A targeted peptide VN_P for procambarus clarkii oocyte and a design and screening method and application thereof

Abstract

The present application belongs to the technical field of biotechnology, and particularly relates to a target peptide VN_P of procambarus clarkii oocyte and a design and screening method and application thereof. The present application discloses a design and screening method of the target peptide VN_P of the procambarus clarkii oocyte, which comprises the following steps: (1) predicting the interaction site between the procambarus clarkii vitellogenin and the vitellogenin receptor by using AlphaFold3; (2) according to the principle that the number of hydrogen bonds and salt bridges in the continuous 20 amino acids is greater than 4, the short peptide sequence is intercepted; (3) using the biological membrane interference technology, according to the binding rate constant and the dissociation rate constant of the short peptide and the vitellogenin receptor, the target peptide of the procambarus clarkii oocyte is screened out. This breakthrough lays a technical foundation for establishing a gene editing element delivery method with oocyte targeting, and is expected to be applied in the field of gene editing of the procambarus clarkii.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a red swamp crayfish oocyte-targeting peptide VN_P, its design and screening methods, and its applications. Background Technology

[0002] Efficient targeted delivery of oocytes remains a technological bottleneck for gene editing and reproductive regulation in crustaceans. Most shrimp and crab fertilized eggs have low survival rates after in vitro hatching, and are encased in an opaque, hard shell, making it difficult to observe cleavage grooves and blastomeres. This hinders the delivery of gene-editing elements via microinjection into fertilized eggs, impeding the application of gene-editing breeding in shrimp and crab aquatic animals. While existing cell-penetrating peptides (CPPs) can penetrate cell membranes, they lack oocyte-specific recognition capabilities and cannot be targeted for delivery under in vivo conditions. The vitellogenin receptor (VgR) in redclaw crayfish is a key receptor mediating oocyte endocytosis, but its ligand-binding domain has not been developed for delivery systems.

[0003] To address the aforementioned issues, this application filed on April 15, 2025, with patent number CN119978074A, concerning a redclaw crayfish oocyte-targeting penetration peptide VgSP and its in vivo delivery method. This invention provides both a redclaw crayfish oocyte-targeting penetration peptide VgSP and its applications. However, it lacks information regarding the pathway for obtaining the peptide, as well as operational methods for designing and screening the targeted peptide.

[0004] Based on this, this application obtained a VgR-specific binding peptide VN_P3 through screening and established an in vivo delivery method, thus solving the above-mentioned technical problems. Summary of the Invention

[0005] To address the problems existing in the prior art, this invention provides a technical solution for the design and screening of VN_P, a target peptide for red swamp crayfish oocytes, and its application.

[0006] The present invention is implemented using the following technical solutions: The first aspect of this invention provides a method for designing and screening VN_P, a targeting peptide for redclaw crayfish oocytes, comprising the following steps: R.1 Using AlphaFold3 to predict the interaction sites between vitellogenin and vitellogenin receptor in red swamp crayfish; R.2 Short peptide sequences are truncated based on the principle that the number of hydrogen bonds and salt bridges interacting within 20 consecutive amino acids is greater than 4. R.3 utilizes biomembrane interference technology to screen for red swamp crayfish oocyte-targeting peptides based on the binding rate constant and dissociation rate constant of short peptides to vitellogenin receptors.

[0007] Furthermore, the prediction of the interaction sites specifically involves: obtaining the amino acid sequences of redclaw crayfish vitellogenin and vitellogenin receptor from the GenBank database as input; generating five models for each complex, and selecting the model with the highest predicted local distance difference test score for further analysis; using PyMOL to visualize the predicted interface residues, and defining residues within 4 Å in distance as interaction sites.

[0008] The second aspect of the present invention provides a red swamp crayfish oocyte targeting peptide VN_P obtained by screening using the above-described method.

[0009] A third aspect of the present invention provides a red swamp crayfish oocyte targeting peptide VN_P3 obtained by screening using the above-described method, wherein the amino acid sequence of the short peptide is shown in SEQ ID NO.3.

[0010] The fourth aspect of this invention provides the application of the above-mentioned red swamp crayfish oocyte targeting peptide VN_P3 in red swamp crayfish oocyte targeting.

[0011] The fifth aspect of this invention provides the application of the aforementioned red swamp crayfish oocyte-targeting peptide VN_P3 as a vector for delivering functional molecules into oocytes.

[0012] Compared with the prior art, the beneficial effects of the present invention are: This invention provides a method for designing and screening the redclaw crayfish oocyte-targeting peptide VN_P, ultimately identifying the short peptide VN_P3, which can be recognized by receptors on the surface of redclaw crayfish oocytes and mediated to enter the oocytes. It also demonstrates the application of this short peptide as a carrier for delivering functional molecules into oocytes. This breakthrough lays the technical foundation for establishing a method for delivering gene-editing elements with oocyte targeting capabilities, and holds promise for application in the field of redclaw crayfish gene editing. Attached Figure Description

[0013] Figure 1 This is a diagram showing the interaction results between VTG and VgR; blue represents VTG protein, and red represents VgR protein. Figure 2 The graph represents the biolayer interferometry (BLI) techniques for VN_P1, VN_P2, VN_P3, VN_P4 and VgR1, VgR2, with the horizontal axis representing time and the vertical axis representing the response value. Figure 3 The results of ovarian fluorescence scanning after VN_P3 injection. Detailed Implementation

[0014] The following detailed description is provided with reference to the embodiments: This embodiment is implemented based on the technical solution of the present invention, and provides detailed implementation methods and specific operation processes, but the protection scope of the present invention is not limited to the following embodiments.

[0015] Example 1: Design and screening of VN_P, a target peptide for red swamp crayfish oocytes Predicting the interaction sites between vitellogenin (VTG) and vitellogenin receptor (VgR) in red swamp crayfish using AlphaFold3 (default parameters). Figure 1 The amino acid sequences of VTG (GenBank accession number AAG17936.1) and VgR (GenBank accession number XP_053635320.2) of red claw crayfish were obtained from the GenBank database as input. Five models were generated for each complex, and the model with the highest predicted local distance difference test (pLDDT) score was selected for further analysis. The predicted interface residues were visualized using PyMOL (version 2.5.4), and residues within 4 Å were defined as interaction sites. Hydrogen bonds and salt bridges at the interaction interfaces were calculated using the HBPLUS plugin (Standard Geometry Standard).

[0016] Based on the hydrogen bonds and salt bridges at the protein-protein interaction interface, short peptides with more than four hydrogen bonds and salt bridges interacting within a consecutive 20 amino acids were screened as target peptides, namely VN_P1: AIQHGDPVIFQAEGPVTAKISS (SEQ ID NO.1), VN_P2: SSSILSGIAKIQRDLRSRLLH (SEQ ID NO.2), VN_P3: EVRIASYIAAVRCAEKWDFEK (SEQ ID NO.3), and VN_P4: YDYHTPKKDPSMVTQLDRIMSQ (SEQ ID NO.4). The target peptides VN_P1, VN_P2, VN_P3, and VN_P4 were synthesized by Nanjing Genscript Biotech Co., Ltd., and 5-FAM was modified at the N-terminus to allow for observation of the distribution of FAM-labeled peptides in living or fixed cells using fluorescence microscopy / confocal microscopy. Their molecular weight, purity, and solubility information are listed in Table 1.

[0017] Table 1. Molecular weight, purity, and solvent of VN_P short peptides .

[0018] Because the VgR protein contains a signal peptide and a transmembrane domain, it cannot be fully expressed. Therefore, this invention splits it into two proteins, VgR1 (N-terminus, 771 amino acids, 86.31 Kda) and VgR2 (C-terminus, 751 amino acids, 77.52 Kda), based on their domains. These proteins are then expressed separately in prokaryotes using the petsumo expression vector, synthesized by Wuhan Jinkairui Biotechnology Co., Ltd. The expression process includes denaturation and renaturation of inclusion bodies. The nucleotide sequence of protein VgR1 is shown in SEQ ID NO.5, and its amino acid sequence is shown in SEQ ID NO.6. The nucleotide sequence of protein VgR2 is shown in SEQ ID NO.7, and its amino acid sequence is shown in SEQ ID NO.8.

[0019] Using biolayer interferometry (BLI), the binding rate constants (Ka(1 / Ms)), dissociation rate constants, and affinity constants of four VN_P short peptides to VgR1 and VgR2 receptors were detected. Figure 2 Table 2 shows the BLI sensing maps of four VN_P short peptides with VgR1 and VgR2 receptors. The affinity data for VN_P short peptides with VgR1 and VgR2 receptors are also presented. Response represents the detected signal response value. The rise in response value indicates that molecules are binding to the chip surface, and the biolayer is thickening. The fall in response value indicates that molecules are dissociating from the chip surface, and the biolayer is thinning. The stable phase of response value indicates that binding and dissociation have reached a dynamic equilibrium. Ka is the binding rate constant, representing the binding rate of the complex. Kdis is the dissociation rate constant, reflecting the stability of the complex. KD is the equilibrium dissociation constant, reflecting the strength of the interaction. Full X ^2 This represents the error between the actual curve and the fitted curve. The closer the error is to 0, the higher the reliability of the value. (Full R) ^2 The variance is represented by a value closer to 1, indicating a more reliable result. Generally, a value > 0.8 is considered reliable. After screening, it was found that VN_P3 can bind to the VgR receptor and is recognized by the N-terminus of VgR.

[0020] Table 2 Affinity data of VN_P short peptides to VgR receptors .

[0021] Example 2: Injection and detection of VN_P3 short peptide The VN_P3 short peptide was dissolved in formic acid and then diluted with enzyme-free water to a final formic acid concentration of 0.1% and a VN_P3 short peptide concentration of 1 μg / ul. At a dose of 1 μg / g shrimp body weight, the VN_P3 short peptide was injected into the female redclaw crayfish from the base of the third walking leg. After injection, the injection site was pressed for 2 minutes until hemolymph flow ceased. One side of the eyestalk was quickly removed using red-hot forceps, and the wound was cauterized to promote rapid healing. Forty-eight hours after eyestalk removal, the injected individuals were euthanized under cold shock. Ovarian tissue was harvested and sectioned. The sections were stained with DAPI and analyzed using fluorescence scanning. Blue fluorescence signals indicated the location of cell nuclei, and green fluorescence signals indicated the location of the short peptide. Figure 3 As shown, a green signal of the VN_P3 short peptide appears in the oocyte.

[0022] Finally, it should be noted that the above examples are merely some specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments and many variations are possible. All variations that can be directly derived or conceived by those skilled in the art from the disclosure of the present invention should be considered within the scope of protection of the present invention.

Claims

1. VN_P3, a target peptide for redclaw crayfish oocytes, characterized in that, The amino acid sequence of the target peptide is shown in SEQ ID NO.

3.

2. The application of the redclaw crayfish oocyte-targeting peptide VN_P3 as described in claim 1 in the preparation of redclaw crayfish oocyte-targeting products.

3. The application of the red swamp crayfish oocyte-targeting peptide VN_P3 as described in claim 1 in the preparation of a vector for delivering functional molecules into oocytes.

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