Lrrc71 antibody as well as preparation method and application thereof
By performing multi-site subcutaneous immunization on Lrrc71 knockout mice, a highly specific and high-affinity Lrrc71 antibody was prepared, solving the problems of false positives and poor binding spectra of existing antibodies, and realizing the effective detection and functional study of Lrrc71 protein.
Patent Information
- Application Number
- CN202511056854.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-30
- Publication Date
- 2025-12-12
AI Technical Summary
Existing Lrrc71 antibodies suffer from false positives and poor binding profiles, as well as insufficient affinity and stability, making them ineffective for detecting Lrrc71 protein.
By administering multi-site subcutaneous immunizations to adult Lrrc71 systemic knockout mice, using Lrrc71 protein fragments of 138-304 amino acids for immunization, and combining this with the immune tolerance deficiency state of gene knockout animals, highly specific and high-affinity antibodies were induced.
We have achieved the preparation of Lrrc71 antibodies with high specificity and high affinity, which can effectively detect the expression level of Lrrc71 protein, providing a basis for the diagnosis and treatment of male infertility.
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Figure CN121108328A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biotechnology, specifically relating to an Lrrc71 antibody, its preparation method, and its application. Background Technology
[0002] Leucine-rich repeats (LRRs) are a family of amino acids widely found in viruses and eukaryotes. Composed of 20-29 amino acids, they are typically folded into an arc or horseshoe shape and have been extensively studied due to their important roles in tumorigenesis and cell proliferation. In recent years, more research has revealed their crucial functions in sperm flagellar structure. For example, Lrrc23 has been found to be located within the radial striations complex of mouse sperm flagella, maintaining the normal structure and function of the mouse sperm flagellum; Lrrc46 is located in the midpiece of the sperm flagellum and is essential for sperm cell elongation and flagellar development.
[0003] The leucine-rich repeat 71 (Lrrc71), a member of the LRR family, is highly conserved in mammalian evolution and is located on the long arm of chromosome 1. Currently, there are very few commercially available anti-mouse Lrrc71 antibodies, and those that are available do not specifically bind to the Lrrc71 protein, leading to false positives. Therefore, developing novel anti-mouse Lrrc71 antibodies would be beneficial for in-depth research into the function of this protein in sperm flagella, and would provide a theoretical basis for developing drug compositions or kits for detecting the Lrrc71 protein in clinical practice for male infertility patients suffering from point mutations or deletions in this gene. Summary of the Invention
[0004] Technical problems to be solved:
[0005] The technical problems addressed by this application are false positives of antibodies and poor antibody affinity, stability, and binding spectrum in the enrichment, purification, and detection of Lrrc71 protein. It provides Lrrc71 antibodies, their preparation methods, and applications.
[0006] Technical solution:
[0007] To achieve the above objectives, this application provides the following technical solution:
[0008] An Lrrc71 antibody, wherein the immunized protein fragment in the antibody is a 138-304 amino acid fragment.
[0009] Furthermore, the protein fragment immunized by the Lrrc71 antibody is as follows: kav keiyirgwkvedrilgifsk clpslsqlqa inlwkvgltd ktlttfiall plcsstlrkv slegnpipeq sfsklmgldstivhlslrnn nindhgaqll gqalstlqns nrtlvslnla fnhigdvgag yiadglrlnr sllwlslahnhiqdkgalkl aevl.
[0010] A method for preparing any of the above-mentioned Lrrc71 antibodies involves subcutaneously immunizing mice with Lrrc71 protein fragments obtained through in vitro recombinant expression and purification via multiple injections.
[0011] Furthermore, the mice are adult Lrrc71 systemic knockout mice.
[0012] Furthermore, the immunized mice were injected subcutaneously at multiple points for 12 weeks.
[0013] Furthermore, the protein fragment is administered via subcutaneous multi-point injection immunization six times, with the injection method being once every two weeks.
[0014] Furthermore, serum was collected and purified one week after the 12-week immunization cycle of the mice was completed using an enucleation method.
[0015] This application also discloses the use of any of the above-mentioned Lrrc71 antibodies in pharmaceutical compositions or kits for detecting Lrrc71 protein.
[0016] Furthermore, the efficacy of the Lrrc71 antibody was verified using wild-type and completely knocked-out Lrrc71 gene sperm to rule out false positive results.
[0017] The technical principle of this application is: by utilizing the "lack of immune tolerance" state of the gene knockout animal itself, it recognizes the protein as a completely foreign antigen, thereby inducing the production of highly specific and high-affinity antibodies. Therefore, the antibodies produced by immunizing the protein with a gene knockout mouse have higher specificity.
[0018] Beneficial effects:
[0019] This application provides an Lrrc71 antibody, its preparation method, and its application, which have the following advantages compared with the prior art:
[0020] 1. The invention described herein can be used to detect the localization and expression level of Lrrc71 protein during scientific research;
[0021] 2. The invention described herein can be used as a study of an inhibitor to suppress the function of Lrrc71 protein;
[0022] 3. Given the homology between the Lrrc71 sequence and the human sequence, this invention can be applied to the expression level of Lrrc71 protein in male sperm, thereby providing a means of detection for the treatment and diagnosis of clinical male infertility or low reproductive capacity. Attached Figure Description
[0023] Figure 1 For the immunofluorescence verification of Lrrc71 antibody in epididymal tail sperm in Example 2 of this application, the left figure is a distribution and localization map of Lrrc71 protein in wild-type (WT) mouse sperm tail; the right figure is a localization map of Lrrc71 protein in Lrrc71 gene knockout (KO) mouse sperm tail, and the scale bar in the figure is 2μm;
[0024] Figure 2 The images shown are immunoblotting verification diagrams of Lrrc71 antibody in testes and sperm proteins in Example 2 of this application. Figure A shows the expression level of Lrrc71 protein in testicular tissue of WT and KO groups, with α-Tubulin protein as the internal reference protein. Figure B shows the verification of the expression level of Lrrc71 protein in epididymal tail sperm of WT and KO groups, with odf2 as the internal reference protein. Detailed Implementation
[0025] The preferred embodiments of the present invention will now be described in detail with reference to specific examples. It should be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from its spirit and essence.
[0026] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.
[0027] The experimental mice used in the following examples were all purchased from Cyagen Biosciences Co., Ltd.
[0028] Example 1:
[0029] An Lrrc71 antibody, wherein the immunized protein fragment in the antibody is a 138-304 amino acid fragment.
[0030] The protein fragment immunized with the Lrrc71 antibody is kav keiyirgwkv edrilgifsk clpslsqlqainlwkvgltd ktlttfiall
[0031] plcsstlrkv slegnpipeq sfsklmglds tivhlslrnn nindhgaqll gqalstlqnsnrtlvslnla fnhigdvgag yiadglrlnr
[0032] sllwlslahn hiqdkgalkl aevl.
[0033] The method for preparing the Lrrc71 antibody involves subcutaneously immunizing mice with the protein fragment obtained after in vitro recombinant expression and purification of the Lrrc71 antibody at multiple sites. The specific steps are as follows:
[0034] The first step was in vitro recombinant expression of the 138-304 fragment sequence of the Lrrc71 protein: First, the gene fragment encoding the 138-304 fragment sequence of Lrrc71 was determined. Then, primers with restriction enzyme sites were designed and used as a template to amplify the sequence of this fragment using mouse testicular cDNA. The sequence was then digested with enzymes and ligated into a plasmid with a GST tag. After the plasmid was confirmed to be correct by sequencing, the constructed plasmid was transfected into BL21 cells, and the expression of the protein fragment was induced by the addition of IPTG. Subsequently, the protein was extracted from E. coli, purified by GST tag beads, and the GST tag was removed to obtain the purified protein fragment. Finally, the concentration of the protein was measured using a BCA kit and recorded.
[0035] The second step involved immunizing mice with the recombinant protein fragment: 100 μg of recombinant protein fragment per mouse was mixed with complete adjuvant, resulting in a volume of approximately 300 μL. This mixture was injected subcutaneously into adult mice with a complete knockout of the Lrrc71 gene in six divided doses, designated as week 0. Subsequently, the mice were immunized every two weeks with a protein dose of 50 μg per mouse, mixed with incomplete adjuvant, until five immunizations were completed. Within one week, serum was collected from the mice by enucleation. The collected serum was then purified using a Protein A kit.
[0036] Example 2:
[0037] The application of an Lrrc71 antibody in a pharmaceutical composition or a kit for detecting Lrrc71 protein. The efficacy of the Lrrc71 antibody is verified using wild-type and completely knocked-out Lrrc71 gene sperm to exclude false positive results. The specific antibody verification method is as follows:
[0038] The first step, sperm collection from mice: Wild-type (WT) and Lrrc71 gene knockout (KO) mice were euthanized, and the epididymal tail was removed by opening the abdominal cavity. Mature sperm were isolated by cutting the epididymal tail into pieces and placing it in a 37°C benchtop solution; sperm were then collected using the swim-up method.
[0039] The second step was immunofluorescence staining of mouse sperm: A portion of the collected sperm was smeared onto an adhesive glass slide, followed by fixation with 4% paraformaldehyde, infiltration with 0.3% Triton X-100, blocking with 5% BSA, overnight incubation with primary antibody at 4°C, incubation with secondary antibody at room temperature, nuclear staining, and image acquisition via confocal fluorescence imaging. The results showed that the sperm tail flagella of the WT group exhibited fluorescent signals of Lrrc71 protein, while the sperm tail flagella of the KO group did not. Figure 1 As shown;
[0040] The third step was immunoblotting of mouse testicular and sperm proteins: Proteins were extracted from the testes and epididymal tails of wild-type (WT) and Lrrc71 gene knockout (KO) mice. The proteins were denatured, subjected to SDS-PAGE electrophoresis, transferred to a membrane, blocked, incubated with primary antibody, incubated with secondary antibody, and then imaged. The results are as follows: Figure 2 The results showed that Lrrc71 protein exhibited a distinct band in wild-type (WT) testes, but not in the KO group; similarly, WT sperm also showed a distinct band in the epididymal tail, while KO sperm did not. Figure 2 As shown in B.
[0041] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.
Claims
1. An Lrrc71 antibody, characterized in that, The protein fragment immunized in the antibody is a 138-304 amino acid fragment.
2. The Lrrc71 antibody according to claim 1, characterized in that: The protein fragment immunized with the Lrrc71 antibody is shown in SEQ ID NO.1: kav keiyirgwkv edrilgifsk clpslsqlqa inlwkvgltd ktlttfiallplcsstlrkv slegnpipeq sfsklmgldstivhlslrnn nindhgaqll gqalstlqns nrtlvslnlafnhigdvgag yiadglrlnr sllwlslahn hiqdkgalkl aevl.
3. A method for preparing the Lrrc71 antibody according to claim 1 or 2, characterized in that: Mice were subcutaneously immunized by multiple injections of the Lrrc71 protein fragment, which was recombinantly expressed and purified in vitro.
4. The method for preparing Lrrc71 antibody according to claim 3, characterized in that: The mice were adult Lrrc71 systemic knockout mice.
5. The method for preparing Lrrc71 antibody according to claim 3, characterized in that: The protein fragment was injected subcutaneously at multiple sites into immunized mice over a period of 12 weeks.
6. The method for preparing Lrrc71 antibody according to claim 3, characterized in that: The protein fragment is administered via subcutaneous injection at multiple sites for immunization six times, with the injection method being once every two weeks.
7. The method for preparing Lrrc71 antibody according to claim 5, characterized in that: One week after the 12-week immunization cycle of the mice ended, serum was collected and purified by enucleation.
8. The use of the Lrrc71 antibody according to claim 1 or 2 in a pharmaceutical composition or a kit for detecting Lrrc71 protein.
9. The application according to claim 8, characterized in that: The efficacy of Lrrc71 antibodies was verified using wild-type and completely knocked-out Lrrc71 gene sperm to rule out false positive results.