Antibody for detecting Wuzhu muuseup sheep GDF9 as well as preparation method and application of antibody

Abstract

The invention relates to an antibody for detecting Wuzhu muuseup sheep GDF9 as well as a preparation method and application of the antibody. The invention discloses an antibody for detecting GDF9 (Growth Factor Factor 9) of a wuzhu muuseup sheep. The antibody comprises a monoclonal antibody A which can be combined with an epitope of the Wuzhu sheep GDF9, and the amino acid sequence of the epitope is as shown in SEQ ID NO: 1; a light chain full-length sequence of the antibody is shown as SEQ ID NO: 2, a heavy chain full-length sequence of the antibody is shown as SEQ ID NO: 3, a sequence of a light chain variable region is shown as SEQ ID NO: 4, and a sequence of a heavy chain variable region is shown as SEQ ID NO: 5; the nucleic acid aptamer B can be combined with another epitope of the cytokine, and the amino acid sequence of the epitope is shown as SEQ ID NO: 6; and the sequence of the nucleic acid aptamer B is as shown in SEQ ID NO: 7. The antibody can form dual recognition, the specificity of the antibody is improved, the cell factor and other cell factors can be effectively distinguished, and false positive is avoided.

Description

Technical Field

[0001] The present invention relates to the technical field of immunoassay, and particularly relates to an antibody for detecting GDF9 of Wuzhumuqin sheep, a preparation method thereof, and an application thereof. Background Art

[0002] Sheep are important livestock economic animals, and their reproductive performance directly affects the yield and quality of mutton, wool, and sheep milk. Wuzhumuqin sheep is a typical grassland meat sheep, with characteristics such as large body size, fast growth, good meat quality, roughage tolerance, and strong adaptability. However, the reproductive performance of Wuzhumuqin sheep is average, mainly with a single fetus per birth.

[0003] Cytokines are a class of small-molecule proteins with a wide range of biological activities. They carry out signal transduction between cells and regulate various physiological functions such as cell growth, differentiation, proliferation, apoptosis, and immune response. Cytokines play an important role in the reproductive process of animals, such as affecting ovarian development, ovulation, fertilization, implantation, and the growth and differentiation of embryos. GDF9 (Growth Differentiation Factor 9) is an important cytokine, mainly secreted by oocytes, and plays an important regulatory role in follicle growth and differentiation, oocyte maturation and ovulation, embryo implantation and development, etc.

[0004] In order to study the mechanism of action of this cytokine in the reproductive process of Wuzhumuqin sheep, it is necessary to quantitatively detect it to analyze its expression levels and change rules in different physiological stages and different tissues. Currently, the commonly used detection method is enzyme-linked immunosorbent assay (ELISA). The key to this method is to use an antibody with strong specificity to recognize and capture this cytokine. However, due to the high similarity of the structure and function of this cytokine to other cytokines, such as GDF8, GDF15, and bone morphogenetic protein 15 (BMP15), which have a high amino acid sequence similarity to GDF9, especially in the mature region, they may cross-react with the antibody of GDF9.

[0005] Therefore, there is an urgent need for a method capable of preparing an antibody for detecting this cytokine with strong specificity, so as to improve the sensitivity and accuracy of detection, and provide an effective tool for the research and improvement of the reproductive performance of Wuzhumuqin sheep. Summary of the Invention

[0006] Based on this, it is necessary to provide an antibody for detecting GDF9 of Wuzhumuqin sheep, a preparation method thereof, and an application thereof. The antibody for detecting GDF9 of Wuzhumuqin sheep is suitable for detecting the expression levels and change rules of GDF9 of Wuzhumuqin sheep by methods such as ELSIA, and has good specificity.

[0007] The present invention adopts the following technical solution: An antibody for detecting GDF9 of Wuzhumuqin sheep, which is composed of the following two parts:

[0008] One part is a murine monoclonal antibody A that can bind to an epitope of Ujumqin sheep GDF9, and the amino acid sequence of this epitope is shown in SEQ ID NO:1. The full-length sequence of the light chain of the antibody is shown in SEQ ID NO:2, the full-length sequence of the heavy chain is shown in SEQ ID NO:3, the sequence of the light chain variable region is shown in SEQ ID NO:4, and the sequence of the heavy chain variable region is shown in SEQ ID NO:5.

[0009] The other part is a nucleic acid aptamer B that can bind to another epitope of the cytokine, and the amino acid sequence of this epitope is shown in SEQ ID NO:6. The sequence of nucleic acid aptamer B is shown in SEQ ID NO:7.

[0010] The two parts of the antibody are conjugated through a linker C, and the linker C can be a biotin-streptavidin system or other substances that can achieve stable connection of the two parts.

[0011] The present invention can also provide an application method of the antibody for detecting Ujumqin sheep GDF9 in the preparation of reagents or kits for detecting Ujumqin sheep GDF9, including the following steps:

[0012] Step 1: Using Ujumqin sheep GDF9 cytokine as an antigen to immunize mice to obtain spleen cells of the mice, hybridizing them with murine myeloma cells to obtain hybridoma cells, screening and identifying them to obtain a hybridoma cell line that can secrete monoclonal antibody A that can bind to an epitope of Ujumqin sheep GDF9 cytokine, and purifying it to obtain monoclonal antibody A;

[0013] Step 2: Using Ujumqin sheep GDF9 cytokine as a target molecule, adopting the systematic evolution of ligands by exponential enrichment (SELEX) technology to screen out nucleic acid aptamer B that can bind to another epitope of Ujumqin sheep GDF9 cytokine from a randomly synthesized nucleic acid library, obtaining the sequence of nucleic acid aptamer B through PCR amplification and sequencing, synthesizing and purifying it to obtain nucleic acid aptamer B;

[0014] Step 3: Conjugating monoclonal antibody A and nucleic acid aptamer B with linker C respectively to obtain a conjugate, and separating and purifying it through methods such as gel filtration chromatography to obtain the antibody, which is the antibody for detecting Ujumqin sheep GDF9 cytokine described above.

[0015] Compared with the prior art, the beneficial effects of the present invention are:

[0016] The antibody of the present invention is composed of a monoclonal antibody and a nucleic acid aptamer, both of which can bind to different epitopes of the GDF9 cytokine of Wuzhumuqin sheep, forming dual recognition, improving the specificity of the antibody, being able to effectively distinguish this cytokine from other cytokines, and avoiding the occurrence of false positives;

[0017] The preparation method of the antibody of the present invention uses the GDF9 cytokine of Wuzhumuqin sheep as an antigen, simultaneously screens out a monoclonal antibody and a nucleic acid aptamer, and then couples the two to obtain the antibody. This method is simple, efficient, and feasible, and can mass-produce antibodies with good stability and high activity;

[0018] The antibody of the present invention can be used in detection methods such as ELISA to quantitatively analyze the expression level and change pattern of the GDF9 cytokine of Wuzhumuqin sheep, providing an effective tool for the research and improvement of the reproductive performance of Wuzhumuqin sheep. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 To detect the binding ability of 33 monoclonal antibodies A1 - A33 against the GDF9 cytokine of Wuzhumuqin sheep by ELISA method.

[0020] Figure 2 It is the secondary structure diagram of the nucleic acid aptamer B.

[0021] Figure 3 It is the electrophoresis comparison diagram before and after the coupling of the nucleic acid aptamer B and the monoclonal antibody A.

[0022] Figure 4 It is the ELISA result of the antibody for detecting the GDF9 standard product of the coupled GDF9 cytokine of Wuzhumuqin sheep.

[0023] Figure 5 It is the specific result comparison diagram of the monoclonal antibody A and the antibody for detecting the coupled GDF9 cytokine of Wuzhumuqin sheep for detecting GDF8, GDF15, and bone morphogenetic protein 15 (BMP15). DETAILED DESCRIPTION OF THE INVENTION

[0024] To facilitate the understanding of the present invention, the present invention will be described more comprehensively below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure content of the present invention more thorough and comprehensive.

[0025] In the embodiments of the present invention, unless otherwise specified, all raw material components are commercially available products well-known to those skilled in the art; in the embodiments of the present invention, if not specifically specified, the technical means used are all conventional means well-known to those skilled in the art.

[0026] The present invention screened and obtained an antibody for detecting the GDF9 cytokine of Wuzhumuqin sheep, and its sequence is as follows:

[0027] The amino acid sequence of the binding epitope of GDF9 protein and part of monoclonal antibody A: QKPLQK (SEQ ID NO: 1).

[0028] The full-length sequence of the light chain of monoclonal antibody A:

[0029] DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSVQAEDLAVYFCHQYHSGYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 2).

[0030] The full-length sequence of the heavy chain of monoclonal antibody A:

[0031] QVQLQESGPGLVKPSETLSLTCTVSGGSISSGYASWVRQAPGQGLEWMGRIYPGNGDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARHYYGSSHWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 3).

[0032] The sequence of the variable region of the light chain of monoclonal antibody A:

[0033] LAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSVQAEDLAVYFCHQYHSGYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGT(SEQ ID NO:4).

[0034] Amino acid sequence of the binding epitope of the GDF9 protein and nucleic acid aptamer B part:

[0035] KPSETLSLTCTVSGGSISSGYASWVRQAPGQGLEWMGRIYPGNGDTRYSPSFQGQVTI

[0036] SADKSISTAYLQWSSLKASDTAMYYCARHYYGSSHWYFDVWGQGTLVTVSS(SEQ ID NO:5).

[0037] Amino acid sequence of the binding epitope of the GDF9 protein and nucleic acid aptamer B part:

[0038] MYTLKG(SEQ ID NO:6).

[0039] Nucleic acid aptamer B sequence:

[0040] GGGAGGACGATGCGGAATTCGCTATGCCACTAGGGTTCCGAGGGACCCTAGTTACGACTCCTCCCGGAGGACGATGCGGAATTCGCTATGCCACTAGGGTTCCGAGGGACCCTAGTTACGACTCCTCCCGGAGGACGATGCGGAATTCGCTATGCCACTAGGGTTCCGAGGGACCCTAGTTACGACTCCTCC(SEQ ID NO:7).

[0041] The above-mentioned antibody for detecting the GDF9 cytokine of Wuzhumuqin sheep can specifically recognize the GDF9 of Wuzhumuqin sheep and is suitable for immunological detection.

[0042] Example 1

[0043] The purpose of this example is to prepare monoclonal antibody A, and the specific steps are as follows:

[0044] Step 1: Use the recombinant protein of this cytokine (hereinafter referred to as this recombinant protein) to immunize mice. The specific method is as follows: Mix this recombinant protein with an adjuvant (such as Freund's adjuvant) at a ratio of 1:1, intraperitoneally inject 0.2 ml into each mouse, immunize 4 times in total, with an interval of 2 weeks each time. 3 days after the last immunization, collect the orbital blood of the mice, and detect the antibody titer in the mouse serum by the double antibody sandwich ELISA method. Select the mice with high antibody titers for the preparation of hybridoma cells.

[0045] Step 2: Anesthetize the selected mice with ether, take their spleens, wash them with sterile PBS, mix the splenocytes with mouse myeloma cells (such as SP2 / 0 cells) at a ratio of 1:1, perform cell fusion with 50% PEG, dilute the cells with serum-free DMEM medium after fusion, aliquot the cells into 96-well plates, 200 μl per well, add two wells of SP2 / 0 cells to each plate as negative controls, and place the plate in an incubator at 37°C with 5% CO2 for culture.

[0046] Step 3: After 6 days of culture, preliminarily screen positive hybridoma cells by the ELISA method. The specific method is as follows: Aspirate the supernatant in the 96-well plate and add it to another 96-well plate that has been coated with this recombinant protein, add 100 μl per well, incubate at 37°C for 1 hour, then wash 3 times with PBS, add anti-mouse IgG secondary antibody labeled with horseradish peroxidase (HRP), add 100 μl per well, incubate at 37°C for 1 hour, wash 3 times with PBS again, add TMB chromogenic reagent, incubate at room temperature for 15 minutes, add 2 M H2SO4 to terminate the reaction, and measure the absorbance (OD value) with an enzyme-linked immunosorbent assay reader. Select the wells with high OD values as positive hybridoma cells, and amplify and subclone them.

[0047] Step 4: Identify the subcloned hybridoma cells. The specific method is as follows: Detect the antibody titer in the supernatant of the subclone by the double antibody sandwich ELISA method, detect the recognition ability of the antibody of the subclone to this recombinant protein by immunoblotting (Western blot), detect the binding ability of the antibody of the subclone to the natural protein of this cytokine (hereinafter referred to as this natural protein) by competitive ELISA. Select the subclone with high antibody titer, strong recognition ability, and high binding ability as the production cell line of monoclonal antibody A (such as Figure 1 shown), freeze and resuscitate it, culture it with serum-free DMEM medium, collect the supernatant, purify monoclonal antibody A by protein A or protein G affinity chromatography, detect the purity of monoclonal antibody A by SDS-PAGE method, measure the concentration of monoclonal antibody A by Bradford method, and store it in a refrigerator at -20°C for standby.

[0048] Example 2

[0049] The objective of this embodiment is to prepare nucleic acid aptamer B, and the specific steps are as follows:

[0050] Step 1: Screen out nucleic acid aptamer B that can bind to another epitope of the cytokine from a randomly synthesized nucleic acid library. The specific method is: immobilize the recombinant protein on the inner wall of a glass tube with an affinity chromatography resin (such as Ni-NTA resin) to form a stationary phase. Dissolve a randomly synthesized nucleic acid library (such as a single-stranded DNA library containing 10 14 different sequences) in a solution containing salt and buffer to form a mobile phase. Pass the mobile phase through the stationary phase to allow the nucleic acids in the nucleic acid library to bind to the recombinant protein. Then wash with solutions containing different concentrations of salt to remove non-specifically bound nucleic acids. Finally, elute with a solution containing a high concentration of salt, and collect the eluate, which is the nucleic acid aptamer obtained from the first round of screening.

[0051] Step 2: Perform PCR amplification on the nucleic acid aptamer obtained from the first round of screening to obtain double-stranded DNA. Then use the alkali hydrolysis method under alkaline conditions or the alkaline phosphatase method to remove one of the strands to obtain single-stranded DNA, which is the nucleic acid library for the second round of screening. Repeat Step 1 to perform the second round of screening to obtain the nucleic acid aptamer obtained from the second round of screening.

[0052] Step 3: Repeat Step 2 for 10 rounds of screening. The nucleic acid aptamer obtained by screening is the required nucleic acid aptamer B (as Figure 2 shown).

[0053] Step 4: Perform PCR amplification on the nucleic acid aptamer obtained from the last round of screening, cut it with a restriction endonuclease, separate and purify it by agarose gel electrophoresis, determine its sequence with a DNA sequencer, analyze the common characteristics of its sequence, identify the key sequence that binds to the cytokine, synthesize and purify it to obtain nucleic acid aptamer B. Detect the purity of nucleic acid aptamer B by SDS-PAGE method, determine the concentration of nucleic acid aptamer B with an ultraviolet spectrophotometer, and store it in a refrigerator at -20°C for standby.

[0054] Example 3

[0055] The objective of this embodiment is to conjugate monoclonal antibody A and nucleic acid aptamer B with linker C respectively to obtain conjugate products. The specific steps are as follows:

[0056] Step 1: Biotinylate monoclonal antibody A with a biotinylation reagent (such as NHS-LC-biotin). The specific method is as follows: Dissolve monoclonal antibody A in PBS buffer with a pH of 8.0 to a concentration of 1 mg / ml. Add NHS-LC-biotin such that the molar ratio of NHS-LC-biotin to monoclonal antibody A is 20:1. Gently stir at room temperature for 2 hours, then desalt and purify using a biotinylation column (such as Zeba Spin Desalting Column). Collect the biotinylated monoclonal antibody A, determine its concentration using the Bradford method, and store it in a refrigerator at 4°C for later use.

[0057] Step 2: Streptavidinylate nucleic acid aptamer B with a streptavidinylation reagent (such as streptavidin-PEG-NHS). The specific method is as follows: Dissolve nucleic acid aptamer B in PBS buffer with a pH of 7.4 to a concentration of 1 mg / ml. Add streptavidin-PEG-NHS such that the molar ratio of streptavidin-PEG-NHS to nucleic acid aptamer B is 10:1. Gently stir at room temperature for 2 hours, then desalt and purify using a streptavidinylation column (such as Zeba Spin Desalting Column). Collect the streptavidinylated nucleic acid aptamer B, determine its concentration using a UV spectrophotometer, and store it in a refrigerator at 4°C for later use.

[0058] Step 3: Mix the biotinylated monoclonal antibody A and the streptavidinylated nucleic acid aptamer B at a molar ratio of 1:1. Gently stir at room temperature for 30 minutes to allow specific binding between biotin and streptavidin to form a conjugate product (as shown in Figure 3 ). Separate and purify using gel filtration chromatography (such as Sephacryl S-300HR). Collect the conjugate product, which is the antibody described. Detect the purity of the antibody using SDS-PAGE, and determine the protein and nucleic acid concentrations of the antibody using the Bradford method and a UV spectrophotometer respectively. Store it in a refrigerator at -20°C for later use.

[0059] Example 4

[0060] The purpose of this example is to perform ELISA detection using the antibody. The specific steps are as follows:

[0061] Step 1: Dilute the native protein to different concentrations with sodium carbonate buffer (pH 9.6) as standards. Take 100 μl of each concentration and add it to a 96-well plate. Add one concentration of the standard to each well, and at the same time add a blank control, that is, add only sodium carbonate buffer without adding the standard. Seal the plate and incubate overnight at 4°C.

[0062] Step 2: Wash the plate 3 times with PBS for 5 minutes each time, then add 1% bovine serum albumin (BSA) solution, 200 μl per well, as the blocking agent, incubate at 37 °C for 1 hour, and then wash 3 times with PBS for 5 minutes each time;

[0063] Step 3: Dilute the antibody to an appropriate concentration, add 100 μl per well, react with the standard or sample (such as sheep serum, ovarian tissue homogenate, etc.), incubate at 37 °C for 1 hour, and then wash 3 times with PBS for 5 minutes each time;

[0064] Step 4: Add the secondary antibody, anti-mouse IgG labeled with HRP, 100 μl per well, incubate at 37 °C for 1 hour, and then wash 3 times with PBS for 5 minutes each time;

[0065] Step 5: Add the TMB chromogenic agent, 100 μl per well, incubate at room temperature for 15 minutes, add 2 M H2SO4 to terminate the reaction, measure the OD value with an enzyme-linked immunosorbent assay (ELISA) reader, draw a standard curve based on the OD values and concentrations of the standards, and calculate the concentration of the cytokine in the sample according to the OD value of the sample (as Figure 4 shown).

[0066] Example 5

[0067] The purpose of this example is to verify the specificity of the antibody. The specific steps are as follows:

[0068] Step 1: Electrophoretically separate the recombinant protein and other cytokines with similar structures and functions (such as GDF8, GDF15, and BMP15) using SDS-PAGE, then transfer them to a 96-well plate, and block the membrane with a 5% skim milk solution, incubate at room temperature for 1 hour;

[0069] Step 2: Dilute the antibody to an appropriate concentration, add it to the membrane, react with the protein on the membrane, incubate at room temperature for 2 hours, and then wash the membrane 3 times with PBS solution containing 0.05% Tween-20 for 10 minutes each time;

[0070] Step 3: Add the secondary antibody, anti-mouse IgG labeled with HRP, react with the antibody on the membrane, incubate at room temperature for 1 hour, and then wash the membrane 3 times with PBS solution containing 0.05% Tween-20 for 10 minutes each time;

[0071] Step 4: Add the TMB chromogenic agent, 100 μl per well, incubate at room temperature for 15 minutes, add 2 M H2SO4 to terminate the reaction, measure the OD value with an ELISA reader. If only the well with the recombinant protein shows color, it indicates that the antibody has good specificity. If the wells with other cytokines also show color, it indicates that there is cross-reaction with the antibody, and the antibody preparation conditions or screening conditions need to be optimized ( Figure 5 ).

[0072] The embodiments described above merely represent several implementation manners of the present invention. The description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all fall within the protection scope of the present invention. Therefore, the protection scope of the present invention patent shall be subject to the appended claims.

Claims

1. An antibody for detecting GDF9 of Wuzhumuqin sheep, characterized in that, The antibody consists of the following two parts: One part is a murine monoclonal antibody A, which can bind to an epitope of Ujimqin sheep GDF9, and the amino acid sequence of this epitope is shown in SEQ ID NO:1; the full-length sequence of the light chain of the antibody is shown in SEQ ID NO:2, the full-length sequence of the heavy chain is shown in SEQ ID NO:3, the sequence of the variable region of the light chain is shown in SEQ ID NO:4, and the sequence of the variable region of the heavy chain is shown in SEQ ID NO:

5. The other part is a nucleic acid aptamer B, which can bind to another epitope of the cytokine, and the amino acid sequence of this epitope is shown in SEQ ID NO:6; the sequence of the nucleic acid aptamer B is shown in SEQ ID NO:

7.

2. The antibody for detecting GDF9 of Wuzhumuqin sheep according to claim 1, wherein The monoclonal antibody A and the nucleic acid aptamer B are conjugated through a linker C, and this linker C is a biotin-streptavidin system.

3. The method for preparing the antibody for detecting Ujimqin sheep GDF9 as claimed in claim 1, comprising the following steps: Step 1: Using Ujimqin sheep GDF9 cytokine as an antigen to immunize mice, obtaining the spleen cells of the mice, hybridizing them with murine myeloma cells to obtain hybridoma cells, screening and identifying them to obtain a hybridoma cell line that can secrete monoclonal antibody A capable of binding to an epitope of Ujimqin sheep GDF9 cytokine, and purifying it to obtain monoclonal antibody A; Step 2: Using Ujimqin sheep GDF9 cytokine as a target molecule, adopting the systematic evolution of ligands by exponential enrichment (SELEX) technique to screen out a nucleic acid aptamer B capable of binding to another epitope of Ujimqin sheep GDF9 cytokine from a randomly synthesized nucleic acid library, obtaining the sequence of the nucleic acid aptamer B through PCR amplification and sequencing, synthesizing and purifying it to obtain nucleic acid aptamer B; Step 3: Conjugating monoclonal antibody A and nucleic acid aptamer B with linker C respectively to obtain a conjugate product, and separating and purifying it by methods such as gel filtration chromatography to obtain the antibody, which is the antibody for detecting Ujimqin sheep GDF9 cytokine as claimed.

4. The application of the antibody for detecting Ujimqin sheep GDF9 as claimed in claim 1 in the preparation of a reagent or kit for detecting Ujimqin sheep GDF9.

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