Fusion protein used as retrograde tracer and preparation method thereof

Abstract

The invention provides a fusion protein used as a retrograde tracer and a preparation method of the fusion protein. The fusion protein comprises cholera toxin B subunit protein (CTB), mbojin fluorescent protein and a linker used for connecting the cholera toxin B subunit protein and the mbojin fluorescent protein. The fusion protein is a protein (called CTBPro for short) after fusion of CTB and mbaojin, and shows remarkable fluorescence intensity, strong light stability and compatibility with multiple experimental modes. Importantly, the CTBPro can realize accurate retrograde tracing in the central and peripheral nervous systems, so that the neuron morphology is visualized with higher definition and details.

Description

Technical Field

[0001] This invention belongs to the field of medical neurobiology and retrograde tracing technology of the nervous system, specifically relating to a fusion protein used as a retrograde tracer and its preparation method. Background Technology

[0002] Tracing neural circuits is one of the most fundamental needs and research goals in neuroscience, providing crucial insights into the complex connections and functional organization of the nervous system. Over the past few decades, advancements in neural tracing methods have significantly improved our ability to explore these circuits. Early methods utilized proteins such as horseradish peroxidase (HRP) and wheat germ lectin (WGA) as tracers to map neuronal pathways. However, these tracers are limited by inherent limitations, such as the inability to reliably determine the directionality of signal transduction and the complexity of multisynaptic tracing, which often hinder the achievement of experimental goals. Subsequent innovations introduced viral tracers, including adeno-associated virus retrograde serotype (AAVretro) and rabies virus (RV), which enhanced circuit tracing capabilities by enabling transsynaptic propagation. However, the application of viral tracers often affects neuronal activity due to significant neurotoxicity, making functional studies difficult.

[0003] The introduction of cholera toxin B subunit (CTB) as a retrograde tracer marks an advancement in neural circuit mapping, enabling directional tracing without cross-synaptic propagation and simplifying the study of neuronal connectivity. Improvements such as CTB conjugated with colloidal gold nanoparticles (CTB-gold) enhance stability and compatibility with immunohistochemistry, but require complex silver-enhanced staining, limiting their practical application. Chemifluorophore conjugates, such as CTB-Alexa488, offer greater convenience through direct visualization via fluorescence microscopy. However, these tracers face limitations, including low fluorescence intensity, insufficient morphological detail, poor compatibility with advanced techniques such as tissue clearing or correlated light electron microscopy (CLEM), and the inability to achieve in vivo gene expression, thus limiting their wider application in experimental models. Summary of the Invention

[0004] To address the problems and shortcomings of existing technologies, this invention provides a fusion protein for use as a retrograde tracer and its preparation method. This fusion protein is a protein resulting from the fusion of CTB and mBaojin (mBaojin, a green fluorescent protein known for its excellent brightness and photostability), referred to herein as CTBPro. It exhibits significant fluorescence intensity, strong photostability, and compatibility with various experimental modalities. Importantly, CTBPro enables precise retrograde tracing in both the central and peripheral nervous systems, thereby allowing for higher clarity and detail visualization of neuronal morphology.

[0005] According to a first aspect of the invention, a fusion protein is provided for use as a retrograde tracer, the fusion protein comprising cholera toxin B subunit protein (CTB), mbaojin fluorescent protein, and a linker for connecting cholera toxin B subunit protein and mbaojin fluorescent protein.

[0006] The fusion protein provided in this invention is obtained by linking CTB with mbaojin fluorescent protein via a linker. We found that compared to the currently commercially available CTB488, it exhibits significantly higher fluorescence intensity, superior photostability, and compatibility with multiple experimental modalities. Furthermore, CTBPro enables precise retrograde tracing in both the central and peripheral nervous systems, thereby visualizing neuronal morphology with greater clarity and detail. As a protein-based tracer, CTBPro offers the potential for endogenous expression in experimental animals, thus expanding its versatility and applicability in neuroscience research. In summary, compared to existing tracers, CTBPro offers a range of advantages, including enhanced fluorescence and superior versatility, providing a novel and effective option for retrograde tracing.

[0007] Specifically, firstly, this invention uses mbaojin fluorescent protein as a reporter gene, which has extremely high photostability and brightness. Compared to chemically coupled organic dyes (such as Alexa Fluor 488), mbaojin fluorescent protein makes the fusion protein more resistant to photobleaching, allowing for continuous imaging and long-term observation, thereby obtaining clearer and more stable neural signal images, and significantly improving the signal-to-noise ratio and data reliability of the experiment.

[0008] Secondly, this invention utilizes gene fusion technology to achieve the connection of CTB and mbaojin at fixed sites with a strict stoichiometric ratio of 1:1 (pentameric CTB: five mbaojin). This highly homogeneous structure avoids the product heterogeneity problems commonly encountered in chemical conjugation methods, such as random labeling sites and inconsistent labeling efficiencies. This construction method preserves the native conformation and biological activity of the CTB protein to the greatest extent possible. The GM1 ganglioside binding site of the CTB pentamer is not chemically modified or masked, ensuring its ultra-high efficiency and affinity for binding to neuronal receptors. This results in a tracer with higher sensitivity and lower background, enabling effective labeling of weaker neural pathways. Therefore, more precise retrograde tracing can be achieved in the central and peripheral nervous systems.

[0009] Third, because the biological activity of CTB is fully preserved, the fusion protein is taken up by neuronal terminals and initiates retrograde axonal transport with significantly higher efficiency than chemically conjugated products. This means that efficient and extensive neural labeling can be achieved using lower doses of tracers, providing a powerful tool for mapping fine neural networks.

[0010] Fourth, all components of this fusion protein (CTB, mbaojin, and linkers) are proteins, expressed and folded through the cell's own mechanisms, avoiding the cytotoxicity that chemically synthesized dyes may cause. Moreover, CTBPro is a gene-encoded protein (not a chemical dye), which allows researchers to genetically modify experimental animals to enable specific types of neurons to spontaneously and continuously produce this tracer, achieving the most natural, stable, and specific labeling of neural pathways. This is of great significance for further research in neurobiology. Furthermore, as a recombinant protein, its production does not rely on complex chemical conjugation and purification processes, exhibiting good batch-to-batch consistency and stable product quality, greatly ensuring the reproducibility of experimental results. Simultaneously, the genetically engineered production method facilitates large-scale preparation and keeps costs under control.

[0011] It is worth noting that there are few reports in the prior art of linking CTB with fluorescent proteins as a novel tracer. Therefore, this invention provides an innovative design direction for a novel tracer.

[0012] Preferably, the connector is a flexible connector.

[0013] Preferably, the fusion protein further comprises an N-terminal signal peptide. The presence of an N-terminal signal peptide can further optimize expression and improve the quality of fusion protein expression.

[0014] According to a second aspect of the present invention, a gene encoding the above-described fusion protein is provided.

[0015] Preferably, the nucleotide sequence encoding the gene is shown in SEQ ID NO:1.

[0016] According to a third aspect of the present invention, an expression vector is provided, the expression vector comprising the above-described coding gene.

[0017] According to a fourth aspect of the present invention, a host cell is provided, the host cell comprising the above-described expression vector.

[0018] Preferably, the host cell is a mammalian cell.

[0019] According to a fifth aspect of the present invention, there is provided an application of the fusion protein of any of the above claims as a retrograde tracer in neural circuit research.

[0020] Preferably, the application of the fusion protein as a retrograde tracer in neural circuit studies includes at least one of the following aspects: (1) retrograde tracing in the central nervous system; (2) retrograde tracing in the peripheral nervous system. The fusion protein provided by this invention can be used not only in the central nervous system, which is commonly studied, but also in the peripheral nervous system, such as the kidneys, for retrograde tracing, which is of great significance for further research on the interaction between the nervous system and other organ systems.

[0021] Preferably, the fusion protein is used for tissue clearing techniques for visualizing brain circuits, and / or for correlated light electron microscopy (CLEM) techniques for ultrastructural studies. Traditional tracers often suffer fluorescence quenching or signal loss when subjected to these techniques. However, CTBPro exhibits tolerance to the harsh chemical treatments required for electron microscopy preparation, retaining its fluorescence even after treatment with various chemical reagents and high temperatures. Furthermore, compared to traditional tracers such as CTB488, which often lose signal intensity or exhibit diffuse fluorescence upon tissue clearing, CTBPro maintains its brightness and clarity, enabling high-resolution whole-brain imaging.

[0022] According to a sixth aspect of the present invention, a method for preparing the fusion protein of any one of the above claims is provided, characterized by comprising the following steps: (a) linking a gene encoding the fusion protein to a vector to construct an expression vector, transfecting the expression vector into a host cell, and culturing the transfected cell at 37±2°C to express the fusion protein; (b) collecting the cell culture supernatant and concentrating the supernatant; and (c) separating and purifying the fusion protein to obtain the fusion protein.

[0023] Preferably, in step (a), the vector is a plasmid.

[0024] Preferably, in step (a), the host cell is a 293F cell. Preferably, the 293F cell is a suspension-cultured 293F cell with a cell density of 1.8–2.2 × 10⁻⁶ cells. 6 cells / mL.

[0025] Preferably, in step (a), the mass ratio of plasmid to PEI transfection reagent is 1:2.5–3.5. Preferably, after mixing the plasmid DNA and PEI transfection reagent, incubate at room temperature for 13–17 min. If isopropanol is used during plasmid extraction, it is best to determine the plasmid concentration by gel electrophoresis with a marker (molecular weight standard) rather than using Nanodrop.

[0026] Preferably, in step (a), the transfected cells are cultured in a shaker at 200–240 rpm for 3–5 days. For certain proteins that are difficult to secrete, the culture time may be appropriately extended.

[0027] Preferably, in step (b), collecting the cell culture supernatant and concentrating the supernatant includes the following steps:

[0028] (b1) After the culture is completed, centrifuge at 3800-4200g for 8-12 minutes at 4±0.5℃, discard the cell pellet and collect the supernatant;

[0029] (b2) Wash the Flag affinity column 2-3 times with 1x sterile PBS and equilibrate at room temperature; note that the column should be used no more than 3 times in this step to ensure the best results.

[0030] (b3) Pass all collected supernatant through a Flag affinity chromatography column; this step requires keeping the column moist and controlling the flow rate, and repeating this process 2 to 3 times if necessary;

[0031] (b4) Wash the Flag affinity column with sufficient 1×PBS buffer until there are no residual impurities in the effluent; in this step, after washing with PBS, almost all CTB-mbaojin (fusion protein, i.e. target protein, abbreviated as CTBPro) is retained on the column.

[0032] (b5) Add 3C protease to the Flag affinity chromatography column bound to the target protein and react overnight at 4°C and 0.5°C to remove the Flag tag; preferably, in this step, the mass ratio of 3C protease to target protein is 1:90-110; and in this step, if the concentration of target protein is too high, more 3C protease needs to be added and the reaction time needs to be extended, which can be adjusted according to the actual situation; the protein concentration can be estimated by performing SDS-PAGE analysis (180V, about 1 hour) on the liquid inside the column and the liquid after column passage, and staining with Coomassie Brilliant Blue;

[0033] Preferably, the separation and purification to obtain the fusion protein includes the following steps:

[0034] (c1) Elute the target protein off the column with 1×PBS buffer and collect the eluent;

[0035] (c2) The eluent is further purified using a pre-equilibrated molecular sieve chromatography system (such as AKTA pure), and the eluent fraction corresponding to the target protein is collected according to the sample elution pattern.

[0036] (c3) Determine the concentration of the final target protein product, quick-freeze it in liquid nitrogen, and store it in an ultra-low temperature environment of -90 to -70°C for a long time; preferably, the concentration of the final target protein product is determined by Nanodrop or BCA method; preferably, it is determined whether the target protein product needs to be concentrated and packaged according to actual needs; preferably, a concentration column is used for concentration.

[0037] Preferably, in the steps of the above-described method for preparing fusion proteins, samples are taken from relevant steps (such as after transfection, effluent / elution buffer at each purification stage) for SDS-PAGE electrophoresis and Western Blot (if available) analysis to monitor the expression level, purity, and enzyme digestion effect of the target protein.

[0038] Compared with the prior art, the present invention has the following advantages:

[0039] (1) Compared to the conventional CTB488, CTBPro exhibits significantly increased fluorescence intensity, achieving superior performance with only a fraction of the required molar concentration. This enhancement enables clearer and more detailed neuronal labeling without the need for higher laser intensities, thereby reducing the risk of photodamage and preserving the integrity of labeled neurons. The ability to acquire high-resolution images under low-intensity laser conditions further highlights the practical advantages of CTBPro in in vivo studies, where signal clarity and minimal tissue disturbance are crucial.

[0040] (2) CTBPro successfully achieved retrograde tracing in both the central and peripheral nervous systems, highlighting its broad applicability. In the central nervous system, the results obtained using CTBPro were superior to those obtained using CTB488, particularly in terms of morphological detail and resolution. Similarly, in peripheral tracing experiments, even under challenging conditions (such as local injection into the renal parenchyma), CTBPro outperformed CTB488, producing brighter and clearer markings.

[0041] (3) CTBPro is compatible with advanced imaging techniques, including tissue clearing methods and correlated light electron microscopy (CLEM). Traditional tracers often suffer fluorescence quenching or signal loss when subjected to these techniques. However, CTBPro exhibits tolerance to the harsh chemical treatments required for electron microscopy preparation, retaining its fluorescence even after fixation and staining. This is a key advantage for studies requiring comprehensive insights into neuronal structure and connectivity at both the macroscopic and ultrastructural levels using both light and electron microscopy. Furthermore, CTBPro's compatibility with tissue clearing methods, such as the Idisco protocol, further enhances its versatility. While traditional tracers like CTB488 often lose signal intensity or exhibit diffuse fluorescence upon tissue clearing, CTBPro maintains its brightness and clarity, enabling high-resolution whole-brain imaging. This characteristic opens up new possibilities for mapping extensive neural circuits at the whole-brain level, which is crucial for studying large-scale neural networks and their functional organization.

[0042] (4) CTBPro can be expressed endogenously in laboratory animals. As a protein-based tracer, CTBPro can be genetically engineered for in vivo expression, thereby facilitating the study of neuronal connectivity in genetically modified animal models. This feature significantly expands its applicability for exploring neural circuits in a wide range of experimental conditions and different species. Attached Figure Description

[0043] Figure 1 This illustrates the design concept of CTBPro in this invention; the left side shows the structure of CTB, and the right side shows CTBPro of this invention, which is formed by adding mbaojin protein to CTB.

[0044] Figure 2 The structure prediction of CTBPro is given by (a) representing the structure prediction score of CTBPro, (b) representing the predicted binding capacity of the original CTB and GM1 gangliosides, and (c) representing the predicted binding capacity of CTBPro and GM1 gangliosides.

[0045] Figure 3 This refers to the CTBPro (fusion protein) prepared in this invention.

[0046] Figure 4 (a) Bright field images of two samples, CTBPro and CTB488; Figure 4 (b) Images of CTBPro and CTB488 samples under 488nm fluorescence intensity excitation.

[0047] Figure 5 The fluorescence intensity spectra of CTBPro and CTB488 samples under different excitation light are shown.

[0048] Figure 6 To validate the tracing of the central nervous system, the neural projection from the ventral tegmental area (VTA) to the nucleus accumbens (NAc) was selected. (a) is a schematic diagram of the neural anatomical projection, and (b) is a theoretical schematic diagram of the results observed at the brain slice level after injecting CTBPro and CTB488 into the NAc.

[0049] Figure 7 Signals from the NAc brain region at the CTBPro and CTB488 injection sites. Figure 7 (c) and (d) show the signal in the NAc brain region at the CTBPro injection site. (c) was taken at 20x magnification, and (d) was taken at 63x high magnification. Figure 7 (e) and (f) show the signal in the NAc brain region at the CTB488 injection site. (e) was taken at 20x magnification, and (f) was taken at 63x high magnification.

[0050] Figure 8 (i) and (j) show the imaging of the endpoint (VTA) of the brain region tracked by CTBPro during injection. (i) was taken at 20x magnification, and (j) was taken at 63x high magnification. Figure 8 (k) and (l) show the imaging of the endpoint (VTA) of CTB488 in the injected brain region. (k) is a 20x image and (l) is a high-resolution 63x image.

[0051] Figure 9 The image shows the results of immunofluorescence staining of Synaptophysin, a marker of neuronal axonal terminals (presynaptic membrane), using CTBPro(g) and CTB488(h) for tracing, captured at 63x.

[0052] Figure 10 The image shows the results of staining the VTA brain region with the neuronal marker MAP2 using CTBPro(m) and CTB488(n) tracing, captured at 63x resolution.

[0053] Figure 11 The comparison of fluorescence intensity of CTBPro and CTB488 in the NAc and VTA brain regions is shown. (a) shows the comparison of fluorescence intensity of CTB488 and CTBPro in the NAc brain region, and (b) shows the comparison of fluorescence intensity of CTB488 and CTBPro in the VTA brain region.

[0054] Figure 12 (a) shows the anatomical structure of the renal efferent nerve (i.e., the sympathetic nerve), which originates from the celiac ganglion complex and accompanies the renal artery into the kidney; Figure 12 (b) shows the anatomical structure of the renal afferent nerve (i.e., sensory nerve), which can be seen transmitting back from the kidney to the dorsal root ganglion of the spinal cord, and then further afferent through the posterior horn of the spinal cord; Figure 12 (c) is a surgical illustration. An insulin needle is inserted under the kidney capsule, avoiding puncturing the renal parenchyma, and CTBPro or CTB488 is injected.

[0055] Figure 13 This is the section with the richest signal in the celiac ganglion observed under a confocal microscope at 20x magnification; the left side is the section after CTBPro injection, and the right side is the section after CTB488 injection.

[0056] Figure 14 The comparison shows the fluorescence intensity and the number of positive neuron signals corresponding to the section with the richest signal in the celiac ganglion observed under a confocal microscope at 20x magnification; where (a) is the comparison of fluorescence intensity between CTB488 and CTBPro, and (b) is the comparison of the number of positive neuron signals between CTB488 and CTBPro.

[0057] Figure 15 The results of CTB488 and CTBPro were observed under a 63x confocal microscope in positive neurons of the celiac ganglion under reverse tracing; (e) shows the celiac ganglion signal, and (f) shows the co-localization of the positive signal with TH immunofluorescence labeling.

[0058] Figure 16 Comparison of the sections with the richest signals in the dorsal root ganglia of the spinal cord observed under 20x magnification using CTB488 and CTBPro confocal microscopes.

[0059] Figure 17 Comparison of fluorescence intensity corresponding to the section with the richest signal in the dorsal root ganglion of the spinal cord observed under 20x magnification using CTB488 and CTBPro confocal microscopes.

[0060] Figure 18 The results show the dorsal root ganglion signal observed by CTB488 and CTBPro under a 63x confocal microscope; where (h) represents the dorsal root ganglion signal, and (i) represents the positive signal co-localized with TH immunofluorescence labeling.

[0061] Figure 19 This is a schematic diagram of intrarenal injection in a mouse kidney.

[0062] Figure 20 (a) The section with the richest signal of the celiac ganglion observed by CTB488 and CTBPro under a confocal microscope at 20x magnification (intraparenchymal injection method). Figure 20 (b) and (c) show the comparison of fluorescence intensity and number of positive neuron signals corresponding to the cut surface, respectively.

[0063] Figure 21 (c) The celiac ganglion signal observed by CTB488 and CTBPro under a confocal microscope at 63x magnification under intraperitoneal injection. Figure 21 (d) shows the co-localization of positive signals of CTB488 and CTBPro with TH immunofluorescence labeling observed under a confocal microscope at 63x magnification.

[0064] Figure 22 Confocal microscopy images of 293T cells before (a) and after (b) CLEM chemical treatment following CTBPro transfection.

[0065] Figure 23 The results of applying CTBPro to the idisco transparency method are shown. (a) is the axon signal of the right injection site after transparency, and (b) is the soma signal of the contralateral cortex after transparency. Detailed Implementation

[0066] To enable those skilled in the art to better understand the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments.

[0067] Example 1

[0068] (1) Design and characterization of CTBPro

[0069] The CTBPro construct developed in this invention fuses CTB with mbaojin (a highly photostable green fluorescent protein) via a flexible linker. To optimize its expression, an N-terminal signal peptide was added, and mammalian codon optimization was performed. (Reference) Figure 1 , Figure 1 The left side shows the structure of CTB, and the right side shows CTBPro of this invention. Adding the mbaojin protein to CTB creates our CTBPro. The complete DNA sequence of CTBPro is shown in SEQ ID NO. 1 of the sequence listing.

[0070] To investigate whether the fusion design affects the structural integrity or functional properties of CTB, we performed structural prediction using AlphaFold2. Comparison of the predicted CTBPro structure with the known CTB crystal structure showed no discernible changes in the conformation of the CTB domains. Figure 2 (a). Furthermore, molecular docking experiments confirmed that CTBPro retained its ability to bind GM1 gangliosides (a key functional property of CTB), with no observable differences compared to native CTB. (Ref.) Figure 2 (b) and (c).

[0071] (2) Preparation and purification of CTBPro fusion protein

[0072] Follow these steps:

[0073] (a) Transfecting a gene encoding the fusion protein into a host cell and culturing the transfected cells at 37°C to express the fusion protein;

[0074] (a1) Prepare 293F cells for transfection in advance using 1L of culture medium;

[0075] (a2) Count the cells. If the cell density is approximately 2.0 × 10⁶ cells per 1 L of culture medium. 6 If there are 100 cells, then transfection begins;

[0076] (a3) Prepare plasmids at a ratio of 2 mg plasmid per 1 L of cells;

[0077] (a4) Mix the plasmid and PEI transfection reagent at a mass ratio of 1:3 and incubate at room temperature for 15 minutes;

[0078] (a41) Dissolve PEI powder in ddH2O (ultrapure water) to form a solution of 1 mg / ml;

[0079] (a42) Filtered using a 0.22 μm filter membrane;

[0080] (a43) Add the plasmid to the PEI solution and mix thoroughly;

[0081] (a44) Incubate at room temperature (RT) for 15 minutes;

[0082] (a5) Add the transfection reagent containing the plasmid to the cells, transfer them to a shaker, and incubate for 4 days at 37°C and 220 rpm.

[0083] In the above steps (a1) to (a5), the steps can be performed in order or out of order depending on the actual situation; the ratio of plasmid to cell number needs to be confirmed; for some proteins that are difficult to secrete, the culture time can be appropriately extended; if isopropanol is used in the plasmid extraction process, it is best to determine the plasmid concentration by adding a marker (molecular weight standard) by gel electrophoresis, rather than using Nanodrop;

[0084] (b) Collect the cell culture supernatant and concentrate the fusion protein in the supernatant;

[0085] (b1) Centrifuge the culture medium containing cells at 4°C with a centrifugal force of 4000g for 10 minutes, discard the cell precipitate, and collect the supernatant.

[0086] (b2) While centrifuging the cells, remove the Flag column in advance, wash it twice with 1x sterile PBS, and equilibrate it at room temperature; note that the column can only be used a maximum of 3 times in this step to ensure the best results.

[0087] (b3) Pass all collected supernatant through a Flag affinity chromatography column; this step requires keeping the column moist and controlling the flow rate, and repeating this process 2 to 3 times if necessary;

[0088] (b4) After passing all the supernatant through the column, wash the column twice with 1xPBS and finally wash once with 2mL PBS. In this step, after washing with PBS, almost all CTB-mbaojin (fusion protein, i.e. target protein, abbreviated as CTBPro) is retained on the column.

[0089] (b5) Perform SDS-PAGE analysis on the liquid inside the column and the liquid after passing through the column (180V, about 1 hour); after Coomassie brilliant blue staining, estimate the target protein concentration; digest with 3C enzyme at 4°C overnight at a mass ratio of target protein: 3C enzyme = 100:1; in this step, if the protein concentration is too high, more 3C enzyme needs to be added and the reaction time needs to be extended.

[0090] (c) Separate and purify to obtain the fusion protein;

[0091] (c1) After digestion, the column was eluted with 1xPBS 2-3 times;

[0092] (c2)2 Concentrate the eluent to 1-2 mL and load it onto AKTA (molecular sieve). After AKTA is completed, select the receiving tubes near the peak according to the sample elution spectrum and perform SDS-PAGE on them to determine whether the purification is complete and the protein content.

[0093] (c3) Based on the SDS-PAGE results, select the appropriate receiving tube, mix the liquid, and concentrate the protein using a concentration column of appropriate molecular weight according to the amount of target protein. Measure the protein concentration using Nanodrop; if the concentration is low, further concentration can be performed. After confirmation, aliquot the target protein, rapidly freeze it in liquid nitrogen, and store it at -80°C. The concentrated target protein (fusion protein) is as follows: Figure 3 As shown.

[0094] It is recommended to use liquid nitrogen for rapid freezing of proteins to minimize side effects. Additionally, it is suggested to perform SDS-PAGE on the liquids produced at each step to observe the results.

[0095] Example 2

[0096] This embodiment compares the fluorescence intensity of the CTBPro provided by this invention with that of the commercially available CTB488 (purchased from Invitrogen, C34775). The specific procedure is as follows: 2.5 μg / μL of CTBPro and CTB488 were used respectively. The bright-field images of the test samples are shown below. Figure 4 As shown in (a), and the images of these two samples under 488nm fluorescence intensity excitation are as follows. Figure 4 As shown in (b). It is noteworthy that, despite having the same mass concentration of 2.5 μg / μL, CTBPro has a molecular weight approximately three times that of CTB488. Therefore, based on molar concentration, CTBPro requires only one-third the molar concentration of CTB488 to achieve superior fluorescence. To maintain consistency, all subsequent experiments used the same mass concentration (2.5 μg / μL) for both tracers.

[0097] Furthermore, this embodiment also tested the two samples using a quantitative photoluminescence spectrometer, and the results are as follows: Figure 5 As shown, Figure 5 The fluorescence intensities of CTBPro and CTB488 under different excitation lights were detected by steady-state and transient fluorescence spectrometry. It can be seen that the fluorescence intensity of CTBPro is much higher than that of CTB488, increasing by about 3 times.

[0098] Example 3

[0099] Comparison of retrograde tracing effects of CTBPro and CTB488 in the central nervous system

[0100] To verify CTBPro's neuronal tracing capabilities in vivo, and using CTB488 as a control, we investigated a well-defined central nervous system circuit: the projection from the ventral tegmental area (VTA) to the nucleus accumbens (NAc), such as... Figure 6 As shown. Specifically, 100 nL of CTBPro and CTB488 were injected into the NAc of mice, and brain samples were collected for imaging 7 days after injection.

[0101] The results are as follows Figure 7 , 8 As shown in 9 and 10:

[0102] Figure 7 Images (c) and (d) show the signal in the NAc brain region at the CTBPro injection site. Image (c) was taken at 20x magnification, and image (d) was taken at 63x high resolution. Figure 7 (c) It can be seen that the two small images represent the same field of view, except that the first one has added blue cell nucleus signals. Similarly, Figure 7 The two smaller images in (d) also represent a field of view and are the first to have the cell nucleus signal highlighted in blue. Figure 7 (e) and (f) show the signal in the NAc brain region at the CTB488 injection site. (e) was taken at 20x magnification, and (f) was taken at 63x high magnification. Figure 7 (e) and (f) are two small images that represent a single field of view, with the first one showing the blue nucleus signal.

[0103] Compare Figure 7 (c), (d) and Figure 7 As can be seen in (e) and (f), CTBPro effectively labeled the axons in the NAc region, producing clear and strong signals even at a relatively low confocal laser intensity of 0.2%. Although CTB488 successfully traced neuronal projections, its imaging quality was significantly lower than that of CTBPro under the same laser intensity conditions, meaning it failed to achieve comparable resolution in terms of neuronal morphology.

[0104] Figure 8 (i) and (j) show the imaging of the endpoint of CTBPro tracking in the injected brain region; Figure 8 (k) and (l) show the imaging of the endpoint (VTA) of CTB488 in the injected brain region; the magnification of each small image and the blue nuclear signal are shown. Figure 7 (c), (d), (e), and (f) are consistent. It can be observed that CTBPro achieves detailed neuronal labeling, enabling visualization of clear neuronal cell bodies and comprehensive morphological information, including neuronal processes in VTA. However, under this imaging condition, the CTB488 cell body signal appears to be diffuse and lacks clear morphological labeling.

[0105] Furthermore, this embodiment also uses immunofluorescence staining with synaptophysin, a marker of neuronal axonal terminals (presynaptic membrane), and the results are as follows: Figure 9 As shown in (g) and (h) (63x image), (g) shows that the signal labeled by CTBPro in the NAc brain region almost overlaps with that of Synaptophysin, but (h) shows that the overlap of CTB488 seems to be less than that of CTBPro.

[0106] Similarly, in this embodiment, the VTA brain region was also stained with the neuronal marker MAP2, and the results are as follows: Figure 10 As shown in (m) and (n) (photographed at 63x), the (m) image shows that the cell bodies labeled by CTBPro and MAP2 are highly co-labeled, but the (n) image shows that the co-labeling rate of CTB488 is not high.

[0107] This embodiment also tested the fluorescence intensity comparison of CTBPro and CTB488 in the NAc and VTA brain regions, respectively, and the results are as follows: Figure 11 As shown, (a) is a comparison of the fluorescence intensity of CTB488 and CTBPro in the NAc brain region, and (b) is a comparison of the fluorescence intensity of CTB488 and CTBPro in the VTA brain region. It can be found that the fluorescence intensity of CTBPro is better than that of CTB488 in both the NAc and VTA brain regions.

[0108] Example 4

[0109] Comparison of retrograde tracing effects of CTBPro and CTB488 in the peripheral nervous system

[0110] Most studies on neural circuits have been limited to the central nervous system, resulting in a relatively abundant pool of tracers for these circuits. In recent years, neurobiological research has increasingly focused on the interactions between the nervous system and other organ systems, necessitating the development of more efficient peripheral neural tracing technologies. The kidneys play a central role in the urinary and cardiovascular systems, regulating blood pressure and blood volume through neural innervation. However, the neural circuitry of the kidneys remains unclear, particularly in reverse neural tracing, which faces numerous technical limitations. Previous studies have used commercially available CTB488 or CTB555 for targeted intrarenal injection for reverse single-stage tracing. Here, we used CTBPro to perform reverse renal tracing in C57BL / 6J mice and compared it with CTB488, finding that CTBPro has significant advantages in peripheral neural imaging.

[0111] The efferent nerves innervating the kidneys are predominantly sympathetic. Current anatomy considers the splanchnic-celiac-superior mesenteric ganglion complex (SCSMG) to be the location of the postganglionic sympathetic neuron cell bodies innervating the kidneys. Figure 12 (a)). For renal afferent nerves, the neuronal cell bodies are located in the dorsal root ganglion (DRG) of the spinal cord. Figure 12 (b)). Each C57BL / 6J mouse was anesthetized and its kidneys were exposed before being subcapsularly injected with 20 μL of CTBPro and CTB488. Figure 12 (c) After waiting for 5 days, the celiac ganglion and the dorsal root ganglion of the L1 segment of the spinal cord of the mouse were taken, and the signal was observed after making 30μm frozen sections.

[0112] The section with the richest positive signal in a single ganglion was selected, and the single ganglion slice was imaged using a laser confocal microscope, keeping the laser intensity, gain value, and pinhole value consistent between the CTBPro-injected experimental group and the CTB488-injected control group. We selected the section with the richest fluorescence signal in the celiac ganglion and observed it under 20x magnification. We found that CTBPro showed more positive signal and stronger fluorescence intensity compared to CTB488. Specific results are as follows: Figure 13 , 14 As shown. Figure 13 This is the section with the richest signal in the celiac ganglion observed under a 20x confocal microscope; the blue area represents the cell nucleus signal. Figure 14 This is a comparison of corresponding fluorescence intensity and the number of positive neuron signals.

[0113] Similarly, as Figure 15As shown in (e) and (f), under a 63x microscope, we observed that CTBPro exhibited a stronger fluorescence signal in the positive neurons of the celiac ganglion identified by reverse tracing. Figure (e) shows the celiac ganglion signal results of CTB488 and CTBPro, and figure (f) shows the co-localization results of the positive signals of CTB488 and CTBPro with TH immunofluorescence labeling. Through immunofluorescence experiments, we achieved co-localization of CTB signals and TH immunofluorescence signals, confirming that the traced neurons were indeed sympathetic neurons. TH is a specific marker of the sympathetic nervous system.

[0114] We observed a similar phenomenon in the dorsal root ganglia of the spinal cord. Due to the sparse distribution of afferent nerves in the kidneys, the labeling rate was lower compared to efferent nerves. However, CTBPro still showed some labeling ability in the dorsal root ganglia, but CTB488 only displayed vascular-like signals, with almost no neuronal morphology observable. Figure 16 As shown, Figure 16 This is a comparison of the sections with the richest signal in the dorsal root ganglia of the spinal cord observed under 20x magnification using CTB488 and CTBPro confocal microscopes. Furthermore, from... Figure 16 It can also be seen that the experimental group injected with CTBPro clearly showed a stronger fluorescence signal, while the control group injected with CTB488 only showed an autofluorescence signal. Furthermore, the fluorescence intensity of the section with the richest signal in the dorsal root ganglion was compared... Figure 17 As shown, the fluorescence intensity of the CTBPro experimental group was significantly higher than that of the CTB488 control group.

[0115] Furthermore, we used confocal microscopy to observe the signals in the dorsal root ganglion under 63x magnification, and the results are as follows: Figure 18 As shown in (h) and (i), (h) presents the signal results of CTB488 and CTBPro in the dorsal root ganglion, and (i) shows the co-localization results of the positive signals of CTB488 and CTBPro with TH immunofluorescence labeling. These results further confirm that the signal intensity tracked by CTBPro is significantly higher than that of CTB488. Furthermore, the co-localization of the tracked positive signals with TUBB3 using immunofluorescence confirms that it is a neural signal. TUBB3 is a marker of neuronal cytoplasm and processes.

[0116] In addition, we also employed the same method as previous, limited studies on renal neural tracing: intrarenal injection. We selected five injection sites on each kidney, injecting 2 μL of either LTBPro or CTB488 (e.g., intrarenal injection) at each site. Figure 19(As shown). Similarly, after waiting 5 days, celiac ganglia and L1 dorsal root ganglia of the spinal cord were harvested from mice and subjected to 30μm frozen sections for signal observation. The results showed that no positive signal was observed in the dorsal root ganglia of the spinal cord via renal parenchymal injection in our study. Therefore, we only performed subsequent comparative analysis on the celiac ganglia. We selected the section with the richest fluorescence signal in the celiac ganglia and observed it under a 20x microscope. We found that CTBPro showed more positive signal and stronger fluorescence intensity compared to CTB488, such as... Figure 20 As shown in the figure. Similarly, under a 63x microscope, we observed that CTBPro showed a stronger fluorescence signal in the positive neurons of the celiac ganglion identified by reverse tracing; through immunofluorescence experiments, we achieved co-localization of CTB signal and TH immunofluorescence signal, which confirmed that the traced neurons were indeed sympathetic neurons; the above results are as follows. Figure 21 As shown.

[0117] Example 5

[0118] CTBPro Compatibility with Advanced Imaging Technologies

[0119] Given the superior neural tracing performance demonstrated by CTBPro in previous experiments, we sought to evaluate its compatibility with other commonly used methods in neuroscience. Specifically, this embodiment tested whether CTBPro could be integrated with tissue transparency techniques for whole-brain circuit visualization and correlated light electron microscopy (CLEM) for ultrastructural studies. Existing fluorescent tracers have limited compatibility with these advanced imaging methods, which restricts their wider application.

[0120] (1) Chemical treatment capability required for CTBPro to tolerate CLEM

[0121] The specific evaluation process is as follows:

[0122] a) 293T cells were transfected with CTBPro, and its expression was detected using confocal microscopy 60 hours post-transfection. The results showed that strong fluorescence signals were observed in the transfected cells, confirming successful CTBPro expression. Figure 22 As shown in (a);

[0123] b) The 293T cells successfully transfected and expressed in a) were placed on ice for 1 hour in a mixture of 2% PFA and 2.5% glutaraldehyde, washed three times with 0.1M PB solution, treated on ice for 1 hour with 0.5% osmium tetroxide, washed three times with 0.1MPB, washed three times with dd water, treated with 1% uranyl acetate at room temperature for 1 hour, washed three times with dd water, dehydrated with a gradient of alcohols (30%, 50%, 70%, 95%, 100%, 100%, 100%) for 10 minutes each, reacted with ethanol: 812 resin at volume ratios of 2:1, 1:1, and 1:2 at room temperature for 30 minutes each, and incubated overnight at room temperature with 812 resin (replaced every three hours, for a total of three times). Finally, the samples were embedded at 58°C for 14-16 hours.

[0124] c) After the treatment described in b), the results were examined using a confocal microscope as follows: Figure 22 As shown in (b), the results indicate that after the above chemical treatment, a large number of cells still retain fluorescence signals in CTBPro, indicating that it has good tolerance to the chemical treatment process of CLEM samples.

[0125] (2) Compatibility of CTBPro with whole-brain tissue transparency methods used for loop tracing

[0126] The specific evaluation process was as follows: CTBPro was injected into the right cortical region of the mouse brain, and the commonly used Idisco clearing method was applied. Unlike the traditional CTB488 (which failed to provide clear axonal signals and exhibited diffuse fluorescence under tissue clearing conditions; data not shown), CTBPro successfully preserved its fluorescence signal after tissue clearing, and successfully traced the cell body signals of contralateral cortical neurons from the right cortex, such as... Figure 23 As shown in (a) and (b), (a) shows the axon injection site after transparency, and (b) shows the soma signal in the contralateral cortex after transparency. This preservation of signal integrity enables CTBPro to perform high-resolution tracing of axon projections at the whole-brain level.

[0127] These results demonstrate that CTBPro is highly compatible with advanced imaging techniques commonly used in neuroscience. Its ability to resist fluorescence loss during CLEM preparation and to maintain signal integrity after tissue clearing makes CTBPro a versatile neural tracer. These properties provide a new tool for circuit tracing studies and expand the applicability of fluorescent tracers in integrative and multimodal neuroscience research.

[0128] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention, but such modifications or substitutions are all within the scope of protection of the present invention.

Claims

1. A fusion protein used as a retrograde tracer, characterized in that: the fusion protein comprises a cholera toxin B subunit protein, an mbaojin fluorescent protein, and a linker for connecting the cholera toxin B subunit protein and the mbaojin fluorescent protein.

2. The fusion protein of claim 1, wherein: the linker is a flexible linker.

3. The fusion protein of claim 1, wherein: the fusion protein further comprises a signal peptide at the N terminus. 4.A gene encoding the fusion protein according to claim 1.

5. The genetic code of claim 4, wherein: the nucleotide sequence of the gene is shown in SEQ ID NO:

1.

6. An expression vector, characterized by: the expression vector comprises the gene according to claim 4 or 5.

7. A host cell, characterized in that: the host cell comprises the expression vector according to claim 6.

8. The host cell of claim 7, wherein: the host cell is a mammalian cell. 9.Use of the fusion protein according to any one of claims 1 to 3 as a retrograde tracer in neural circuit research.

10. A method of preparing a fusion protein according to any one of claims 1 to 3, characterized in that, comprising the following steps: (a) connecting a gene encoding the fusion protein to a vector to construct an expression vector, transfecting the expression vector into a host cell, and culturing the transfected cell at 37 ± 2 ℃ to express the fusion protein; (b) collecting cell culture supernatant and concentrating the supernatant; (c) isolating and purifying to obtain the fusion protein.

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