阅读说明：本技术 串联片段荧光互补系统及其构建方法及应用 (Tandem fragment fluorescence complementary system and construction method and application thereof ) 是由 陈明海 张先恩 严闯 于 2020-11-09 设计创作，主要内容包括：本申请涉及蛋白质相互作用成像技术领域,本申请提供一种串联片段荧光互补系统及其构建方法及应用。该系统包括第一载体和第二载体。第一载体为包含有序列SEQ ID NO.2和第一基因序列的载体,第二载体为包含有序列SEQ ID NO.3和第二基因序列的载体。利用该系统,可以在同一细胞内共表达第一蛋白-IFN132-IFN132形成的融合蛋白以及IFC133-IFC133-第二蛋白形成的融合蛋白。在第一蛋白和第二蛋白的相互作用下,IFN132-IFN132串联片段和IFC133-IFC133串联片段相互靠近以重构成近红外光敏色素蛋白IFP2.0并被激发以发出荧光。重构的近红外光敏色素蛋白IFP2.0能够在生理温度下成熟,并且发出的荧光亮度更强,有利于成像观察。(The application relates to the technical field of protein interaction imaging, and provides a tandem fragment fluorescence complementation system and a construction method and application thereof. The system includes a first carrier and a second carrier. The first vector is a vector containing a sequence SEQ ID NO.2 and a first gene sequence, and the second vector is a vector containing a sequence SEQ ID NO.3 and a second gene sequence. By using the system, the fusion protein formed by the first protein IFN132-IFN132 and the fusion protein formed by the IFC133-IFC 133-second protein can be co-expressed in the same cell. Under the interaction of the first protein and the second protein, the IFN132-IFN132 tandem fragment and the IFC133-IFC133 tandem fragment are brought into close proximity to reconstitute into the near-infrared photopigment protein IFP2.0 and excited to fluoresce. The reconstructed near-infrared light-sensitive pigment protein IFP2.0 can be matured at physiological temperature, and the emitted fluorescence has stronger brightness, thereby being beneficial to imaging observation.)

1. A tandem fragment fluorescence complementation system based on near-infrared light-sensitive pigment protein IFP2.0 is characterized by comprising a first carrier and a second carrier, wherein the first carrier is a carrier containing a sequence SEQ ID NO.2, and the second carrier is a carrier containing a sequence SEQ ID NO. 3.

2. The tandem fragment fluorescent complementation system of claim 1,

the sequence SEQ ID NO.2 is used for expressing IFN132-IFN132 tandem fragments, and the sequence SEQ ID NO.3 is used for expressing IFC133-IFC133 tandem fragments;

when the IFN132-IFN132 serial fragment and the IFC133-IFC133 serial fragment are close to each other, the near-infrared photosensitive chromoprotein IFP2.0 can be reconstructed.

3. The tandem fragment fluorescence complementation system of claim 2, wherein the first vector further comprises a first gene sequence for expressing a first protein and a first vector for expressing a first fusion protein, wherein the first fusion protein is a fusion protein of the IFN132-IFN132 tandem fragment and the first protein.

4. The tandem fragment fluorescence complementation system of claim 3, wherein the IFN132-IFN132 tandem fragment is a protein fragment formed by two IFN132 fragments in tandem, and the IFN132 fragment is a protein fragment formed by the amino acids from the 1 st to the 132 nd of the photopigment protein IFP 2.0.

5. The tandem fragment fluorescent complementation system of claim 2, wherein the second vector further comprises a second gene sequence for expressing a second protein, and the second vector is used for expressing a second fusion protein, wherein the second fusion protein is a fusion protein of the IFC133-IFC133 tandem fragment and the second protein.

6. The tandem fragment fluorescent complementation system of claim 5, wherein the IFC133-IFC133 tandem fragment is a protein fragment formed by tandem connection of two IFC133 fragments, and the IFC133 fragment is a protein fragment formed by the amino acids from position 133 to position 321 of the photopigment protein IFP 2.0.

7. A method for constructing a series fragment fluorescence complementation system based on near-infrared photosensitive pigment protein IFP2.0 is characterized by comprising the following steps:

performing Polymerase Chain Reaction (PCR) by using the gene sequence of the near-infrared light-sensitive pigment protein IFP2.0 as a template to obtain a sequence SEQ ID NO.2 and a sequence SEQ ID NO. 3;

inserting the sequence SEQ ID NO.2 into the multiple cloning site of the first plasmid by using the double restriction enzyme sites of the first plasmid;

and (2) inserting the sequence SEQ ID NO.3 into the multiple cloning site of the second plasmid by using the double restriction enzyme sites of the second plasmid.

8. The method of claim 7, further comprising:

inserting a nucleotide sequence for expressing a first protein into another multiple cloning site of the first plasmid by using another group of double enzyme cutting sites of the first plasmid to obtain a first vector, wherein the nucleotide sequence for expressing the first protein is positioned at the upstream of the sequence SEQ ID NO. 2;

and inserting a nucleotide sequence for expressing a second protein into another multiple cloning site of the second plasmid by using another group of double enzyme cutting sites of the second plasmid to obtain a second vector, wherein the nucleotide sequence for expressing the second protein is positioned at the downstream of the sequence SEQ ID NO. 3.

9. The method of claim 8,

the sequence SEQ ID NO.2 is used for expressing IFN132-IFN132 tandem fragment, the IFN132-IFN132 tandem fragment is a protein fragment formed by connecting two IFN132 fragments in series, and the IFN132 fragment is a protein fragment formed by amino acids from the 1 st position to the 132 th position of the photosensitive pigment protein IFP 2.0;

the first vector is used for expressing the fusion protein of the IFN132-IFN132 tandem fragment and the first protein;

the sequence SEQ ID NO.3 is used for expressing IFC133-IFC133 serial fragments, the IFC133-IFC133 serial fragments are protein fragments formed by connecting two IFC133 fragments in series, and the IFC133 fragments are protein fragments formed by amino acids from No. 133 to No. 321 of the photopigment protein IFP 2.0;

said second vector is for expressing a fusion protein of said IFC133-IFC133 tandem fragment and said second protein; and

said first protein and said second protein are interacting proteins for bringing said IFN132-IFN132 tandem fragment into proximity with said IFC133-IFC133 tandem fragment.

10. Use of a tandem fragment fluorescence complementation system according to any of the claims 1-7 based on the photopigment protein IFP2.0, characterized in that: the series fragment fluorescence complementation system based on the photopigment protein IFP2.0 is applied to imaging the protein interaction in living bodies and living cells.

Technical Field

The application relates to the technical field of protein interaction imaging, in particular to a tandem fragment fluorescence complementation system based on near-infrared photosensitive pigment protein IFP2.0, a construction method of the system and application.

Background

The interaction between proteins plays an important role in the life process of an organism. For example, many protein interactions are involved in gene regulation, cell signaling, and tumor growth. Monitoring the interaction between these proteins is particularly important for the analysis of life processes. Several fluorescence imaging based methods have been developed and used to study protein-protein interactions over the past decades, such as: fluorescence Resonance Energy Transfer (FRET), imaging techniques based on singlet oxygen triplet energy transfer, and Bimolecular fluorescence complementation (BiFC), among others.

The bimolecular fluorescence complementary system is a fragment complementary system taking fluorescent protein as a material. The basic principle is to split the fluorescent protein into two fragments, each of which is not fluorescent, at the appropriate site of the protein. When two interacting proteins are fused with the two separated fragments which do not fluoresce, the two separated fragments which do not fluoresce will approach each other under the action of the two interacting proteins, thereby recovering the conformation of the complete fluorescent protein and emitting specific fluorescence. The bimolecular fluorescence complementation technology is a simple, visual and sensitive method for detecting the interaction between proteins, and has attracted more and more attention in recent years.

Currently developed bimolecular fluorescence complementation systems include Green Fluorescent Protein (GFP) -based fluorescence complementation systems. However, the fluorescent complementation system of GFP requires low temperature to generate mature complete fluorescent protein and emit fluorescence, thereby limiting the application of the system under physiological conditions. In addition, in the fluorescence imaging process of biological tissues, light with the wavelength of 600nm-1200nm has better tissue permeability and can generate better imaging effect in animal living bodies. The fluorescent complementary system of GFP generates fluorescence with shorter wavelength, usually less than 600nm, thereby limiting the application of GFP-based fluorescent fragment complementary system in vivo imaging of animals.

The currently developed bimolecular fluorescence complementary system also includes a near-infrared light-sensitive chromoprotein-based fluorescence complementary system. Near-infrared photopigment proteins are proteins that absorb infrared or near-infrared light, are capable of maturing under physiological conditions (37 ℃) and produce fluorescence at wavelengths greater than 700 nm. However, the complementary fluorescence intensity generated by the fluorescence complementary system is weak due to the low fluorescence intensity of the photosensitizing chromoprotein, which is not favorable for imaging observation.

Disclosure of Invention

The main purpose of the present application is to provide a series fragment fluorescence complementary system based on near-infrared photosensitive pigment protein IFP2.0, a method for constructing a series fragment fluorescence complementary system based on near-infrared photosensitive pigment protein IFP2.0, and an application of a series fragment fluorescence complementary system based on near-infrared photosensitive pigment protein IFP2.0, which can generate strong complementary fluorescence intensity and is beneficial to imaging observation.

In order to solve the above technical problem, an embodiment of the present application provides a tandem fragment fluorescence complementation system based on near-infrared photopigment protein IFP2.0, including a first vector and a second vector, where the first vector is a vector including a sequence SEQ ID No.2, and the second vector is a vector including a sequence SEQ ID No. 3.

Wherein, the sequence SEQ ID NO.2 is used for expressing IFN132-IFN132 tandem fragment, and the sequence SEQ ID NO.3 is used for expressing IFC133-IFC133 tandem fragment. When the IFN132-IFN132 serial fragment and the IFC133-IFC133 serial fragment are close to each other, the near-infrared photosensitive chromoprotein IFP2.0 can be reconstructed.

Wherein, the first vector further comprises a first gene sequence used for expressing a first protein, the first vector is used for expressing a first fusion protein, and the first fusion protein is a fusion protein of IFN132-IFN132 tandem fragment and the first protein.

Wherein, the IFN132-IFN132 tandem fragment is a protein fragment formed by tandem connection of two IFN132 fragments, and the IFN132 fragment is a protein fragment formed by amino acids from the 1 st position to the 132 th position of the photopigment protein IFP 2.0.

Wherein, the second vector further comprises a second gene sequence used for expressing a second protein, the second vector is used for expressing a second fusion protein, and the second fusion protein is a fusion protein of the IFC133-IFC133 serial fragment and the second protein.

Wherein, IFC133-IFC133 tandem fragment is a protein fragment formed by two IFC133 fragments in tandem, and IFC133 fragment is a protein fragment formed by amino acids from No. 133 to No. 321 of the photopigment protein IFP 2.0.

In order to solve the above technical problem, an embodiment of the present application provides a method for constructing a tandem fragment fluorescence complementation system based on near-infrared photopigment protein IFP2.0, including: performing Polymerase Chain Reaction (PCR) by using the gene sequence of the near-infrared light-sensitive pigment protein IFP2.0 as a template to obtain a sequence SEQ ID NO.2 and a sequence SEQ ID NO. 3; inserting the sequence SEQ ID NO.2 into the multiple cloning site of the first plasmid by using the double restriction enzyme sites of the first plasmid; the sequence SEQ ID NO.3 was inserted into the multiple cloning site of the second plasmid using the double restriction sites of the second plasmid.

Wherein, the method also comprises: inserting a nucleotide sequence for expressing a first protein into another multiple cloning site of the first plasmid by using another group of double enzyme cutting sites of the first plasmid to obtain a first vector, wherein the nucleotide sequence for expressing the first protein is positioned at the upstream of the sequence SEQ ID NO. 2; and inserting the nucleotide sequence for expressing the second protein into another multiple cloning site of the second plasmid by using another group of double enzyme cutting sites of the second plasmid to obtain a second vector, wherein the nucleotide sequence for expressing the second protein is positioned at the downstream of the sequence SEQ ID NO. 3.

Wherein, the sequence SEQ ID NO.2 is used for expressing IFN132-IFN132 serial fragments, the IFN132-IFN132 serial fragments are protein fragments formed by connecting two IFN132 fragments in series, and the IFN132 fragments are protein fragments formed by amino acids from the No.1 to the No. 132 of the photosensitive pigment protein IFP 2.0; the first vector is used for expressing the fusion protein of the IFN132-IFN132 tandem fragment and the first protein; the sequence SEQ ID NO.3 is used for expressing IFC133-IFC133 serial fragments, the IFC133-IFC133 serial fragments are protein fragments formed by connecting two IFC133 fragments in series, and the IFC133 fragments are protein fragments formed by amino acids from No. 133 to No. 321 of the photopigment protein IFP 2.0; the second vector is used for expressing the IFC133-IFC133 serial fragment and the fusion protein of the second protein; the first protein and said second protein are interacting proteins for bringing an IFN132-IFN132 tandem fragment into proximity with an IFC133-IFC133 tandem fragment.

In order to solve the above technical problem, an embodiment of the present application adopts a technical solution of providing an application of the tandem fragment fluorescence complementation system based on the photopigment protein IFP2.0 as described above. Wherein, the tandem fragment fluorescence complementation system based on the photopigment protein IFP2.0 is applied to imaging the protein interaction in living bodies and/or living cells.

Compared with the existing bimolecular fluorescence complementary system, the application provides a tandem fragment fluorescence complementary system based on near-infrared photosensitive pigment protein IFP2.0, which comprises a first carrier and a second carrier. Wherein, the first carrier is a carrier containing a sequence SEQ ID NO.2, and the second carrier is a carrier containing a sequence SEQ ID NO. 3. The system is a fluorescence complementary system based on near-infrared photosensitive pigment protein IFP2.0, and the near-infrared photosensitive pigment protein IFP2.0 can be excited to generate fluorescence with longer wavelength, so that the fluorescence generated by the system has better tissue permeability. And the reconstructed near-infrared light-sensitive pigment protein IFP2.0 can be matured at physiological temperature, so that the interaction between proteins can be researched under the normal physiological temperature condition of cells. Furthermore, the sequence SEQ ID NO.2 can express an IFN132-IFN132 tandem fragment, the sequence SEQ ID NO.3 can express an IFC133-IFC133 tandem fragment, and the IFN132-IFN132 tandem fragment and the IFC133-IFC133 tandem fragment respectively comprise two IFN132 protein fragments and two IFC133 protein fragments, so that the fluorescence brightness emitted by the reconstructed near-infrared photosensitive pigment protein IFP2.0 is stronger, and imaging observation is facilitated.

Drawings

The present application will now be described with reference to the accompanying drawings. The drawings in the present application are for the purpose of illustrating embodiments only. Other embodiments according to the steps described below can be readily made by those skilled in the art without departing from the principles of the present invention.

FIG. 1 is a schematic diagram of the vector pcDNA3.1-IFP2.0 provided in the examples of the present application.

FIG. 2A is a schematic diagram of a first vector comprising a sequence of SEQ ID NO.2 for expression of IFN132-IFN132 tandem fragment provided in the examples of the present application.

FIG. 2B is a schematic diagram of a second vector comprising the sequence SEQ ID NO.3 for expressing IFC133-IFC133 tandem fragment provided in the examples herein.

FIG. 3A is a schematic diagram of a first vector comprising a fusion protein for expression of bJun-IFN132-IFN132 provided in the examples herein.

FIG. 3B is a schematic diagram of a second vector comprising a fusion protein for expressing IFC133-IFC133-bFos provided in the examples herein.

Fig. 4A is a schematic diagram of fluorescence emission of a tandem fragment fluorescence complementation system based on near-infrared photopigment protein IFP2.0 in HEK293T cells, provided in the examples of the present application.

Fig. 4B is a fluorescence efficiency statistical diagram of a tandem fragment fluorescence complementation system based on near-infrared photopigment protein IFP2.0 provided in the present application.

FIG. 4C is a schematic diagram of the tandem fragment fluorescence complementation system based on near infrared photopigment protein IFP2.0 to generate fluorescence in animal living body according to the embodiment of the present application.

Detailed Description

The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. It is to be understood that the specific embodiments described herein are merely illustrative of the application and are not limiting of the application. It should be further noted that, for the convenience of description, only some of the structures related to the present application are shown in the drawings, not all of the structures. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.

The terms "first", "second", etc. in this application are used to distinguish between different objects and not to describe a particular order. Furthermore, the terms "include" and "have," as well as any variations thereof, are intended to cover non-exclusive inclusions. For example, a process, method, system, article, or apparatus that comprises a list of steps or elements is not limited to only those steps or elements listed, but may alternatively include other steps or elements not listed, or inherent to such process, method, article, or apparatus.

Reference herein to "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment can be included in at least one embodiment of the application. The appearances of the phrase in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments. It is explicitly and implicitly understood by one skilled in the art that the embodiments described herein can be combined with other embodiments.

The application provides a tandem fragment fluorescence complementation system based on a photopigment protein IFP2.0, which comprises a first carrier and a second carrier, wherein the first carrier is a carrier containing a sequence SEQ ID NO.2, and the second carrier is a carrier containing a sequence SEQ ID NO. 3.

Specifically, as shown in FIG. 1, FIG. 1 is a schematic diagram of the vector pcDNA3.1-IFP 2.0. The vector pcDNA3.1-IFP2.0 is a vector comprising a sequence SEQ ID NO.1, wherein the sequence SEQ ID NO.1 is a sequence for expressing the near infrared photopigment protein IFP2.0, the near infrared photopigment protein IFP2.0 is a protein formed by 321 amino acids, and the amino acid sequence of the protein is shown as a sequence SEQ ID NO. 4. The vector pcDNA3.1-IFP2.0 can be obtained by inserting the sequence SEQ ID NO.1 into the multiple cloning site of the plasmid pcDNA3.1 through the double-enzyme cleavage site. It is understood that the sequence SEQ ID NO.1 can also be inserted into other eukaryotic expression vectors, such as pEGFP-C1, pEGFP-N1, etc., and the sequence SEQ ID NO.1 can be inserted into different eukaryotic expression vectors by selecting an appropriate double-enzyme cleavage site. In the examples of the present application, the vector pcDNA3.1-IFP2.0 is commercially available (Jinzhi Biotech, Suzhou) in which the sequence SEQ ID NO.1 is inserted into the plasmid pcDNA3.1 via NheI and HindIII cleavage sites.

In the present example, the near infrared photopigment protein IFP2.0 will be split between amino acid 132 and amino acid 133 into two protein fragments, IFN132 fragment and IFC133 fragment, respectively. Wherein, the IFN132 fragment can be understood as a protein fragment containing the amino acids from the 1 st to the 132 th positions of the near-infrared light-sensitive pigment protein IFP2.0 (i.e. the sequence shown in SEQ ID NO. 4); the IFC133 fragment is understood to be a protein fragment comprising the amino acids from position 133 to position 321 of the near infrared photopigment protein IFP 2.0.

After the vector pcDNA3.1-IFP2.0 is obtained, the sequence SEQ ID NO.2 can be obtained by amplification through Polymerase Chain Reaction (PCR) by taking the vector as a template. Wherein, the first upstream primer used for PCR amplification is:

5’-GGGGTACCATGGCTCGGGACCCTCAACCTTTCTTCC-3’，

the first downstream primer is:

5’-TCCCGCCACCTCCACTCCCGCCACCTCCCCATGCCTCAGTAGGCTCAATTCCAGAATC-3’；

the second upstream primer is:

5’-GGAGGTGGCGGGAGTGGAGGTGGCGGGAGTATGGCTCGGGACCCTCAACCTTTCTTC-3’，

the second downstream primer is:

5’-CGGGATCCTTACCATGCCTCAGTAGGCTCGAATTCCAG-3’。

the sequence SEQ ID NO.2 obtained by PCR amplification is a sequence formed by connecting two nucleotide sequences capable of expressing IFN132 fragments in series. That is, the sequence SEQ ID NO.2 is capable of expressing two IFN132 fragments in tandem, i.e., IFN132-IFN132 tandem.

Furthermore, the sequence SEQ ID NO.3 can also be obtained by PCR using the vector pcDNA3.1-IFP2.0 as a template. The third upstream primer used for PCR amplification in this case is:

5’-CTAGCTAGCGCCACCATGGACTCTATTGGACCCCACGCTCTGAG-3’，

the third downstream primer is:

5’-ACTCCCGCCACCTCCACTCCCGCCACCTCCGGCTTCTTTCCTCTGCACCTGCAGGGAC-3’；

the fourth upstream primer is:

5’-GGAGGTGGCGGGAGTGGAGGTGGCGGGAGTGACTCTATTGGACCCCACGCTCTGAG-3’，

the fourth downstream primer is:

5’-CCCAAGCTTGGCTTCTTTCCTCTGCACCTGCAGGGACAGGAGC-3’。

in this case, the sequence SEQ ID NO.3 amplified by PCR is a sequence in which two nucleotide sequences capable of expressing IFC133 fragments are connected in series. That is, the sequence SEQ ID NO.3 is capable of expressing two tandem IFC133 fragments, namely IFC133-IFC133 tandem fragments.

Further, a sequence SEQ ID NO.2 for expressing the IFN132-IFN132 tandem fragment and a sequence SEQ ID NO.3 for expressing the IFC133-IFC133 tandem fragment are respectively inserted into two eukaryotic expression vectors to construct a first vector containing the sequence SEQ ID NO.2 and a second vector containing the sequence SEQ ID NO. 3. The eukaryotic expression vector is not limited in the present application, and may be, for example, pEGFP-C1, pEGFP-N1, pcDNA3.1 or other eukaryotic expression vectors. This example will be described taking plasmid pcDNA3.1 as an example.

Specifically, as shown in FIG. 2A, FIG. 2A is a schematic diagram of a first vector comprising SEQ ID NO. 2. In the embodiment of the application, the sequence SEQ ID NO.2 for expressing the IFN132-IFN132 tandem fragment is inserted into the multiple cloning site of pcDNA3.1 by using double restriction sites KpnI and BamHI to construct a first vector containing the sequence SEQ ID NO. 2.

As shown in FIG. 2B, FIG. 2B is a schematic diagram of a second vector comprising SEQ ID NO. 3. In the embodiment of the application, a second vector containing a sequence SEQ ID NO.3 is constructed by inserting the sequence SEQ ID NO.3 for expressing the IFC133-IFC133 serial fragment into the multiple cloning site of pcDNA3.1 by utilizing double enzyme cutting sites NheI and HindIII.

Further, the first vector also includes a sequence for expressing the first protein, and the second vector also includes a sequence for expressing the second protein. Thus, the first vector may express a first fusion protein of the first protein and an IFN132-IFN132 tandem fragment; the second vector can express a second fusion protein formed by a second protein and an IFC133-IFC133 tandem fragment. Wherein the first protein and the second protein are two interacting proteins.

Specifically, a plasmid containing a first gene sequence is used as a template, corresponding upstream primers and downstream primers are designed, and the first gene sequence is obtained through PCR amplification. Wherein the first gene sequence is for expressing a first protein. And then the first gene sequence is inserted into the multiple cloning site of the first vector by utilizing the double-enzyme cutting site. The first vector constructed at this time can express the first protein IFN132 first fusion protein. It should be noted that the position of insertion of the first gene sequence on the first vector should be upstream of the sequence SEQ ID NO. 2. Thus, in a first fusion protein expressed from a first vector, the first protein can be located at the nitrogen terminus of the IFN132-IFN132 tandem.

Similarly, a plasmid containing the second gene sequence is used as a template, corresponding upstream primers and downstream primers are designed, and the second gene sequence is obtained through PCR amplification. Wherein the second gene sequence is for expressing a second protein. And then the second gene sequence is inserted into the multiple cloning site of the second vector by utilizing the double-enzyme cutting site. The second vector constructed at this time can express a second fusion protein formed by IFC133-IFC 133-second protein. It should be noted that the position of insertion of the second gene sequence on the second vector should be downstream of the sequence SEQ ID NO. 3. Thus, in a second fusion protein expressed from a second vector, the second protein can be located at the carbon-terminus of the IFC133-IFC133 tandem fragment.

It is understood that the application is not limited to a first protein and a second protein that can interact, for example, the first protein can be an FKBP protein and the second protein can be an FRB protein; the first protein can be a Bak protein and the second protein can be a Bcl-XL protein; the first protein may be a bJun protein, the second protein may be a bFos protein, and so forth. In practice, the first protein and the second protein may be determined for a particular subject.

In the examples of the present application, the bJun protein is taken as a first protein, and the bFos protein is taken as a second protein. Obtaining a plasmid pbJun-iRN97 containing a bJun gene sequence, and designing corresponding upstream primers as follows:

5’-CTAGCTAGCGCCACCATGAAGGCGGAGAGGAAGCGCATGAGAAACCGC-3’,

the downstream primer is:

5’-CCCAAGCTTAAACGTTTGCAACTGCTGCGTTAGCATGAG-3’。

the first vector containing the bJun gene sequence and the sequence SEQ ID NO.2 is constructed by inserting the bJun gene sequence into the multiple cloning site of the first vector containing the sequence SEQ ID NO.2 by using double restriction sites NheI and HindIII, as shown in FIG. 3A. In this example, the sequence of the bJun gene is located upstream of the sequence SEQ ID NO. 2. Thus, the first vector can express a first fusion protein bJun-IFN132-IFN132 consisting of a tandem fragment of the bJun protein and IFN132-IFN 132.

Meanwhile, plasmid piRC98-bFos containing the bFos gene sequence is obtained, and corresponding upstream primers are designed as follows:

5’-CGGGATCCGGTCGTGCGCAGTCCATCGGTCGTCG-3’,

the downstream primer is:

5’-CGGAATTCTTAACCCAGGTCGTTCGGGATTTTGCACGCCGGACGG-3’。

the second vector comprising the bFos gene sequence and the sequence of SEQ ID NO.3 was constructed by inserting the bFos gene sequence into the multiple cloning site of the second vector comprising the sequence of SEQ ID NO.3 using BamHI and EcoRI, as shown in FIG. 3B. In this example, the bFos gene sequence is located downstream of the sequence SEQ ID No. 3. Thus, the second vector can express a second fusion protein IFC133-IFC133-bFos consisting of the IFC133-IFC133 tandem fragment and the bFos protein.

Further, after the tandem fragment fluorescence complementation system based on the photopigment protein IFP2.0 is constructed, the fluorescence effect of the system can be detected in a cell line and an animal living body.

Specifically, a first vector containing a first gene sequence and a sequence SEQ ID NO.2, a second vector containing a second gene sequence and a sequence SEQ ID NO.3, a control vector and a reference vector are constructed. Wherein, the contrast carrier is a carrier containing a third gene sequence and a sequence SEQ ID NO.3, and the protein segment expressed by the third gene sequence can be any protein segment which can not generate interaction with the protein segment expressed by the first gene sequence. The method for constructing the control vector can refer to the method for constructing the second vector containing the second gene sequence and the sequence SEQ ID No.3, and the second gene sequence is replaced by the third gene sequence, which is not described herein again. The reference vector can express other fluorescent proteins, and fluorescence generated by other fluorescent proteins can be used as an internal reference of fluorescence brightness.

For convenience of description, the first gene sequence is also referred to as a bJun gene sequence and the second gene sequence is also referred to as a bFos gene sequence, but it is understood that in practical applications, the first gene sequence and the second gene sequence may be determined according to a specific subject. In this example, the first vector constructed was the bJun-IFN132-IFN132 vector, the second vector constructed was the IFC133-IFC133-bFos vector, the control vector constructed was the IFC133-IFC133-mbFos vector, and the reference vector constructed was the pEGFP vector. Wherein the control vector can express a mutant bFos protein fragment, namely an mbFos protein fragment, and the mbFos protein fragment cannot interact with the bJun protein fragment expressed by the first vector. The pEGFP vector can express EGFP, and the EGFP can generate fluorescence under the excitation of 488nm exciting light to be used as an internal reference for detecting the fluorescence effect of the tandem fragment fluorescence complementation system of the embodiment.

Based on the tandem fragment fluorescence complementation system, an experimental group and a control group are set.

The constructed first vector (pbJun-IFN132-IFN132), second vector (pIFC133-IFC133-bFos) and reference vector (pEGFP) were transfected into experimental HEK293T cells, and the transfected experimental HEK293T cells were cultured at 37 ℃ for 24 hours. The first vector (pbJun-IFN132-IFN132), the control vector (pIFC133-IFC133-mbFos) and the reference vector (pEGFP) were transfected into the HEK293T cells of the control group, and the transfected HEK293T cells of the control group were cultured at 37 ℃ for 24 hours.

Further, excitation light with a wavelength of 640nm was used to excite experimental HEK293T cells and control HEK293T cells; exciting experimental HEK293T cells and control HEK293T cells by using 488nm excitation light; in addition, Hoechst staining was performed on experimental HEK293T cells and control HEK293T cells for localization of the nucleus.

As can be seen from fig. 4A, under excitation with 640nm excitation light, the HEK293T cells of the experimental group produced red fluorescence, while the HEK293T cells of the control group did not produce fluorescence. For reference, the experimental group of HEK293T cells and the control group of HEK293T cells both produced green fluorescence under excitation with 488nm excitation light.

This is due to the fact that the first and second vectors in the experimental group produced a bJun-IFN132-IFN132 fusion protein and an IFC133-IFC133-bFos fusion protein, respectively. Wherein the bJun protein and the bFos protein are capable of interacting such that the IFN132-IFN132 tandem fragment and the IFC133-IFC133 tandem fragment are in proximity to each other and are reconfigured to form the complete photopigment protein IFP2.0 that fluoresces upon excitation by excitation light of the corresponding wavelengths. In the control group, the first vector and the control vector respectively express a bJun-IFN132-IFN132 fusion protein and an IFC133-IFC133-mbFos fusion protein. Since the bJun and mbFos proteins do not interact, the IFN132-IFN132 tandem and the IFC133-IFC133 tandem cannot be brought into close proximity to reconstitute the complete photopigment protein IFP2.0, and thus do not produce fluorescence.

That is, in the tandem fragment fluorescence complementation system based on near-infrared photopigment protein IFP2.0 of the present application, a fusion protein of a first protein and an IFN132-IFN132 tandem fragment and a fusion protein of an IFC133-IFC133 tandem fragment and a second protein are expressed in the same cell, and through the interaction between the first protein and the second protein, the IFN132-IFN132 tandem fragment and the IFC133-IFC133 tandem fragment can be brought close to each other to reconstruct and form a complete photopigment protein IFP2.0 capable of emitting fluorescence under the excitation of excitation light.

Further, to examine the fluorescence efficiency of the tandem-fragment fluorescence complementation system provided herein, the fluorescence intensity produced by the photopigment protein IFP2.0 reconstituted by fluorescent molecule complementation in the examples of the present application was compared with the fluorescence intensity produced by EGFP expressed from the reference vector. For example, corresponding regions in 640nm and 488nm excitation channels can be selected from the pictures of HEK293T cells in the experimental group in fig. 4A, and the intensity of red fluorescence and the intensity of green fluorescence can be compared by using the picture processing software. It is to be understood that the present application is not limited to the image processing software for analyzing fluorescence intensity.

FIG. 4B shows a statistical plot of the fluorescent complementation efficiency generated by the tandem fragment fluorescent complementation system based on near-infrared photopigment protein IFP 2.0. As can be seen from the figure, the fluorescence intensity produced by the cells capable of achieving the reconstruction of the photopigment protein IFP2.0 by the tandem-fragment fluorescence complementation system is significantly higher than the fluorescence intensity produced by the cells incapable of achieving the reconstruction of the photopigment protein IFP2.0 by the tandem-fragment fluorescence complementation system.

The application further detects the effect of fluorescence generated by the series fragment fluorescence complementation system based on the near-infrared photosensitive pigment protein IFP2.0 in the living animal body.

Specifically, the first vector (pbJun-IFN132-IFN132), the second vector (pIFC133-IFC133-bFos) and the reference vector (pEGFP) were transfected into the HEK293T cells, and the transfected HEK293T cells were cultured at 37 ℃ for 24 hours. The first vector (pbJun-IFN132-IFN132), the control vector (pIFC133-IFC133-mbFos) and the reference vector (pEGFP) were transfected into HEK293T cells of the control group, and the transfected HEK293T cells of the control group were cultured at 37 ℃ for 24 hours.

The HEK293T cells of the experimental group and the control group were collected and inoculated subcutaneously into nude mice, respectively. Among them, the experimental group HEK293T cells were inoculated subcutaneously into the left side of nude mice, and the control group HEK293T cells were inoculated subcutaneously into the right side of the same nude mice.

FIG. 4C is a schematic diagram of the fluorescence generation in vivo in animals based on the tandem fragment fluorescence complementation system of near-infrared photopigment protein IFP 2.0.

As shown in FIG. 4C, under the excitation of 488nm excitation light, the left subcutaneous side and the right subcutaneous side of the nude mouse can emit green fluorescence. Under excitation of 640nm excitation light, red fluorescence was emitted subcutaneously from the left side of nude mice inoculated with experimental group HEK293T cells, while no fluorescence was emitted subcutaneously from the right side of nude mice inoculated with control group HEK293T cells.

This is due to the fact that the first and second vectors in the experimental group produced a bJun-IFN132-IFN132 fusion protein and an IFC133-IFC133-bFos fusion protein, respectively. Wherein the bJun protein and the bFos protein are capable of interacting such that the IFN132-IFN132 tandem fragment and the IFC133-IFC133 tandem fragment are in proximity to each other and are reconfigured to form the complete photopigment protein IFP2.0 that fluoresces upon excitation by excitation light of the corresponding wavelengths. In the control group, the first vector and the control vector respectively express a bJun-IFN132-IFN132 fusion protein and an IFC133-IFC133-mbFos fusion protein. Since the bJun and mbFos proteins do not interact, the IFN132-IFN132 tandem and the IFC133-IFC133 tandem cannot be brought into close proximity to reconstitute the complete photopigment protein IFP2.0, and thus do not produce fluorescence.

That is, in the tandem fragment fluorescence complementation system based on near-infrared photopigment protein IFP2.0 of the present application, a fusion protein of a first protein and an IFN132-IFN132 tandem fragment and a fusion protein of IFC133-IFC133 and a second protein are expressed in the same cell, and through the interaction between the first protein and the second protein, the IFN132-IFN132 tandem fragment and the IFC133-IFC133 tandem fragment can be close to each other to be reconstructed to form a complete photopigment protein IFP2.0 capable of emitting fluorescence under the excitation of excitation light.

Compared with the existing bimolecular fluorescence complementary system, the application provides a tandem fragment fluorescence complementary system based on near-infrared photosensitive pigment protein IFP2.0, which comprises a first carrier and a second carrier. Wherein, the first carrier is a carrier containing a sequence SEQ ID NO.2, and the second carrier is a carrier containing a sequence SEQ ID NO. 3. The system is a fluorescence complementary system based on near-infrared photosensitive pigment protein IFP2.0, and the near-infrared photosensitive pigment protein IFP2.0 can be excited to generate fluorescence with longer wavelength, so that the fluorescence generated by the system has better tissue permeability. And the reconstructed near-infrared light-sensitive pigment protein IFP2.0 can be matured at physiological temperature, so that the interaction between proteins can be researched under the normal physiological temperature condition of cells. Furthermore, the sequence SEQ ID NO.2 can express an IFN132-IFN132 tandem fragment, the sequence SEQ ID NO.3 can express an IFC133-IFC133 tandem fragment, and the IFN132-IFN132 tandem fragment and the IFC133-IFC133 tandem fragment respectively comprise two IFN132 protein fragments and two IFC133 protein fragments, so that the fluorescence brightness emitted by the reconstructed near-infrared photosensitive pigment protein IFP2.0 is stronger, and imaging observation is facilitated.

The above description is only for the purpose of illustrating the preferred embodiments of the present application and is not intended to limit the scope of the present application, which is defined by the appended claims and their equivalents, and all changes that can be made therein without departing from the spirit and scope of the invention.

SEQUENCE LISTING

<110> Shenzhen advanced technology research institute of Chinese academy of sciences

<120> tandem fragment fluorescence complementary system and construction method and application thereof

<160> 4

<170> PatentIn version 3.1

<210> 1

<211> 963

<212> DNA

<213> Artificial sequence

<400> 1

ATGGCTCGGGACCCTCAACCTTTCTTCCCTCCTCTGTATCTCGGAGGACCAGAGATCACTACTGAGAACTGCGAGCGGGAACCTATACATATACCAGGTTCTATTCAACCTCATGGAGCACTGCTCACAGCCGATGGACATTCTGGCGAGGTGCTGCAAGTTTCACTCAACGCAGCCACTTTCCTGGGCCAGGAACCAACTGTTCTGCGGGGACAGACCCTGGCAGCCCTGCTGCCAGACCAGTGGCCTGCTCTGCAGACTGCCCTGCCTCCAGGCTGCCAGGACGCCCTGCAGTATCGCGCCACCCTGGATTGGCCCGCCGCCGGACACCTGAGCCTGACCGTGCATAGGGTGGCCGAGCTGCTGATTCTGGAATTCGAGCCTACTGAGGCATGGGACTCTATTGGACCCCACGCTCTGAGAAATGCAATGTTCGCTCTGGAGAGTGCTCCTAATCTCCGGGCACTGGCTGAAGTCGCAACCCAAACAGTCCGGGAACTGTCAGGTTTCGACCGGGTGATGCTGTACAAGTTTGCACCAGACGCAACAGGAGAGGTTATTGCCGAAGCAAGGCGCGAGGGCATGCAGGCTTACCTCGGGCATAGGTTTCCCGCATCCACCACCCCTGCACAAGCTAGGGCCCTCTACACAAGACACCTGCTCCGGCTGACCGCAGACACCAGGGCTGCAGCAGTGCCCCTCGACCCCGTGCTGAATCCCCAGACAAATGCTCCTACACCTCTGGGCGGAGCTGTCCTCAGAGCTACATCCCCAATGCACATGCAGTACCTGAGGAATATGGGAGTGGGCTCCTCCCTGAGCGTCAGCGTCGTGGTCGGCGGCCAGCTGTGGGGACTGATTGTCTGCCACCATCAGACACCCTACGTGCTGCCACCAGATCTGCGGACCACCCTGGAGTATCTGGGGAGGCTCCTGTCCCTGCAGGTGCAGAGGAAAGAAGCC

<210> 2

<211> 822

<212> DNA

<213> Artificial sequence

<400> 2

ATGGCTCGGGACCCTCAACCTTTCTTCCCTCCTCTGTATCTCGGAGGACCAGAGATCACTACTGAGAACTGCGAGCGGGAACCTATACATATACCAGGTTCTATTCAACCTCATGGAGCACTGCTCACAGCCGATGGACATTCTGGCGAGGTGCTGCAAGTTTCACTCAACGCAGCCACTTTCCTGGGCCAGGAACCAACTGTTCTGCGGGGACAGACCCTGGCAGCCCTGCTGCCAGACCAGTGGCCTGCTCTGCAGACTGCCCTGCCTCCAGGCTGCCAGGACGCCCTGCAGTATCGCGCCACCCTGGATTGGCCCGCCGCCGGACACCTGAGCCTGACCGTGCATAGGGTGGCCGAGCTGCTGATTCTGGAATTCGAGCCTACTGAGGCATGGGGAGGTGGCGGGAGTGGAGGTGGCGGGAGTATGGCTCGGGACCCTCAACCTTTCTTCCCTCCTCTGTATCTCGGAGGACCAGAGATCACTACTGAGAACTGCGAGCGGGAACCTATACATATACCAGGTTCTATTCAACCTCATGGAGCACTGCTCACAGCCGATGGACATTCTGGCGAGGTGCTGCAAGTTTCACTCAACGCAGCCACTTTCCTGGGCCAGGAACCAACTGTTCTGCGGGGACAGACCCTGGCAGCCCTGCTGCCAGACCAGTGGCCTGCTCTGCAGACTGCCCTGCCTCCAGGCTGCCAGGACGCCCTGCAGTATCGCGCCACCCTGGATTGGCCCGCCGCCGGACACCTGAGCCTGACCGTGCATAGGGTGGCCGAGCTGCTGATTCTGGAATTCGAGCCTACTGAGGCATGG

<210> 3

<211> 1164

<212> DNA

<213> Artificial sequence

<400> 3

GACTCTATTGGACCCCACGCTCTGAGAAATGCAATGTTCGCTCTGGAGAGTGCTCCTAATCTCCGGGCACTGGCTGAAGTCGCAACCCAAACAGTCCGGGAACTGTCAGGTTTCGACCGGGTGATGCTGTACAAGTTTGCACCAGACGCAACAGGAGAGGTTATTGCCGAAGCAAGGCGCGAGGGCATGCAGGCTTACCTCGGGCATAGGTTTCCCGCATCCACCACCCCTGCACAAGCTAGGGCCCTCTACACAAGACACCTGCTCCGGCTGACCGCAGACACCAGGGCTGCAGCAGTGCCCCTCGACCCCGTGCTGAATCCCCAGACAAATGCTCCTACACCTCTGGGCGGAGCTGTCCTCAGAGCTACATCCCCAATGCACATGCAGTACCTGAGGAATATGGGAGTGGGCTCCTCCCTGAGCGTCAGCGTCGTGGTCGGCGGCCAGCTGTGGGGACTGATTGTCTGCCACCATCAGACACCCTACGTGCTGCCACCAGATCTGCGGACCACCCTGGAGTATCTGGGGAGGCTCCTGTCCCTGCAGGTGCAGAGGAAAGAAGCCGGAGGTGGCGGGAGTGGAGGTGGCGGGAGTGACTCTATTGGACCCCACGCTCTGAGAAATGCAATGTTCGCTCTGGAGAGTGCTCCTAATCTCCGGGCACTGGCTGAAGTCGCAACCCAAACAGTCCGGGAACTGTCAGGTTTCGACCGGGTGATGCTGTACAAGTTTGCACCAGACGCAACAGGAGAGGTTATTGCCGAAGCAAGGCGCGAGGGCATGCAGGCTTACCTCGGGCATAGGTTTCCCGCATCCACCACCCCTGCACAAGCTAGGGCCCTCTACACAAGACACCTGCTCCGGCTGACCGCAGACACCAGGGCTGCAGCAGTGCCCCTCGACCCCGTGCTGAATCCCCAGACAAATGCTCCTACACCTCTGGGCGGAGCTGTCCTCAGAGCTACATCCCCAATGCACATGCAGTACCTGAGGAATATGGGAGTGGGCTCCTCCCTGAGCGTCAGCGTCGTGGTCGGCGGCCAGCTGTGGGGACTGATTGTCTGCCACCATCAGACACCCTACGTGCTGCCACCAGATCTGCGGACCACCCTGGAGTATCTGGGGAGGCTCCTGTCCCTGCAGGTGCAGAGGAAAGAAGCC

<210> 4

<211> 321

<212> PRT

<213> IFP2.0

<400> 4

MARDPQPFFPPLYLGGPEITTENCEREPIHIPGSIQPHGALLTADGHSGEVLQVSLNAATFLGQEPTVLRGQTLAALLPDQWPALQTALPPGCQDALQYRATLDWPAAGHLSLTVHRVAELLILEFEPTEAWDSIGPHALRNAMFALESAPNLRALAEVATQTVRELSGFDRVMLYKFAPDATGEVIAEARREGMQAYLGHRFPASTTPAQARALYTRHLLRLTADTRAAAVPLDPVLNPQTNAPTPLGGAVLRATSPMHMQYLRNMGVGSSLSVSVVVGGQLWGLIVCHHQTPYVLPPDLRTTLEYLGRLLSLQVQRKEA