Suppressin antibody and application thereof

Abstract

The invention relates to the technical field of antibody preparation, particularly discloses a statin antibody and application thereof, and researches that a synthetic swine statin antigen is used for immunizing a Japanese big-ear rabbit to prepare a polyclonal antibody, and the polyclonal antibody is injected into the abdominal cavity of a mouse to promote the ovulation number of the female mouse to be obviously increased, so that the efficiency of biological purification through IVF (in-vitro fertilization) is improved. On one hand, the statin level in mice can be directly reduced; on the other hand, the ovary local microenvironment is influenced, and the superovulation level is more effectively improved. The purpose of higher superovulation is achieved by inhibiting the effect of the statin through intraperitoneal injection of the statin antibody, the efficiency is effectively improved by 50% or above, and an unexpected effect is achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of antibody preparation, and particularly relates to an inhibin antibody and its application. Background Art

[0002] After being carefully artificially raised and managed, laboratory animals, through strict microbial control, ensure the clarity of their genetic background and the transparency of their sources, and are widely used in fields such as scientific research, teaching, production, and verification. Especially mice, due to the advantages of microbial control, have become the preferred model in experiments. In order to achieve high standards of microbial control, in vitro fertilization technology (IVF) is routinely used for the purification of mice. However, the ovulation amount of conventional female mice is often insufficient to meet experimental requirements. Therefore, the application of superovulation technology is crucial for increasing the ovulation amount and the number of reproductive mice.

[0003] Biological purification technologies such as IVF are the key to constructing specific pathogen-free (SPF) or germ-free mouse populations, aiming to eliminate potential pathogens in mice. Nevertheless, traditional IVF technology has problems such as limited egg numbers and difficulty in obtaining high-quality eggs, which limit its efficiency and scale. Inhibin plays an important role in animal reproductive regulation. Although its connection with reproduction has been studied, the existing technology still fails to fully meet the growing demand for high-quality and large numbers of experimental mice. Summary of the Invention

[0004] The purpose of the present invention is: aiming at the problems existing in the prior art.

[0005] In order to achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0006] The first object of the present invention is to protect an inhibin antibody, and the antigen amino acid sequence for preparing this antibody is as shown in SEQ ID NO.1.

[0007] Among them, the antigen amino acid sequence for preparing this antibody is as shown in SEQ ID NO.1: FRPSQHTRSRQVTSA-C-KLH.

[0008] The second object of the present invention is to protect an inhibin antibody, the antigen amino acid sequence for preparing this antibody is as shown in SEQ ID NO.2, and the amino acid at the C-terminal end of this antigen is used as the coupling site to be coupled with a carrier protein.

[0009] Among them, the antigen amino acid sequence for preparing this antibody is as shown in SEQ ID NO.2: FRPSQHTRSRQVTSA.

[0010] Furthermore, the carrier protein is ovalbumin, keyhole limpet hemocyanin, tetanus toxoid, bovine serum albumin or human serum albumin.

[0011] The third object of the present invention is to protect the use of an inhibin antibody as described above in the preparation of a drug for increasing the ovulation amount of female mice.

[0012] Furthermore, the dosage of the inhibin antibody injected into each female mouse each time is 0.2 - 0.8 ml, and the concentration of the inhibin antibody is 0.8 - 4 mg / ml.

[0013] The fourth object of the present invention is to protect an inhibin vaccine, which comprises the above antigen sequence.

[0014] The fifth object of the present invention is to protect a reagent for increasing the ovulation amount of female mice, which comprises the above inhibin vaccine.

[0015] The sixth object of the present invention is to protect a method for preparing the above inhibin antibody, which comprises the following steps:

[0016] Using CTC resin as a raw material, synthesizing an oligopeptide sequence with an amino acid sequence as shown in SEQ ID NO.2 by Fmoc solid-phase peptide synthesis method;

[0017] Coupling the synthesized peptide with a carrier protein by using MBS, and using the amino acid at the C-terminal end of the antigen peptide as the coupling site.

[0018] In summary, due to the adoption of the above technical solutions, the beneficial effects of the present invention are as follows:

[0019] The present invention provides an inhibin antibody and its application. By immunizing Japanese white rabbits with a synthesized porcine inhibin antigen to prepare polyclonal antibodies, and injecting the polyclonal antibodies into the abdominal cavity of mice, the ovulation number of female mice is significantly increased, thereby improving the efficiency of biological purification by IVF. On the one hand, it can directly reduce the inhibin level in mice; on the other hand, it affects the local microenvironment of the ovary and more effectively improves the superovulation level. By injecting inhibin antibody into the abdominal cavity to inhibit the action of inhibin, the purpose of higher superovulation is achieved, and the efficiency is effectively increased by more than 50%, achieving unexpected effects. Description of the Drawings

[0020] Figure 1 It is a diagram showing the SDS-PAGE results of the antibody.

[0021] Figure 2 It is a diagram showing the ovulation numbers under different conditions. PMSG + hCG: Conventional superovulation method; AIS + PMSG + hCG; Inhibin antibody superovulation method (Example 1); Hyper Ova + hCG: Control superovulation method.

[0022] P+h Superovulation Method: At 5:00 pm on the first day, mice were intraperitoneally injected with 10 IU of PMSG, and 46 - 48 hours later, they were intraperitoneally injected with 10 IU of hCG.

[0023] AIS+PMSG+hCG Superovulation Method: At 5:00 pm on the first day, mice were intraperitoneally injected with 100 μL of AIS and 10 IU of PMSG, and 46 - 48 hours later, they were intraperitoneally injected with 10 IU of hCG.

[0024] Hyper Ova+hCG Superovulation Method: At 5:00 pm on the first day, mice were intraperitoneally injected with 100 μL of Hyper Ova, and 46 - 48 hours later, they were intraperitoneally injected with 10 IU of hCG.

[0025] Figure 3 It is a diagram of the situation of obtaining two - cell embryos after IVF. The left is superovulation using AIS, and the right is using the conventional superovulation method. Detailed Implementation Modes

[0026] In order to make the objectives, technical solutions and advantages of the present invention clearer and more understandable, the following further elaborates on the present invention in combination with embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0027] Example 1

[0028] Biological purification technologies such as IVF are crucial for constructing specific pathogen - free (SPF) or germ - free mouse populations, aiming to eliminate potential pathogens in mice. Nevertheless, traditional IVF technologies have problems such as limited egg numbers and difficulty in obtaining high - quality eggs, which limit their efficiency and scale. Inhibin plays an important role in animal reproductive regulation. Although its connection with reproduction has been studied, the existing technologies still cannot fully meet the growing demand for high - quality and large numbers of experimental mice.

[0029] Example 1

[0030] This example provides an inhibin antibody, and the amino acid sequence of the antibody is shown as SEQ ID NO.1.

[0031] Sequence SEQ ID NO.1: FRPSQHTRSRQVTSA - C - KLH.

[0032] Specific preparation process:

[0033] Using CTC resin as the raw material, an oligopeptide sequence with the amino acid sequence shown as SEQ ID NO.2, FRPSQHTRSRQVTSA, was synthesized by the Fmoc solid - phase peptide synthesis method;

[0034] The synthetic peptide was conjugated to the carrier protein using MBS, and the amino acid at the C-terminal end of the antigen peptide was used as the conjugation site.

[0035] In some embodiments, the carrier protein is ovalbumin, keyhole limpet hemocyanin, tetanus toxoid, bovine serum albumin, or human serum albumin.

[0036] Use of an inhibin antibody as described above in the preparation of a medicament for increasing the ovulation amount of female mice.

[0037] In some embodiments, the dose of inhibin antibody injected into each female mouse each time is 0.2 - 0.8 ml, and the concentration of the inhibin antibody is 0.8 - 4 mg / ml.

[0038] In some embodiments, an inhibin vaccine is provided, and the inhibin vaccine comprises the above-mentioned antibody.

[0039] In some embodiments, a reagent for increasing the ovulation amount of female mice is provided, comprising the above-mentioned inhibin vaccine.

[0040] The more detailed exploration experiment process of this embodiment is as follows:

[0041] (I) Experimental materials

[0042] Experimental animals:

[0043] Female C57BL / 6 mice, 3 - 10 weeks old, weighing 15 - 25 g, were purchased from a supplier compliant with experimental animal management regulations and were housed in a SPF-level animal room with compliant control.

[0044] Japanese white rabbits, 2.5 - 3 kg, were used for antibody preparation and were separately housed in a similar standard animal house to ensure no risk of disease infection.

[0045] Reagents and equipment:

[0046] High-purity porcine inhibin antigen (≥90%), complete Freund's adjuvant, incomplete Freund's adjuvant, analytical pure saturated ammonium sulfate solution, ultracentrifuge, laminar flow hood, stereomicroscope, micromanipulator, precision microinjector, various cell culture media (fertilization culture medium, embryo development culture medium, etc.), pregnant mare serum gonadotropin (PMSG), human chorionic gonadotropin (hCG), were all purchased from internationally renowned biological reagent manufacturers and professional instrument manufacturers to ensure reliable quality and stable performance.

[0047] (II) Preparation process of rabbit-derived anti-porcine inhibin antibody

[0048] Antigen preparation:

[0049] Three amino acid sequences were designed according to the sequence and were entrusted to a company for synthesis and conjugation with KLH protein to form a complete antigen.

[0050] The peptide is synthesized according to the amino acid sequence, and then the synthesized peptide is coupled to keyhole limpet hemocyanin (KLH) to form a complete antigen.

[0051] Immunization program:

[0052] Place porcine inhibin antigen and complete Freund's adjuvant in an ice bath at a volume ratio of 1:1, and slowly stir with a homogenizer to emulsify into a stable emulsion. Select 2 rabbits for each antigen, and inject the emulsified antigen into the popliteal lymph node, subcutaneous plantar area, and subcutaneous dorsal area of each rabbit for the first immunization, for a total of 6 points. The injection volume for each point is about 0.2-0.3ml, and the total injection volume for a single rabbit is 1.2-1.8ml.

[0053] Two weeks after the first immunization, the first booster immunization was performed. Porcine inhibin and incomplete Freund's adjuvant were emulsified in the same volume ratio and injected subcutaneously on the back and soles of the feet. The injection volume at each point was adjusted to 0.15-0.25 ml, and the total injection volume for a single rabbit was 0.9-1.5 ml. After that, the booster immunization operation was repeated every 2 weeks, for a total of 4 booster immunizations.

[0054] Antibody Monitoring and Purification:

[0055] 10-14 days after each booster immunization, a small amount of blood is drawn from the rabbit's ear vein, and the titer of anti-porcine inhibin antibodies in the serum is detected using enzyme-linked immunosorbent assay (ELISA). When the titer reaches 1:12000 or above, it is determined to meet the conditions for large-scale blood collection.

[0056] After the rabbit is anesthetized, the whole blood of the immune rabbit is collected by carotid artery bleeding, and quickly transferred into a sterile anticoagulant centrifuge tube. After standing for 1 hour at 37°C, centrifuge at 3500rpm for 20 minutes to collect the upper serum. The serum is purified by saturated ammonium sulfate precipitation method, and after dialysis and desalination, the purified antibody solution is obtained. The main band is clear after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) detection. The test results are as follows Figure 1 shown.

[0057] (III) Experimental steps of IVF in female mice

[0058] Mouse grouping and pretreatment:

[0059] Thirty-five 4-week-old female C57BL / 6 mice were randomly divided into 7 groups, 3 mice in each group, and marked as experimental group and control group. They were adaptively raised for 1 week, during which their feeding, drinking, activity and other status were observed daily, and any mice with abnormal signs were eliminated.

[0060] Antibody injection and ovulation induction:

[0061] The experimental group mice were given rabbit anti-porcine inhibin antibody by intraperitoneal injection in the proestrus period. According to the weight of the mice, 0.3 ml of 1.2 mg / ml antibody and 5-10 IU PMSG solution were injected. The control group mice were injected with the same amount of sterile saline at the same time.

[0062] 46-48 hours after injection, both groups of mice were injected with hCG intraperitoneally at the same time, with a dose of 5-10 IU per mouse. 13 hours after hCG injection, the mice were quickly placed in a carbon dioxide euthanasia chamber and killed, the abdominal cavity was opened in an ultra-clean workbench, and the fallopian tubes were removed with fine forceps and scissors and placed in the operating fluid.

[0063] Egg collection and IVF operation:

[0064] Under a stereo microscope, use micro forceps to carefully puncture the enlarged part of the fallopian tube, collect the oozing eggs and cumulus cell clusters, place the cell clusters in culture medium, add 3-5 microliters of pre-capacitated high-quality sperm, and place in a 37°C, 5% CO2 incubator for in vitro fertilization.

[0065] 24 hours after fertilization, the two-cell embryos and unfertilized eggs were picked out, and the embryonic development morphology, cell number and other indicators were observed and recorded. Figure 2 and Figure 3 As shown, statistical analysis showed that 150 two-cell embryos were obtained in the experimental group, and the proportion of fertilized eggs developing into two-cell embryos was 93.2%. In the control group, 81 two-cell embryos were obtained in the experimental group, and the proportion of fertilized eggs developing into two-cell embryos was 82.7%. The difference was highly statistically significant (P<0.01).

[0066] The study found that this method can effectively improve the efficiency of super ovulation by 85% compared with the existing method (the number of effectively divided oocytes is increased from 81 to 150, an increase of 85.2%), and the prepared antibodies are of stable quality and easy to use.

[0067] 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 should be included in the protection scope of the present invention.

Claims

1. An inhibin antibody, characterized in that, The antigen amino acid sequence for preparing the antibody is shown as SEQ ID NO.

1.

2. An inhibin antibody, characterized in that, The antigen amino acid sequence for preparing the antibody is shown as SEQ ID NO.2, and the amino acid at the C-terminal end of the antigen is used as the coupling site to couple with the carrier protein.

3. The inhibin antibody according to claim 2, characterized in that, The carrier protein is ovalbumin, keyhole limpet hemocyanin, tetanus toxoid, bovine serum albumin or human serum albumin.

4. Use of an inhibin antibody according to any one of claims 1-3 in the preparation of a drug for increasing the ovulation amount of female mice.

5. The application according to claim 4, characterized in that, The dosage of the inhibin antibody injected into each female mouse each time is 0.2-0.8 ml, and the concentration of the inhibin antibody is 0.8-4 mg / ml.

6. An inhibin vaccine, characterized in that, The inhibin vaccine comprises the antigen sequence according to any one of claims 1-3.

7. A reagent for increasing the ovulation amount of female mice, characterized in that, Comprises the inhibin vaccine according to claim 6.

8. A method for preparing an inhibin antibody according to claim 2 or 3, characterized in that, Comprises the following steps: Using CTC resin as the raw material, synthesizing an oligopeptide sequence with the amino acid sequence shown as SEQ ID NO.2 by the Fmoc solid-phase polypeptide synthesis method; Coupling the synthetic peptide with the carrier protein using MBS, and using the amino acid at the C-terminal end of the antigen peptide as the coupling site.

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