Porcine sperm specific antibody as well as preparation method and application thereof

Abstract

The invention discloses a pig sperm specific antibody. The coding sequence of the antibody is shown as any one of SEQ ID No: 1-3. According to the method, the nano antibody library is prepared by immunizing alpaca with whole pig sperms, and then the antibody capable of being specifically combined with the X or Y sperms can be screened out through the library, so that sorting of the X and Y sperms is realized. The screened antibody is a nano antibody, the size of the nano antibody is only 10% of that of a traditional antibody, the structure is simpler, the advantages of high stability, strong specificity and low immunogenicity are achieved, large-scale expression of the antibody can be achieved, the production cost of the antibody is greatly reduced, and industrial application is facilitated.

Description

Technical Field

[0001] The present invention relates to the field of antibody preparation and animal breeding, and in particular to a pig sperm-specific antibody and a preparation method and application thereof. Background Art

[0002] Sex control is one of the core technologies in reproductive biology research, and it is of great significance for animal breeding, genetic disease prevention and control, and animal husbandry production. In order to solve practical problems such as the number and output of livestock and poultry in animal husbandry, it is particularly necessary to use sex control technology to promote the development of animal husbandry. Sex control technology is a modern biotechnology that selects and separates X and Y sperm through human participation, and finally obtains offspring of the desired sex, which has very important theoretical and practical significance. First, sex control technology can give full play to the growth rate and meat quality of male animals, and the reproduction and lactation performance of female animals, thereby obtaining huge economic benefits; secondly, it can enhance the selection intensity of excellent traits, accelerate the breeding process, and reduce breeding costs to obtain the greatest genetic progress; in addition, it can also overcome the phenomenon of twin infertility by controlling the sex of offspring, and eliminate the hazards of sex-linked harmful genes. Sex-controlled semen can be prepared into frozen semen, and sex can be selected for insemination according to needs, so as to finally achieve the purpose required for production. Therefore, efficient and accurate separation of sex-controlled semen has become one of the key research directions of sex control technology. In animal husbandry production, the research of sex control technology has very important practical production value.

[0003] At present, the main methods for preparing sex-controlled semen of pigs (pig X and Y sperm sorting) include Percoll density gradient centrifugation, flow cytometer sorting, and immunoassay to sort X and Y sperm. Regarding the Percoll density gradient centrifugation method: Cao Shizhen et al. showed that the sex ratio of the offspring of the semen obtained by using Percoll discontinuous density gradient to separate the qualitative semen of dairy cows was significantly different, among which the female proportion of the offspring of the qualitative semen rich in X sperm was more than 60%. The Percoll density gradient centrifugation technology is simple to operate and low in cost, but during the centrifugation process, sperm cells are prone to produce high concentrations of reactive oxygen species (ROS), which damages sperm vitality and reduces sperm integrity. Regarding the flow cytometer sorting method: Xiong Xianrong et al. added allura red on the basis of using flow cytometry to eliminate sperm with incomplete plasma membrane staining, which can significantly enhance the vitality of yak sperm after sorting and improve the conception rate of sex-controlled frozen semen to a certain extent. However, Zeng Youquan et al. found that the litter size was 91.67% after the Y sperm was separated for conception, but the litter size was decreasing. Flow cytometer sorting of X and Y sperm is a sex control technology currently used on a large scale in actual production, but it is rarely used in sorting pig semen. The main reason is that the amount of semen required for pig insemination is very large, and the efficiency of flow sorting is limited, which makes it difficult to meet the requirements of the number of pig insemination. In addition, the longer the flow sorting lasts, the greater the loss of sperm. Therefore, flow cytometer sorting of X and Y sperm is not suitable for the actual production of pig sex control needs. Regarding the immunological method of sorting X and Y sperm: Umehara et al. showed that TLR7 / 8 encoded by the X chromosome is expressed in the middle and tail of X sperm, but not in Y sperm. Umehara et al. further used TLR7 / 8 to separate mouse sperm. When R848 ligand was added, the motility of X and Y sperm of mice was significantly different. The solution showed a stratified state. The motility of X sperm was inhibited and was in the lower layer. When R848 was removed, the motility of X sperm was restored and still had fertilization ability. Fa Ren et al. obtained the same results when using TLR7 / 8 receptors in the study of separating X and Y sperm of dairy goats, and proved that TLR7 / 8 can directly regulate the ATP level of dairy goats to affect the motility of X sperm. Liu Weidong's research showed that TLR7 / 8 agonist R848 can significantly reduce the motility of X sperm in beef cattle sperm, but has no obvious effect on the integrity of sperm acrosome and plasma membrane. R848 was used to sort beef cattle fresh semen. After sorting, the purity of Y sperm can reach 88.6%, and the purity of X sperm can reach 72.5%. Although the accuracy of TRL7 / 8 sperm sorting is not high, 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 have poor effects on pig sperm sorting. A study by Wu Changhua et al. (2023), a graduate student at South China Agricultural University, showed that there was almost no difference in the expression of R848 and R837 ligands on pig X / Y sperm, so the separation purpose could not be achieved. At present, relevant research has been carried out on the separation of sexed semen from cattle, sheep, and mice, but there is still a blank in the research on pig sperm sorting, mainly because the number of pigs required for insemination is large, and traditional flow sorting technology cannot meet the requirements of pig semen sexing sorting in production practice.

[0004] Therefore, it is urgent to develop a new antibody and X, Y sperm sorting method that can be used to efficiently sort X, Y sperm to meet the needs of sex control of pig semen. Summary of the invention

[0005] The purpose of the present invention is to provide a new pig sperm-specific antibody that can efficiently and specifically bind to X or Y sperm, thereby achieving efficient separation of X and Y sperm and rapid and stable sorting of pig semen to solve the above problems.

[0006] According to the first aspect of the present invention, there is provided a porcine sperm-specific antibody, the coding sequence of which is shown in any one of SEQ ID No: 1-3. The antibody is a nano antibody, which is only 10% of the size of a traditional antibody, has a simpler structure, and has the advantages of high stability, strong specificity, and low immunogenicity. It can achieve large-scale expression of antibodies, greatly reduce the cost of antibody production, and facilitate industrial application.

[0007] According to the second aspect of the present invention, there is provided a pig sperm-specific antibody, which is an antibody optimized according to the codon usage preference of the cell eukaryotic expression system, and the coding sequence of the optimized antibody is shown in SEQ ID No: 4. The antibody coding sequence is a sequence optimized according to the codon usage preference of the cell eukaryotic expression system. After the optimization, the sequence can specifically bind to pig sperm and can be better used for the sorting of pig semen. Moreover, the antibody is a nano antibody, which is only 10% of the size of a traditional antibody, has a simpler structure, and has the advantages of high stability, strong specificity, and low immunogenicity. It can realize large-scale expression of antibodies, greatly reduce the cost of antibody production, and facilitate industrial application.

[0008] According to the third aspect of the present invention, there is provided a pig sperm-specific antibody, which is a recombinantly expressed antibody fused with an exogenous Fc fragment, and the amino acid sequence of the recombinantly expressed antibody is shown in SEQ ID No: 5. The recombinantly expressed antibody is fused and expressed by the screened nanobody and the Fc fragment of the exogenous (rabbit source) IGg antibody to form a VHH-Fc antibody, which can induce sperm agglutination reaction after binding to sperm, and the fused and expressed Fc fragment can be used to assemble magnetic beads coupled with streptavidin or protein A / G, and made into magnetic beads for pig sperm immunosorting.

[0009] According to the fourth aspect of the present invention, a method for preparing pig sperm-specific antibodies is provided, which comprises the following steps: S1: immunizing alpacas with pig whole sperm to prepare a pig sperm-specific nanoantibody library; S2: panning the library prepared in step S1; S3: screening antibodies that specifically bind to sperm from the panned library. This method is the first to use pig whole sperm as an immunogen to immunize alpacas to prepare a nanoantibody library, and the library capacity reaches 3.4×10 8 pfu, the antibody library has a higher abundance, a higher coverage of whole sperm antigens, and a more complete range of antibodies, thereby improving the success rate of capturing pig sex control-specific antibodies, efficiently screening out positive antibodies, and easily obtaining the coding sequence of each antibody, which can achieve large-scale expression of antibodies and greatly reduce the cost of antibody production.

[0010] In certain embodiments, the above method may further include the following steps: S1: immunizing alpacas with whole pig sperm, and isolating peripheral blood cells of alpacas after immunization; S2: extracting RNA from peripheral blood cells, reversely 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 a pig sperm antibody gene; S4: transforming the recombinant phage vector into Escherichia coli and expanding the culture to obtain a pig sperm antibody phage display library; S5: using pig X / Y sperm to pan the phage display library obtained in step S4 to construct a pig sperm-specific nanoantibody library: using X sperm for negative screening, using Y sperm for positive screening, and panning to construct a Y sperm-specific nanoantibody library; using Y sperm for negative screening, using X sperm for positive screening, and panning to construct an X sperm-specific nanoantibody library.

[0011] According to a fifth aspect of the present invention, there is provided a porcine sperm-specific antibody prepared by the above preparation method. Thus, the above method can simply, quickly, efficiently and stably obtain positive antibodies with good specificity, and the antibody has high sorting efficiency, strong specificity and little damage to sperm.

[0012] According to the sixth aspect of the present invention, there is provided the use of pig sperm-specific antibodies in the sorting of pig sperm X and Y. Thus, through this application, efficient sorting of pig sperm can be achieved, laying the foundation for achieving sex control of pig semen in production.

[0013] According to the seventh aspect of the present invention, there is provided the use of a porcine sperm-specific antibody in the preparation of a product for porcine sperm sorting. Thus, the product for sperm sorting (such as a kit, immunomagnetic beads, etc.) prepared by the antibody can sort sperm quickly and efficiently to achieve pig sex control.

[0014] According to the eighth aspect of the present invention, a method for sorting pig X and Y sperm is provided, which comprises the following steps: S1: biotinylating the recombinant expression antibody with an amino acid sequence as shown in SEQ ID No: 5, and then labeling the biotinylated recombinant expression antibody on streptavidin magnetic beads to prepare biotin immunomagnetic beads; S2: diluting the semen to be sorted and adding immunomagnetic beads, incubating at room temperature, standing on a magnetic frame after incubation, and then collecting the sperm in the supernatant that is not bound to the magnetic beads, and then resuspending the sperm enriched by the immunomagnetic beads after removing the supernatant to obtain sperm of the other sex. Thus, biotin immunomagnetic beads are prepared by the antibody, and magnetic sorting is performed, and the supernatant that is not bound to the magnetic beads is sperm of one sex, and the sperm enriched by the magnetic beads is sperm of another sex, thereby making it possible to simply, conveniently and efficiently sort pig sperm and achieve sex control of pig semen.

[0015] In certain embodiments, the conditions for sperm sorting are: the diameter of the magnetic beads is 300 nm or 1 μm; the incubation time at room temperature is 30 minutes or more; and the sperm density is more than 350 million / mL.

[0016] According to the ninth aspect of the present invention, a method for sorting pig X and Y sperm is provided, the method comprising the following steps: S1: fixing the recombinant expression antibody with the amino acid sequence shown in SEQ ID No: 5 to ProteinA / G magnetic beads to construct ProteinA / G immunomagnetic beads; S2: adding ProteinA / G immunomagnetic beads after diluting the semen to be sorted, collecting the sperm in the supernatant that is not enriched with the magnetic beads, and collecting the sperm enriched with the magnetic beads separately to achieve the sorting of X and Y sperm. Thus, through magnetic sorting, pig sperm can be sorted simply, conveniently and efficiently to achieve sex control of pig semen.

[0017] The beneficial effects of the present invention are as follows: a nano antibody library is prepared by immunizing alpacas with whole pig sperm, and then antibodies that can specifically bind to X or Y sperm can be screened through the library to achieve the sorting of X and Y sperm. Moreover, the screened antibodies are nano antibodies, which are only 10% of the size of traditional antibodies, have a simpler structure, and have the advantages of high stability, strong specificity, and low immunogenicity. The large-scale expression of antibodies can be achieved, which greatly reduces the cost of antibody production and is convenient for industrial application. The screened antibodies are applied to the sex control of pig semen. Since the antibodies have different binding abilities to X and Y sperm, they can induce agglutination reactions of sperm of a specific sex. For example, antibody W17 can induce agglutination of X sperm, and the enrichment efficiency of X sperm reaches 80%. Therefore, W17 antibody immunomagnetic beads can be used to efficiently enrich X sperm (the unenriched ones are Y sperm), thereby realizing efficient sorting of pig X and Y sperm. Moreover, the antibody and its sorting method are highly efficient in separating X and Y sperm with little damage to sperm, which is more advantageous than sperm flow sorting technology, does not rely on expensive equipment and professional technicians, and is conducive to establishing an economical and efficient pig semen sex control technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is the result of monoclonal antibody abundance analysis of pig sperm panning library;

[0019] Figure 2 This is the result of monoclonal antibody binding specificity analysis of the pig sperm panning library;

[0020] Figure 3 Figure 1 is the result of differential antibody flow cytometry analysis;

[0021] Figure 4 This is the result map of differential antibody binding position location;

[0022] Figure 5 This is the result of W17 biotin immunomagnetic beads diameter screening;

[0023] Figure 6 This is the result diagram of W17 biotin immunomagnetic beads incubation time optimization;

[0024] Figure 7 Results graph for sperm density optimization for incubation;

[0025] Figure 8 This is the result diagram of optimizing the dosage of biotin immunomagnetic beads;

[0026] Fig. 9 This is the result of enrichment of semen from different pig strains using W17 biotin immunomagnetic beads;

[0027] Fig.10 The effect of protein A / G immunomagnetic beads on pig semen sorting;

[0028] Fig.11Results of immunomagnetic beads' ability to agglutinate pig sperm. DETAILED DESCRIPTION

[0029] The invention will be further described in detail below in conjunction with the accompanying drawings.

[0030] Example 1 Construction and selection of nanoantibody library from whole-sperm pig immunized alpaca

[0031] 1.1 Construction of nanoantibody library of alpaca immunized with pig sperm

[0032] Select healthy alpacas, take 10 million fresh pig sperm, centrifuge at 800g for 10 minutes at room temperature, remove the supernatant, add 1mL PBS to wash, repeat twice, resuspend with 1mL PBS after washing, and inject subcutaneously to immunize the alpaca. 14 days after immunization, do booster immunization in the same way for 3 consecutive times. During the immunization process, alpaca serum is separated, and the titer of pig sperm-specific antibodies in the serum is monitored by immunofluorescence technology. After the antibody titer is stable, the alpaca peripheral blood cells (PBMC) are separated, total RNA is extracted, reverse transcribed into cDNA, and the alpaca antibody heavy chain variable region (VHH) coding sequence is amplified using cDNA as a template, and cloned into the phage plasmid pCom3XSS ( The plasmid was electroporated into TG1 Escherichia coli (Apark Biotech, P001) and cultured to obtain the antibody bacterial library. The library capacity was determined to be 3.4×10 8 pfu, the positive rate was 96%, and the diversity was 100%. The bacterial library was added to 100 mL of 2×TY medium until OD600 = 0.1, and cultured at 37°C 250rpm until OD600 = about 0.5. The helper phage M13KO7 (New England Biolabs (NEB, N0315S) was added at a ratio of 1:20 (bacterial number: helper phage number) to rescue and expand the culture. The progeny phage was purified using PEG / NaCl solution, and the primary antibody phage library was purified. The library titer was determined to be 1.2×10 14 pfu / mL.

[0033] 1.2 Selection of nanoantibodies specific for pig sperm

[0034] Take 10 million fresh pig sperm, wash 3 times with PBS, add 2 mL of PBS to resuspend, add 10 million sperm / well to 6-well cell culture, centrifuge at 1200g for 10 minutes, aspirate the supernatant, dry at 37-50℃, fix with 4% paraformaldehyde for 15 minutes, block with 3% BSA solution for 1 hour, wash 3 times with PBS, use the fixed well without sperm as blank control, add pig sperm immunized alpaca nanoantibody library for the first round of panning, recover and amplify the positive phages in the first round of screening, and use the obtained screening antibody library for the next round of panning. Using the same method, flow sorted pig X sperm and Y sperm were fixed to 12-well cell culture plates, and X sperm was used for negative screening and Y sperm was used for positive screening, or Y sperm was used for negative screening and X sperm was used for positive screening. 4-7 rounds of panning were continued, and the number of positive phages in each round of panning was determined. The positive ratio of each round of panning was calculated by dividing the number of bound phages by the number of input phages to evaluate the antibody enrichment effect.

[0035] The nanoantibody library was screened for 7 rounds using pig Y sperm for positive screening. The library was effectively enriched. In the 7th round (the library dosage was 5.76×10 7 pfu, number of bound phages: 5.04×10 4 , positive ratio 8.75×10 -4 ) panning relative to the first round (input library dosage 3.40×10 11 pfu, number of bound phages: 5.06×10 5 , positive ratio 1.49×10 -6 ) The panning antibody library was enriched 587 times (Table 1).

[0036] Table 1 Results of panning of pig Y sperm-specific nanoantibodies

[0037] Selection rounds Input library dosage / pfu Number of binding phages / pfu Positive ratio Round 1 <![CDATA[3.40×10 11 ]]> <![CDATA[5.06×10 5 ]]> <![CDATA[1.49×10 -6 ]]> Round 2 <![CDATA[5.06×10 8 ]]> <![CDATA[1.83×10 5 ]]> <![CDATA[3.62×10 -4 ]]> Round 3 <![CDATA[1.83×10 8 ]]> <![CDATA[3.78×10 5 ]]> <![CDATA[2.07×10 -3 ]]> Round 4 <![CDATA[3.78×10 8 ]]> <![CDATA[3.50×10 5 ]]> <![CDATA[9.26×10 -4 ]]> Round 5 <![CDATA[3.50×10 8 ]]> <![CDATA[8.64×10 4 ]]> <![CDATA[2.47×10 -4 ]]> Round 6 <![CDATA[8.64×10 7 ]]> <![CDATA[5.76×10 4 ]]> <![CDATA[6.67×10 -4 ]]> Round 7 <![CDATA[5.76×10 7 ]]> <![CDATA[5.04×10 4 ]]> <![CDATA[8.75×10 -4 ]]>

[0038] Note: Positive ratio = number of binding phages in the Nth round of screening / amount of library input.

[0039] The nanoantibodies were selected for four rounds using pig X sperm for positive screening. The library was also effectively enriched. In the fourth round (the library dosage was 5.76×10 8 pfu, number of bound phages: 1.05×10 6 , positive ratio 1.82×10 -3 ) panning compared to the first round (input library dosage 3.40×10 11 pfu, number of bound phages: 5.06×10 5 , positive ratio 1.49×10 -6) The antibody library was enriched by 1221 times. In order to reduce the capacity of the X sperm-specific library and reduce the difficulty of antibody identification, 100 times the capacity of the fourth round of screening library was used for phage screening in the fifth round, and 2.64×10 positive phages were obtained. 4 (Table 2).

[0040] Table 2 Results of panning of pig X sperm-specific nanoantibodies

[0041] Selection rounds Input library dosage / pfu Number of binding phages / pfu Positive ratio Round 1 <![CDATA[3.40×10 11 ]]> <![CDATA[5.06×10 5 ]]> <![CDATA[1.49×10 -6 ]]> Round 2 <![CDATA[5.06×10 8 ]]> <![CDATA[4.08×10 5 ]]> <![CDATA[8.06×10 -4 ]]> Round 3 <![CDATA[4.08×10 8 ]]> <![CDATA[5.76×10 5 ]]> <![CDATA[1.41×10 -3 ]]> Round 4 <![CDATA[5.76×10 8 ]]> <![CDATA[1.05×10 6 ]]> <![CDATA[1.82×10 -3 ]]> Round 5 <![CDATA[1.05×10 8 ]]> <![CDATA[2.60×10 4 ]]> <![CDATA[2.48×10 -4 ]]>

[0042] Note: Positive ratio = Nth round of screening, combined with the number of phages / amount of library input Example 2 Functional verification of pig sperm-specific nanoantibody library and screening of specific antibodies

[0043] The antibody library panning eluate obtained in Example 2 was used to infect TG1 Escherichia coli, and then coated on a 2×TY solid culture plate containing ampicillin. After overnight culture, a monoclonal colony was picked and commissioned to BGI for first generation sequencing to analyze the antibody coding sequence information and abundance. The results of monoclonal colony sequencing and abundance analysis are shown in Figure 2. Figure 1 As shown in: After pig sperm selection, positive antibodies were effectively enriched, with the highest abundance being 13.19%. Finally, 47 nanoantibodies with the highest sequence abundance were selected for subsequent specific detection.

[0044] Take the flow sorted pig X and Y sperm, then centrifuge at 800g for 10 minutes, remove the supernatant, resuspend with CBS coating solution (pH9.6), add 300,000 sperm per well to a 96-well ELISA plate, and coat at 4°C overnight. Gently remove the supernatant, dry at 50°C, fix with 4% paraformaldehyde for 15 minutes, block with 3% BSA for 1 hour, wash 3 times with PBST, add phage wells without display antibody as negative control, and no antibody wells as blank control. Add the same amount of the same strain of antibody to the X sperm and Y sperm coated wells, incubate at room temperature for 2 hours, wash 3 times with PBST, add secondary antibody anti-M13Bacteriophage (HRP) (11973-MM05T-H, Sino Biological Inc), incubate at room temperature for 1 hour, wash 6 times with PBST, develop with TMB colorimetric solution, measure OD370 absorbance, and screen antibodies that differentially bind to X and Y sperm. The binding specificity of 47 highly abundant antibodies was detected by ELISA technology, and the ratio of the OD370 value of sperm X to the OD370 value of sperm Y (OD370-X / Y) greater than or equal to 1.3 was used as the specificity screening standard. Figure 2 The results showed that there were 11 strains of antibodies with stronger binding ability to X sperm, namely W9, W10, W14, W17, W23, W37, W56, W57, W81, S92, and S94.

[0045] Immunofluorescence technology and flow cytometry were used to analyze the binding position and specificity of differential antibodies on the surface of sperm membrane. 10 million fresh pig sperm were taken, washed 3 times with PBS, fixed with 4% paraformaldehyde at room temperature for 15 minutes, blocked with 3% BSA for 1 hour, washed 3 times with PBST, and the phage that did not display the antibody was used as a control. The screened differential antibodies were added, incubated at room temperature for 2 hours, washed 3 times with PBST, and the secondary antibody anti-M13 Bacteriophage (FITC) (11973-MM05T-P; SinoBiological Inc.) was added, incubated at room temperature for 1 hour, washed 10 times with PBST, and resuspended with 1mL of PBS. 700μL of antibody-incubated sperm were taken to detect the fluorescence ratio using a flow cytometer to analyze the antibody specificity. Take another 300 μL of antibody-incubated sperm in a 48-well cell culture plate, centrifuge at 1200 g for 10 minutes, remove the supernatant, add DAPI staining solution, incubate at room temperature for 20 minutes, and observe the binding position of the antibody on the sperm membrane surface under a fluorescence microscope. Figure 3 As shown in the figure: 11 antibodies (W9, W10, W14, W17, W23, W37, W56, W57, W81, S92, S94) all have high affinity for pig sperm, among which W17 has the strongest specificity, followed by W37 and W57, so W17 antibody was selected for subsequent research. Figure 4 As shown, the binding sites of the 11 antibodies were all located on the mitochondrial sheath of the sperm tail, indicating that the antibodies can specifically recognize and bind to sperm.

[0046] Through analysis of the first generation sequencing results, the coding sequences of W17, W37 and W57 antibodies are shown in SEQ ID No: 1-3:

[0047] W17 antibody coding sequence (SEQ ID No: 1):

[0048] GATGTGCAGCTGCAGGAGTCTGGAGGAGGCTTGGTGCAGGCTGGGGGGTCTCTGAGACTCACCTGTGCAGCCTCTAGAAGCATCGACAATATCCTTACCATGGGCTGGTACCGCCAGGCTCCAGGGAAGCAGCGCGAGTCGGTCGCGCGAATTCCTAATGGTAGTACTACAATCTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACAGTCTATCTACAAATGAATAGCCTGAAACCTGAGGACACAGCCGTCTATTACTGTGCAGCCGGGTATGAGGACAGCGACTACAAAGGGAATGACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCAGC

[0049] W37 antibody coding sequence (SEQ ID No:2):

[0050] GATGTGCAGCTGCAGGAGTCTGGAGGAGGATTGGTGCAAACTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGAAACATCTTCAGTTTCCATTCCTGGGCCTGGTACCGCCAGCCTCCAGGGAAGCAGCGCGACTTGGTCGCACGGTTTAATAGTGGTGGTGGCACAAACTATGCAGACTCCGTGAAGGACCGATTCACCATCTCCAGAGACGTCGCCAAGAAAACAATGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACAGCCGTCTATAGCTGTTATGCATTTGGGGGCGACTATGACGATGGCTACAGGTATTTCGAAGTTTGGGGCCAGGGCACCCAGGTCACCGTCTCCAGC

[0051] W57 antibody coding sequence (SEQ ID No:3):

[0052] GATGTGCAGCTGCAGGAGTCTGGGGGAGGCTTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTGGAAGTGCCTTCACTCTCGATGCCATGGGCTGGTACCGCCAGGCTCCAAGGAAACAGCGGGAGTTGGTCGCAACTGTTGTTAGTGGTGGGAGCACAAACTATGCGGACT CCGTGAAGGGCCGATTCACCATCTCCGAGGACGTCGCCAAGAACACGTTCAATCTGCAAATGAACAGCCTGAAACCTGAGGACACAGCCACCTACTACTGCCACGCCGAGGGCACATCCTACAGTGGGCCTTACCCAAGGAAGTATAATTCATGGGGCCAGGGGGACCCAGGTCACCGTCTCCAGC

[0053] Example 3. Recombinant expression of nanobodies and preparation of immunomagnetic beads

[0054] According to the codon usage preference of the CHO cell eukaryotic expression system, the coding sequence of the W17 antibody (SEQ ID No: 1) was codon optimized, and 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, and the plasmid was transfected into CHO cells for fusion expression. The recombinant expressed antibody (amino acid sequence as shown in SEQ ID No: 5) was purified from the cell culture medium, and the antibody concentration was 2.01 mg / mL using a micro-spectrophotometer, and a total of 6.31 mg of recombinant expressed antibody was obtained. The protein purity was detected using SDS-PAGE technology and SEC-HPLC technology: SDS-PAGE test results showed that the purity of the recombinant expressed antibody was greater than 95%, the reduced molecular weight was 39 kDa, and the non-reduced molecular weight was 78 kDa, which was consistent with the size of the target protein; SEC-HPLC test results showed that the purity of the recombinant expressed antibody was 99.73%. The endotoxin content was detected using an endotoxin detection kit, and the endotoxin content of the recombinant expressed antibody was less than 1 EU / mg, meeting the requirements for the preparation of immunomagnetic beads.

[0055] Preparation of biotin immunomagnetic beads: The recombinant expressed antibodies were biotinylated using a biotin coupling kit (Abcam, UK). According to the instructions of streptavidin (Beaver Biotechnology, Suzhou), the biotinylated recombinant antibodies were labeled on streptavidin magnetic beads with diameters of 300 nm, 1 μm, 2.8 μm, and 5 μm, respectively, to prepare biotin immunomagnetic beads with four different diameters.

[0056] Preparation of protein A / G immunomagnetic beads: According to the operating procedures of the BeaverBeads ProteinA / G Antibody Purification Kit (Beaver Bio, 20202), the recombinant expressed antibody was fixed to the ProteinA / G magnetic beads to construct ProteinA / G immunomagnetic beads, which were named PW-17.

[0057] The codon-optimized W17 antibody coding sequence is as SEQ ID No: 4 shows: GATGTTCAGCTCCAGGAAAGCGGCGGAGGCCTCGTACAAGCAGGCGGCTCCCTGAGACTCACCTGTGCAGCTTCCAGGTCTATCGACAACATCCTTACCATGGGATGGTACAGGCAAGCTCCAGGTAAGCAGAGGGAAAGCGTAGCACGCATTCCAAACGGATCAACCACCATTTA CGCAGACTCTGTTAAGGGCCGTTTCACTATTTCTCGGGACAATGCCAAAAACACCGTGTACCTTCAGATGAACTCTCTGAAACCCGAAGACACAGCCGTTTATTATTGCGCCGCTGGGTACGAAGACTCAGACTATAAGGGAAACGATTACTGGGGGCAGGGCACCCAAGTGACTGTGAGTTCC

[0058] The amino acid sequence of the W17 recombinantly expressed antibody is shown in SEQ ID No: 5: MHSSALLCCLVLLTGVRA DVQLQ GGGLVQAGGSLRLTCAASRSIDNILTMGWYRQAPGKQRESVARIPNGSTTIYADSVKGRFTISRDNAKNTVYLQMNS LKPEDTAVYYCAAGYEDSDYKGNDYWGQGTQVTVSS APSTASKPTCPPPELLGGPSVFIFPPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIE KTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK

[0059] (Note: The italic part is the signal peptide sequence, the underlined and bold part is the W17 amino acid sequence, and the rest is the rabbit Fc amino acid sequence.)

[0060] Example 4. Optimization of incubation conditions for enriching pig sperm using W17 biotin immunomagnetic beads 4.1 Screening of biotin immunomagnetic beads diameter

[0061] Take 5 1.5mL centrifuge tubes, add 12.5 million pig spermatozoa to each tube, centrifuge at 800g for 10 minutes, gently remove the supernatant, and resuspend with 500μL sperm dilution and preservation solution. Using magnetic beads with unlabeled antibodies as a control, add 20μL of 300nm, 1μm, 2.8μm and 5μm W17 immunomagnetic beads to 4 of the tubes, and gently rotate and incubate at room temperature for 2 hours. After the incubation, place the centrifuge tube in a magnetic rack for 5 minutes, remove the supernatant, wash once with sperm dilution and preservation solution, add 100μL PBS and resuspend to obtain immunomagnetic bead-enriched spermatozoa. Take 20 μL of enriched sperm, centrifuge and remove the supernatant, add 30 μL of alkaline sperm lysis solution, lyse at 65°C for 20 minutes, add 30 μL of neutralizing solution to neutralize, add sterile water to dilute to 120 μL, purify sperm genomic DNA with MicroElute Genomic DNA Kit (OMEGA, D3096), and use absolute quantitative PCR technology to detect the ratio of enriched sperm X and Y sperm. The results of magnetic bead size optimization are as follows Figure 5 As shown in the figure: All four W17 immunomagnetic beads can effectively enrich X sperm, among which 1μm magnetic beads have the best enrichment effect, with an enrichment efficiency of 72.19%, followed by 300nm magnetic beads, with an enrichment efficiency of 59.51%; but since 300nm magnetic beads have better dispersibility, 300nm W17 magnetic beads were selected for subsequent optimization experiments.

[0062] 4.2 Incubation time screening

[0063] In the same way, 300 nm W17 immunomagnetic beads were used to enrich pig sperm, and the incubation time was set to 30 minutes, 60 minutes, 90 minutes, 120 minutes, 150 minutes, and 180 minutes to explore the optimal incubation time for immune antibodies. The incubation time optimization results are shown in Figure 2. Figure 6 As shown: A good enrichment effect can be obtained by incubating the immunomagnetic beads with sperm for 30 minutes, with an enrichment efficiency of 75.70%. The enrichment effect is optimal when incubated for 90 minutes, with an enrichment efficiency of 82.95%. The enrichment effect is not further optimized by further extending the incubation time.

[0064] 4.3 Sperm density screening

[0065] The sperm density was set to 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 experiment was carried out using 300 nm W17 magnetic beads, and the magnetic bead-enriched sperm was taken to detect the sperm enrichment effect, so as to explore the optimal sperm density for immunomagnetic bead enriched sperm. The results of sperm density optimization incubation are as follows Figure 7 As shown: When the sperm density reaches 350 million / mL, W17 immunomagnetic beads begin to show an enrichment effect on X sperm, with an enrichment efficiency of 62.42%. When the sperm density increases to 450 million / mL, the enrichment efficiency is 84.10%. When the sperm density continues to increase, the enrichment efficiency stabilizes at around 80%.

[0066] 4.4 Screening of magnetic beads dosage

[0067] The sperm density was set to 33.19 million / mL, and 10 μL, 20 μL, 30 μL, 40 μL, 50 μL, 60 μL, 70 μL, 80 μL, 90 μL, 100 μL, 110 μL, 120 μL, and 130 μL of 300 nm W17 immunomagnetic beads were used to enrich sperm to find the optimal amount of magnetic beads. The results of the optimization of the amount of magnetic beads are shown in the figure. Figure 8 As shown: the fixed incubation sperm density is 3319 million / mL, and 79.50% enrichment efficiency of X sperm can be achieved by using 10 μL of W17 immunomagnetic beads. Continuing to increase the amount of magnetic beads, the enrichment efficiency is not further improved.

[0068] In summary, the conditions for using biotin magnetic beads for sperm sorting are: the diameter of the magnetic beads is 300nm or 1μm; the incubation time at room temperature is 30 minutes or more; the sperm density is above 350 million / mL.

[0069] Example 5: Enrichment of different strains of pig sperm using W17 immunomagnetic beads

[0070] Semen of 8 lean pig strains of Wens Group were collected, including Duroc pig strains S21, S22, S23, S29, Large White pig strains W64 and W65, and Landrace pig strains W55 and W57, and then the sperm density was diluted to 25 million / mL. Three portions of 500 μL of semen of each pig strain were taken, centrifuged at 800g for 10 minutes, and gently resuspended with 500 μL sperm dilution and preservation solution. To increase the number of enriched sperm, 20 μL of unlabeled antibody magnetic beads were added to the control group, 20 μL of 300nm W17 biotin immunomagnetic beads were added to the experimental group 1, and 20 μL of 1μm W17 biotin immunomagnetic beads were added to the experimental group 2. The mixture was incubated at room temperature for 30 minutes, and then placed on a magnetic stand. The immunomagnetic beads were collected to enrich sperm, and the absolute quantitative PCR technology was used to analyze the proportion of X and Y sperm in the enriched sperm.

[0071] The quantitative results of sperm enrichment by biotin immunomagnetic beads are as follows Fig. 9 As shown: Immunomagnetic beads W17 have a stable enrichment effect on 8 types of lean pig X sperm, the average enrichment efficiency of 300nm immunomagnetic beads is 77.86%, and the average enrichment efficiency of 1μm immunomagnetic beads is 78.43%. Among them, the collection efficiency of Duroc pig X sperm: 300nm immunomagnetic beads is the highest 85.92%, the lowest is 64.45%, and the average is 78.88%; 1μm immunomagnetic beads is the highest 83.02%, the lowest is 69.83%, and the average is 77.98%. The enrichment efficiency of Large White pig X sperm: 300nm immunomagnetic beads is the highest 81.06%, the lowest is 76.61%, and the average is 78.84%; 1μm immunomagnetic beads is the highest 81.19%, the lowest is 78.97%, and the average is 80.08%. The enrichment efficiency of Landrace X sperm was as follows: the highest efficiency of 300nm immunomagnetic beads was 84.45%, the lowest was 65.23%, and the average was 74.84%; the highest efficiency of 1μm magnetic beads was 78.75%, the lowest was 76.61%, and the average was 77.68%.

[0072] Semen from two large white pigs, named YY1 and YY2, was collected to test the effect of protein A / G immunomagnetic beads PW-17 on pig X / Y sperm sorting. The results are as follows Fig.10 As shown: PW-17 magnetic beads can effectively enrich X sperm, among which the enrichment efficiency of X sperm in YY1 semen is 76.45%, and the enrichment efficiency of X sperm in YY2 semen is 82.73%, and the average enrichment efficiency is 79.59%.

[0073] In summary, since the W17 antibody has different binding abilities to X and Y sperm, it can induce agglutination of sperm of a specific sex. For example, antibody W17 can induce agglutination of X sperm (e.g. Fig.11As shown), the enrichment efficiency of X sperm is 80%. Therefore, X sperm can be efficiently enriched by W17 antibody immunomagnetic beads, and X sperm is enriched, while Y sperm is in the unenriched supernatant, so that X and Y sperm of pigs can be efficiently sorted (separated) conveniently and efficiently. Moreover, the antibody and its sorting method have high efficiency in separating X and Y sperm and little damage to sperm, which is more advantageous than sperm flow sorting technology, does not rely on expensive equipment and professional technicians, and is conducive to establishing economical and efficient gender control technology. Moreover, the antibody is a nano antibody, which is only 10% of the size of traditional antibodies, has a simpler structure, has the advantages of high stability, strong specificity, and low immunogenicity, can realize large-scale expression of antibodies, greatly reduce the cost of antibody production, and is convenient for industrial application.

[0074] The above are only some embodiments of the present invention. For those skilled in the art, several modifications and improvements can be made without departing from the inventive concept of the present invention, which all belong to the protection scope of the invention.

Claims

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

2. Pig sperm specific antibodies, including: The antibody is an antibody optimized according to the codon usage preference of the eukaryotic expression system of the cell, and the coding sequence of the optimized antibody is shown in SEQ ID No:

4.

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

5.

4. A method for preparing a porcine sperm-specific antibody, wherein: The method comprises the following steps: S1: Immunize alpacas with whole pig sperm to prepare a pig sperm-specific nanoantibody library; S2: panning the library prepared in step S1; S3: Screening sperm-specific antibodies from the panned library.

5. The preparation method according to claim 4, wherein: The step S1 also includes the following steps: S1: Immunize alpacas with pig whole sperm, and separate alpaca peripheral blood cells after immunization; S2: Extract RNA from peripheral blood cells, reverse transcribe to obtain cDNA, and amplify 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 pig sperm antibody gene; S4: Transform the recombinant phage vector into Escherichia coli and expand the culture to obtain the porcine whole sperm antibody phage display library; S5: Use pig X / Y sperm to pan the phage display library obtained in step S4 to construct a pig single sex sperm-specific nanoantibody library: use X sperm for negative screening and Y sperm for positive screening, perform panning, and construct a Y sperm-specific nanoantibody library; use Y sperm for negative screening and X sperm for positive screening, perform panning, and construct an X sperm-specific nanoantibody library.

6. A porcine sperm-specific antibody prepared according to the method described in claim 4 or 5.

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

8. Use of the antibody according to any one of claims 1 to 3 in the preparation of a product for pig sperm sorting.

9. A method for sorting pig X and Y sperm, wherein: The method comprises the following steps: S1: biotinylating the recombinant expressed antibody having an amino acid sequence as shown in SEQ ID No: 5, and then labeling the biotinylated recombinant expressed antibody onto streptavidin magnetic beads to prepare biotin immunomagnetic beads; S2: Dilute the semen to be sorted and add biotin immunomagnetic beads, incubate at room temperature, and let it stand on a magnetic rack after incubation. Then collect the sperm in the supernatant that is not bound to the magnetic beads, and 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 comprises the following steps: S1: Immobilizing the recombinantly expressed 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: Dilute the semen to be sorted and add ProteinA / G immunomagnetic beads. Collect the sperm in the supernatant that are not enriched with the magnetic beads, and collect the sperm enriched with the magnetic beads separately to achieve the sorting of X and Y sperm.

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