Protein Y targeting to mucous layer and enhancing MUC5AC expression, and coding gene and application thereof
Patent Information
- Application Number
- CN202511264778.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-05
- Publication Date
- 2026-01-20
Smart Images
Figure CN121362239A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the technical field of biomedical engineering, and particularly relates to a protein Y targeting mucus layer and enhancing expression of MUC5AC, a coding gene thereof and application. BACKGROUND
[0002] Host mucus layer, as the first physical and biochemical barrier against pathogens and environmental stimuli, widely covers the mucosal epithelial surface of respiratory tract, digestive tract and reproductive tract. The layer is mainly composed of highly glycosylated mucin, among which MUC5AC, as an important gel-forming mucin, is secreted by mucosal epithelial cells and plays a key role in lubrication, cell protection, pathogen interception and immune regulation through its complex network structure and glycosylation modification. However, the mucus layer has a dynamic updating feature, which is replaced through secretion and cilia clearance mechanism, resulting in short retention time and low penetration efficiency of exogenous therapeutic molecules such as proteins, polypeptides, nucleic acids and traditional drugs on the mucosal surface, thereby significantly limiting their bioavailability and therapeutic effect. At present, in order to improve the retention and delivery efficiency of drugs and biological macromolecules in the mucus layer, some studies have been devoted to developing mucus-adhesive carrier systems. For example, using chitosan, alginate and other bioadhesive polymers, or modifying the surface of nanoparticles to enhance the interaction with mucus components. Although these strategies have improved local retention to some extent, there are still problems such as insufficient targeting, easy removal by mucus clearance mechanism, or inability to actively regulate the mucosal microenvironment. Especially, there is a lack of delivery carriers that can specifically recognize the main components of mucus (such as MUC5AC) and have the ability to actively regulate biological functions. Therefore, developing a protein carrier that can not only efficiently target the mucus layer, but also actively enhance the function of the mucus barrier, has important value for promoting the development of mucosal drug delivery systems and mucosal immune strategies. Under this background, the present application proposes a new protein Y, which can not only specifically target MUC5AC protein in the mucus layer, but also enhance the expression and secretion of MUC5AC, thereby strengthening the mucus barrier and forming a positive feedback to enhance its own retention, providing a new strategy for mucosal targeted delivery. SUMMARY
[0003] Technical problems solved: The present application aims to solve the technical deficiencies existing in the existing mucosal targeting delivery strategy. In the prior art, although some carrier materials can be temporarily retained in the mucus layer by non-specific adsorption or physical entrapment, there are still problems such as weak targeting, easy to be removed by mucus self-cleaning mechanism, and inability to actively adjust the function of mucosal microenvironment. In particular, there is a lack of functional protein carriers that can specifically recognize key components of mucus (such as MUC5AC) and actively enhance the integrity of the mucus barrier and form a retention-enhancing effect. In view of the above defects, the present application provides a protein Y for targeting the mucus layer and enhancing MUC5AC expression, its coding gene and application. The protein can also actively up-regulate the transcription and expression level of MUC5AC, thereby thickening and strengthening the mucus gel layer, further enhancing the retention and enrichment of the protein itself and the therapeutic molecules carried by the protein in the mucosal site, forming a positive feedback cycle, and ultimately significantly improving the delivery efficiency and action time of drugs, vaccines or other bioactive molecules in the mucosal local.
[0004] Technical scheme: A protein Y, the amino acid sequence of which is shown as SEQ ID NO: 1.
[0005] A gene encoding the protein Y, the nucleotide sequence of which is shown as SEQ ID NO: 2.
[0006] A recombinant vector comprising the above-mentioned gene.
[0007] The above-mentioned vector is a prokaryotic expression vector pET-28(+).
[0008] A host cell comprising the above-mentioned recombinant vector.
[0009] The above-mentioned host cell is Escherichia coli BL21 (DE3).
[0010] A pharmaceutical composition comprising the above-mentioned protein Y and a pharmaceutically acceptable carrier.
[0011] Application of the above-mentioned protein Y in the preparation of a composition for improving the adhesion of molecules in the mucus layer.
[0012] The above-mentioned composition is a mucosal drug delivery carrier.
[0013] Application of the above-mentioned protein Y in the preparation of a composition for enhancing the expression of MUC5AC in mucosal epithelial cells.
[0014] Beneficial effects: Laser confocal microscope (CLSM) observation confirmed that protein Y can significantly adhere to the surface of human laryngeal epithelial cells (Hep-2) and human lung epithelial cells (A549) MUC5AC. With the help of surface plasmon resonance (SPR) technology, it is further confirmed that protein Y can bind to MUC5AC in a concentration gradient-dependent manner. Unexpectedly, the present application also found that after protein Y binds to MUC5AC on the surface of the cell, it can up-regulate the mRNA transcription and protein expression level of MUC5AC in a concentration gradient-dependent manner. The up-regulation of MUC5AC expression helps to thicken and strengthen the mucus layer, thereby promoting the retention of protein Y in the mucosa area, forming a positive feedback. Therefore, protein Y is an excellent mucosa-targeted delivery carrier, which can be used to improve the delivery efficiency of drugs, vaccines, antibacterial peptides or other active molecules in the respiratory tract, digestive tract, reproductive tract and other mucous layer-containing areas. BRIEF DESCRIPTION OF DRAWINGS
[0015] Figure 1 : Laser confocal microscope was used to observe the adhesion of protein Y to MUC5AC on the surface of Hep-2 (A) and A549 (B) cells; DAPI was used to label the cell nucleus, Anti-MUC5AC (green) was used to label MUC5AC, and red fluorescent protein Y was used to label protein Y.
[0016] Figure 2 : Surface plasmon resonance (SPR) technology was used to analyze the binding characteristics of protein Y and MUC5AC.
[0017] Figure 3 : Protein Y enhances the expression (A) and transcription (B) of MUC5AC in a concentration gradient-dependent manner. Hep-2 and A549 cells were treated with different concentrations of protein Y, and then the expression and transcription of MUC5AC were detected by Western Blot and qPCR, respectively. DETAILED DESCRIPTION
[0018] The following examples can enable a person skilled in the art to more fully understand the present application, but do not limit the present application in any way. Modifications and substitutions of the strategies, methods, steps or conditions of the present application, without departing from the spirit and essence of the present application, all belong to the scope of the present application.
[0019] Unless otherwise specified, the examples are carried out according to the conventional experimental conditions, such as Sambrook et al. Molecular Cloning Laboratory Manual (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual, 2001), or according to the conditions recommended by the manufacturer's instructions.
[0020] Example 1: Construction of the expression vector of protein Y The Nanjing Genscript Biotech Co., Ltd. was commissioned to synthesize the gene sequence (SEQ ID NO: 2) encoding protein Y based on the amino acid sequence of protein Y, and cloned it into the pET-28(+) expression vector.
[0021] Example 2: Expression and purification of protein Y The constructed recombinant plasmid was transformed into *E. coli* BL21(DE3) competent cells. Subsequently, it was transferred to 200 mL LB liquid medium at a 1:100 ratio, Kans antibiotic was added, and the cells were incubated at 37 °C with shaking at 180 rpm. OD was then calculated. 600 When the concentration reached 0.5, 1 mM IPTG was added, and the mixture was transferred to a 16 °C shaker for overnight induction. The next day, bacterial cells were collected by centrifugation at 10,000 g for 10 min, washed twice with PBS, and resuspended in 20 mL of supernatant dissolution buffer (20 mM PBS, 150 mM NaCl, 10 mM ME, pH 7.0). The mixture was then placed in an ice box and sonicated to lyse the bacteria. The lysate was centrifuged at 10,000 g for 20 min, and the supernatant was collected. A 1 mL His-tagged Ni-NTA affinity chromatography column was used for subsequent purification. A small amount of purified protein was taken, 5×SDS loading buffer was added, and the sample was boiled in water for 10 min. After centrifugation, the supernatant was collected for SDS-PAGE electrophoresis.
[0022] Example 3: Laser confocal microscopy analysis of adhesion ability High-purity protein was ultrafiltered, concentrated, and the solvent was replaced with PBS. The ultrafiltered protein was incubated with Hep-2 and A549 cells at 37 °C and 5% CO2 for 2.5 hours, followed by washing three times with DMEM. The samples were then fixed with 4% paraformaldehyde for 15 minutes, followed by washing three times with PBS; then blocked with 5% BSA for 1 hour, followed by washing three times with PBS. The samples were then incubated overnight with Anti-MUC5AC and His primary antibody at 4 °C, followed by washing three times with PBS; then incubated with goat anti-rabbit-FITC secondary antibody (green) and goat anti-mouse-TRITC secondary antibody (red protein) at room temperature for 1 hour. Finally, the cell nuclei were incubated with DAPI (blue) in a dark room for 10 minutes.
[0023] The results of laser confocal microscopy observation show that ( Figure 1 The colocalization of green fluorescent signals (representing MUC5AC) and red fluorescent signals (representing the protein) around cells treated with protein Y indicates that protein Y can adhere to MUC5AC on the cell surface.
[0024] Example 4: Surface Plasmon Resonance (SPR) analysis of binding affinity MUC5AC was immobilized on the CM7 chip surface. Protein Y was serially diluted and allowed to flow over the chip surface at a gradient of concentrations. The SPR analysis results ( Figure 2 ) showed that the binding response of protein Y to MUC5AC increased significantly with increasing concentration, and the calculated equilibrium dissociation constant (KD) value was 3.7 × 10 -7 M, indicating that protein Y and MUC5AC had high affinity and the binding was concentration-dependent.
[0025] Example 5: Protein Y enhances expression of MUC5AC Different concentrations of protein Y were incubated with Hep-2 and A549 cells at 37 ℃, 5% CO2 for 2.5 hours, followed by washing the cells with DMEM for 3 times. The expression level and transcription level of MUC5AC were detected by Western blot (WB) and qPCR, respectively.
[0026] The specific steps are as follows: WB: The cells were lysed with sodium dodecyl sulfate (SDS) sample buffer. Equal amounts of total protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to a polyvinylidene fluoride (PVDF) membrane. The PVDF membrane was blocked with a solution containing 5% skim milk and incubated at 37 ℃ for 1 hour. Then, the PVDF membrane was incubated with a 1:2000 diluted primary antibody (MUC5AC and Tubulin) at 4 ℃ overnight, and then treated with a 1:2000 diluted horseradish peroxidase (HRP)-labeled goat anti-mouse IgG. Each step was separated by washing the membrane with a PBS solution containing 0.05% Tween 20 for 10 minutes, and this operation was repeated three times. Finally, the signal was detected using ECL + reagent substrate.
[0027] qPCR: Total RNA was extracted from the sample using TRIzol reagent according to the instructions. Total RNA was reverse transcribed into cDNA using the HiScript II cDNA synthesis kit. qPCR was performed on the QuantStudio 6 Flex instrument using cDNA as a template, and the transcription level of the selected genes was verified using ChamQ Universal SYBR qPCR Master Mix. The transcription level of the housekeeping gene GAPDH was used as an internal control to standardize the expression of the target genes. The results ( Figure 3) The results showed that the transcription and expression levels of MUC5AC in the protein Y treatment group were significantly up-regulated in a concentration-dependent manner. This indicates that protein Y has a unique function of increasing MUC5AC expression.
[0028] Although the present application is explained in detail with reference to the examples given, the technical solution of the present application can be modified or replaced equivalently according to the needs without departing from the spirit and scope of the technical solution of the present application.
Claims
1. A protein Y, characterized by, The amino acid sequence of which is shown as SEQ ID NO:
1.
2. A gene encoding the protein Y according to claim 1, characterized in that, The nucleotide sequence of which is shown as SEQ ID NO:
2.
3. A recombinant vector, characterized in that, The gene according to claim 2.
4. The recombinant vector of claim 3, wherein, The vector is a prokaryotic expression vector pET-28(+).
5. A host cell, characterized in that, The host cell comprises the recombinant vector according to claim 3 or 4.
6. The host cell of claim 5, wherein, The host cell is Escherichia coli BL21 (DE3).
7. A pharmaceutical composition, characterized by, The protein Y according to claim 1 and a pharmaceutically acceptable carrier.
8. Use of the protein Y according to claim 1 in the preparation of a composition for improving the adhesion of a molecule in the mucus layer.
9. Use according to claim 8, characterized in that, The composition is a mucosal administration carrier.
10. Use of the protein Y according to claim 1 in the preparation of a composition for enhancing the expression of MUC5AC in mucosal epithelial cells.