Porcine sperm-specific antibody, and preparation method therefor and use thereof

Abstract

Provided is a porcine sperm-specific antibody. The coding sequence of the antibody is shown in any one of SEQ ID NOs: 1-3. An alpaca is immunized with whole porcine sperm to construct a nanobody library, and then an antibody that can specifically bind to X or Y sperm can be selected from the library, thereby achieving the sorting of X and Y sperm. Moreover, the selected antibody is a nanobody, the size thereof is only 10% of that of a conventional antibody, and the structure thereof is simpler.

Description

porcine sperm-specific antibodies, their preparation methods and applications Technical Field

[0001] This invention relates to the fields of antibody preparation and animal breeding, and particularly to a porcine sperm-specific antibody, its preparation method, and its application. Background Technology

[0002] Sex control is a core technology in reproductive biology research, playing a vital role in animal breeding, genetic disease prevention and control, and livestock production. To address practical issues such as livestock quantity and yield, utilizing sex control technology to promote livestock development is essential. Sex control technology is a modern biotechnology that, through artificial selection and separation of X and Y sperm, ultimately yields offspring of the desired sex, possessing significant theoretical and practical implications. Firstly, sex control technology can fully utilize the growth rate and meat quality of male animals, and the reproductive and lactation performance of female animals, thereby achieving substantial economic benefits. Secondly, it can enhance the selection intensity of superior traits, accelerate the breeding process, reduce breeding costs, and maximize genetic progress. Furthermore, controlling the sex of offspring can overcome phenomena such as twin infertility and eliminate the harmful effects of sex-linked genes. Sex-controlled semen can be prepared into frozen semen and inseminated with selected sex for optimal production. Therefore, efficient and accurate separation of sex-controlled semen has become a key research direction in sex control technology, and its research has significant practical production value in livestock production.

[0003] Currently, the main methods for preparing sex-controlled semen from pigs (sorting of pig X and Y sperm) include Percoll density gradient centrifugation, flow cytometry sorting, and immunoassay for X and Y sperm sorting. Regarding Percoll density gradient centrifugation: Studies by Cao Shizhen et al. have shown that using Percoll discontinuous density gradient separation of bovine semen resulted in significant differences in the sex ratio of offspring, with semen rich in X sperm showing a female proportion exceeding 60%. While Percoll density gradient centrifugation is simple and low-cost, the process can lead to the production of high concentrations of reactive oxygen species (ROS) in sperm cells, damaging sperm motility and reducing sperm integrity. Regarding flow cytometry sorting: Xiong Xianrong et al. added Allura Red to their flow cytometry setup to stain and eliminate sperm with incomplete plasma membranes, significantly enhancing the motility of sorted yak sperm and potentially improving the conception rate of sex-controlled frozen semen to some extent. However, after Zeng Quanyou et al. used flow cytometry to separate pig sperm for insemination, the litter size was 100% with Y sperm and 91.67% with X sperm, but the litter size showed a downward trend. Flow cytometry for X and Y sperm separation is currently a widely used sex control technology in actual production, but its use in separating pig semen is relatively limited. This is mainly because the volume of semen required for pig insemination is very large, while the efficiency of flow cytometry is limited, making it difficult to meet the required number of inseminations. Furthermore, the longer the flow cytometry process, the greater the sperm loss. Therefore, flow cytometry for X and Y sperm separation is not currently suitable for the sex control needs of pigs in actual production. Regarding the immunoassay method for X and Y sperm separation: Studies by Umehara et al. have shown that the TLR7 / 8 encoded by the X chromosome is expressed in the midsection and tail of X sperm, but not in Y sperm. Umehara et al. further used TLR7 / 8 to isolate mouse sperm. Under conditions with the addition of R848 ligand, the motility of X and Y sperm in mice differed significantly, and the solution showed a stratified state. The motility of X sperm was inhibited, and it was located in the lower layer. When R848 was removed, the motility of X sperm was restored, and it still possessed fertilization capacity. Fa Ren et al. obtained the same results when using the TLR7 / 8 receptor in their study of isolating X and Y sperm in dairy goats, and demonstrated that TLR7 / 8 can directly regulate the ATP levels of dairy goats to affect X sperm motility. Liu Weidong's research showed that in beef bovine semen, the TLR7 / 8 agonist R848 could significantly reduce the motility of X sperm, but had no significant effect on sperm acrosome integrity and plasma membrane integrity. Furthermore, using R848 to sort fresh beef bovine semen, the purity of Y sperm reached 88.6%, and the purity of X sperm reached 72.5%. Although the TRL7 / 8 sperm sorting method has a low accuracy rate, it is simple and convenient to operate, does not damage sperm during the test, does not require expensive equipment, and saves production costs.However, R848 and R837 are not very effective in separating boar sperm. A study by Wu Changhua et al. (2023), graduate students at South China Agricultural University, showed almost no difference in the expression of R848 and R837 ligands in boar X / Y sperm, thus failing to achieve the separation objective. Currently, related research has been conducted on separating sex-controlled semen from cattle, sheep, and mice, but research on separating boar sperm is still lacking. This is mainly because the large quantity required for boar insemination makes traditional flow cytometry unable to meet the requirements of sex-controlled semen separation in production practice.

[0004] Therefore, there is an urgent need to develop a new antibody and a method for efficiently separating X and Y sperm to meet the needs of sex control in pig semen. Technical issues

[0005] Currently, relevant research has been carried out on the separation of sex-controlled semen from cattle, sheep, and mice, but research on the sorting of pig semen is still in its infancy. This is mainly because pig insemination requires a large quantity of semen, and traditional flow cytometry cannot meet the requirements of sex-controlled semen sorting in production practice.

[0006] Therefore, there is an urgent need to develop a new antibody and a new method for efficiently separating X and Y sperm, so as to achieve efficient separation of X and Y sperm and rapid and stable sorting of boar semen to meet the needs of sex control in boar semen. Technical solutions

[0007] The purpose of this invention is to provide a novel porcine sperm-specific antibody that can efficiently and specifically bind to X or Y sperm, thereby achieving efficient separation of X and Y sperm and enabling rapid and stable sorting of porcine semen to solve the aforementioned problems.

[0008] According to a first aspect of the present invention, a porcine sperm-specific antibody is provided, the coding sequence of which is shown in any one of SEQ ID No: 1-3. This antibody is a nanobody, only 10% the size of a conventional antibody, with a simpler structure, and possesses advantages such as high stability, strong specificity, and low immunogenicity. It enables large-scale antibody expression, significantly reducing antibody production costs and facilitating industrial application.

[0009] According to a second aspect of the present invention, a porcine sperm-specific antibody is provided. This antibody is optimized based on the codon usage preferences of a eukaryotic expression system, and the coding sequence of the optimized antibody is shown in SEQ ID No:4. This antibody coding sequence, optimized according to the codon usage preferences of a eukaryotic expression system, can specifically bind to porcine sperm, thus enabling better sorting of porcine semen. Furthermore, this antibody is a nanobody, only 10% the size of traditional antibodies, with a simpler structure, and possesses advantages such as high stability, strong specificity, and low immunogenicity. It allows for large-scale antibody expression, significantly reducing antibody production costs and facilitating industrial application.

[0010] According to a third aspect of the present invention, a porcine sperm-specific antibody is provided, which is a recombinant expression antibody fused with an exogenous Fc fragment, the amino acid sequence of which is shown in SEQ ID No:5. This recombinant expression antibody is formed by fusing a selected nanobody with the Fc fragment of an exogenous (rabbit-derived) IGg antibody to form a VHH-Fc antibody. Upon binding to sperm, it can induce sperm agglutination. The fused Fc fragment can be used to aggregate and couple streptavidin or protein A / G magnetic beads, and to prepare magnetic beads specifically for porcine sperm immunosorting.

[0011] According to a fourth aspect of the present invention, a method for preparing porcine sperm-specific antibodies is provided, the method comprising the following steps: S1: immunizing alpacas with whole porcine sperm to prepare a porcine sperm-specific nanobody library; S2: panning the library prepared in step S1; S3: screening the panned library for antibodies that specifically bind to sperm. This method is the first to use whole porcine sperm as an immunogen to immunize alpacas to prepare a nanobody library, achieving a library capacity of 3.4 × 10⁻⁶. 8 PFU has a higher antibody library abundance, higher coverage of whole sperm antigens, and a more complete range of antibody types, thereby improving the capture success rate of porcine sex control specific antibodies. It can efficiently screen out positive antibodies and easily obtain the coding sequence of each antibody, enabling large-scale antibody expression and greatly reducing antibody production costs.

[0012] In some embodiments, the above method may further include the following steps: S1: Immunizing alpacas with whole porcine sperm, and then isolating alpaca peripheral blood cells after immunization; S2: Extracting RNA from peripheral blood cells, reverse transcribing to obtain cDNA, and amplifying the VHH coding sequence of the antibody; S3: Cloning the amplified VHH coding sequence into a phage plasmid vector to prepare a recombinant phage vector carrying the porcine sperm antibody gene; S4: Transforming the recombinant phage vector into E. coli and expanding its culture to obtain a porcine sperm antibody phage display library; S5: Using porcine X / Y sperm to pan the phage display library obtained in step S4 to construct a porcine sperm-specific nanobody library: using X sperm for negative selection and Y sperm for positive selection, and panning to construct a Y sperm-specific nanobody library; using Y sperm for negative selection and X sperm for positive selection, and panning to construct an X sperm-specific nanobody library.

[0013] According to a fifth aspect of the present invention, a porcine sperm-specific antibody prepared using the above-described preparation method is provided. Thus, the above method provides a simple, rapid, efficient, and stable way to obtain positive antibodies with high specificity, high sorting efficiency, strong specificity, and minimal damage to sperm.

[0014] According to a sixth aspect of the present invention, the application of porcine sperm-specific antibodies in the sorting of porcine X and Y sperm is provided. Thus, through this application, efficient sorting of porcine sperm can be achieved, laying the foundation for sex control of porcine semen in production.

[0015] According to a seventh aspect of the present invention, the use of a porcine sperm-specific antibody in the preparation of products for porcine sperm sorting is provided. Thus, products for sperm sorting prepared using this antibody (such as kits, immunomagnetic beads, etc.) can rapidly and efficiently sort sperm, enabling sex control in pigs.

[0016] According to an eighth aspect of the present invention, a method for sorting pig X and Y sperm is provided. The method includes the following steps: S1: Biotinylating a recombinant expression antibody with an amino acid sequence as shown in SEQ ID No:5, and then labeling the biotinylated recombinant expression antibody onto streptavidin magnetic beads to prepare biotinylated immunomagnetic beads; S2: Diluting the semen to be sorted and adding the immunomagnetic beads, incubating at room temperature, and then placing it on a magnetic rack. Sperm that did not bind to the magnetic beads in the supernatant are then collected. The sperm enriched by the immunomagnetic beads after removing the supernatant are then resuspended to obtain sperm of the other sex. Thus, by preparing biotinylated immunomagnetic beads from this antibody and performing magnetic sorting, sperm of one sex are found in the supernatant that did not bind to the magnetic beads, while sperm of the other sex are enriched by the magnetic beads. This allows for simple, convenient, and efficient sorting of pig sperm and achieves sex control of pig semen.

[0017] In some embodiments, the conditions for sperm sorting are: magnetic bead diameter of 300 nm or 1 μm; room temperature incubation time of 30 minutes or more; and sperm density of 350 million / mL or more.

[0018] According to a ninth aspect of the present invention, a method for sorting pig X and Y sperm is provided. The method includes the following steps: S1: Immobilizing a recombinant expression antibody with an amino acid sequence as shown in SEQ ID No:5 onto Protein A / G magnetic beads to construct Protein A / G immunomagnetic beads; S2: Diluting the semen to be sorted and adding Protein A / G immunomagnetic beads, collecting sperm that are not enriched with the magnetic beads from the supernatant, and collecting sperm that are enriched with the magnetic beads separately, thereby achieving the sorting of X and Y sperm. Thus, through magnetic sorting, pig sperm can be sorted simply, conveniently, and efficiently, achieving sexual control of pig semen. Beneficial effects

[0019] A nanobody library was prepared by immunizing alpacas with whole porcine sperm. This library was then used to screen for antibodies that specifically bind to X or Y sperm, enabling the separation of X and Y sperm. The screened antibodies are nanobodies, only 10% the size of traditional antibodies, with a simpler structure, high stability, strong specificity, and low immunogenicity. This allows for large-scale antibody expression, significantly reducing production costs and facilitating industrial applications. When applied to sex control in porcine semen, the different binding abilities of the antibodies to X and Y sperm induce agglutination reactions in sperm of specific sexes. For example, antibody W17 induces X sperm agglutination, achieving an enrichment efficiency of 80%. Therefore, X sperm can be efficiently enriched using W17 antibody immunomagnetic beads (unenriched sperm are Y sperm), thus achieving efficient sorting of pig X and Y sperm. Moreover, the separation of X and Y sperm using this antibody and its sorting method is highly efficient and causes little damage to sperm, which is more advantageous than sperm flow cytometry technology. It does not rely on expensive equipment and professional technicians, which is conducive to establishing an economical and efficient pig semen sex control technology. Attached Figure Description

[0020] Figure 1 shows the results of monoclonal antibody abundance analysis of the porcine sperm panning library;

[0021] Figure 2 shows the results of monoclonal antibody binding specificity analysis of the porcine sperm panning library;

[0022] Figure 3 shows the results of differential antibody flow cytometry analysis.

[0023] Figure 4 shows the results of the differential antibody binding site localization.

[0024] Figure 5 shows the results of diameter screening for W17 biotin-immunoma magnetic beads.

[0025] Figure 6 shows the results of optimized incubation time for W17 biotin-immunoma magnetic beads.

[0026] Figure 7 shows the results of optimized sperm density during incubation.

[0027] Figure 8 shows the results of the optimized dosage of biotin immunomagnetic beads.

[0028] Figure 9 shows the enrichment results of W17 biotin-based immunomagnetic beads on semen from different pig breeds.

[0029] Figure 10. Effect of protein A / G immunomagnetic beads on bovine semen sorting;

[0030] Figure 11. Results of the agglutination ability of immunomagnetic beads on porcine sperm. Embodiments of the present invention

[0031] The invention will now be described in further detail with reference to the accompanying drawings.

[0032] Example 1: Construction and Panning of a Pig Whole Sperm Immunization Alpaca Nanobody Library

[0033] 1.1 Construction of a library of porcine sperm-immunized alpaca nanobody

[0034] Healthy alpacas were selected, and 10 million fresh boar sperm were collected. These sperm were centrifuged at 800g for 10 minutes at room temperature, the supernatant was removed, and the cells were washed with 1 mL of PBS. This process was repeated twice. After washing, the cells were resuspended in 1 mL of PBS and subcutaneously injected into the alpacas for immunization. A booster immunization was performed 14 days later, for a total of three consecutive days. During immunization, alpaca serum was isolated, and the titer of boar sperm-specific antibodies in the serum was monitored using immunofluorescence. After the antibody titer stabilized, alpaca peripheral blood cells (PBMCs) were isolated, total RNA was extracted, and reverse transcribed into cDNA. The coding sequence of the variable region (VHH) of the alpaca antibody heavy chain was amplified using the cDNA as a template and cloned into the phage plasmid pCom3XSS (Apak Biotechnology, P001). The plasmid was electroporated into TG1 Escherichia coli (Apak Biotechnology, P021), and the culture was expanded to obtain an antibody bacterial library. The library size was determined to be 3.4 × 10⁻⁶. 8 PFU was detected with a positive rate of 96% and a diversity of 100%. The bacterial library was added to 100 mL of 2×TY medium until the OD600 reached 0.1, and cultured at 37℃ and 250 rpm until the OD600 reached approximately 0.5. Helper phage M13KO7 (New England Biolabs (NEB, N0315S)) was added at a ratio of 1:20 (bacterial count: helper phage count) for rescue, and the culture was expanded. The progeny phages were purified using PEG / NaCl solution, and the resulting primary antibody phage library was determined to have a titer of 1.2 × 10⁻⁶. 14 pfu / mL.

[0035] 1.2 Screening of Targeted Swine Sperm-Specific Nanobodies

[0036] 10 million fresh bovine sperm were collected, washed three times with PBS, resuspended in 2 mL of PBS, and added to 6-well cell cultures at a ratio of 10 million sperm per well. The cells were centrifuged at 1200 g for 10 minutes, the supernatant was removed, and the cells were dried at 37-50 °C. The cells were fixed with 4% paraformaldehyde for 15 minutes and blocked with 3% BSA solution for 1 hour. After washing three times with PBS, the wells without sperm fixation were used as blank controls. A bovine sperm-immunized alpaca nanobody library was added for the first round of panning. Positive phages from the first round of screening were recovered and amplified, and the resulting screening antibody library was used for the next round of panning. Using the same method, flow cytometry-sorted porcine X sperm and Y sperm were immobilized into 12-well cell culture plates. X sperm were used for negative selection and Y sperm for positive selection, or Y sperm for negative selection and X sperm for positive selection. This process was repeated for 4-7 rounds. The number of positive phages in each round was measured. The positive rate of each round was calculated by dividing the number of bound phages by the number of phages introduced, in order to evaluate the antibody enrichment effect.

[0037] Seven rounds of positive screening were performed on the nanobody library using porcine Y sperm, resulting in effective enrichment of the library. The seventh round (using 5.76 × 10⁻⁶ sperm samples) yielded the best results. 7 PFU and the number of phages bound to the virus were 5.04 × 10⁻⁶. 4 The positive rate was 8.75 × 10⁻⁶. -4 The selection process was compared to the first round (3.40 x 10 documents were used). 11 PFU and the number of phages bound to the virus were 5.06 × 10⁻⁶. 5 The positive rate was 1.49 × 10⁻⁶. -6 The antibody library was enriched 587 times (Table 1).

[0038] Table 1. Results of the selection of porcine Y sperm-specific nanobodies

[0039] Selection round | Library usage / PFU-bound phage count / PFU positivity rate | Round 1 | 3.40 × 10⁻⁶ 11 5.06×10 5 1.49×10 -6 Round 2: 5.06 × 10 8 1.83×10 5 3.62×10 -4 Round 3 1.83×10 8 3.78×10 5 2.07×10 -3 Round 4: 3.78 × 10 8 3.50×10 5 9.26×10 -4 Round 5 3.50×10 88.64×10 4 2.47×10 -4 Round 6: 8.64 × 10 7 5.76×10 4 6.67×10 -4 Round 7: 5.76 × 10 7 5.04×10 4 8.75×10 -4

[0040] Note: Positive rate = Nth round of screening, number of phages / amount of library used.

[0041] Four rounds of positive screening using porcine X sperm were conducted to select nanobodies, and the library was effectively enriched. In the fourth round (5.76 × 10⁻⁶ sperm samples were used), the nanobody was successfully enriched. 8 PFU and phage binding count: 1.05 × 10⁻⁶ 6 The positive rate was 1.82 × 10⁻⁶. -3 The selection process compared to the first round (using 3.40 x 10 documents) 11 PFU and the number of phages bound to the virus were 5.06 × 10⁻⁶. 5 The positive rate was 1.49 × 10⁻⁶. -6 The antibody library was enriched 1221-fold. To reduce the size of the X sperm-specific library and simplify antibody identification, phages were added at 100 times the size of the fourth-round screening library for a fifth round of screening, yielding 2.64 × 10⁻⁶ positive phages. 4 (Table 2).

[0042] Table 2. Results of the selection of porcine X sperm-specific nanobodies

[0043] Selection round | Library usage / PFU-bound phage count / PFU positivity rate | Round 1 | 3.40 × 10⁻⁶ 11 5.06×10 5 1.49×10 -6 Round 2: 5.06 × 10 8 4.08×10 5 8.06×10 -4 Round 3 4.08×10 8 5.76×10 5 1.41×10 -3 Round 4: 5.76 × 10 8 1.05×10 6 1.82×10 -3 Round 5 1.05×10 8 2.60×10 4 2.48×10 -4

[0044] Note: Positive rate = Nth round of screening, number of phages / amount of library used.

[0045] Example 2: Functional validation of a porcine sperm-specific nanobody library and screening of specific antibodies.

[0046] The antibody library eluent obtained in Example 2 was used to infect TG1 Escherichia coli, plated on 2×TY solid culture plates containing ampicillin, and cultured overnight. Single colonies were picked and sent to BGI Genomics for first-generation sequencing to analyze antibody coding sequence information and abundance. The results of single-clone colony sequencing and abundance analysis are shown in Figure 1. After screening with porcine sperm, positive antibodies were effectively enriched, with the highest abundance reaching 13.19%. Finally, 47 nanobodies with the highest sequence abundance were selected for subsequent specificity detection.

[0047] Pig X and Y sperm were sorted by flow cytometry, then centrifuged at 800g for 10 minutes. The supernatant was removed, and the cells were resuspended in CBS coating buffer (pH 9.6). 300,000 sperm cells per well were added to a 96-well ELISA plate and incubated overnight at 4°C. The supernatant was gently removed, and the cells were dried at 50°C. The plates were fixed with 4% paraformaldehyde for 15 minutes, blocked with 3% BSA for 1 hour, and washed three times with PBST. Wells containing phage cells without antibody were used as negative controls, and wells without antibody were used as blank controls. The same amount of the same antibody strain was added to both X and Y sperm wells, and the plates were incubated at room temperature for 2 hours. After washing three times with PBST, the secondary antibody anti-M13 Bacteriophage (HRP) (11973-MM05T-H, Sino Biological Inc) was added, and the plates were incubated at room temperature for 1 hour. After washing six times with PBST, the plates were developed with TMB chromogenic buffer, and the OD370 absorbance was measured to screen for differentially binding antibodies to X and Y sperm. The binding specificity of 47 antibodies with high abundance was detected using ELISA technology. The ratio of X sperm OD370 value to Y sperm OD370 value (OD370-X / Y) greater than or equal to 1.3 was used as the specificity screening criterion. As shown in Figure 2, 11 antibodies with stronger binding ability to X sperm were identified: W9, W10, W14, W17, W23, W37, W56, W57, W81, S92, and S94.

[0048] Immunofluorescence and flow cytometry were used to analyze the binding site and specificity of differentially expressed antibodies on the sperm membrane surface. One hundred million fresh porcine sperm cells were collected, washed three times with PBS, fixed with 4% paraformaldehyde at room temperature for 15 minutes, blocked with 3% BSA for 1 hour, and washed three times with PBST. Using phages that did not display antibodies as a control, the selected differentially expressed antibodies were added, and the mixture was incubated at room temperature for 2 hours, washed three times with PBST, and then incubated with the secondary antibody anti-M13 Bacteriophage (FITC) (11973-MM05T-P; Sino Biological Inc.) for 1 hour at room temperature. The mixture was washed 10 times with PBST and resuspended in 1 mL of PBS. 700 μL of the antibody was used to incubate the sperm cells, and the fluorescence ratio was detected by flow cytometry to analyze antibody specificity. Separately, 300 μL of antibody was incubated in a 48-well cell culture plate, centrifuged at 1200g for 10 minutes, the supernatant was discarded, DAPI staining solution was added, and the plate was incubated at room temperature for 20 minutes. The binding sites of the antibodies on the sperm membrane surface were observed under a fluorescence microscope. Flow cytometry analysis results are shown in Figure 3: all 11 antibodies (W9, W10, W14, W17, W23, W37, W56, W57, W81, S92, and S94) showed high affinity for porcine sperm. Among them, W17 showed the strongest specificity, followed by W37 and W57. Therefore, antibody W17 was selected for subsequent studies. Fluorescence observation results are shown in Figure 4. The binding sites of all 11 antibodies were located on the mitochondrial sheath of the sperm tail, indicating that the antibodies can specifically recognize and bind to sperm.

[0049] Analysis of the first-generation sequencing results revealed the coding sequences for the W17, W37, and W57 antibodies as shown in SEQ ID No: 1-3:

[0050] W17 antibody coding sequence (SEQ ID No:1): GATGTGCAGCTGCAGGAGTCTGGAGGAGGCTTGGTGCAGGCTGGGGGGTCTCTGAGACTCACCTGTGCAGCCTCTAGAAGCATCGACAATATCCTTACCATGGGCTGGTACCGCCAGGCTCCAGGGAAGCAGCGCGAGTCGGTCGCGCGAATTCCTAATGGTAGTACTACAATCTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACAGTCTATCTACAAATGAATAGCCTGAAACCTGAGGACACAGCCGTCTATTACTGTGCAGCCGGGTATGAGGACAGCGACTACAAAGGGAATGACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCAGC

[0051] W37 antibody coding sequence (SEQ ID No:2): GATGTGCAGCTGCAGGAGTCTGGAGGAGGATTGGTGCAAACTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGAAACATCTTCAGTTTCCATTCCTGGGCCTGGTACCGCCAGCCTCCAGGGAAGCAGCGCGACTTGGTCGCACGGTTTAATAGTGGTGGTGGCACAAACTATGCAGACTCCGTGAAGGACCGATTCACCATCTCCAGAGACGTCGCCAAGAAAACAATGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACAGCCGTCTATAGCTGTTATGCATTTGGGGGCGACTATGACGATGGCTACAGGTATTTCGAAGTTTGGGGCCAGGGCACCCAGGTCACCGTCTCCAGC

[0052] W57 antibody coding sequence (SEQ ID No:3):

[0053] GATGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTGGAAGTGCCTTCACTCTCGATGCCATGGGCTGGTACCGCCAGGCTCCAAGGAAACAGCGGGAGTTGGTCGCAACTGTTGTTAGTGGTGGGAGCACAAACTATGCGGACT CCGTGAAGGGCCGATTCACCATCTCCGAGGACGTCGCCAAGAACACGTTCAATCTGCAAATGAACAGCCTGAAACCTGAGGACACAGCCACCTACTACTGCCACGCCGAGGGCACATCCTACAGTGGGCCTTACCCAAGGAAGTATAATTCATGGGGCCAGGGGGACCCAGGTCACCGTCTCCAGC

[0054] Example 3: Recombinant Expression of Nanobodies and Preparation of Immunomagnetic Beads

[0055] The coding sequence of the W17 antibody (SEQ ID No:1) was codon-optimized according to the codon usage preference of the CHO cell eukaryotic expression system. The optimized sequence (as shown in SEQ ID No:4) was fused with the Fc fragment of the rabbit IGg antibody to construct a fusion expression plasmid. The plasmid was transfected into CHO cells for fusion expression. The recombinant expressed antibody (amino acid sequence shown in SEQ ID No:5) was purified from the cell culture medium. The antibody concentration was measured to be 2.01 mg / mL using a micro-spectrophotometer, yielding a total of 6.31 mg of recombinant expressed antibody. Protein purity was determined using SDS-PAGE and SEC-HPLC techniques: SDS-PAGE results showed that the purity of the recombinant expressed antibody was greater than 95%, with a reduced molecular weight of 39 kDa and a non-reduced molecular weight of 78 kDa, consistent with the target protein size; SEC-HPLC results showed that the purity of the recombinant expressed antibody was 99.73%. Endotoxin content was detected using an endotoxin detection kit. The endotoxin content of the recombinant expressed antibody was less than 1 EU / mg, meeting the requirements for immunomagnetic bead preparation.

[0056] Preparation of biotin-conjugated immunomagnetic beads: Recombinant expression antibodies were biotinylated using a biotin-conjugated kit (Abcam, UK). Following the streptavidin instructions (Beaver Biotechnology, Suzhou), the biotinylated recombinant antibodies were labeled onto streptavidin magnetic beads with diameters of 300 nm, 1 μm, 2.8 μm, and 5 μm, respectively, to prepare biotin-conjugated immunomagnetic beads of four diameters.

[0057] Preparation of Protein A / G Immunomagnetic Beads: Following the operating procedure of the BeaverBeads Protein A / G Antibody Purification Kit (Beaver Biotechnology, 2020), the recombinant expression antibody was immobilized onto Protein A / G magnetic beads to construct Protein A / G immunomagnetic beads, which were named PW-17.

[0058] The codon-optimized coding sequence for the W17 antibody is shown in SEQ ID No:4:

[0059] GATGTTCAGCTCCAGGAAAGCGGCGGAGGCCTCGTACAAGCAGGCGGCTCCCTGAGACTCACCTGTGCAGCTTCCAGGTCTATCGACAACATCCTTACCATGGGATGGTACAGGCAAGCTCCAGGTAAGCAGAGGGAAAGCGTAGCACGCATTCCAAACGGATCAACCACCATTTACGCA GACTCTGTTAAGGGCCGTTTCACTATTTCTCGGGACAATGCCAAAAACACCGTGTACCTTCAGATGAACTCTCTGAAACCCGAAGACACAGCCGTTTATTATTGCGCCGCTGGGTACGAAGACTCAGACTATAAGGGAAACGATTACTGGGGGCAGGGCACCCAAGTGACTGTGAGTTCC

[0060] The amino acid sequence of the W17 recombinant expression antibody is shown in SEQ ID No:5:

[0061] MHSSALLCCLVLLTGVRADVQLQESGGGLVQAGGSLRLTCAASRSIDNILTMGWYRQAPGKQRESVARIPNGSTTIYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAGYEDSDYKGNDYWGQGTQVTVSSAPSTASKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVV DVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK

[0062] (Note: The italicized part is the signal peptide sequence, the underlined and bolded part is the W17 amino acid sequence, and the rest is the rabbit Fc amino acid sequence.)

[0063] Example 4: Optimization of incubation conditions for enriching porcine sperm with W17 biotin-based immunomagnetic beads.

[0064] 4.1 Screening of Biotin-Immunomagnetic Bead Diameter

[0065] Five 1.5 mL centrifuge tubes were used, each containing 12.5 million porcine sperm cells. The tubes were centrifuged at 800 g for 10 minutes, the supernatant was gently aspirated, and the sperm were resuspended in 500 μL of sperm dilution and preservation solution. Using unlabeled magnetic beads as a control, 20 μL of W17 immunomagnetic beads (300 nm, 1 μm, 2.8 μm, and 5 μm) were added to four of the tubes, respectively. The tubes were gently incubated at room temperature for 2 hours. After incubation, the centrifuge tubes were placed on a magnetic rack and allowed to stand for 5 minutes. The supernatant was aspirated, the tubes were washed once with sperm dilution and preservation solution, and the mixture was resuspended in 100 μL of PBS to obtain immunomagnetically enriched sperm. Take 20 μL of enriched sperm, centrifuge to remove the supernatant, add 30 μL of alkaline sperm lysis buffer, lyse at 65℃ for 20 minutes, add 30 μL of neutralization solution to neutralize, and dilute with sterile water to 120 μL. Purify sperm genomic DNA using the MicroElute Genomic DNA Kit (OMEGA, D3096). Quantitative PCR was used to detect the ratio of X to Y sperm in the enriched sperm. The results of magnetic bead size optimization are shown in Figure 5: all four types of W17 immunomagnetic beads effectively enriched X sperm, with 1 μm beads showing the best enrichment effect (72.19%), followed by 300 nm beads (59.51%). However, due to the better dispersibility of 300 nm beads, 300 nm W17 beads were selected for subsequent optimization experiments.

[0066] 4.2 Incubation Time Screening

[0067] Using the same method, porcine sperm was enriched with 300nm W17 immunomagnetic beads. Incubation times were set to 30, 60, 90, 120, 150, and 180 minutes to explore the optimal incubation time for the immunoantibody. The results of the optimized incubation time are shown in Figure 6: Co-incubation of the immunomagnetic beads and sperm for 30 minutes yielded a good enrichment effect with an efficiency of 75.70%. The optimal enrichment effect was achieved at 90 minutes, with an efficiency of 82.95%. Further extending the incubation time did not further optimize the enrichment effect.

[0068] 4.3 Sperm density screening

[0069] Sperm density was set at 2 million / mL, 2.5 million / mL, 3 million / mL, 3.5 million / mL, 4 million / mL, 4.5 million / mL, 5 million / mL, 6 million / mL, 6.5 million / mL, 7 million / mL, 7.5 million / mL, 8.5 million / mL, 9 million / mL, 10 million / mL, 20 million / mL, 30 million / mL, 40 million / mL, 50 million / mL, 60 million / mL, 70 million / mL, 80 million / mL, 90 million / mL, 100 million / mL, 110 million / mL, 120 million / mL, and 130 million / mL. Sperm enrichment assays were performed using 300nm W17 magnetic beads. Sperm enriched by the magnetic beads were collected to test the enrichment effect, in order to explore the optimal sperm density for sperm enrichment by immunomagnetic beads. The results of sperm density optimization during incubation are shown in Figure 7: When the sperm density reached 350 million / mL, the W17 immunomagnetic beads began to show an enrichment effect on X sperm, with an enrichment efficiency of 62.42%. When the sperm density increased to 450 million / mL, the enrichment efficiency was 84.10%. With further increases in sperm density, the enrichment efficiency stabilized at around 80%.

[0070] 4.4 Screening of Magnetic Bead Usage

[0071] The sperm density was set to 33.19 million / mL, and sperm were enriched using 10μL, 20μL, 30μL, 40μL, 50μL, 60μL, 70μL, 80μL, 90μL, 100μL, 110μL, 120μL, and 130μL of 300nm W17 immunomagnetic beads to determine the optimal bead dosage. The results of the optimized bead dosage are shown in Figure 8: With a fixed sperm density of 33.19 million / mL, 10μL of W17 immunomagnetic beads was sufficient to achieve an enrichment efficiency of 79.50% for X sperm. Further increasing the bead dosage did not improve the enrichment efficiency.

[0072] In summary, the conditions for using biotinylated magnetic beads for sperm sorting are: a bead diameter of 300 nm or 1 μm; an incubation time of 30 minutes or more at room temperature; and a sperm density of 350 million / mL or higher.

[0073] Example 5: Enrichment of different strains of boar semen using W17 immunomagnetic beads

[0074] Semen was collected from eight lean-type pig breeds of Wens Foodstuff Group, including Duroc breeds S21, S22, S23, and S29, Large White breeds W64 and W65, and Landrace breeds W55 and W57. The sperm density was then diluted to 25 million / mL. Three 500 μL aliquots of semen from each breed were centrifuged at 800g for 10 minutes and gently resuspended in 500 μL of sperm dilution and preservation solution. To increase the number of enriched sperm, 20 μL of unlabeled antibody-coated magnetic beads were added to the control group, 20 μL of 300 nm W17 biotin-coated immunomagnetic beads were added to experimental group 1, and 20 μL of 1 μm W17 biotin-coated immunomagnetic beads were added to experimental group 2. The mixtures were incubated at room temperature for 30 minutes, then allowed to stand on a magnetic rack. The immunomagnetic beads were collected to enrich the sperm, and the ratio of X to Y sperm in the enriched sperm was analyzed using absolute quantitative PCR.

[0075] The quantitative results of biotin-enriched sperm populations using immunomagnetic beads are shown in Figure 9. Immunomagnetic beads W17 showed stable enrichment effects on eight lean porcine X sperm types. The average enrichment efficiency of 300nm immunomagnetic beads was 77.86%, and that of 1μm immunomagnetic beads was 78.43%. Specifically, for Duroc porcine X sperm, the enrichment efficiency was highest at 85.92% and lowest at 64.45% with an average of 78.88% for 300nm immunomagnetic beads; and highest at 83.02% and lowest at 69.83% with an average of 77.98% for 1μm immunomagnetic beads. For Large White porcine X sperm, the enrichment efficiency was highest at 81.06% and lowest at 76.61% with an average of 78.84% for 300nm immunomagnetic beads; and highest at 81.19% and lowest at 78.97% with an average of 80.08% for 1μm immunomagnetic beads. Enrichment efficiency of Landrace pig X sperm: 300nm immunomagnetic beads had the highest efficiency of 84.45%, the lowest efficiency of 65.23%, and an average efficiency of 74.84%; 1μm magnetic beads had the highest efficiency of 78.75%, the lowest efficiency of 76.61%, and an average efficiency of 77.68%.

[0076] Semen samples were collected from two Large White pigs and named YY1 and YY2 to test the sorting effect of protein A / G immunomagnetic beads PW-17 on pig X / Y sperm. The results are shown in Figure 10: PW-17 beads can effectively enrich X sperm, with an enrichment efficiency of 76.45% for X sperm in YY1 semen and 82.73% for YY2 semen, with an average enrichment efficiency of 79.59%.

[0077] In summary, due to the different binding abilities of W17 antibodies to X and Y sperm, they can induce agglutination reactions in sperm of specific sexes. For example, antibody W17 can induce X sperm agglutination (as shown in Figure 11), with an enrichment efficiency of up to 80%. Therefore, X sperm can be efficiently enriched using W17 antibody immunomagnetic beads, resulting in the enrichment of X sperm, while the unenriched supernatant contains Y sperm. This allows for convenient and efficient sorting (separation) of pig X and Y sperm. Moreover, this antibody and its sorting method offer high efficiency in separating X and Y sperm with minimal sperm damage, providing greater advantages than sperm flow cytometry. It does not rely on expensive equipment or specialized technicians, facilitating the establishment of an economical and efficient sex control technology. Furthermore, this antibody is a nanobody, only 10% the size of traditional antibodies, with a simpler structure, high stability, strong specificity, and low immunogenicity. It enables large-scale antibody expression, significantly reducing antibody production costs and facilitating industrial application.

[0078] The above descriptions are merely some embodiments of the present invention. For those skilled in the art, various modifications and improvements can be made without departing from the inventive concept of the present invention, and all such modifications and improvements fall within the scope of protection of the invention.

Claims

1. Porcine sperm-specific antibodies, among which, The coding sequence of the antibody is shown in any one of SEQ ID No:1-3.

2. Porcine sperm-specific antibodies, among which, The antibody is an antibody optimized according to the codon usage preferences of the eukaryotic expression system, and the coding sequence of the optimized antibody is shown in SEQ ID No:

4.

3. Porcine sperm-specific antibodies, among which, The antibody is a recombinant expression antibody fused with an exogenous Fc fragment, and the amino acid sequence of the recombinant expression antibody is shown in SEQ ID No:

5.

4. A method for preparing porcine sperm-specific antibodies, wherein, The method includes the following steps: S1: Prepare a library of bovine sperm-specific nanobodies by immunizing alpacas with whole bovine sperm; S2: Screen the library prepared in step S1; S3: Select antibodies that specifically bind to sperm from the pooled library.

5. The preparation method according to claim 4, wherein, Step S1 further includes the following steps: S1: Immunize alpacas with whole pig sperm, and then separate alpaca peripheral blood cells after immunization; S2: RNA was extracted from peripheral blood cells, reverse transcribed to obtain cDNA, and the VHH coding sequence of the antibody was amplified; S3: The amplified VHH coding sequence was cloned into a phage plasmid vector to prepare a recombinant phage vector carrying the porcine sperm antibody gene; S4: Transform the recombinant phage vector into Escherichia coli and expand its culture to obtain a porcine whole sperm antibody phage display library; S5: Using pig X / Y sperm, the phage display library obtained in step S4 is panned to construct a pig single-sex sperm-specific nanobody library: negative screening is performed with X sperm and positive screening is performed with Y sperm, and panning is performed to construct a Y sperm-specific nanobody library; negative screening is performed with Y sperm and positive screening is performed with X sperm, and panning is performed to construct an X sperm-specific nanobody library.

6. The porcine sperm-specific antibody prepared by the method according to claim 4 or 5.

7. The use of the antibody according to any one of claims 1-3 in the sorting of pig X and Y sperm.

8. The use of the antibody according to any one of claims 1-3 in the preparation of a product for sorting porcine sperm.

9. A method for sorting pig X and Y sperm, wherein, The method includes the following steps: S1: Biotinylate the recombinant expression antibody with the amino acid sequence shown in SEQ ID No:5, and then label the biotinylated recombinant expression antibody onto streptavidin magnetic beads to prepare biotinylated immunomagnetic beads; S2: Dilute the semen to be sorted and add biotinylated immunomagnetic beads. Incubate at room temperature. After incubation, place it on a magnetic rack and collect the sperm that did not bind to the magnetic beads from the supernatant. Then, resuspend the sperm enriched by the immunomagnetic beads after removing the supernatant to obtain sperm of the other sex.

10. A method for sorting pig X and Y sperm, wherein, The method includes the following steps: S1: The recombinant expression antibody with the amino acid sequence shown in SEQ ID No:5 was immobilized onto Protein A / G magnetic beads to construct Protein A / G immunomagnetic beads; S2: Dilute the semen to be sorted and add Protein A / G immunomagnetic beads. Collect the sperm that are not enriched with the magnetic beads in the supernatant and collect the sperm that are enriched with the magnetic beads separately to achieve the sorting of X and Y sperm.

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