Cherax quadricarinatus oocyte targeted penetrating peptide VgSP and in-vivo delivery method thereof

Abstract

The invention discloses a cherax quadricarinatus oocyte targeted penetrating peptide VgSP and a living body delivery method thereof, and belongs to the technical field of biology. The invention provides the cherax quadricarinatus oocyte targeted penetrating peptide VgSP on one hand, and provides application of the cherax quadricarinatus oocyte targeted penetrating peptide VgSP on the other hand. The invention provides a polypeptide VgSP capable of being recognized by a cherax quadricarinatus oocyte surface receptor and mediated into an oocyte, and an injection and detection method of the polypeptide in sub-adult and adult female cherax quadricarinatus. The breakthrough lays a technical foundation for establishing a gene editing element delivery method with oocyte targeting, and is expected to be applied to the field of cherax quadricarinatus gene editing.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and specifically relates to a red claw crayfish ( Cheraxquadricarinatus ) Oocyte-targeted penetrating peptides and in vivo delivery methods thereof, for efficiently delivering functional molecules into oocytes. Background Art

[0002] Efficient targeted delivery of oocytes is a technical bottleneck for gene editing and reproductive regulation in crustaceans. Most shrimp and crab fertilized eggs have a low survival rate after in vitro hatching, and the fertilized eggs are covered with an opaque hard shell, so the cleavage furrow and blastomeres cannot be observed. It is difficult to deliver gene editing elements through microinjection of fertilized eggs, which hinders the application of gene editing breeding in shrimp and crab aquatic animals. Although existing cell-penetrating peptides (CPPs) can penetrate the cell membrane, they lack oocyte-specific recognition ability and cannot be delivered in vivo. The vitellogenin receptor (VgR) of red claw crayfish is a key receptor that mediates oocyte endocytosis, but its ligand binding domain has not been developed for delivery systems. The present invention solves the above technical problems by screening to obtain a VgR-specific binding peptide VgSP and establishing an in vivo delivery method. Summary of the invention

[0003] In view of the problems existing in the prior art, the object of the present invention is to design and provide a technical solution of a red claw crayfish oocyte targeted penetrating peptide VgSP and a method for its in vivo delivery.

[0004] The present invention is specifically implemented by the following technical solutions: The first aspect of the present invention provides a polypeptide VgSP, the amino acid sequence of the polypeptide is shown as SEQ ID NO.1.

[0005] The second aspect of the present invention provides the use of the above polypeptide VgSP as a red claw crayfish oocyte targeting penetrating peptide.

[0006] Furthermore, in this application, the polypeptide can specifically bind to the vitellogenin receptor VgR of red claw crayfish and enter the oocyte through receptor-mediated endocytosis. It carries a histone tag and can be identified by immunofluorescence targeting histones.

[0007] The third aspect of the present invention provides a method for in vivo delivery of a targeted penetrating peptide VgSP to oocytes of red claw crayfish, the method comprising: selecting sub-adult female crayfish, removing a unilateral eyestalk of the sub-adult female red claw crayfish, and inducing synchronization of oocyte development; 60 to 78 hours after the eyestalk is removed, injecting the polypeptide VgSP into the hemolymph at a dose of 400 to 600 ng / g shrimp body weight; 42 to 54 hours after the injection, confirming the enrichment of VgSP in the oocyte by immunofluorescence detection.

[0008] Furthermore, the subadult female shrimp is a 4-month-old individual; the unilateral eyestalk is removed 60 to 78 hours before injection; the eyestalk is removed by instantaneously burning the broken limb part with high-temperature tweezers to achieve rapid hemostasis; the injection is performed using a micro-injection syringe with a bevel angle of 45°, the injection site is the membrane area of ​​the third foot base, and pressing for 2 minutes after injection ensures hemolymph closure.

[0009] The fourth aspect of the present invention provides a method for in vivo delivery of a red claw crayfish oocyte targeted penetrating peptide VgSP, the method comprising: selecting an adult female crayfish, injecting the polypeptide VgSP into the hemolymph of the adult female red claw crayfish at a dose of 400 to 600 ng / g shrimp body weight; immediately removing a unilateral eyestalk to promote oocyte development; 42 to 54 hours after injection, detecting the localization of VgSP in the developing oocyte by immunofluorescence.

[0010] Furthermore, the adult female shrimp is an individual whose oocytes are in the early stage of development; the unilateral eyestalk is removed immediately after injection; the eyestalk is removed by instantaneously burning the broken limb part with high-temperature tweezers to achieve rapid hemostasis; the injection is performed using a micro-injection syringe with a bevel angle of 45°, the injection site is the membrane area of ​​the third foot base, and pressing for 2 minutes after injection ensures the closure of the hemolymph.

[0011] The present invention provides a polypeptide VgSP that can be recognized by a receptor on the surface of red claw crayfish oocytes and mediate entry into oocytes, as well as a method for injecting and detecting the polypeptide in sub-adult and adult female red claw crayfish. This breakthrough lays a technical foundation for establishing a gene editing element delivery method with oocyte targeting, which is expected to be applied in the field of gene editing in red claw crayfish. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 Surface Plasmon Resonance (SPR) analysis of VgSP and VgR, with the horizontal axis representing time and the vertical axis representing the response value; Figure 2 His-Tag immunofluorescence results of ovaries without VgSP injection and ovaries of subadult and adult shrimp injected with VgSP. DETAILED DESCRIPTION

[0013] The following is a detailed description in conjunction with the embodiments: This embodiment is implemented on the premise of the technical solution of the present invention, and a detailed implementation method and a specific operation process are given, but the protection scope of the present invention is not limited to the following embodiments.

[0014] Example 1: Acquisition of Red Claw Oocyte Targeting Penetrating Peptide VgSP and Analysis of Its Interaction with VgR The red claw crayfish oocyte targeting penetrating peptide VgSP of the present invention is synthesized by Nanjing GenScript Biotechnology Co., Ltd., with a molecular weight of 2914.43, an amino acid sequence as shown in SEQ ID NO.1, a purity of 96.5% by high performance liquid chromatography, a total amount of 4.6 mg, and an ultrapure water solvent. The binding rate constant (Ka (1 / Ms)) with the VgR receptor is 7.68×10 2 The dissociation rate constant (Kd (1 / s)) is 5.61×10 -4 , medium binding speed and high stability. Figure 1 Surface Plasmon Resonance (SPR) analysis was performed for VgSP and VgR.

[0015] Table 1 Affinity data of ligands for analytes Receptor Name Ligand name Chi² (RU²) Ka(1 / Ms) Kd(1 / s) KD (M) Rmax (RU) VxD VxP 9.25E+02 7.68E+02 5.61E-04 7.31E-07 19.4 Example 2: Functional verification of red claw crayfish oocyte surface receptor recognition polypeptide VgSP 1. In vivo injection in subadult female red claw crayfish Subadult female shrimps were 4-month-old individuals. Before injection, high-temperature (500°C) forceps were used to remove the unilateral eyestalk to achieve rapid hemostasis. After 72 hours, VgSP was injected using a microsyringe with a bevel angle of 45°. The injection dose was 500 ng / g body weight. The injection site was the membrane area of ​​the third foot coxa. After injection, pressure was pressed for 2 minutes to ensure hemolymph closure. 48 hours after injection, the injected individuals were killed by cold shock, and the ovarian tissue was removed and preserved in 4% paraformaldehyde for more than 24 hours. Paraffin sections were prepared, and the sections were stained with DAPI and analyzed by immunofluorescence. The primary antibody used was His-tag (12698S) from CST, with a dilution ratio of 1:200, and the secondary antibody used was Alexa Fluor 488 goat anti-rabbit (PN0108) from Pinofi Bio, with a dilution ratio of 1:200. The blue fluorescent signal shows the location of the cell nucleus, and the green fluorescent signal is the location of the polypeptide. Figure 2 As shown, the results showed that after the eyestalks of sub-adult shrimp were removed, some oocytes in the ovaries began to develop, and green signals of VgSP polypeptide appeared in the developing oocytes, proving that the VgSP polypeptide successfully entered the developing oocytes. At the same time, VgSP polypeptide signals that were still in transport also appeared in the follicular cells around the oocytes.

[0016] 2. In vivo injection in adult female red claw crayfish In adult female red claw crayfish, injection should be performed before the oocyte enters the middle stage of development. Use a microsyringe with a needle tip bevel angle of 45° to inject the VgSP polypeptide into the body from the base of the third walking leg of the red claw crayfish at a dose of 500 ng / g shrimp body weight. Press the injection site for 2 minutes after injection until the hemolymph stops flowing out. Use red-hot tweezers to quickly remove the eyestalk on one side of the red claw crayfish, and burn the wound to make it heal quickly. 48 hours after the removal of the eyestalk, the injected individual was killed by cold shock, and the ovarian tissue was removed and stored in 4% paraformaldehyde for more than 24 hours. Paraffin sections were prepared, and the sections were stained with DAPI and analyzed by immunofluorescence. The primary antibody used CST's His-tag (12698S) at a dilution ratio of 1:200, and the secondary antibody used Pinofi Bio AlexaFluor 488 goat anti-rabbit (PN0108) at a dilution ratio of 1:200. The blue fluorescent signal shows the location of the cell nucleus, and the green fluorescent signal is the location of the polypeptide. Figure 2 As shown, the results showed that the development of oocytes in the ovaries of adult red claw crayfish was basically synchronized after the eyestalks were removed, and a large area of ​​VgSP polypeptide green signal appeared in the oocytes.

[0017] Finally, it should be noted that the above examples are only some specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments, and there are many variations. All variations that can be directly derived or associated with the content disclosed by a person skilled in the art should be considered as the protection scope of the present invention.

Claims

1. A polypeptide VgSP, characterized in that The amino acid sequence of the polypeptide is shown in SEQ ID NO.

1.

2. Use of the polypeptide VgSP as claimed in claim 1 as a targeting penetrating peptide for red claw crayfish oocytes.

3. The use according to claim 2, characterized in that The polypeptide can specifically bind to the vitellogenin receptor VgR of red claw crayfish and enter the oocyte through receptor-mediated endocytosis. It carries a histone tag and can be identified by immunofluorescence targeting histones.

4. A method for in vivo delivery of red claw crayfish oocyte targeting penetrating peptide VgSP, characterized in that: Subadult female shrimps are selected, and the unilateral eyestalk of the subadult female red claw crayfish is removed to induce synchronization of oocyte development; 60 to 78 hours after the eyestalk is removed, the polypeptide VgSP according to claim 1 is injected into the hemolymph at a dose of 400 to 600 ng / g shrimp body weight; 42 to 54 hours after the injection, the enrichment of VgSP in the oocytes is confirmed by immunofluorescence detection.

5. The method according to claim 4, characterized in that The subadult female shrimp is 4 months old; the unilateral eyestalk is removed 60 to 78 hours before injection; the eyestalk is removed by instantaneously burning the broken limb with high-temperature tweezers to achieve rapid hemostasis; the injection is performed using a micro-injection syringe with a bevel angle of 45°, the injection site is the membrane area of ​​the third foot base, and pressing for 2 minutes after injection ensures hemolymph closure.

6. A method for in vivo delivery of red claw crayfish oocyte targeting penetrating peptide VgSP, characterized in that: Adult female shrimps are selected, and the polypeptide VgSP described in claim 1 is injected into the hemolymph of adult female red claw crayfish at a dose of 400-600 ng / g shrimp body weight; the unilateral eyestalk is immediately removed to promote the development of oocytes; 42-54 hours after the injection, the localization of VgSP in the developing oocytes is detected by immunofluorescence.

7. The method according to claim 6, characterized in that The adult female shrimp is an individual whose oocytes are in the early stage of development; the unilateral eyestalk is removed immediately after injection; the eyestalk is removed by instantaneously burning the broken limb part with high-temperature tweezers to achieve rapid hemostasis; the injection is performed using a micro-injection syringe with a bevel angle of 45°, the injection site is the membrane area of ​​the third foot base, and pressing for 2 minutes after injection ensures the closure of the hemolymph.

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