Preparation method of novel testicular long sperm marker detection kit
By detecting the expression pattern of Vha68-3 protein in the testicular tissue of Drosophila, a testicular long sperm tail marker detection kit was prepared, which solved the problem of difficult to judge the expression distribution of long sperm tail in the prior art, and achieved the testicular length Accurate judgment of differentiation of the morphogenic sperm extension process.
Patent Information
- Application Number
- CN202411710668.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-27
- Publication Date
- 2025-02-14
AI Technical Summary
The prior art is difficult to effectively judge the expression distribution of the long sperm tail of the testicles in the fruit fly, which affects the differentiation of the long sperm extension process in the testicles.
By detecting the specific expression of Vha68-3 protein in testicular tissues, a new testicular elongate sperm tail marker detection kit was prepared. The kit uses immunofluorescence staining to analyze and detect the expression pattern of Vha68-3 protein.
Effectively judging the differentiation of the elongated sperm extension process in the testicles provides accurate detection of the expression distribution of the tail of the elongated sperm in the testicles, and has important application value.
Smart Images
Figure CN119438585A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of biomedicine, and in particular to a method for preparing a novel testicular elongated sperm marker detection kit. Background Art
[0002] The elongation process of long sperm in Drosophila testis includes a series of highly complex and conservative biological events, among which the assembly of axoneme, the formation of individualized complexes and the maintenance of mitochondrial derivatives are key events, providing an important structural basis for male fertility. In the late stage of sperm growth, the individualized complex structure based on actin is formed around the nucleus of the long sperm and moves along the tail of the long sperm, during which a large cystic bulge is formed and most of the cytoplasm is gathered into the waste bag at the bottom of the long sperm capsule. Some regulatory factors have been confirmed to be involved in the elongation process of long sperm. V-ATPases are a common transporter protein. They were originally found in the lysosomes and vacuoles of fungi and plants. They are ATP-driven proton pumps and are ubiquitous in many organisms. Studies have found that V-ATPases are highly conserved from lower animals to higher animals and are involved in a variety of biological processes, including intracellular transport, biomolecule synthesis and processing, pH homeostasis and nutrient uptake, bone remodeling, tumor cell invasion and sperm maturation. These functions are mainly determined by the diversity of V-ATPase in cellular localization. Recent studies have found that V-ATPase also plays an important role in the reproductive system. Through whole genome screening studies, it was found that V-ATPase-related factors Vha16-1, Vha44, Vha13 and VhaAC45 play a key role in the maintenance and differentiation of testicular germ stem cells and cyst stem cells in Drosophila. The present invention found that Vha68-3 is a 68kD subunit of vacuolar H+ATPase, which can be used as a testis-specific expression protein and is highly enriched in the testis elongated sperm tail structure. This characteristic can be used to prepare a testis elongated sperm tail structure detection kit. Summary of the invention
[0003] Technical problem to be solved: The present invention provides a method for preparing a novel testicular elongated sperm marker detection kit. Vha68-3 protein is one of the key proteins identified by the previous research of this research group. It is highly enriched in the tail of testicular elongated sperm. By detecting the expression pattern of Vha68-3 protein, the expression distribution of the tail of testicular elongated sperm can be effectively determined.
[0004] Technical solution: The present invention discloses a method for detecting a marker of testicular elongated sperm tails, which detects the specific expression of Vha68-3 protein in testicular tissue as a method for detecting testicular elongated sperm tails. The Vha68-3 protein is used in the preparation of a novel testicular elongated sperm tail marker detection kit, which detects the expression pattern of Vha68-3 protein in testicular tissue as a marker for the extension process of the novel testicular elongated sperm tails.
[0005] Preferably, the kit uses the specific expression of Vha68-3 protein in testicular tissue as a method for detecting testicular elongated sperm tails.
[0006] Preferably, the kit uses a Vha68-3 protein-specific antibody to detect the expression level of amino acids ASNEDRARNSGSLNQD-C at positions 18-33 of the Vha68-3 protein in testicular tissue.
[0007] Preferably, the sample tissue is Drosophila testis tissue.
[0008] Preferably, the kit uses an immunofluorescence staining method to analyze and detect the expression pattern of amino acids ASNEDRARNSGSLNQD-C at positions 18-33 of the Vha68-3 protein in a testicular tissue sample.
[0009] Compared with the prior art, the present invention has at least the following outstanding advantages: 1. The present invention utilizes Vha68-3 as a testis-specific expression protein. Vha68-3 is a 68kD subunit of vacuolar H+ATPase and is highly enriched in the testis elongated sperm tail structure. This characteristic can be used to prepare a testis elongated sperm tail structure detection kit; 2. This detection kit can effectively determine the differentiation status of the elongation process of testicular elongated sperm by detecting the expression pattern of Vha68-3 protein in testicular tissue. As a new testicular-specific expressed protein, the kit prepared using Vha68-3 protein can be effectively used to detect the expression distribution of the tail of testicular elongated sperm, which has important application value for the elongation process of testicular elongated sperm. BRIEF DESCRIPTION OF THE DRAWINGS
[0010] Figure 1 The expression pattern of Vha68-3 protein in Drosophila testis tissue; Figure 2 The expression pattern of Orb and Orb2 in Drosophila testis tissue. DETAILED DESCRIPTION
[0011] To make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the following will be combined with the attached Figure 1~2The technical solutions of the embodiments of the present invention are described clearly and completely. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the described embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field belong to the scope of protection of the present invention.
[0012] The present invention discloses a method for detecting a testicular elongated sperm tail marker, which detects the specific expression of Vha68-3 protein in testicular tissue as a method for detecting the testicular elongated sperm tail. The Vha68-3 protein is used in the preparation of a novel testicular elongated sperm tail marker detection kit, which uses the expression pattern of the Vha68-3 protein in testicular tissue as a marker for the extension process of the novel testicular elongated sperm tail. The expression pattern of the 18th to 33rd amino acids of the Vha68-3 protein ASNEDRARNSGSLNQD-C (sequence number seq-1) in the testicular tissue sample is analyzed by immunofluorescence staining to effectively judge the differentiation of the testicular elongated sperm extension process.
[0013] The present invention utilizes Vha68-3 as a testis-specific expression protein. Vha68-3 is a 68kD subunit of vacuolar H+ATPase and is highly enriched in the tail structure of testis elongated sperm. This characteristic can be used to prepare a testis elongated sperm tail structure detection kit. The detection kit can effectively judge the differentiation of the testis elongated sperm elongation process by detecting the expression pattern of the Vha68-3 protein in testis tissue. As a novel testis-specific expression protein, the Vha68-3 protein can be used to prepare a kit effectively applied to the detection of the expression distribution of the testis elongated sperm tail, and has important application value for the testis elongated sperm elongation process.
[0014] The specific test process of the embodiment of the present invention is as follows: (A) Expression pattern of Vha68-3 in Drosophila testis tissue: (1) Rearing and maintenance of fruit fly strains: The fruit fly strain is raised in a special incubator, the culture temperature is set to 25°C, and the relative humidity is set to 40-60%. According to the fruit fly breeding conditions, a new tube is switched every 8-10 days. The fruit fly strain used in the present invention is w1118. Female fruit flies lay eggs after mating, go through growth stages such as larvae and pupae, and finally emerge into adults. Male fruit flies within two days of emergence are collected and immunofluorescence staining experiments of testicular tissue are performed.
[0015] (2) Immunofluorescence staining of testicular tissue: Collect newly born male fruit flies within two days, dissect their testicular tissue in PBS solution, and place the dissected testes in PCR tubes, with about 20 testes in each PCR tube; use 4% PFA to fix the testicular tissue for 25 minutes; use 1% PBST for permeabilization, wash once every 5 minutes, and the total permeabilization time is 15 minutes; use 5% BSA for blocking, and block for 30 minutes at room temperature; add antibodies prepared with 5% BSA to the PCR tubes, incubate at room temperature for 1 hour, or incubate overnight at 4°C; after recovering the primary antibody, wash with 1% PBST for three times, each for 5 minutes; then add the secondary antibody prepared with 5% BSA, and incubate at room temperature for 1 hour in the dark; after discarding the secondary antibody, wash with 1% PBST for three times, each for 5 minutes; add 1X Hoechst dilution solution prepared in PBS (volume ratio 1:800) was treated in the dark at room temperature for 10 minutes; the testicular tissue in the PCR tube was transferred to a glass slide using a pipette, the testicles were arranged using anatomical forceps, and the slides were sealed with an antifade agent.
[0016] like Figure 1 As shown, the expression pattern of Vha68-3 protein in Drosophila testis tissue was observed by immunofluorescence staining. The staining results showed that Vha68-3 protein was highly expressed in the long sperm tail structure of Drosophila testis.
[0017] (II) Analysis of protein expression patterns of elongated sperm markers in the testis group: like Figure 2 As shown, the study showed that Orb and Orb2 proteins are key proteins in the elongation process of testicular long sperm. Immunofluorescence staining found that Orb and Orb2 are highly enriched in the tail of long sperm in the testis, and their expression simulates the expression pattern of Vha68-3 protein in the structure of long sperm tail in the testis.
[0018] The above are preferred embodiments of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.
Claims
1. A method for detecting markers of testicular elongated sperm tails, characterized in that: The specific expression of Vha68-3 protein in testicular tissue was detected as a method for detecting the elongated sperm tail in the testis.
2. A method for preparing a novel testicular elongated sperm tail marker detection kit, characterized in that: The kit uses the specific expression of Vha68-3 protein in testicular tissue as a method for detecting the elongated sperm tail in the testis.
3. The preparation method of the novel testicular elongated sperm tail marker detection kit according to claim 2, characterized in that: The kit uses a Vha68-3 protein-specific antibody to detect the expression level of amino acids ASNEDRARNSGSLNQD-C at positions 18 to 33 of the Vha68-3 protein in testicular tissue.
4. The preparation method of the novel testicular elongated sperm tail marker detection kit according to claim 2, characterized in that: The sample tissue is Drosophila testis tissue.
5. The preparation method of the novel testicular elongated sperm tail marker detection kit according to claim 3, characterized in that: The kit uses an immunofluorescence staining method to analyze and detect the expression pattern of amino acids ASNEDRARNSGSLNQD-C at positions 18-33 of the Vha68-3 protein in testicular tissue samples.