WO2023005109A1 - 基于光敏色素蛋白miRFP670nano的双分子荧光互补系统 - Google Patents

基于光敏色素蛋白miRFP670nano的双分子荧光互补系统 Download PDF

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WO2023005109A1
WO2023005109A1 PCT/CN2021/138127 CN2021138127W WO2023005109A1 WO 2023005109 A1 WO2023005109 A1 WO 2023005109A1 CN 2021138127 W CN2021138127 W CN 2021138127W WO 2023005109 A1 WO2023005109 A1 WO 2023005109A1
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张先恩
陈明海
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Shenzhen Institute of Advanced Technology of CAS
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    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
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    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/65—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression using markers
    • G—PHYSICS
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
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    • C12N2800/107—Plasmid DNA for vertebrates for mammalian

Definitions

  • the present application relates to the technical field of protein interaction imaging, in particular to a bimolecular fluorescence complementation system based on phytochrome protein miRFP670nano, and a construction method of the bimolecular fluorescence complementation system based on phytochrome protein miRFP670nano.
  • FRET fluorescence resonance energy transfer
  • BiFC bimolecular fluorescence complementary technology
  • Bimolecular fluorescence complementation system is a kind of fragment complementation system using fluorescent protein as material.
  • the basic principle is to split the fluorescent protein into two non-fluorescent fragments at the appropriate site.
  • the split non-fluorescent fragments will approach each other, thereby restoring integrity
  • the conformation of the fluorescent protein emits specific fluorescence.
  • Bimolecular fluorescence complementation technology is a simple, intuitive and sensitive method to detect protein-protein interactions, and has received more and more attention in recent years.
  • the currently developed bimolecular fluorescence complementation system includes the fluorescence complementation system based on green fluorescent protein (GFP) and the fluorescence complementation system based on near-infrared phytochrome protein.
  • GFP green fluorescent protein
  • HPC near-infrared phytochrome protein
  • the fluorescent complementation system of GFP needs to be produced at low temperature to produce mature and complete fluorescent protein and emit fluorescence, which limits the application of this system under physiological conditions.
  • light with a wavelength of 600nm-1200nm has better tissue permeability, and can produce better imaging effects in living animals.
  • the fluorescence wavelength generated by the GFP fluorescence complementation system is relatively short, usually less than 600nm, which limits the imaging application of the GFP-based fluorescence fragment complementation system in vivo.
  • the near-infrared phytochrome protein absorbs infrared or near-infrared light and matures under physiological conditions (37° C.), producing fluorescence with a wavelength greater than 650 nm.
  • both near-infrared phytochrome protein and GFP protein have relatively large protein molecular weight (27-35kDa). Fluorescent protein fragments are large and may somewhat hinder the protein-protein interactions to be studied. Therefore, it is of great significance to develop a near-infrared fluorescent complementary system with a smaller molecular weight and mature under physiological conditions.
  • a technical solution adopted in the embodiment of the present application is to provide a bimolecular fluorescence complementation system based on the phytochrome protein miRFP670nano, including a first carrier and a second carrier, wherein the first carrier contains the sequence SEQ The vector of ID NO. 2, the second vector is the vector containing the sequence of SEQ ID NO. 3.
  • sequence SEQ ID NO. 2 is used to express the miRN107 protein fragment
  • sequence SEQ ID NO. 3 is used to express the miRC108 protein fragment; when the miRN107 protein fragment and the miRC108 protein fragment are close to each other, they can be reconfigured to form a phytochrome Protein miRFP670nano.
  • the first vector further includes a first gene sequence, the first gene sequence is used to express the first protein, the first vector is used to express the first fusion protein, and the first fusion protein is the first protein and miRN107 protein fragment fusion protein.
  • the miRN107 protein fragment is a protein fragment composed of amino acids 1 to 107 of the phytochrome protein miRFP670nano.
  • the second vector further includes a second gene sequence, the second gene sequence is used to express the second protein, the second vector is used to express the second fusion protein, the second fusion protein is the second protein and miRC108 protein fragment fusion protein.
  • the miRC108 protein fragment is a protein fragment composed of amino acids 108 to 147 of the phytochrome protein miRFP670nano.
  • another technical solution adopted in the embodiment of the present application is to provide a method for constructing a bimolecular fluorescence complementation system based on the phytochrome protein miRFP670nano, including: using the gene sequence of the phytochrome protein miRFP670nano as a template to perform polymerase Chain reaction to obtain the sequence SEQ ID NO. 2 and the sequence of SEQ ID NO. 3; using the double restriction site of the first plasmid, inserting the sequence of SEQ ID NO. 2 into the multiple cloning site of the first plasmid to obtain the first vector; using the double enzyme of the second plasmid Insert the sequence SEQ ID NO. 3 into the multiple cloning site of the second plasmid to obtain the second vector.
  • the method before inserting the sequence SEQ ID NO. 2 into the multiple cloning site of the first plasmid by using the double restriction site of the first plasmid, the method further includes: obtaining the expression of the first plasmid by PCR.
  • the first gene sequence of the protein and the second gene sequence for expressing the second protein by overlapping PCR, the first tandem gene in which the first gene sequence is connected in series with the sequence SEQ ID NO. 2 is obtained; by overlapping PCR, the second gene is obtained The second tandem gene whose sequence is tandem with the sequence SEQ ID NO. 3.
  • using the double restriction site of the first plasmid inserting the sequence SEQ ID NO. 2 into the multiple cloning site of the first plasmid, comprising: using the double restriction site of the first plasmid, inserting the first The tandem gene is inserted into the multiple cloning site of the first plasmid; using the double restriction site of the second plasmid, the sequence SEQ ID NO. 3 is inserted into the multiple cloning site of the second plasmid, including: utilizing the double restriction site of the second plasmid site to insert the second tandem gene into the multiple cloning site of the second plasmid.
  • the sequence SEQ ID NO. 2 is used to express the miRN107 protein fragment of the phytochrome protein miRFP670nano, and the miRN107 protein fragment is a protein fragment composed of amino acids 1 to 107 of the phytochrome protein miRFP670nano;
  • the first The vector is used to express the fusion protein of the first protein and the miRN107 protein fragment;
  • the sequence SEQ ID NO. 3 is used to express the miRC108 protein fragment of the phytochrome protein miRFP670nano, and the miRC108 protein fragment is composed of position 108 to 147 of the phytochrome protein miRFP670nano
  • the second vector is used to express the fusion protein of the second protein and the miRC108 protein fragment.
  • another technical solution adopted in the embodiment of the present application is to provide an imaging application of a bimolecular fluorescence complementation system based on the phytochrome protein miRFP670nano in the interaction between proteins.
  • the bimolecular fluorescence complementary system based on phytochrome protein miRFP670nano is the above-mentioned bimolecular fluorescent complementary system based on phytochrome protein miRFP670nano.
  • the present application provides a bimolecular fluorescence complementation system based on phytochrome protein miRFP670nano, including a first carrier and a second carrier.
  • the first vector is a vector comprising the sequence of SEQ ID NO. 2
  • the second vector is a vector comprising the sequence of SEQ ID NO. 3.
  • the phytochrome protein miRFP670nano with a complete conformation consists of only 147 amino acids, has a small molecular weight, and has little effect on the protein-protein interaction to be studied.
  • the smaller molecular weight phytochrome protein miRFP670nano is more conducive to the closeness of the proteins to be studied.
  • the phytochrome protein miRFP670nano can produce fluorescence at physiological temperature (37°C), which is conducive to the detection of fluorescent signals generated in living cells and in vivo.
  • the phytochrome protein miRFP670nano can generate light with a wavelength of 600nm-1200nm in the process of fluorescence imaging of biological tissues, has better tissue permeability, and can produce better imaging effects in living animals.
  • 1A is a schematic diagram of the first vector containing the sequence SEQ ID NO. 2 for expressing the first protein fragment miRN107 of the phytochrome protein miRFP670nano provided in the examples of the present application.
  • 1B is a schematic diagram of the second vector containing the sequence SEQ ID NO. 3 of the second protein fragment miRC108 used to express the phytochrome protein miRFP670nano provided in the examples of the present application.
  • Fig. 2A is a schematic diagram of the first vector for expressing the bJun-miRN107 fusion protein provided in the embodiment of the present application.
  • Fig. 2B is a schematic diagram of the second vector for expressing the miRC108-bFos fusion protein provided by the embodiment of the present application.
  • Figure 3A-3B shows the fluorescence brightness produced by HEK293T cells transfected with the first vector and the second vector provided in the examples of the present application and the fluorescence produced by the HEK293T cells transfected with the first vector and the control vector provided in the examples of the present application Brightness contrast graph for brightness.
  • Figure 3C shows the fluorescence brightness produced by nude mice after HEK293T cells were transfected with the first vector and the second vector provided in the examples of the present application and inoculated under the skin of nude mice, which is the same as that of HEK293T cells transfected with the first vector provided in the examples of the present application.
  • the brightness contrast graph of the fluorescence brightness produced by the nude mice after the vector and the control vector were inoculated subcutaneously in the nude mice.
  • the application provides a bimolecular fluorescent complementary system based on phytochrome protein miRFP670nano, including a first carrier and a second carrier, wherein the first carrier is a carrier containing the sequence SEQ ID NO. 2, and the second carrier is a carrier containing the sequence SEQ ID NO. 3 carrier.
  • FIG. 1A is a schematic diagram of a first vector comprising SEQ ID NO. 2
  • FIG. 1B is a schematic diagram of a second vector comprising SEQ ID NO. 3.
  • the phytochrome protein miRFP670nano is a protein composed of 147 amino acids, its gene sequence is shown in the sequence SEQ ID NO. 1, and its amino acid sequence is shown in the sequence SEQ ID NO. 4.
  • the vector pUC57-miRFP670nano was constructed, which contains the sequence SEQ ID NO. 1 for expressing the phytochrome protein miRFP670nano.
  • the sequence of SEQ ID NO. 1 can be inserted into the multiple cloning site of the plasmid pUC57 through double restriction sites to obtain the vector pUC57-miRFP670nano.
  • sequence SEQ ID NO. 1 can also be inserted into other eukaryotic expression vectors, such as pEGFP-C1, pEGFP-N1, pcDNA3.1, etc.
  • the appropriate dual enzymes can be selected
  • the cleavage site inserts the sequence SEQ ID NO. 1, and the application does not limit the selection of the eukaryotic expression vector and the selection of the enzyme cleavage site.
  • the vector pUC57-Akaluc was purchased commercially (Suzhou Jinweizhi Biotechnology Co., Ltd.).
  • the phytochrome protein miRFP670nano will be split into two protein fragments between the 107th amino acid and the 108th amino acid (counting from the nitrogen terminal), namely the miRN107 protein fragment and the miRC108 protein fragment .
  • the miRN107 protein fragment can be understood as a protein fragment containing amino acids from No. 1 to No. 107 of the phytochrome protein miRFP670nano (ie, the sequence shown in SEQ ID NO. 4);
  • the miRC108 protein fragment can be understood as containing A protein fragment of amino acids 108 to 147 of the phytochrome protein miRFP670nano.
  • sequences SEQ ID NO. 2 and SEQ ID NO. 3 can be amplified by polymerase chain reaction (PCR).
  • the PCR amplification sequence SEQ is carried out using the vector pUC57-miRFP670nano as a template
  • suitable upstream and downstream primers can be designed according to the actual situation.
  • the upstream primer used when amplifying the sequence SEQ ID NO. 2 is:
  • Downstream primers are:
  • the sequence SEQ ID NO. 2 obtained by PCR amplification is a nucleotide sequence capable of expressing miRN107 protein fragments. Then, using the double restriction sites NheI and HindIII , the sequence SEQ ID NO. 2 for expressing the miRN107 protein fragment was inserted into the multiple cloning site of pcDNA3.1, and the first sequence containing the sequence SEQ ID NO. 2 was constructed. carrier.
  • upstream primer used when amplifying the sequence SEQ ID NO. 2 is:
  • Downstream primers are:
  • the sequence SEQ ID NO. 3 obtained by PCR amplification is a nucleotide sequence capable of expressing miRC108 protein fragments. Then, using the double enzyme cutting sites NheI and HindIII , the sequence SEQ ID NO. 3 for expressing the miRC108 protein fragment was inserted into the multiple cloning site of pcDNA3.1, and a second sequence containing the sequence SEQ ID NO. 3 was constructed. carrier.
  • the present application does not limit the eukaryotic expression vectors, for example, eukaryotic expression vectors such as pEGFP-C1, pEGFP-N1, pcDNA3.1, etc.
  • the first vector further includes a first gene for expressing the first protein
  • the second vector further includes a second gene for expressing the second protein.
  • the first vector can express the first fusion protein formed by the first protein and miRN107 protein fragment
  • the second vector can express the second fusion protein formed by the second protein and miRC108 protein fragment.
  • the first protein and the second protein are two interacting proteins.
  • the first gene and the sequence SEQ ID NO. 2 can form the first tandem gene by overlapping PCR, and the tandem gene as a whole is inserted into the plasmid to obtain the first vector, as shown in Figure 2A;
  • the second gene and The sequence of SEQ ID NO. 3 can form a second tandem gene by overlapping PCR, and insert the tandem gene as a whole into a plasmid to obtain a second vector, as shown in FIG. 2B .
  • the first gene is obtained by PCR amplification.
  • the first gene is used to express the first protein.
  • the plasmid containing the sequence SEQ ID NO. 2 as a template for example, using the plasmid pUC57-miRFP670nano as a template
  • design corresponding upstream primers and downstream primers and obtain the sequence SEQ ID NO. 2 by PCR amplification.
  • overlap PCR overlap PCR
  • the first tandem gene of the first gene-SEQ ID NO. 2 tandem can be obtained.
  • the first tandem gene is inserted into the expression vector by using the double enzyme cutting site to obtain the first vector.
  • the upstream of the NO. 2 gene sequence can also connect the first gene in series to the downstream of the SEQ ID NO. 2 gene sequence.
  • appropriate primers can be designed according to the upstream and downstream sequences of the two gene sequences. For example, when deciding to connect the first gene in series to the upstream of the SEQ ID NO. 2 gene sequence, the upstream primer used when amplifying the first gene sequence can be used as the upstream primer in the overlap PCR to amplify SEQ ID NO.
  • the downstream primer used in the gene sequence is used as the downstream primer in the overlap PCR, and at the same time, there is a certain complementary sequence between the downstream primer used in the amplification of the first gene sequence and the upstream primer used in the amplification of the SEQ ID NO. 2 gene sequence .
  • the downstream primer used when amplifying the first gene sequence can be used as the downstream primer in the overlap PCR, and the SEQ ID NO.
  • the upstream primer used in the overlap PCR is used as the upstream primer in the overlap PCR, and at the same time, there is a certain complementary sequence between the upstream primer used when amplifying the first gene sequence and the downstream primer used when amplifying the SEQ ID NO. 2 gene sequence.
  • the second gene is obtained by PCR amplification.
  • the second gene is used to express the second protein.
  • the plasmid containing SEQ ID NO. 3 as a template for example, using the plasmid pUC57-miRFP670nano as a template
  • design corresponding upstream primers and downstream primers and obtain the sequence SEQ ID NO. 3 by PCR amplification.
  • the second gene obtained by PCR and the sequence SEQ ID NO. 3 as a template to carry out overlap PCR.
  • the second tandem gene in which the second gene is tandem with SEQ ID NO. 3 can be obtained.
  • the second tandem gene is inserted into the expression vector by using the double enzyme cutting site to obtain the second vector.
  • the second gene can also be connected in series to the downstream of the SEQ ID NO. 3 gene sequence.
  • appropriate primers can be designed according to the upstream and downstream sequences of the two gene sequences. For example, when deciding to connect the second gene in series to the upstream of the SEQ ID NO. 3 gene sequence, the upstream primer used when amplifying the second gene sequence can be used as the upstream primer in the overlap PCR to amplify SEQ ID NO.
  • the downstream primer used in the gene sequence is used as the downstream primer in the overlap PCR, and at the same time, there is a certain complementary sequence between the downstream primer used in the amplification of the second gene sequence and the upstream primer used in the amplification of the SEQ ID NO. 3 gene sequence .
  • the downstream primer used when amplifying the second gene sequence can be used as the downstream primer in the overlap PCR, and the SEQ ID NO.
  • the upstream primer used during overlap PCR is used as the upstream primer during overlap PCR, and at the same time, there is a certain complementary sequence between the upstream primer used when amplifying the second gene sequence and the downstream primer used when amplifying the SEQ ID NO. 3 gene sequence.
  • the present application does not limit the first protein and the second protein that can interact.
  • the first protein can be FKBP protein, and the second protein can be FRB protein; the first protein can be Bak protein, and the second protein can be The protein can be Bcl-XL protein; the first protein can be bJun protein, the second protein can be bFos protein and so on.
  • the first protein and the second protein can be determined according to specific research objects.
  • the system provided by the present application can also be used to determine whether the two proteins as research objects have an interaction.
  • bJun protein is used as the first protein
  • bFos protein is used as the second protein as an example for description.
  • the first vector is constructed.
  • a plasmid containing the bJun gene sequence such as plasmid pbJun-iRN97, and design the corresponding upstream primer bJun-F as:
  • the downstream primer bJun-R is:
  • the downstream primer miRN107-R is:
  • sequence SEQ ID NO. 2 was obtained, which is the gene sequence used to express the miRN107 protein fragment.
  • first tandem gene bJun-SEQ ID NO. 2 was inserted into the multiple cloning site of the pcDNA3.1 vector by using the double enzyme cutting sites NheI and HindIII to construct the first vector.
  • the first vector can express the first fusion protein bJun-miRN107 composed of bJun protein and miRN107 protein fragments.
  • the downstream primer bFos-R is:
  • the second gene namely the bFos gene sequence, is obtained.
  • the downstream primer miRC108-R is:
  • sequence SEQ ID NO. 3 was obtained, which is the gene sequence used to express the miRC108 protein fragment.
  • the second vector can express the second fusion protein miRC108-bFos composed of bFos protein and miRC108 protein fragment.
  • the fluorescence effect of the system can be detected in cell lines and living animals.
  • the first vector containing the first tandem gene (the first gene and the sequence of SEQ ID NO. 2)
  • the second vector containing the second tandem gene (the sequence of SEQ ID NO. 3 and the second gene)
  • Control vector is a vector comprising the third gene in series with the sequence of SEQ ID NO. 3
  • the protein fragment expressed by the third gene can be any protein fragment that cannot interact with the protein fragment expressed by the first gene sequence.
  • the reference vector can express other fluorescent proteins, and the fluorescence produced by other fluorescent proteins can be used as an internal reference for fluorescence brightness.
  • the bJun gene sequence is also used as the first gene sequence and the bFos gene sequence is used as the second gene sequence as an example, but it can be understood that in practical applications, the first gene sequence and the second gene sequence can be determined according to the specific research object sequence.
  • the first vector constructed is the bJun-SEQ ID NO. 2 vector
  • the second vector constructed is the SEQ ID NO. 3-bFos vector
  • the constructed control vector is the SEQ ID NO. 3-mbFos vector
  • the constructed reference vector is pEGFP vector.
  • control vector can express a mutated bFos protein fragment, that is, the mbFos protein fragment, and the mbFos protein fragment cannot interact with the bJun protein fragment expressed by the first vector, so it can be used as a control vector.
  • the pEGFP vector can express EGFP, and the EGFP can generate fluorescence under the excitation of 488nm excitation light, which is used as an internal reference for detecting the fluorescence effect of the bimolecular fluorescence complementation system of this embodiment.
  • the constructed first vector pbJun-SEQ ID NO. 2
  • the second carrier SEQ ID NO. 3-bFos
  • the reference vector pEGFP
  • the constructed first vector pbJun-SEQ ID NO. 2
  • control vector SEQ ID NO. 3-mbFos
  • the reference vector pEGFP
  • Figure 3B shows the fluorescence brightness produced by HEK293T cells transfected with the first vector and the second vector provided in the examples of the present application and the fluorescence brightness produced by the HEK293T cells transfected with the first vector and the control vector provided in the examples of the present application Brightness comparison chart.
  • the fluorescence brightness produced by cells transfected with the first vector bJun-miRN107 and the second vector miRC108-bFos (cells in the experimental group) was significantly higher than that transfected with the first vector bJun-miRN107 and the control vector miRC108-
  • the fluorescence brightness produced by the mbFos cells (control group cells) was about 13 times higher than that produced by the control group cells.
  • Figure 3C shows the fluorescence brightness produced by nude mice after HEK293T cells were transfected with the first vector and the second vector provided in the examples of the present application and inoculated under the skin of nude mice, which is the same as that of HEK293T cells transfected with the first vector provided in the examples of the present application.
  • the brightness contrast graph of the fluorescence brightness produced by the nude mice after the vector and the control vector were inoculated subcutaneously in the nude mice.
  • EGFP as an internal reference can emit green fluorescence from the left subcutaneous and right subcutaneous of nude mice; the left subcutaneous of nude mice inoculated with HEK293T cells of the experimental group can emit green fluorescence Fluorescence, while the right subcutaneous of nude mice inoculated with HEK293T cells in the control group did not emit fluorescence.
  • the first vector and the second vector in the experimental group expressed bJun-miRN107 fusion protein and miRC108-bFos fusion protein, respectively.
  • the bJun protein and the bFos protein can interact to make the miRN107 protein fragment and the miRC108 protein fragment close to each other, and restructure to form a complete phytochrome protein miRFP670nano, which can produce fluorescence under the excitation of the corresponding wavelength of excitation light.
  • the first vector and the control vector respectively expressed bJun-miRN107 fusion protein and miRC108-mbFos fusion protein.
  • the miRN107 protein fragment and the miRC108 protein fragment cannot approach each other to reconstitute the complete phytochrome protein miRFP670nano, thereby failing to produce fluorescence.
  • the fusion protein of the first protein and the miRN107 protein fragment and the fusion protein of the miRC108 protein fragment and the second protein are expressed in the same cell, Through the interaction between the first protein and the second protein, the miRN107 protein fragment and the miRC108 protein fragment can be approached to restructure to form a complete phytochrome protein miRFP670nano that can emit fluorescence under the excitation of excitation light.
  • the present application provides a bimolecular fluorescence complementation system based on phytochrome protein miRFP670nano, including a first carrier and a second carrier.
  • the first vector is a vector comprising the sequence of SEQ ID NO. 2
  • the second vector is a vector comprising the sequence of SEQ ID NO. 3.
  • the phytochrome protein miRFP670nano with a complete conformation consists of only 147 amino acids, has a small molecular weight, and has little effect on the protein-protein interaction to be studied.
  • the smaller molecular weight phytochrome protein miRFP670nano is more conducive to the closeness of the proteins to be studied.
  • the phytochrome protein miRFP670nano can produce fluorescence at physiological temperature (37°C), which is conducive to the detection of fluorescent signals generated in living cells and in vivo.
  • the phytochrome protein miRFP670nano can generate light with a wavelength of 600nm-1200nm in the process of fluorescence imaging of biological tissues, has better tissue permeability, and can produce better imaging effects in living animals.

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Abstract

本申请涉及蛋白质相互作用成像技术领域，本申请提供一种基于光敏色素蛋白miRFP670nano的双分子荧光互补系统及其构建方法和应用。该系统包括第一载体和第二载体，其中，所述第一载体为包含有序列SEQ ID NO. 2的载体，所述第二载体为包含有序列SEQ ID NO. 3的载体。该系统中，光敏色素蛋白miRFP670nano仅由147个氨基酸构成，分子量较小，对待研究的蛋白质间相互作用影响较小。同时，光敏色素蛋白miRFP670nano能够在生理温度（37℃）条件下产生荧光，有利于检测活细胞及活体内产生的荧光信号，并且产生的荧光波长位于600nm-1200nm之间，具有较好的组织通透性，能够在动物活体中产生较好的成像效果。

Description

基于光敏色素蛋白miRFP670nano的双分子荧光互补系统 技术领域
本申请涉及蛋白质相互作用成像技术领域，特别是涉及一种基于光敏色素蛋白miRFP670nano的双分子荧光互补系统，以及基于光敏色素蛋白miRFP670nano的双分子荧光互补系统的构建方法。
 背景技术
蛋白质之间的相互作用在生物体的生命过程中发挥了重要的作用。比如在基因调控、细胞信号转导以及肿瘤的生长发育过程中均涉及到许多蛋白质之间的相互作用。监测这些蛋白质间的相互作用对于生命过程的解析尤为重要。在过去的几十年里有一些基于荧光成像的方法被发展并用于研究蛋白质间的相互作用，比如：荧光共振能量转移（fluorescence resonance energy transfer, FRET）、基于单重态氧三重态能量转移的成像技术以及双分子荧光互补技术（Bimolecular fluorescence complementation，BiFC）等。
双分子荧光互补系统是一种以荧光蛋白作为材料的片段互补系统。其基本原理是在荧光蛋白的合适位点将其拆分成各自不发荧光的两个片段。当两个相互作用的蛋白分别与拆分的不发荧光的两个片段相互融合，在这两个相互作用蛋白的作用下，拆分的不发荧光两个片段就会相互靠近，从而恢复完整的荧光蛋白的构象而发出特异性的荧光。双分子荧光互补技术是一种检测蛋白质间相互作用的简便、直观、灵敏的方法，近些年得到越来越多的关注。
目前发展的双分子荧光互补系统包括基于绿色荧光蛋白(GFP)的荧光互补系统和基于近红外光敏色素蛋白的荧光互补系统。
但是，GFP的荧光互补系统需要在低温条件下才能使产生成熟的完整荧光蛋白并发出荧光，从而限制了该系统在生理条件下的应用。并且，在生物组织的荧光成像过程中，600nm-1200nm波长的光具有比较好的组织通透性，能够在动物活体中产生较好的成像效果。而GFP的荧光互补系统产生的荧光波长较短，通常小于600nm，从而限制了基于GFP的荧光片段互补系统在动物活体内的成像应用。
而近红外光敏色素蛋白是吸收红外或近红外光，并在生理条件(37℃)下成熟，产生的荧光波长大于650nm。但是，近红外光敏色素蛋白和GFP蛋白都具有比较大的蛋白分子量（27-35kDa）。荧光蛋白片段较大，可能会在一定程度上阻碍待研究的蛋白质间相互作用。因此，发展较小分子量的且可在生理条件下成熟的近红外荧光互补系统具有十分重要的意义。
 技术问题
为解决上述技术问题，本申请实施例采用的一个技术方案是提供一种基于光敏色素蛋白miRFP670nano的双分子荧光互补系统，包括第一载体和第二载体，其中，第一载体为包含有序列SEQ ID NO. 2的载体，第二载体为包含有序列SEQ ID NO. 3的载体。
 技术解决方案
在一些实施例中，序列SEQ ID NO. 2用于表达miRN107蛋白片段，序列SEQ ID NO. 3用于表达miRC108蛋白片段；当miRN107蛋白片段和miRC108蛋白片段相互靠近时，可重构形成光敏色素蛋白miRFP670nano。
在一些实施例中，第一载体进一步包括第一基因序列，第一基因序列用于表达第一蛋白，第一载体用于表达第一融合蛋白，第一融合蛋白为第一蛋白与miRN107蛋白片段的融合蛋白。
在一些实施例中，miRN107蛋白片段为由光敏色素蛋白miRFP670nano的第1号位至第107号位氨基酸构成的蛋白片段。
在一些实施例中，第二载体进一步包括第二基因序列，第二基因序列用于表达第二蛋白，第二载体用于表达第二融合蛋白，第二融合蛋白为第二蛋白与miRC108蛋白片段的融合蛋白。
在一些实施例中，miRC108蛋白片段为由光敏色素蛋白miRFP670nano的第108号位至第147号位氨基酸构成的蛋白片段。
为解决上述技术问题，本申请实施例采用的另一个技术方案是提供一种基于光敏色素蛋白miRFP670nano的双分子荧光互补系统的构建方法，包括：以光敏色素蛋白miRFP670nano的基因序列为模板进行聚合酶链式反应得到序列SEQ ID NO. 2和序列SEQ ID NO. 3；利用第一质粒的双酶切位点，将序列SEQ ID NO. 2插入第一质粒的多克隆位点，以得到第一载体；利用第二质粒的双酶切位点，将序列SEQ ID NO. 3插入第二质粒的多克隆位点，以得到第二载体。
在一些实施例中，在利用第一质粒的双酶切位点，将序列SEQ ID NO. 2插入第一质粒的多克隆位点之前，该方法还包括：通过PCR，获得用于表达第一蛋白的第一基因序列以及用于表达第二蛋白的第二基因序列；通过重叠PCR，获得第一基因序列与序列SEQ ID NO. 2串联的第一串联基因；通过重叠PCR，获得第二基因序列与序列SEQ ID NO. 3串联的第二串联基因。
在一些实施例中，利用第一质粒的双酶切位点，将序列SEQ ID NO. 2插入第一质粒的多克隆位点，包括：利用第一质粒的双酶切位点，将第一串联基因插入第一质粒的多克隆位点；利用第二质粒的双酶切位点，将序列SEQ ID NO. 3插入第二质粒的多克隆位点，包括：利用第二质粒的双酶切位点，将第二串联基因插入第二质粒的多克隆位点。
在一些实施例中，序列SEQ ID NO. 2用于表达光敏色素蛋白miRFP670nano的miRN107蛋白片段， miRN107蛋白片段为由光敏色素蛋白miRFP670nano第1号位至第107号位氨基酸构成的蛋白片段；第一载体用于表达第一蛋白和miRN107蛋白片段的融合蛋白；序列SEQ ID NO. 3用于表达光敏色素蛋白miRFP670nano的miRC108蛋白片段，miRC108蛋白片段为由光敏色素蛋白miRFP670nano第108号位至第147号位氨基酸构成的蛋白片段；第二载体用于表达第二蛋白和miRC108蛋白片段的融合蛋白。
为解决上述技术问题，本申请实施例采用的又一个技术方案是提供一种基于光敏色素蛋白miRFP670nano的双分子荧光互补系统在蛋白质间相互作用的成像应用。其中，基于光敏色素蛋白miRFP670nano的双分子荧光互补系统为以上所述的基于光敏色素蛋白miRFP670nano的双分子荧光互补系统。
 有益效果
相比于现有的双分子荧光互补系统，本申请提供一种基于光敏色素蛋白miRFP670nano的双分子荧光互补系统，包括第一载体和第二载体。其中，第一载体为包含有序列SEQ ID NO. 2的载体，第二载体为包含有序列SEQ ID NO. 3的载体。在该系统中，拥有完整构象的光敏色素蛋白miRFP670nano仅由147个氨基酸构成，分子量较小，对待研究的蛋白质间相互作用影响较小。也就是说，若待研究的蛋白质间具有相互作用，较小分子量的光敏色素蛋白miRFP670nano更有利于待研究的蛋白质相互靠近。同时，光敏色素蛋白miRFP670nano能够在生理温度（37℃）条件下产生荧光，有利于检测活细胞及活体内产生的荧光信号。进一步地，光敏色素蛋白miRFP670nano在生物组织的荧光成像过程中，能够产生600nm-1200nm波长的光，具有较好的组织通透性，能够在动物活体中产生较好的成像效果。
 附图说明
本申请将结合附图对实施方式进行说明。本申请的附图仅用于描述实施例，以展示为目的。在不偏离本发明的原理的条件下，本领域技术人员能够轻松地通过以下描述根据所述步骤做出其他实施例。
图1A为本申请实施例提供的包含有用于表达光敏色素蛋白miRFP670nano的第一蛋白片段miRN107的序列SEQ ID NO. 2的第一载体示意图。
图1B为本申请实施例提供的包含有用于表达光敏色素蛋白miRFP670nano的第二蛋白片段miRC108的序列SEQ ID NO. 3的第二载体示意图。
图2A为本申请实施例提供的包含有用于表达bJun-miRN107融合蛋白的第一载体示意图。
图2B为本申请实施例提供的包含有用于表达miRC108-bFos融合蛋白的第二载体示意图。
图3A-3B为HEK293T细胞转染了本申请实施例提供的第一载体和第二载体所产生的荧光亮度与HEK293T细胞转染了本申请实施例提供的第一载体和对照载体所产生的荧光亮度的亮度对比图。
图3C为HEK293T细胞转染了本申请实施例提供的第一载体和第二载体接种至裸鼠皮下后，裸鼠所产生的荧光亮度，与HEK293T细胞转染了本申请实施例提供的第一载体和对照载体接种至裸鼠皮下后，裸鼠所产生的荧光亮度的亮度对比图。
 本发明的实施方式
下面将结合本申请实施例中的附图，对本申请实施例中的技术方案进行清楚、完整地描述。可以理解的是，此处所描述的具体实施例仅用于解释本申请，而非对本申请的限定。另外还需要说明的是，为了便于描述，附图中仅示出了与本申请相关的部分而非全部结构。基于本申请中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本申请保护的范围。
本申请中的术语“第一”、“第二”等是用于区别不同对象，而不是用于描述特定顺序。此外，术语“包括”和“具有”以及它们任何变形，意图在于覆盖不排他的包含。例如包含了一系列步骤或单元的过程、方法、系统、产品或设备没有限定于已列出的步骤或单元，而是可选地还包括没有列出的步骤或单元，或可选地还包括对于这些过程、方法、产品或设备固有的其它步骤或单元。
在本文中提及“实施例”意味着，结合实施例描述的特定特征、结构或特性可以包含在本申请的至少一个实施例中。在说明书中的各个位置出现该短语并不一定均是指相同的实施例，也不是与其它实施例互斥的独立的或备选的实施例。本领域技术人员显式地和隐式地理解的是，本文所描述的实施例可以与其它实施例相结合。
本申请提供基于光敏色素蛋白miRFP670nano的双分子荧光互补系统，包括第一载体和第二载体，其中，第一载体为包含有序列SEQ ID NO. 2的载体，第二载体为包含有序列SEQ ID NO. 3的载体。
具体地，如图1A和图1B所示，图1A为包含有SEQ ID NO. 2的第一载体的示意图，图1B为包含有SEQ ID NO. 3的第二载体的示意图。
首先，获得包含有光敏色素蛋白miRFP670nano的基因的载体。光敏色素蛋白miRFP670nano是由147个氨基酸形成的蛋白，其基因序列如序列SEQ ID NO. 1所示，其氨基酸序列如序列SEQ ID NO. 4所示。在本申请实施例中，构建了载体pUC57-miRFP670nano，其包含有用于表达光敏色素蛋白miRFP670nano的序列SEQ ID NO. 1。例如，可以通过双酶切位点将序列SEQ ID NO. 1插入到质粒pUC57的多克隆位点中，即可得到载体pUC57-miRFP670nano。可以理解的是，序列SEQ ID NO. 1也可插入其他的真核表达载体，例如pEGFP-C1，pEGFP-N1，pcDNA3.1等，在不同的真核表达载体中，可选择合适的双酶切位点将序列SEQ ID NO. 1插入，本申请对真核表达载体的选择以及酶切位点的选择不做限制。在本申请实施例中，载体pUC57-Akaluc为商业购得（苏州金唯智生物科技有限公司）。
在本申请实施例中，光敏色素蛋白miRFP670nano将在107号位氨基酸和108号位氨基酸之间（从氮端开始计数），被拆分成两个蛋白片段，分别为miRN107蛋白片段和miRC108蛋白片段。其中，miRN107蛋白片段可以理解为包含有光敏色素蛋白miRFP670nano（即如SEQ ID NO. 4所示的序列）的第1号位至第107号位氨基酸的蛋白片段；miRC108蛋白片段可以理解为包含有光敏色素蛋白miRFP670nano的第108号位至第147号位氨基酸的蛋白片段。
获得载体pUC57-miRFP670nano后，以该载体为模板，通过聚合酶链式反应（PCR）可扩增得到序列SEQ ID NO. 2和SEQ ID NO. 3。
在本申请实施例中，以载体pUC57-miRFP670nano为模板进行PCR扩增序列SEQ ID NO. 2时，可根据实际情况设计合适的上下游引物。例如，本实施例中，扩增序列SEQ ID NO. 2时用到的上游引物为：
5’- CTAGCTAGCGCCACCATGGCAAACCTGGACAAGATGCTG-3’；
下游引物为：
5’- CCCAAGCTTTTAGCCCTGCAGGATGGGCACGGCC- 3’。
通过PCR扩增得到的序列SEQ ID NO. 2为能够表达miRN107蛋白片段的核苷酸序列。然后，利用双酶切位点 NheI和 HindIII，将用于表达miRN107蛋白片段的序列SEQ ID NO. 2插入到pcDNA3.1的多克隆位点，构建成包含有序列SEQ ID NO. 2的第一载体。
进一步地，以载体pUC57-miRFP670nano为模板进行PCR扩增序列SEQ ID NO. 3时，可根据实际情况设计合适的上下游引物。例如，本实施例中，扩增序列SEQ ID NO. 2时用到的上游引物为：
5’-
CTAGCTAGCGCCACCATGAAGAAGCTGTGGGGCCTGTTGGTG-3’；
下游引物为：
5’- CCCAAGCTTTTAGCTCTGCTGGATGGCGATGCCCACCAC - 3’。
通过PCR扩增得到的序列SEQ ID NO. 3为能够表达miRC108蛋白片段的核苷酸序列。然后，利用双酶切位点 NheI和 HindIII，将用于表达miRC108蛋白片段的序列SEQ ID NO. 3插入到pcDNA3.1的多克隆位点，构建成包含有序列SEQ ID NO. 3的第二载体。
本申请对真核表达载体不做限制，例如可以为pEGFP-C1、pEGFP-N1、pcDNA3.1等真核表达载体。
进一步地，第一载体还包括第一基因，用于表达第一蛋白，第二载体还包括第二基因，用于表达第二蛋白。这样，第一载体可表达第一蛋白与miRN107蛋白片段形成的第一融合蛋白；第二载体可表达第二蛋白与miRC108蛋白片段形成的第二融合蛋白。其中，第一蛋白与第二蛋白为两个相互作用的蛋白。
具体地，第一基因与序列SEQ ID NO. 2可以通过重叠PCR的方式形成第一串联基因，以串联基因作为整体，插入至质粒中得到第一载体，如图2A所示；第二基因与序列SEQ ID NO. 3可以通过重叠PCR的方式形成第二串联基因，以串联基因作为整体，插入至质粒中得到第二载体，如图2B所示。
例如，以包含有第一基因序列的质粒为模板，设计相应的上游引物和下游引物，通过PCR扩增获得第一基因。其中，第一基因用于表达第一蛋白。以包含有序列SEQ ID NO. 2的质粒为模板（例如以质粒pUC57-miRFP670nano为模板），设计相应的上游引物和下游引物，通过PCR扩增获得序列SEQ ID NO. 2。再以PCR得到的第一基因和序列SEQ ID NO. 2为模板，进行重叠PCR（overlap PCR）。经重叠PCR后，可得到第一基因-SEQ ID NO. 2串联的第一串联基因。再利用双酶切位点，将第一串联基因插入到表达载体中，得到第一载体。
其中，可以通过overlap PCR将第一基因串联至SEQ ID NO. 2基因序列的上游，也可以将第一基因串联至SEQ ID NO. 2基因序列的下游。实际应用中，可根据两个基因序列的上下游顺序，设计合适的引物。例如，当决定将第一基因串联至SEQ ID NO. 2基因序列的上游时，可将扩增第一基因序列时使用的上游引物作为overlap PCR时的上游引物，将扩增SEQ ID NO. 2基因序列时使用的下游引物作为overlap PCR时的下游引物，同时使扩增第一基因序列时使用的下游引物和扩增SEQ ID NO. 2基因序列时使用的上游引物之间具有一定的互补序列。当决定将第一基因串联至SEQ ID NO. 2基因序列的下游时，可将扩增第一基因序列时使用的下游引物作为overlap PCR时的下游引物，将扩增SEQ ID NO. 2基因序列时使用的上游引物作为overlap PCR时的上游引物，同时使扩增第一基因序列时使用的上游引物和扩增SEQ ID NO. 2基因序列时使用的下游引物之间具有一定的互补序列。
类似地，以包含有第二基因的质粒为模板，设计相应的上游引物和下游引物，通过PCR扩增获得第二基因。其中，第二基因用于表达第二蛋白。以包含有SEQ ID NO. 3的质粒为模板（例如以质粒pUC57-miRFP670nano为模板），设计相应的上游引物和下游引物，通过PCR扩增获得序列SEQ ID NO. 3。再以PCR得到的第二基因和序列SEQ ID NO. 3为模板，进行overlap PCR。经overlapPCR后，可得到第二基因与SEQ ID NO. 3串联的第二串联基因。再利用双酶切位点，将第二串联基因插入到表达载体中，得到第二载体。
其中，可以通过overlap PCR将第二基因串联至SEQ ID NO. 3基因序列的上游，也可以将第二基因串联至SEQ ID NO. 3基因序列的下游。实际应用中，可根据两个基因序列的上下游顺序，设计合适的引物。例如，当决定将第二基因串联至SEQ ID NO. 3基因序列的上游时，可将扩增第二基因序列时使用的上游引物作为overlap PCR时的上游引物，将扩增SEQ ID NO. 3基因序列时使用的下游引物作为overlap PCR时的下游引物，同时使扩增第二基因序列时使用的下游引物和扩增SEQ ID NO. 3基因序列时使用的上游引物之间具有一定的互补序列。当决定将第二基因串联至SEQ ID NO. 3基因序列的下游时，可将扩增第二基因序列时使用的下游引物作为overlap PCR时的下游引物，将扩增SEQ ID NO. 3基因序列时使用的上游引物作为overlap PCR时的上游引物，同时使扩增第二基因序列时使用的上游引物和扩增SEQ ID NO. 3基因序列时使用的下游引物之间具有一定的互补序列。
可以理解的是，本申请对可以互相作用的第一蛋白和第二蛋白不做限制，例如第一蛋白可以为FKBP蛋白，第二蛋白可以为FRB蛋白；第一蛋白可以为Bak蛋白，第二蛋白可以为Bcl-XL蛋白；第一蛋白可以为bJun蛋白，第二蛋白可以为bFos蛋白等。实际应用中，可以根据具体的研究对象确定第一蛋白和第二蛋白。又或者，在未知两个蛋白是否具有相互作用时，也可采用本申请提供的系统来进行判断作为研究对象的两个蛋白之间是否具有相互作用。
在本申请实施例中，将以bJun蛋白作为第一蛋白，bFos蛋白作为第二蛋白为例进行描述。
首先，构建第一载体。获取包含有bJun基因序列的质粒，例如质粒pbJun-iRN97，设计相应的上游引物bJun-F为：
5’- CTAGCTAGCGCCACCATGAAGGCGGAGAGGAAGCGCATGAGAAACCGC -3’
下游引物bJun-R为：
5’- ACTCCCGCCACCTCCACTCCCGCCACCTCCAAACGTTTGCAACTGCTGCGTTAG -3’。
经过PCR，得到第一基因，即bJun基因序列。
获取包含有可表达miRN107蛋白片段的质粒pUC57-miRFP670nano，设计相应的上游引物miRN107-F为：
5’- GGAGGTGGCGGGAGTGGAGGTGGCGGGAGTATGGCAAACCTGGACAAGATGCTG -3’；
下游引物miRN107-R为：
5’- CCCAAGCTTTTAGCCCTGCAGGATGGGCACGGCC -3’。
经过PCR，得到序列SEQ ID NO. 2，即用于表达miRN107蛋白片段的基因序列。
进一步地，以bJun-F作为上游引物，miRN107-R作为下游引物，对第一基因bJun基因序列和序列SEQ ID NO. 2进行overlap PCR，得到第一串联基因bJun-SEQ ID NO. 2。再利用双酶切位点 NheI和 HindIII，把第一串联基因bJun-SEQ ID NO. 2插入到pcDNA3.1载体的多克隆位点上，构建得到第一载体。
此时，第一载体可表达由bJun蛋白和miRN107蛋白片段构成的第一融合蛋白bJun- miRN107。
然后，构建第二载体。获取包含有bFos基因序列的质粒，例如质粒piRC98-bFos，设计相应的上游引物bFos-F为：
5’- GGAGGTGGCGGGAGTGGAGGTGGCGGGAGTGGTCGTGCGCAGTCCATCGGTCG -3’
下游引物bFos-R为：
5’- CCCAAGCTTTTAACCCAGGTCGTTCGGGATTTTGCACGCCGGACGG -3’。
经过PCR，得到第二基因，即bFos基因序列。
然后，获取包含有可表达miRC108蛋白片段的质粒pUC57-miRFP670nano，设计相应的上游引物miRC108-F为：
5’- CTAGCTAGCGCCACCATGAAGAAGCTGTGGGGCCTGTTGGTG -3’；
下游引物miRC108-R为：
5’- ACTCCCGCCACCTCCACTCCCGCCACCTCCGCTCTGCTGGATGGCGATGCCCACCAC -3’。
经过PCR，得到序列SEQ ID NO. 3，即用于表达miRC108蛋白片段的基因序列。
进一步地，以miRC108-F作为上游引物，bFos-R作为下游引物，对第二基因bFos基因序列和序列SEQ ID NO. 3进行overlap PCR，得到第二串联基因SEQ ID NO. 3-bFos。再利用双酶切位点 NheI和 HindIII，把第二串联基因SEQ ID NO. 3-bFos插入到pcDNA3.1载体的多克隆位点上，构建得到第二载体。
此时，第二载体可表达由bFos蛋白和miRC108蛋白片段构成的第二融合蛋白miRC108-bFos。
进一步地，构建上述光敏色素蛋白miRFP670nano的双分子荧光互补系统之后，可在细胞系以及动物活体中检测该系统的荧光效果。
具体地，构建包含有第一串联基因（第一基因以及序列SEQ ID NO. 2）的第一载体、包含有第二串联基因（序列SEQ ID NO. 3以及第二基因）的第二载体、对照载体、以及参考载体。其中，对照载体为包含有第三基因与序列SEQ ID NO. 3相串联的载体，第三基因表达的蛋白片段可以为任何无法与第一基因序列表达的蛋白片段产生相互作用的蛋白片段。构建对照载体的方法可参考上述的构建包含有第二基因序列以及序列SEQ ID NO. 3的第二载体的方法，将第二基因序列替换为第三基因序列即可，在此不做赘述。参考载体可表达其他荧光蛋白，其他荧光蛋白产生的荧光可作为荧光亮度的内参。
为了方便描述，同样以bJun基因序列作为第一基因和bFos基因序列作为第二基因序列为例，但可以理解是，在实际应用中，可以根据具体的研究对象确定第一基因序列和第二基因序列。在本实施例中，构建的第一载体为bJun-SEQ ID NO. 2载体，构建的第二载体为SEQ ID NO. 3-bFos载体，构建的对照载体为SEQ ID NO. 3-mbFos载体，构建的参考载体为pEGFP载体。其中，对照载体可表达突变的bFos蛋白片段，即mbFos蛋白片段，而mbFos蛋白片段无法与第一载体表达出的bJun蛋白片段进行相互作用，因此可作为对照载体。pEGFP载体可表达EGFP，该EGFP能够在488nm激发光的激发下产生荧光，以作为检测本实施例的双分子荧光互补系统的荧光效果的内参。
基于上述的蛋白片段互补系统，设置实验组和对照组。
向实验组的HEK293T细胞中，转染构建好的第一载体（pbJun-SEQ ID NO. 2）、第二载体（SEQ ID NO. 3 -bFos）以及参考载体（pEGFP），在37℃条件下培养转染后的实验组HEK293T细胞24小时。然后，将实验组的细胞收集，并将细胞接种在裸鼠的皮下。
向对照组的HEK293T细胞中，转染构建好的第一载体（pbJun-SEQ ID NO. 2）、对照载体（SEQ ID NO. 3-mbFos）以及参考载体（pEGFP），在37℃条件下培养转染后的对照组HEK293T细胞24小时。然后，将对照组的细胞收集，并将细胞接种在裸鼠的皮下。
通过图3A可以看出，在激发光的激发下，作为参照地，实验组的HEK293T细胞和对照组的HEK293T细胞均产生了绿色荧光（EGFP发出的荧光）。而实验组的HEK293T细胞产生了红色荧光，但对照组的HEK293T细胞没有产生荧光。
图3B为HEK293T细胞转染了本申请实施例提供的第一载体和第二载体所产生的荧光亮度与HEK293T细胞转染了本申请实施例提供的第一载体和对照载体所产生的荧光亮度的亮度对比图。如图中所示，转染了第一载体bJun-miRN107和第二载体miRC108-bFos的细胞（实验组细胞）产生的荧光亮度显著高于转染了第一载体bJun-miRN107和对照载体miRC108-mbFos的细胞（对照组细胞）产生的荧光亮度，实验组细胞产生的荧光亮度约为对照组细胞产生亮度的13倍。
图3C为HEK293T细胞转染了本申请实施例提供的第一载体和第二载体接种至裸鼠皮下后，裸鼠所产生的荧光亮度，与HEK293T细胞转染了本申请实施例提供的第一载体和对照载体接种至裸鼠皮下后，裸鼠所产生的荧光亮度的亮度对比图。
如图所示，作为内参的EGFP，在相应激发光的激发下，裸鼠的左侧皮下和右侧皮下均能够发出绿色荧光；接种了实验组的HEK293T细胞的裸鼠的左侧皮下能够发出荧光，而接种了对照组的HEK293T细胞的裸鼠的右侧皮下没有荧光发出。
这是由于实验组中的第一载体和第二载体分别表达了bJun-miRN107融合蛋白和miRC108-bFos融合蛋白。其中bJun蛋白和bFos蛋白能够相互作用，使miRN107蛋白片段和miRC108蛋白片段相互靠近，并重构形成完整的光敏色素蛋白miRFP670nano，在相应波长的激发光的激发下，可产生荧光。而在对照组中，第一载体和对照载体分别表达出bJun-miRN107融合蛋白和miRC108-mbFos融合蛋白。由于bJun蛋白和mbFos蛋白不能相互作用，因此，无法使miRN107蛋白片段和miRC108蛋白片段相互靠近以重构成完整的光敏色素蛋白miRFP670nano，从而无法产生荧光。
也就是说，在本申请的基于光敏色素蛋白miRFP670nano的双分子荧光互补系统中，在同一细胞内，表达出第一蛋白与miRN107蛋白片段的融合蛋白以及miRC108蛋白片段与第二蛋白的融合蛋白，通过第一蛋白与第二蛋白的相互作用，可以使miRN107蛋白片段和miRC108蛋白片段靠近从而重构形成完整的、能够在激发光的激发下发出荧光的光敏色素蛋白miRFP670nano。
相比于现有的双分子荧光互补系统，本申请提供一种基于光敏色素蛋白miRFP670nano的双分子荧光互补系统，包括第一载体和第二载体。其中，第一载体为包含有序列SEQ ID NO. 2的载体，第二载体为包含有序列SEQ ID NO. 3的载体。在该系统中，拥有完整构象的光敏色素蛋白miRFP670nano仅由147个氨基酸构成，分子量较小，对待研究的蛋白质间相互作用影响较小。也就是说，若待研究的蛋白质间具有相互作用，较小分子量的光敏色素蛋白miRFP670nano更有利于待研究的蛋白质相互靠近。同时，光敏色素蛋白miRFP670nano能够在生理温度（37℃）条件下产生荧光，有利于检测活细胞及活体内产生的荧光信号。进一步地，光敏色素蛋白miRFP670nano在生物组织的荧光成像过程中，能够产生600nm-1200nm波长的光，具有较好的组织通透性，能够在动物活体中产生较好的成像效果。
以上仅为本申请的较佳实施方式，并非因此限制本申请的专利范围，凡是利用本申请说明书及附图内容所作的等效结构或等效流程变换，或直接或间接运用在其他相关的技术领域，均同理包括在本申请的专利保护范围内。

Claims (11)

  1. 一种基于光敏色素蛋白miRFP670nano的双分子荧光互补系统，其特征在于，包括第一载体和第二载体，其中，所述第一载体为包含有序列SEQ ID NO. 2的载体，所述第二载体为包含有序列SEQ ID NO. 3的载体。
  2. 根据权利要求1所述的双分子荧光互补系统，其特征在于，
    所述序列SEQ ID NO. 2用于表达miRN107蛋白片段，所述序列SEQ ID NO. 3用于表达miRC108蛋白片段；
    当所述miRN107蛋白片段和所述miRC108蛋白片段相互靠近时，可重构形成所述光敏色素蛋白miRFP670nano。
  3. 根据权利要求2所述的双分子荧光互补系统，其特征在于，所述第一载体进一步包括第一基因序列，所述第一基因序列用于表达第一蛋白，所述第一载体用于表达第一融合蛋白，所述第一融合蛋白为所述第一蛋白与所述miRN107蛋白片段的融合蛋白。
  4. 根据权利要求3所述的双分子荧光互补系统，其特征在于，所述miRN107蛋白片段为由所述光敏色素蛋白miRFP670nano的第1号位至第107号位氨基酸构成的蛋白片段。
  5. 根据权利要求2所述的双分子荧光互补系统，其特征在于，所述第二载体进一步包括第二基因序列，所述第二基因序列用于表达第二蛋白，所述第二载体用于表达第二融合蛋白，所述第二融合蛋白为所述第二蛋白与所述miRC108蛋白片段的融合蛋白。
  6. 根据权利要求5所述的双分子荧光互补系统，其特征在于，所述miRC108蛋白片段为由所述光敏色素蛋白miRFP670nano的第108号位至第147号位氨基酸构成的蛋白片段。
  7. 一种基于光敏色素蛋白miRFP670nano的双分子荧光互补系统的构建方法，其特征在于，包括：
    以所述光敏色素蛋白miRFP670nano的基因序列为模板进行聚合酶链式反应得到序列SEQ ID NO. 2和序列SEQ ID NO. 3；
    利用第一质粒的双酶切位点，将所述序列SEQ ID NO. 2插入所述第一质粒的多克隆位点，以得到第一载体；
    利用第二质粒的双酶切位点，将所述序列SEQ ID NO. 3插入所述第二质粒的多克隆位点，以得到第二载体。
  8. 根据权利要求7所述的方法，其特征在于，在所述利用第一质粒的双酶切位点，将所述序列SEQ ID NO. 2插入所述第一质粒的多克隆位点之前，所述方法还包括：
    通过PCR，获得用于表达第一蛋白的第一基因序列以及用于表达第二蛋白的第二基因序列；
    通过重叠PCR，获得第一基因序列与序列SEQ ID NO. 2串联的第一串联基因；
    通过重叠PCR，获得第二基因序列与序列SEQ ID NO. 3串联的第二串联基因。
  9. 根据权利要求8所述的方法，其特征在于，
    所述利用第一质粒的双酶切位点，将所述序列SEQ ID NO. 2插入所述第一质粒的多克隆位点，包括：利用第一质粒的双酶切位点，将所述第一串联基因插入所述第一质粒的多克隆位点；
    所述利用第二质粒的双酶切位点，将所述序列SEQ ID NO. 3插入所述第二质粒的多克隆位点，包括：利用第二质粒的双酶切位点，将所述第二串联基因插入所述第二质粒的多克隆位点。
  10. 根据权利要求9所述的方法，其特征在于，
    所述序列SEQ ID NO. 2用于表达所述光敏色素蛋白miRFP670nano的miRN107蛋白片段，所述miRN107蛋白片段为由所述光敏色素蛋白miRFP670nano第1号位至第107号位氨基酸构成的蛋白片段；
    所述第一载体用于表达所述第一蛋白和所述miRN107蛋白片段的融合蛋白；
    所述序列SEQ ID NO. 3用于表达所述光敏色素蛋白miRFP670nano的miRC108蛋白片段，所述miRC108蛋白片段为由所述光敏色素蛋白miRFP670nano第108号位至第147号位氨基酸构成的蛋白片段；
    所述第二载体用于表达所述第二蛋白和所述miRC108蛋白片段的融合蛋白。
  11. 一种基于光敏色素蛋白miRFP670nano的双分子荧光互补系统在蛋白质间相互作用的成像应用，其特征在于，所述基于光敏色素蛋白miRFP670nano的双分子荧光互补系统为如权利要求1-6中任意一项所述的基于光敏色素蛋白miRFP670nano的双分子荧光互补系统。
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