载体的构建的方法可以参见如下，包括如下步骤：
S10，以真核表达质粒pDisplay为载体，将具有序列表SEQ.ID.No.3的碱基序列的Rluc8蛋白编码基因克隆至质粒pDisplay的多克隆位点(MSC)，形成Rluc8表达质粒pDisplay-Rluc8-MCS，其质粒的图谱可以参见图3所示；
S20，将受体蛋白的黄色荧光蛋白的编码基因克隆到步骤S10所获得的表达质粒pDisplay-Rluc8-MCS的多克隆位点(MSC)中与Rluc8蛋白编码基因末端衔接，形成荧光素酶和荧光蛋白的融合体表达质粒，其质粒的图谱可以参见图4所示；
S30，在融合体表达质粒中Rluc8蛋白编码基因和黄色荧光蛋白的编码基因之间插入MMP13特异识别和降解的肽段底物的编码基因，形成融合表达质粒，其质粒的图谱可以参见图5所示。
本发明所获得的融合表达质粒，其本身可以在真核细胞内自行表达荧光蛋白，表达之后的产物即为BRET探针，即可直接进行检测；比如软骨细胞SW1353的体外检测中，可以将上述构建的融合表达质粒转染至软骨细胞SW1353；然后进行培养，让其在此软骨细胞SW1353内表达荧光蛋白，通过流式细胞仪或荧光酶标仪检测三个BRET融合体的相对荧光强度。
其中，上述具有序列表SEQ.ID.No.3的碱基序列的Rluc8蛋白编码基因与Rluc8蛋白氨基酸序列的编码对应表：
Figure PCTCN2015072618-appb-000001
Figure PCTCN2015072618-appb-000002
并且，在上述构建的质粒的步骤实施中，其采用的真核质粒pDisplay作为表达载体，实施中如果能有能够在骨细胞中有良好转染和表达的其它表达载体质粒也可以进行采用。同时，在本发明中采用的上述质粒pDisplay，并且其pDisplay的多克隆位点中具有BglII、PstI、HindIII、SalI多个酶切位点；在上述供体蛋白基因、受体蛋白基因和多肽底物的表达序列，三者分次的插入可以分别利用这BglII、PstI、HindIII、SalI四个酶切位点进行，实现准确的定位和衔接。
为了使本发明的上述方法步骤实施中的细节和过程能进一步易于本领域是人员的理解和改进，同时为使本发明探针的进步性的效果有更深入的理解，以下通过详细的实施例进行具体说明：
实施例1：
S10，将pDisplay载体进行双酶切(BgIII+PstI)，琼脂糖凝胶电泳后回收线性化载体；
以具有序列表SEQ.ID.No.3的碱基序列的Rluc8蛋白编码基因为模板，PCR扩增Rluc8的基因序列，Rluc8的基因扩增引物如下：
Figure PCTCN2015072618-appb-000003
上述反向引物中的“TCCCCGCGGCCCAAGCTTAAAACTGCAGT”代表MCS(多克隆位点)，包括三个酶切位点(PstI+HindIII+SalI)。
PCR中分别能引入相应的酶切位点，后将PCR产物进行双酶切(BgIII+PstI)后，最终与切好的线性化载体连接，转化后测序鉴定序列的正确性，最终以测序正确的具有序列表SEQ.ID.No.17的碱基序列的Rluc8表达质粒pDisplay-Rluc8-MCS进行下一步工作。
S20，在步骤S10之后，筛选重组成功的pDisplay-Rluc8-MCS质粒，进行扩增、质粒大量提取，双酶切(HindIII和SalI)，胶回收双酶切后线性化载体；
将黄色荧光蛋白编码基因进行扩增，扩增中需要特异性设计引物，引入特定酶切的位点；因此根据黄色荧光蛋白的类型设计引物如下：
引物名称 碱基序列 序列表序列号
YFP-F(HindIII) CCCAAGCTTAGCAAGGGCGAGGAGCTGTTCA SEQ.ID.No.10
YFP-R(SalI) ACGCGTCGACGGCCATCCACGTTGTACAGCTC SEQ.ID.No.11
cpVensu-F(HindIII) CCCAAGCTTGACGGCGGCGTGCAGCT SEQ.ID.No.12
cpVenus-R(SalI) ACGCGTCGACCTCGATGTTGTGGCGGA SEQ.ID.No.13
cpYFP-F(HindIII) CCCAAGCTTTACAACAGCGACAACGTCTATAT SEQ.ID.No.14
cpYFP-R(SalI) ACGCGTCGACGTTGTACTCCAGCTTGTGCCCCAGG SEQ.ID.No.15
同时，结合上述pDisplay质粒扩增之后具有BglII、PstI、HindIII、SalI的酶切位点。针对不同的受体蛋白编码基因：YFP基因、cpYFP基因、cpVenus基因；然后分别设计上表中的对应引物对，在荧光蛋白的编码基因的扩增之后引入了HindIII和SalI的酶切位点，而之后便可以通过双酶切(HindIII、SalI)插入至表达质粒pDisplay-Rluc8-MCS的HindIII、SalI的酶切位点之间；便可以分别得到pDisplay-Rluc8-YFP、pDisplay-Rluc8-cpYFP或pDisplay-Rluc8-cpVenus的荧光素酶和荧光蛋白的融合体表达质粒；
本实施例中以cpYFP作为黄色荧光蛋白进行，在该步骤中构建完成的质粒的碱基序列中，可以采用测序进行验证，重组后质粒中Rluc8-cpYFP片段的正 确序列结果可以参见序列表SEQ.ID.No.18。
S30、在步骤S20之后，筛选插入成功的融合体表达质粒，之后进一步将人工合成的多肽底物的编码基因与上述融合体表达质粒进行重组；
当然，其中采用多肽底物的表达序列基因其序列信息如下表：
Figure PCTCN2015072618-appb-000004
在该步骤中，根据编码MMP13的底物多肽“GPLGMRGL”序列的上述引物，在引物退火配对后经PstI和HindIII酶切后，直接与用PstI和HindIII双酶切的琼脂糖凝胶电泳回收的pDisplay-Rluc8-YFP、pDisplay-Rluc8-cpYFP或pDisplay-Rluc8-cpVenus进行连接。
在设计的人工合成表达的引物中“GGTCCGCTGGGTATGCGTGGTCTG”用于编码MMP13的底物多肽GPLGMRGL，同时两端分别直接设计添加具有HindIII和PstI的酶切位点，使其能通过HindIII和PstI的双酶切，便能将这一多肽底物编码基因插入至质粒的HindIII和PstI的酶切位点之间。
当然，上述重组根据受体的黄色荧光蛋白的种类的不同，最终可以得到三个重组载体即为获得BRET探针的融合表达质粒，分别可以表示为：pDisplay-Rluc8-GPLGMRGL-YFP、pDisplay-Rluc8-GPLGMRGL-cpYFP、pDisplay-Rluc8-GPLGMRGL-cpVenus；当然，重组质粒的结果是否准确，可以用测序进行验证。本次实施例中采用pDisplay-Rluc8-GPLGMRGL-cpYFP进行。
S40，将步骤S30所获得的BRET探针的融合表达质粒，采用脂质体转染法转染软骨细胞SW1353的细胞株；转染的步骤可以根据参见脂质体转染试剂盒操作说明使用转染试剂来转染哺乳动物SW1353软骨细胞，在转染以前放置一天使6孔板的每个孔的密度达到2*105个细胞。转染之后，上述构建的表达质粒本身具有能够在真核细胞内表达的能力自行生成BRET探针。
S50，通过流式细胞仪或荧光酶标仪检测BRET融合体的相对荧光强度的变化信息；具体：
S51，流式细胞仪检测：转染后36小时以上，将细胞用磷酸盐缓冲液(PBS)洗涤两次，再用0.25％胰蛋白酶消化成单个细胞，离心收集细胞后再用PBS洗涤两次，悬浮于500μl的PBS。加入终浓度为5μM腔肠素，在37度反应约10min后，进行流式检测(计数大约20000个细胞)。
S52，转染后的细胞接种于96孔板中，每个孔添加终浓度为5μM腔肠素，使用多功能酶标仪的荧光模块分析每次转染的大约100000个细胞，采用综合读数10min，收集通过两个不同波长(分别为475nm，525nm)范围的滤光片的光。
同时，在转染前后，可以分别观察细胞膜表面、从细胞膜上表达的BRET探针、以及表达的BRET探针是否定位于真核细胞膜表面时荧光情况，其结果可以参见图6所示。
S53，转染后的细胞接种接于腔室载玻片中，每个腔室添加终浓度为5μM腔肠素，由探针所表达的生物发光供体海肾荧光素酶利用O2和腔肠荧光素催化发光反应，产生发射光，传递给黄色荧光蛋白使之产生发射光，然后使用激光共聚集显微镜检测细胞的相对荧光强度。
同时，还可以采用上述探针，转染至加入不同MMP13浓度处理的软骨细胞，然后观测BRET荧光相对强度，其结果参见图7。从图7中，可以看出随着MMP13浓度增加，BRET荧光相对强度发生衰减。
并且，同时采用FACS流式细胞仪检测也能检测到MMP13活性，其细胞仪测试的细胞数量和MMP13添加量，对BRET荧光位移发生改变产生的荧光强度变化关系，所绘制的曲线参见图8所示。从曲线中也可以看出，随着MMP13添加，BRET荧光位移发生改变，荧光强度减弱。
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包括在本发明的保护范围之内。

Claims (10)

  1. 一种检测细胞外基质MMP13的BRET探针，包括BRET生物发光供体和BRET受体荧光蛋白，其特征在于，所述BRET生物发光供体和BRET受体荧光蛋白之间通过MMP13特异性识别降解的多肽底物连接；
    其中，所述BRET生物发光供体为具有序列表SEQ.ID.No.1的氨基酸序列的Rluc8蛋白，所述BRET受体荧光蛋白为黄色荧光蛋白。
  2. 如权利要求1所述的检测细胞外基质MMP13的BRET探针，其特征在于，所述多肽底物具有序列表SEQ.ID.No.2氨基酸序列。
  3. 如权利要求2所述的检测细胞外基质的MMP13的BRET探针，其特征在于，所述黄色荧光蛋白为YFP、cpYFP、cpVenus中的一种。
  4. 一种权利要求1所述的检测细胞外基质MMP13的BRET探针的编码基因，其特征在于，包括顺次连接的BRET生物发光供体编码基因、MMP13特异性识别降解的多肽底物编码基因和BRET受体荧光蛋白编码基因；
    所述BRET生物发光供体编码基因为具有序列表SEQ.ID.No.3碱基序列的Rluc8蛋白编码基因。
  5. 如权利要求4所述的检测细胞外基质MMP13的BRET探针的编码基因，其特征在于，所述多肽底物编码基因为具有序列表SEQ.ID.No.4碱基序列。
  6. 如权利要求4所述的检测细胞外基质MMP13的BRET探针的编码基因，其特征在于，
    所述BRET受体荧光蛋白编码基因为具有序列表SEQ.ID.No.5碱基序列的YFP基因；
    或，所述BRET受体荧光蛋白编码基因为具有序列表SEQ.ID.No.6碱基序列的cpYFP基因；
    或，所述BRET受体荧光蛋白编码基因为具有序列表SEQ.ID.No.7碱基序列的cpVensu基因。
  7. 一种包含权利要求4至6任一项所述的检测细胞外基质MMP13的BRET探针的编码基因的真核细胞表达质粒。
  8. 如权利要求7所述的包含检测细胞外基质MMP13的BRET探针的编码基因的真核细胞表达质粒，所述真核细胞表达载体为pDisplay载体。
  9. 一种权利要求8所述的包含检测细胞外基质MMP13的BRET探针的编码基因的表达质粒的构建方法，其特征在于，包括如下步骤：
    将具有序列表SEQ.ID.No.3的碱基序列的Rluc8蛋白编码基因克隆至质粒pDisplay的多克隆位点，得到Rluc8表达质粒；
    将黄色荧光蛋白的编码基因克隆至所述Rluc8表达质粒中与Rluc8蛋白编码基因末端衔接，形成生物发光与荧光蛋白融合的表达质粒；
    将具有序列表SEQ.ID.No.4碱基序列的多肽底物编码基因插入至所述融合体表达质粒中Rluc8蛋白编码基因和黄色荧光蛋白的编码基因之间。
  10. 如权利要求9所述的包含检测细胞外基质MMP13的BRET探针的编码基因的表达载体的构建方法，其特征在于，
    将所述具有序列表SEQ.ID.No.3的碱基序列的Rluc8蛋白的编码基因克隆至质粒pDisplay的多克隆位点步骤包括：
    以具有序列表SEQ.ID.No.8碱基序列的用RLuc8-F上游引物和具有序列表SEQ.ID.No.9碱基序列的下游引物RLuc8-R扩增所述Rluc8蛋白编码基因；
    分别将Rluc8蛋白编码基因的扩增产物和pDisplay质粒用BgIII和PstI进 行双酶切后，进行连接；
    和/或，将所述黄色荧光蛋白的编码基因克隆至所述Rluc8表达质粒中与Rluc8蛋白编码基因末端衔接步骤包括：
    将所述黄色荧光蛋白的编码基因PCR扩增；其中，
    当所述黄色荧光蛋白为具有序列表SEQ.ID.No.5碱基序列的YFP基因时，PCR中采用的引物为具有序列表SEQ.ID.No.10碱基序列的上游引物YFP-F和具有序列表SEQ.ID.No.11碱基序列的下游引物YFP-R；
    当所述黄色荧光蛋白为具有序列表SEQ.ID.No.6碱基序列的cpYFP基因时，PCR中采用的引物为具有序列表SEQ.ID.No.12碱基序列的上游引物cpYFP-F和具有序列表SEQ.ID.No.13碱基序列的下游引物cpYFP-R；
    当所述黄色荧光蛋白为具有序列表SEQ.ID.No.7碱基序列的cpVensu基因时，PCR中采用的引物为具有序列表SEQ.ID.No.14碱基序列的上游引物cpVensu-F和具有序列表SEQ.ID.No.15碱基序列的下游引物cpVensu-R；
    分别将Rluc8蛋白编码基因的扩增产物和Rluc8表达质粒用HindIII和SalI进行双酶切后，进行连接；
    和/或，将所述具有序列表SEQ.ID.No.4碱基序列的肽段底物编码基因克隆插入至所述融合体表达质粒中Rluc8蛋白编码基因和黄色荧光蛋白的编码基因之间步骤包括：
    将具有序列表SEQ.ID.No.16碱基序列的多肽底物的编码基因引物和融合体表达质粒分别用PstI和HindIII双酶切后，进行连接。
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