Extracellular vesicles specifically labeled with fluorescent protein mKate2 and their construction method and application

Abstract

This invention discloses a fluorescent protein mKate2-specifically labeled extracellular vesicle, its construction method, and its application. The invention designs an mKate2-VSVG fusion gene, replacing the extracellular region of the vesicular stomatitis virus glycoprotein G (VSVG) gene with the mKate2 gene while retaining the transmembrane and intracellular regions of the VSVG gene. The mKate2 red fluorescent protein is specifically expressed in EVs. Leveraging the superior in vivo and in vitro tracking capabilities of the mKate2 far-infrared fluorescent protein, the function of EVs can be better and more accurately tracked and studied in vivo and in vitro, especially in vivo imaging, such as the distribution of EVs in various tissues and organs of mice, metabolic processes, and the specific targeting ability of EVs.

Description

Technical Field

[0001] This invention relates to an extracellular vesicle specifically labeled with fluorescent protein mKate2, its construction method and application, belonging to the field of biomedical technology. Background Technology

[0002] Extracellular vesicles (EVs) are lipid bilayer nanovesicles with a diameter of 30–100 nm secreted by cells. They possess the ability to transfer nucleic acids, lipids, and proteins, mediating intercellular communication. In recent years, researchers have discovered that EVs are closely related to various physiological and pathological processes in cells, but their specific roles within these processes remain unclear. Because EVs are nanoscale in size, they are difficult to observe directly using conventional methods. Therefore, EV tracking and labeling methods have become fundamental tools in EV research. Based on the different media used in EV tracking and labeling methods, they can be categorized into fluorescent dyes, fluorescent proteins, luciferases, and physical labeling methods. These labeling methods can label EVs on their outer membrane, inner membrane, or interior in different ways.

[0003] Based on the different media used in EV tracer labeling methods, EV tracer labeling methods can be divided into fluorescent dyes, fluorescent proteins, and physical labeling methods. These labeling methods can label the outer membrane, inner membrane, or interior of EVs in different ways.

[0004] (1) Fluorescent dyes: Fluorescent dyes are one of the commonly used methods for EV tracking and labeling. Initially, most fluorescent dyes were used for cell imaging. These fluorescent dyes label cell membrane lipid components or membrane proteins by binding to fluorescent groups with different functions. Based on the characteristics of fluorescent dyes and EVs, fluorescent dyes that can also be used for EV tracking and labeling have been discovered. Commonly used ones include carbonyl cyanide dye, PKH dye, CFSE, and calcein acetylated methyl ester. The method of labeling EVs with fluorescent dyes is often used for EV uptake analysis and biodistribution studies. It has the advantages of simple operation, easy dye acquisition, and no impact on EV bioactivity. However, fluorescent dyes may not be accurate enough for long-term EV tracking, that is, the existence time of the dye may be longer than the existence time of the labeled EVs. After EVs are taken up by cells, they will degrade in the cells, but fluorescent dyes are relatively stable and not easily degraded. Therefore, fluorescent dyes can only be used for short-term EV studies. In addition, current EV extraction methods cannot completely separate EVs from certain lipoprotein particles or small proteins. Using fluorescent dyes to label EVs is not specific and may label certain lipoprotein particles or small proteins, interfering with experimental results.

[0005] (2) Fluorescent Proteins: Fluorescent proteins are commonly used reporter proteins that emit corresponding fluorescent signals under excitation light of a specific wavelength. By fusing fluorescent proteins with protein molecules on or inside the EV membrane, specific fusion proteins can be constructed. The fluorescent signals emitted by these fusion proteins are then detected to trace the EVs. Commonly used fluorescent proteins include GFP, EGFP, RFP, and mCherry. The method of labeling EVs with fluorescent proteins can be used for quantitative analysis, qualitative analysis, uptake analysis, and biodistribution studies of EVs. It has advantages such as high specificity, stability, high efficiency, and safety and controllability, which are unmatched by other labeling methods.

[0006] (3) Physical labeling method: With the development of technology, researchers use the physical properties of certain substances to label EVs for EV tracking studies. Examples include quantum dots (QD), radioactive isotopes, and SPIO (a commonly used contrast agent in magnetic resonance imaging). Although this method has higher sensitivity and resolution than other methods, it is usually complex to operate or requires special instruments. In addition, the use of radioactive isotope labeling in this method will generate ionizing radiation.

[0007] The optimal detection optical window for in vivo optical imaging is 600-1000nm. Light in this wavelength range has the advantages of strong tissue penetration, minimal damage to animal tissues, low tissue background, and low photobleaching effect, making it more suitable for in vivo fluorescence imaging of deep tissues and organs. Far-infrared fluorescent proteins, i.e., red fluorescent proteins with a maximum emission wavelength >650nm, catalyze the formation of chromophores due to their special structure and possess many excellent characteristics: (1) Easy to detect, high sensitivity, and stable fluorescence properties. The maturation of fluorescent protein chromophores does not require the addition of substrates or other auxiliary factors. They can emit fluorescence under excitation light alone, and single-cell labeling is clear and easily observed under a fluorescence microscope; (2) The coding sequence of fluorescent protein genes is short, making it easy to construct vectors; (3) Low cytotoxicity, allowing direct labeling of live cells or live tissues; (4) Compared to GFP, its excitation and emission wavelengths are longer, located in the near-red region (650-900nm). It has lower light absorption and scattering in animal tissues, higher penetration, and is more suitable for deep imaging of animal live tissues, making it a more ideal fluorescent labeling molecule for in vivo imaging.

[0008] Early far-infrared fluorescent proteins such as HcRed, mPlum, and AQ143 had low fluorescence intensity, only 10% of that of EGFP, limiting their applications. In 2007, Shcherbo et al. screened a high-brightness monomeric far-infrared fluorescent protein and named it mKate. Further modification yielded mKate2, which is three times brighter than mKate and ten times brighter than mPlum, exhibiting high stability and low toxicity. As a novel far-infrared fluorescent protein, mKate2 has an excitation wavelength of 588 nm and an emission wavelength of 635 nm, demonstrating higher specificity and less background interference compared to existing fluorescent proteins used to label EVs. Based on these findings, mKate2 is currently the best far-infrared fluorescent protein reporter gene for in vivo fluorescence imaging. However, the mKate2 gene is not present in mammalian cells and cannot be expressed in extracellular vesicles. Therefore, to effectively express the mKate2 gene in mammalian cells and localize it in extracellular vesicles, in vitro vector construction and specific modification are required. Summary of the Invention

[0009] To address the aforementioned technical problems, this invention designs an mKate2-VSVG fusion gene, which replaces the extracellular region of the vesicular stomatitis virus glycoprotein G (VSVG) gene with the mKate2 gene while retaining the transmembrane and intracellular regions of the VSVG gene. The mKate2 red fluorescent protein is specifically expressed in EVs. Leveraging the superior in vivo and in vitro tracing capabilities of the mKate2 far-infrared fluorescent protein, the function of EVs can be better and more accurately tracked and studied in vivo and in vitro, especially in vivo imaging, such as the distribution of EVs in various tissues and organs of mice, metabolic processes, and the specific targeting ability of EVs.

[0010] The first objective of this invention is to provide an extracellular vesicle specifically labeled with the fluorescent protein mKate2, wherein the extracellular vesicle expresses the mKate2-VSVG fusion protein, wherein the mKate2-VSVG fusion protein is formed by replacing the extracellular region of vesicular stomatitis virus glycoprotein G (VSVG) with the fluorescent protein mKate2.

[0011] Furthermore, the amino acid sequence of the mKate2-VSVG fusion protein is shown in SEQ ID NO.1.

[0012] Furthermore, the mKate2-VSVG fusion protein has a signal peptide at its front end.

[0013] Furthermore, the amino acid sequence of the signal peptide is shown in SEQ ID NO.2.

[0014] A second objective of this invention is to provide a method for constructing the aforementioned extracellular vesicles, comprising the following steps:

[0015] S1. Amplify to obtain the mKate2 gene fragment;

[0016] S2, the full-length gene fragments of the transmembrane and intracellular regions of VSVG synthesized in vitro;

[0017] S3. The mKate2 gene fragment from step S1 and the full-length transmembrane and intracellular regions of the VSVG gene fragment from step S2 are ligated into a vector to obtain a recombinant expression vector.

[0018] S4. The recombinant expression vector is transferred into the host cell, and after culturing, the cell culture supernatant is collected. The extracellular vesicles are prepared according to the exosome preparation method.

[0019] Furthermore, the carrier is a pCMV-Flag carrier.

[0020] Furthermore, the host cell is a 293T cell or a dendritic cell.

[0021] Further, the method for preparing exosomes includes the following steps: centrifuging the cell culture supernatant at 8000-15000g for 20-40min to obtain the supernatant; filtering the supernatant through a micron-membrane and then ultracentrifuging at 80000-120000g for 80-100min to obtain the precipitate; resuspending the precipitate in a buffer solution and then ultracentrifuging at 80000-120000g for 80-100min to remove the supernatant, thereby obtaining the extracellular vesicles.

[0022] A third objective of this invention is to provide the application of the aforementioned extracellular vesicles in extracellular vesicle tracking and labeling.

[0023] The beneficial effects of this invention are:

[0024] This invention designs an mKate2-VSVG fusion gene, which replaces the extracellular region of the vesicular stomatitis virus glycoprotein G (VSVG) gene with the mKate2 gene, while retaining the transmembrane and intracellular regions of the VSVG gene. The mKate2 red fluorescent protein is specifically expressed in EVs. With the superior in vivo and in vitro tracking capabilities of the mKate2 far-infrared fluorescent protein, the function of EVs can be better and more accurately tracked and studied in vivo and in vitro, especially in vivo imaging, such as the distribution of EVs in various tissues and organs of mice, metabolic processes, and the specific targeting ability of EVs. Attached Figure Description

[0025] Figure 1To replace the extracellular region of vesicular stomatitis virus glycoprotein G (VSVG) with the mKate2 gene, a fusion vector of mKate2 and VSVG was formed. After transfection into cells for expression, the resulting EVs expressed the mKate2-VSVG fusion protein, in which the extracellular region is the mKate2 protein, and the transmembrane and intracellular regions are the transmembrane and intracellular regions of the VSVG protein.

[0026] Figure 2 To validate mKate2-labeled extracellular vesicles (EVs-mKate2-VSVG) using Western blot, CD9, CD63, and Alix were used as marker proteins for EVs.

[0027] Figure 3 To add EVs-mKate2-VSVG to HeLa cells, the proportion of EVs-mKate2-VSVG uptake by HeLa cells was observed and fluorescence images were captured at 2, 6 and 8 hours (left); after digestion, the cells were fixed with 4% paraformaldehyde and the proportion of fluorescent cells was detected by cell flow cytometry (right).

[0028] Figure 4 To investigate the effects of intraperitoneal injection of extracted EVs-mKate2-VSVG and control group (EVs-mCherry-VSVG, i.e., mCherry-VSVG fluorescent fusion protein expressed in EVs) into mice, each mouse received 150 μg. After 24 hours, the fluorescence intensity was detected by in vivo imaging (PerkinElmer IVIS Spectrum small animal in vivo optical three-dimensional imaging system). The results showed that the in vivo fluorescence intensity of the EVs-mKate2-VSVG injection group was significantly higher than that of the EVs-mCherry-VSVG injection group.

[0029] Figure 5 To investigate the effects of intraperitoneal injection of extracted EVs-mKate2-VSVG and control group (EVs-mCherry-VSVG) on mice, each mouse received 150 μg. After 24 hours, the imaging of mouse tissues and organs was examined. The results showed that the fluorescence intensity in the lungs, liver, kidneys and small intestine of the EVs-mKate2-VSVG injection group was significantly higher than that in the EVs-mCherry-VSVG injection group.

[0030] Figure 6 For enzyme-linked immunosorbent assay (ELISA) detection Figure 5The expression of inflammatory factors in the serum of mice was investigated. The results showed that the expression of IL-6, TNF-α and IFN-β in the serum of mice injected with EVs-mKate2-VSVG was not significantly different from that in the untreated group (Mock) and the EVs-NC group derived from 293T cells, indicating that EVs-mKate2-VSVG did not induce an inflammatory response. Detailed Implementation

[0031] The present invention will be further described below with reference to specific embodiments, so that those skilled in the art can better understand and implement the present invention, but the embodiments are not intended to limit the present invention.

[0032] Example 1:

[0033] The mKate2-VSVG fusion gene was designed by replacing the extracellular region of the vesicular stomatitis virus glycoprotein G (VSVG) gene with the mKate2 gene, while retaining the transmembrane and intracellular regions of the VSVG gene. The mKate2-VSVG fusion gene was synthesized by a gene synthesis company; the synthesized mKate2-VSVG fusion gene was ligated with enzymes and cloned into the expression vector pCMV-Flag to construct the pCMV-mKate2-VSVG fusion gene vector, which was then sequenced for verification. Figure 1 ).

[0034] Example 2:

[0035] 293T cell lines were plated (100mm dishes) at a density of approximately 60-70%. The pCMV-mKate2-VSVG fusion gene vector was transfected into cells via PEI (plasmid to PEI mass ratio 1:3). After 6 hours, the cells were cultured in EV-free medium for another 48 hours. The cell culture supernatant was then collected. The collected supernatant was first centrifuged at 10,000g, 4°C for 30 minutes to remove cell debris. The supernatant was then filtered through a 0.22-micron membrane and centrifuged at 100,000g, 4°C for 90 minutes using an ultracentrifuge (Beckman, Germany). The supernatant was carefully removed, and an appropriate amount of PBS was added. The EVs precipitate was resuspended and mixed by pipetting. The cells were then centrifuged again at 100,000g, 4°C for 90 minutes. The supernatant was discarded, and the cells were resuspended in an appropriate amount of PBS to obtain mKate2-labeled extracellular vesicles (EVs-mKate2-VSVG). The results were verified by Western blot. Figure 2 ).

[0036] Example 3:

[0037] Extracted EVs-mKate2-VSVG was added to HeLa cells (12-well plate, cell density 90%), with 50 μg added to each well. The proportion of HeLa cells taking up EVs-mKate2-VSVG was observed and fluorescence images were captured at 2, 6, and 8 hours. After cell digestion, the cells were fixed with 4% paraformaldehyde, and the proportion of fluorescent cells was detected by flow cytometry (excitation wavelength 588 nm, emission wavelength 635 nm). Figure 3 ).

[0038] Example 4:

[0039] Extracted EVs-mKate2-VSVG and a control group (EVs-mCherry-VSVG, i.e., mCherry-VSVG fluorescent fusion protein expressed in EVs) were intraperitoneally injected into mice, 150 μg per mouse. After 24 hours, fluorescence intensity was detected using in vivo imaging (PerkinElmer IVIS Spectrum small animal in vivo optical 3D imaging system). The results showed that the in vivo fluorescence intensity of the EVs-mKate2-VSVG injection group was significantly higher than that of the EVs-mCherry-VSVG injection group. Figure 4 Afterwards, imaging of mouse tissues and organs was examined. The results showed that the fluorescence intensity in the lungs, liver, kidneys, and small intestine of mice injected with EVs-mKate2-VSVG was significantly higher than that in mice injected with EVs-mCherry-VSVG. Figure 5 Simultaneously, the expression of inflammatory factors in mouse serum was detected by enzyme-linked immunosorbent assay (ELISA). The results showed that the expression of IL-6, TNF-α, and IFN-β in the serum of mice in the EVs-mKate2-VSVG injection group was not significantly different from that in the untreated group (Mock) and the EVs-NC group derived from 293T cells, indicating that EVs-mKate2-VSVG did not induce an inflammatory response. Figure 6 ).

[0040] We previously used the Lamp2b gene to fuse with mKate2 for expression. Since the Lamp2b gene has been shown to be expressed in extracellular vesicles, the gene fused with it should theoretically also be expressed in extracellular vesicles. However, through various experiments, the Lamp2b gene fused with mKate2 could not be localized in extracellular vesicles. It is speculated that the mKate2 protein may affect the effective folding of the Lamp2b protein.

[0041] The above-described embodiments are merely preferred embodiments provided to fully illustrate the present invention, and the scope of protection of the present invention is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are all within the scope of protection of the present invention. The scope of protection of the present invention is defined by the claims.

Claims

1. A method for constructing extracellular vesicles specifically labeled with the fluorescent protein mKate2, characterized in that, Includes the following steps: S1. Amplify to obtain the mKate2 gene fragment; S2, the full-length gene fragments of the transmembrane and intracellular regions of VSVG synthesized in vitro; S3. The mKate2 gene fragment from step S1 and the full-length transmembrane and intracellular regions of the VSVG gene fragment from step S2 are ligated into a vector to obtain a recombinant expression vector. S4. The recombinant expression vector is transferred into the host cells. After culturing, the cell culture supernatant is collected. The cell culture supernatant is centrifuged at 8000-15000g for 20-40 min to obtain the supernatant. The supernatant is filtered through a micron membrane and then centrifuged at 80000-120000g for 80-100 min to obtain the precipitate. The precipitate is resuspended in a buffer solution and then centrifuged at 80000-120000g for 80-100 min to remove the supernatant, thus obtaining the extracellular vesicles. The extracellular vesicles express the mKate2-VSVG fusion protein, the amino acid sequence of which is shown in SEQ ID NO.1, and the mKate2-VSVG fusion protein has a signal peptide at its front end. The host cell is a 293T cell.

2. The construction method according to claim 1, characterized in that, The amino acid sequence of the signal peptide is shown in SEQ ID NO.

2.

3. The construction method according to claim 1, characterized in that, The carrier is a pCMV-Flag carrier.

4. The application of extracellular vesicles prepared by the construction method according to any one of claims 1-3 in extracellular vesicle tracking and labeling.

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