Preparation and use method of immunomagnetic beads for separating X and Y sperms

Abstract

The invention relates to the technical field of sex control, in particular to preparation and use methods of an immunomagnetic bead for separating X and Y sperms, the immunomagnetic bead is coupled with an SRY polyclonal antibody and can be specifically combined with the Y sperms in seminal fluid, and then the Y sperms are adsorbed on the wall of an EP tube under the action of magnetic force, so that the effect of separating the X sperms from the Y sperms is achieved. According to the method, the X sperms and the Y sperms can be accurately separated by utilizing the specific recognition effect of the anti-SRY antibody on the Y sperms, the purity of the X sperms or the Y sperms in the separated semen is relatively high, the separated semen A contains more than 70% of the X sperms, and the separated semen B contains more than 80% of the Y sperms. And reliable technical support is provided for sex control in the fields of reproductive medicine and animal reproduction.

Description

Technical Field

[0001] The present invention relates to the technical field of sex control, and in particular to a preparation method and a use method of immunomagnetic beads for separating X and Y sperm. Background Art

[0002] The study of sex control is of great significance in many fields, especially in the animal husbandry industry. Through sex control technology, animal husbandry can more effectively manage the sex ratio of animal offspring, so as to maximize the production traits restricted or affected by sex, such as growth rate, meat quality and lactation capacity, so as to maximize economic benefits. In addition, sex control can also help increase the intensity of selection, speed up the breeding process, overcome certain reproductive obstacles, such as the infertility of opposite-sex twins in cattle embryo transplantation, and eliminate the harm of sex-linked harmful genes.

[0003] At present, the more mature methods of sex control mainly include sperm separation and embryo sex identification. Among them, sperm separation is to use the differences between X and Y sperm in terms of DNA content, volume, weight or surface charge, etc., to separate X and Y sperm by means of flow cytometric separation, sedimentation, centrifugation, electrophoresis or immunological separation, and then obtain offspring of the desired sex. The most mature method for separating X and Y sperm is flow cytometric separation, which is based on the slight difference in DNA content between X and Y sperm. Although the technology is advanced and widely used, this method also has obvious disadvantages. Among them, the high cost, complex operation and potential damage to sperm of flow cytometers are the main limiting factors. In addition, this method has high requirements for experimental conditions, including sperm quality, vitality, temperature and pH value during the processing process, etc., all of which may affect the separation effect and the subsequent function of sperm. Therefore, it is particularly important to explore a simpler, more efficient and less damaging separation method for sperm.

[0004] The SRY gene is only located on the Y chromosome. Studies have found that SRY (or at least some fragments or epitopes thereof) is expressed on the surface of Y sperm. The specific protein difference between X sperm and Y sperm may be used to separate X and Y sperm. The present invention combines immunological method and magnetic bead method to prepare an immunomagnetic bead for separating X and Y sperm. The immunomagnetic bead is coupled with SRY polyclonal antibody, which can specifically bind to Y sperm in semen, and then adsorbs Y sperm to the EP tube wall through magnetic force, thereby achieving the separation effect of X sperm and Y sperm. Summary of the invention

[0005] In view of the above-mentioned deficiencies in the prior art, the object of the present invention is to overcome the shortcomings and deficiencies of the existing X and Y sperm separation methods and to provide a method for preparing and using immunomagnetic beads for separating X and Y sperm that is simple, rapid and less harmful to sperm.

[0006] A method for preparing immunomagnetic beads for separating X and Y sperm, the reagents required for the preparation include:

[0007] Activation buffer: pH 6.0, 0.01 mol / L NaH2PO4 Tween;

[0008] Coupling buffer: pH 7.4, 0.01 mol / L Tween phosphate;

[0009] Carbodiimide EDC;

[0010] N-hydroxysuccinimide NHS;

[0011] Carboxyl magnetic beads: 50-500nm particle size;

[0012] Blocking solution: 1% bovine serum albumin, BSA;

[0013] Storage solution: pH 7.4, 0.01 mol / L phosphate Tween containing 0.02% Na3N, 0.1% BSA;

[0014] Polyclonal anti-SRY antibody;

[0015] Sperm culture medium: Tris 2.4g, citric acid 1.4g, glucose 0.8g, BSA 1.0g, add double distilled water to make up to 100mL;

[0016] The preparation method comprises the following steps:

[0017] S1. Activation: Prepare 50 mg / ml EDC and 50 mg / ml NHS with activation buffer; add 1.5 ml activation buffer, 0.5 ml EDC and 0.5 ml NHS to the EP tube containing magnetic beads, mix well and incubate at room temperature for 15 min; place the EP tube on the magnetic rack for 30-60 seconds to fix the magnetic beads on the wall of the EP tube, and discard the liquid with a pipette;

[0018] S2, coupling: Take an appropriate amount of SRY polyclonal antibody and the treated magnetic beads, add 2 ml of coupling buffer, mix well; incubate at room temperature for 90 minutes or at 4°C overnight on a rotator; place the EP tube on a magnetic rack for 30-60 seconds to fix the magnetic beads on the wall of the EP tube, and discard the liquid with a pipette;

[0019] S3. Blocking and preservation: Add 5 ml of blocking solution to the EP tube, mix well and incubate on a rotator at room temperature for 30 minutes; place the EP tube on a magnetic rack for 30-60 seconds to fix the magnetic beads on the wall of the EP tube, use a pipette to discard the liquid, and repeat 3-4 times; add 1 ml of preservation solution to the EP tube, mix well and store at 2-8°C.

[0020] Preferably, in step S1, EDC and NHS should be prepared and used immediately, kept away from light, and stored in ice before use.

[0021] Preferably, the polyclonal anti-SRY antibody is an anti-SRY antibody suitable for humans, pigs, cattle, sheep, dogs or cats, and the host species is rabbit or mouse, depending on the immunogen.

[0022] Preferably, the method of use comprises the following steps:

[0023] a. Centrifuge the semen at 800r / min for 5min, remove the supernatant, and resuspend the sperm to 1.0x107 / mL in sperm culture medium;

[0024] b. Add 10 μL of the immunomagnetic beads prepared above for separating X and Y sperm to the EP tube containing 500 μL of resuspended semen, mix well, and incubate at 37°C for 30 min;

[0025] c. Place the EP tube on the magnetic rack for 30-60 seconds to fix the magnetic beads on the wall of the EP tube, and use a pipette to aspirate the semen to obtain semen A, which is semen enriched with X sperm; remove the EP tube from the magnetic rack, and use 500 μL sperm culture medium to resuspend the magnetic beads to obtain semen B, which is semen enriched with Y sperm.

[0026] Preferably, the SRY antibody-immunomagnetic beads prepared by the present invention are used for separation of X and Y sperms from fresh semen, frozen semen, and semen collected from the epididymis.

[0027] The beneficial effects of the present invention are:

[0028] High specificity and accuracy: The present invention utilizes the specific recognition of anti-SRY antibodies on Y sperm to accurately separate X and Y sperm. The purity of X or Y sperm in the separated semen is high. The separated semen A contains more than 70% X sperm, and semen B contains more than 80% Y sperm. It provides reliable technical support for sex control in the field of reproductive medicine and animal reproduction.

[0029] Easy operation: The preparation process and use of the entire immunomagnetic beads are relatively simple, do not require complex equipment and professional technicians, and are easy to promote and apply. The method is time-saving, simple to operate, and does not damage sperm.

[0030] Little impact on sperm activity: During the preparation and use of immunomagnetic beads, the selected reagents and conditions are optimized to minimize the impact on sperm activity and ensure that the separated sperm has high vitality and can be used for subsequent assisted reproductive technology or animal breeding.

[0031] Wide applicability: The immunomagnetic beads of the present invention are suitable for the separation of semen of multiple species such as humans, cattle, sheep, pigs, dogs, cats, etc., and have a wide range of applications. DETAILED DESCRIPTION

[0032] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. The components of the embodiments of the present invention described and shown herein can be arranged and designed in various different configurations.

[0033] Example 1

[0034] A method for preparing immunomagnetic beads for separating X and Y sperm, the reagents required for the preparation include:

[0035] Activation buffer: pH 6.0, 0.01 mol / L NaH2PO4 Tween;

[0036] Coupling buffer: pH 7.4, 0.01 mol / L Tween phosphate;

[0037] Carbodiimide EDC;

[0038] N-hydroxysuccinimide NHS;

[0039] Carboxyl magnetic beads: 50-500nm particle size;

[0040] Blocking solution: 1% bovine serum albumin, BSA;

[0041] Storage solution: pH 7.4, 0.01 mol / L phosphate Tween containing 0.02% Na3N, 0.1% BSA;

[0042] Polyclonal anti-SRY antibody;

[0043] Sperm culture medium: Tris 2.4g, citric acid 1.4g, glucose 0.8g, BSA 1.0g, add double distilled water to make up to 100mL;

[0044] The preparation method comprises the following steps:

[0045] Use activation buffer to prepare 50mg / ml EDC and 50mg / ml NHS; add 1.5ml activation buffer, 0.5ml EDC and 0.5ml NHS to the EP tube containing magnetic beads, mix well and incubate at room temperature for 15min; place the EP tube on the magnetic rack for 60s to fix the magnetic beads on the wall of the EP tube, and use a pipette to discard the liquid. Take an appropriate amount of rabbit anti-SRY polyclonal antibody (the immunogen is the internal synthetic peptide of canine SRY) and mix with the above-treated magnetic beads, add 2ml coupling buffer, mix well; place on a rotator and incubate at room temperature for 90min; place the EP tube on the magnetic rack for 60s to fix the magnetic beads on the wall of the EP tube, and use a pipette to discard the liquid. Add 5 ml of blocking solution to the EP tube, mix well and incubate on a rotator at room temperature for 30 min; place the EP tube on a magnetic rack for 60 s to fix the magnetic beads on the wall of the EP tube, and use a pipette to discard the liquid (repeat 4 times); add 1 ml of preservation solution to the EP tube, mix well and store at 4°C.

[0046] A method for using immunomagnetic beads for separating canine X and Y sperm, comprising the steps of: collecting fresh canine semen with a motility of 95% and a density of 2.5x10 8 / mL, centrifuge at 800r / min for 5min, remove the supernatant, and resuspend the sperm in sperm culture medium to 1.0x10 7 / mL. Take 500μL of resuspended semen in an EP tube, add 10μL of the SRY antibody-coupled immunomagnetic beads prepared above for canine X and Y sperm separation to the tube, mix well, and incubate at 37°C for 30min; place the EP tube on a magnetic rack for 60s to fix the magnetic beads on the wall of the EP tube, and use a pipette to aspirate the semen (semen A, semen enriched with X sperm). Remove the EP tube from the magnetic rack and use 500μL of sperm culture medium to resuspend the magnetic beads (semen B, semen enriched with Y sperm). Use fluorescent quantitative PCR to detect the ratio of X sperm and Y sperm in fresh semen, semen A and semen B. The results are shown in Table 1.

[0047] Table 1 Ratio of X sperm and Y sperm in semen

[0048] Sperm ratio Fresh Essence Semen A Semen B X sperm ratio 48.61±1.38 76.95±2.87 15.69±0.94 Y sperm ratio 50.96±1.66 23.68±2.66 84.43±2.75

[0049] As shown in Table 1, the separated semen A contains more than 70% X sperm, and the semen B contains more than 80% Y sperm, indicating that the immunomagnetic beads of the present invention can effectively separate X and Y sperm in canine semen.

[0050] Example 2

[0051] A method for preparing immunomagnetic beads for separating X and Y sperm, comprising the steps of:

[0052] Use activation buffer to prepare 50mg / ml EDC and 50mg / ml NHS; add 1.5ml activation buffer, 0.5ml EDC and 0.5ml NHS to the EP tube containing magnetic beads, mix well and incubate at room temperature for 15min; place the EP tube on the magnetic rack for 60s to fix the magnetic beads on the wall of the EP tube, and use a pipette to discard the liquid. Take an appropriate amount of mouse anti-SRY polyclonal antibody (the immunogen is the internal synthetic peptide of bovine SRY) and mix with the above-treated magnetic beads, add 2ml coupling buffer, mix well; place on a rotator and incubate overnight at 4℃; place the EP tube on the magnetic rack for 60s to fix the magnetic beads on the wall of the EP tube, and use a pipette to discard the liquid. Add 5 ml of blocking solution to the EP tube, mix well and incubate on a rotator at room temperature for 30 min; place the EP tube on a magnetic rack for 60 s to fix the magnetic beads on the wall of the EP tube, and use a pipette to discard the liquid (repeat 4 times); add 1 ml of preservation solution to the EP tube, mix well and store at 4°C.

[0053] A method for using immunomagnetic beads for separating bovine X and Y sperm, comprising the steps of: rapidly thawing a tube of 0.5 mL bovine frozen semen at 37°C for 30 seconds, testing the semen after thawing, and finding that the motility is 65% and the density is 1.3x10 8 / mL, centrifuge at 800r / min for 5min, remove the diluent, and resuspend the sperm in sperm culture medium to 1.0x10 7 / mL. Take 500μL of resuspended semen in an EP tube, add 10μL of the SRY antibody-coupled immunomagnetic beads prepared above for separation of bovine X and Y sperm to the tube, mix well, and incubate at 37°C for 30min; place the EP tube on a magnetic rack for 60s to fix the magnetic beads on the wall of the EP tube, and use a pipette to aspirate the semen (semen A, semen enriched with X sperm). Remove the EP tube from the magnetic rack and use 500μL of sperm culture medium to resuspend the magnetic beads (semen B, semen enriched with Y sperm). Fluorescence quantitative PCR was used to detect the ratio of X sperm and Y sperm in fresh semen, semen A and semen B. The results are shown in Table 2.

[0054] Table 2 Ratio of X sperm and Y sperm in semen

[0055] Sperm ratio Fresh Essence Semen A Semen B X sperm ratio 47.92±1.72 78.11±1.96 16.01±0.98 Y sperm ratio 51.83.±1.65 21.72±2.03 83.93±1.65

[0056] As shown in Table 2, the separated semen A contains more than 70% X sperm and semen B contains more than 80% Y sperm, indicating that the immunomagnetic beads of the present invention can effectively separate X and Y sperm in bovine semen.

[0057] The immunomagnetic beads prepared by the present invention are used to separate X and Y sperms and are applicable to multiple species such as humans, cattle, sheep, pigs, dogs, cats, etc. They have high separation efficiency and accuracy, and have little effect on the activity of sperms. The method is time-consuming, simple to operate, and does not damage sperms, providing an effective technical means for sex control in the field of reproductive medicine and animal reproduction.

[0058] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for preparing immunomagnetic beads for separating X and Y sperm, characterized in that: The reagents required for preparation include: Activation buffer: pH 6.0, 0.01 mol / L NaH2PO4 Tween; Coupling buffer: pH 7.4, 0.01 mol / L Tween phosphate; Carbodiimide EDC; N-hydroxysuccinimide NHS; Carboxyl magnetic beads: 50-500nm particle size; Blocking solution: 1% bovine serum albumin, BSA; Storage solution: pH 7.4, 0.01 mol / L phosphate Tween containing 0.02% Na3N, 0.1% BSA; Polyclonal anti-SRY antibody; Sperm culture medium: Tris 2.4g, citric acid 1.4g, glucose 0.8g, BSA 1.0g, add double distilled water to make up to 100mL; The preparation method comprises the following steps: S1. Activation: Prepare 50 mg / ml EDC and 50 mg / ml NHS with activation buffer; add 1.5 ml activation buffer, 0.5 ml EDC and 0.5 ml NHS to the EP tube containing magnetic beads, mix well and incubate at room temperature for 15 min; place the EP tube on the magnetic rack for 30-60 seconds to fix the magnetic beads on the wall of the EP tube, and discard the liquid with a pipette; S2, coupling: Take an appropriate amount of SRY polyclonal antibody and the treated magnetic beads, add 2 ml of coupling buffer, mix well; incubate at room temperature for 90 minutes or at 4°C overnight on a rotator; place the EP tube on a magnetic rack for 30-60 seconds to fix the magnetic beads on the wall of the EP tube, and discard the liquid with a pipette; S3. Blocking and preservation: Add 5 ml of blocking solution to the EP tube, mix well and incubate on a rotator at room temperature for 30 minutes; place the EP tube on a magnetic rack for 30-60 seconds to fix the magnetic beads on the wall of the EP tube, use a pipette to discard the liquid, and repeat 3-4 times; add 1 ml of preservation solution to the EP tube, mix well and store at 2-8°C.

2. The method for preparing immunomagnetic beads for separating X and Y sperm according to claim 1, characterized in that: In step S1, EDC and NHS should be prepared and used immediately, stored in the dark, and kept in ice before use.

3. The method for preparing immunomagnetic beads for separating X and Y sperm according to claim 1, characterized in that: Depending on the immunogen, polyclonal anti-SRY antibodies are anti-SRY antibodies suitable for humans, pigs, cattle, sheep, dogs or cats, and the host species are rabbits or mice.

4. The method for preparing immunomagnetic beads for separating X and Y sperm according to claim 1, characterized in that: The method of use includes the following steps: a. Centrifuge the semen at 800r / min for 5min, remove the supernatant, and resuspend the sperm in sperm culture medium to 1.0x10 7 Pieces / mL; b. Add 10 μL of the immunomagnetic beads prepared above for separating X and Y sperm to the EP tube containing 500 μL of resuspended semen, mix well, and incubate at 37°C for 30 min; c. Place the EP tube on the magnetic rack for 30-60 seconds to fix the magnetic beads on the wall of the EP tube, and use a pipette to aspirate the semen to obtain semen A, which is semen enriched with X sperm; remove the EP tube from the magnetic rack, and use 500 μL sperm culture medium to resuspend the magnetic beads to obtain semen B, which is semen enriched with Y sperm.

5. The method for preparing immunomagnetic beads for separating X and Y sperm according to claim 1, characterized in that: The SRY antibody-immunomagnetic beads prepared by the present invention are used for separating X and Y sperms from fresh sperm, frozen sperm, and semen collected from the epididymis.