A colloidal gold immunochromatographic preparation based on AMH nanobodies and its application

Abstract

This invention belongs to the field of bioengineering technology, specifically relating to the preparation and application of colloidal gold immunochromatographic assay based on AMH nanobodies. This invention discloses two AMH nanobodies, the amino acid sequences of which are shown in SEQ ID NO.1 and SEQ ID NO.2, and the nucleotide molecules encoding these amino acid sequences are shown in SEQ ID NO.3 and SEQ ID NO.4. This invention also discloses a method for preparing colloidal gold immunochromatographic assay based on AMH nanobodies. The results of the examples show that the molecular weight of both AMH nanobodies is approximately 15 kDa, consistent with the size of nanobodies. The purified nanobodies can be used for the preparation of colloidal gold immunochromatographic assay, and the colloidal gold can be used for the detection of AMH in the serum of sheep, donkeys, horses, and camels.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to the preparation and application of colloidal gold immunochromatography based on AMH nanobodies. Background Technology

[0002] Single-domain antibodies are naturally occurring, biologically active, specific antibodies found in camel-like animals (alpacas, camels) and cartilaginous fish, lacking heavy chains. The antigen-binding site (VHH) of a single-domain antibody possesses independent antigen recognition capabilities, and independently expressed VHHs are also known as nanobodies. Compared to traditional antibodies, nanobodies offer advantages such as small molecular weight, simple structure, and stable physicochemical properties. These superior properties enable nanobodies to bind to some hidden antigenic epitopes, making them particularly suitable for targets where antibodies are difficult to obtain. Furthermore, nanobodies have a simple structure, are easy to express in vitro, and have a simple production process.

[0003] Anti-Müllerian hormone ( Anti-müllerian Hormone AMH (Amyotrophic Lateral Breast Milk) is a disulfide-linked homodimeric glycoprotein and a member of the transforming growth factor β family. AMH is primarily expressed in the gonads and participates in processes such as Müllerian duct degeneration, granulosa cell differentiation, primordial follicle recruitment, and cavitary follicle sensitivity. It is a key factor in inducing sex differentiation and regulating follicle development, playing an important biological role in the development of testicular interstitial cells and oocytes. In applications, it serves as a reliable endocrine marker for cryptorchidism, oocyte and embryo quality in animals such as equines. It can be used to assess reproductive lifespan and ovarian follicle reserve in adult animals, and as a reliable marker for selecting superior donors in embryo transfer.

[0004] Therefore, developing a portable method for detecting AMH serum levels for ranch testing has significant application value. Summary of the Invention

[0005] The purpose of this invention is to provide a portable method for detecting AMH levels in animal serum. The two nanobodies provided by this invention can be paired for the preparation of colloidal gold immunochromatography and used for the detection of AMH levels in the serum of sheep, donkeys, horses, and camels.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] The present invention provides an AMH nanobody, the amino acid sequence of which is shown in SEQ ID NO.1 or SEQ ID NO.2.

[0008] Preferably, the AMH nanobody can specifically bind to the AMH protein.

[0009] The present invention also provides a nucleotide molecule encoding the above-mentioned AMH nanobody, wherein the nucleoside sequence of the nucleotide molecule is shown in SEQ ID NO.3 or SEQ ID NO.4.

[0010] The present invention also provides a kit for detecting AMH protein expression levels, the kit comprising the above-mentioned nanobody.

[0011] The present invention also provides a recombinant plasmid comprising the above-described nucleotide sequence.

[0012] Preferably, the plasmid comprises the above-described nucleotide sequence and the pMCO-AOXα vector.

[0013] The present invention also provides an expression strain comprising the above-mentioned recombinant plasmid.

[0014] The present invention also provides the application of the above-mentioned AMH nanobody, the above-mentioned recombinant plasmid, or the above-mentioned expression strain in the preparation of reagents for detecting AMH serum levels.

[0015] The present invention also provides the application of the above-mentioned AMH nanobody, the above-mentioned recombinant plasmid, or the above-mentioned expression strain in the preparation of colloidal gold immunochromatographic test strips or kits for detecting AMH serum levels.

[0016] The present invention also provides the application of the above-mentioned AMH nanobody in detecting AMH levels in animal serum, wherein the animals are sheep, donkeys, horses and camels.

[0017] The beneficial effects of this invention are:

[0018] The nanobody screening method described in this invention utilizes phage display technology for screening. Phage display technology can display expressed exogenous polypeptides or proteins as fusion proteins on the surface of phages, and then screen phages expressing specific proteins through affinity enrichment.

[0019] The AMH nanobody developed using phage display technology can specifically bind to the AMH protein.

[0020] The AMH nanobody described in this invention has a molecular weight of approximately 15 kDa, which is consistent with the size of nanobodies. It has advantages such as small molecular weight, stable physicochemical characteristics, high affinity, simple structure, and ease of recombinant expression preparation.

[0021] The AMH nanobody described in this invention can specifically bind to the AMH protein and can be used to detect AMH in serum using colloidal gold immunoassay strips. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 The image shows the results of Western blotting for the detection of purified AMH (M: protein marker; 1, 2, 3 and 4: 20% elution buffer; 5: 30% elution buffer; 6: 40% elution buffer; 7: 100% elution buffer).

[0024] Figure 2 AMH assay results purified for Coomassie Brilliant Blue detection (M: protein marker; 1, 2, 3, and 4: 20% elution buffer; 5: 30% elution buffer; 6: 40% elution buffer; 7: 100% elution buffer).

[0025] Figure 3 This is a schematic diagram of the assembly of the reaction pad for the colloidal gold test strip;

[0026] Figure 4 The detection of AMH in the serum of sheep, horse, donkey and camel using colloidal gold prepared for AMH nanobodies (where A: sheep serum; B: donkey serum; C: horse serum; D: camel serum). Detailed Implementation

[0027] This invention provides two types of AMH nanobodies, the amino acid sequences of which are shown in SEQ ID NO.1 or SEQ ID NO.2.

[0028] AMH-VHH1-A1-AA: QVQLVESGGGLVQPEGSLTLSCVASGFTFSSNDMNWVRQAPGKGLEWVSTINRGGGGTYYATSVKGRFTISRDNAKNTLYLQMNSLKTEDTAVYYCATDPPPGGRWLLSLLHSQGTQVTVSSCHY (SEQID NO.1);

[0029] AMH-VHH1-A10-AA:QVQLVESGGGSVRAGGSLRLSCAASGYTYSSNCMGWFRQAPGKEREGVAAIDSRGGRTFYAHSVKGRFTISQDNAKNTVYLQMNSLKPEDTAMYYCAAGLGRDPMGCDSYEYNDWGQGTEVIVSS (SEQ ID NO. 2).

[0030] The nucleotide sequences encoding the amino acid sequence of the AMH nanobody are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively:

[0031] AMH-VHH1-A1:CAGGTGCAGCTCGTGGAGTCTGGGGGAGGCTTGGTGCAGCCTGAGGGGTCTCTGACACTCTCCTGTGTAGCCTCTGGATTCACATTCAGTAGTAACGACATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTCGAGTGGGTCTCAACTATTAATCGTGGTGGTGGTGGCACATACTATGCA ACCTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGCTCTATCTGCAAATGAACAGCCTGAAAACTGAGGACACTGCCGTGTATTACTGCGCCACAGATCCACCCCCGGGTGGTAGGTGGCCTCCTATCACTACTGCACAGCCAGGGGACCCAGGTCACCGTCTCCTCATGCCATTAT (SEQ ID NO.3);

[0032] AMH-VHH1-A10:CAGGTGCAGCTCGTGGAGTCTGGGGGAGGCTCGGTGCGGGCTGGAGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGATACACCTACAGTAGCAACTGCATGGGCTGGTTCCGCCAGGCTCCAGGGAAGGCGCGAGGGGGTCGCAGCTATTGATTCGCGTGGTGGACGCACATTCTATGCC CACTCCGTGAAGGGCCGATTCACCATCTCCCAAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACTGCCATGTACTACTGTGCGGCGGGTCTTGGTCGAGACCCGATGGGTTGCGATTCATATGAGTATAACGACTGGGGCCAGGGGGACCGAGGTCATCGTCTCCTCA (SEQ ID NO.4).

[0033] This invention provides two screening methods for AMH nanobodies as follows:

[0034] 1) The AMH protein expressed in prokaryotes was used to prepare a natural non-immune nanobody library (which can be prepared according to the method disclosed in Chinese patent CN201910058785.0) and the first round of washing was used to obtain AMH-VHH1;

[0035] The coating concentration of AMH protein in the first round of washing was 20 μg-30 μg / mL;

[0036] 2) The AMH-VHH1 obtained in step 1) is washed in the second, third and fourth rounds to obtain the phage solution;

[0037] The AMH protein coating concentration after the second round of washing was 10 μg / mL;

[0038] The coating concentration of AMH protein in the third round of washing was 8 μg / mL;

[0039] The coating concentration of AMH protein in the fourth round of washing was 5 μg / mL;

[0040] 3) Mix the phage fluid obtained in step 2) with TG1 bacterial solution, infect the bacteria, and then culture to obtain the bacterial strain;

[0041] 4) Mix the strain obtained in step 3) with KM13 helper phage and infect it. Perform a first shaking culture (35~42℃) and a first centrifugation (7500~8500 g) on ​​the resulting infected material. Resuspend the first precipitate in liquid medium and perform a second shaking culture (28~32℃) and a second centrifugation (2000~2100 g). Mix the second supernatant with blocking solution, incubate and then perform indirect ELISA detection to detect the reactivity of the second supernatant with AMH protein, so as to determine whether the strain is reactive with AMH protein.

[0042] 5) Extract the plasmid (pCANTAB5) from the strain that reacts with the AMH protein in step 4), sequence it using universal primers for the plasmid, obtain the VHH nanobody gene sequence, and confirm it as the VHH sequence. Ligate the correct VHH nanobody fragment to the yeast expression vector (pMCO-AOXα) to obtain the recombinant plasmid.

[0043] The plasmid primers (Table 1) include upstream plasmid primer (SEQ ID NO.5) and downstream plasmid primer (SEQ ID NO.6).

[0044] Table 1 Primer Information

[0045]

[0046] 6) The recombinant plasmid obtained in step 5) was transformed into Pichia pastoris GS115 to obtain a nanobody expression strain. After methanol induction of the nanobody expression strain, the protein of the induced nanobody expression strain was extracted. The protein was identified by SDS-PAGE and Western blotting. Based on the molecular weight and his-tag, it was identified as an AMH nanobody.

[0047] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0048] Unless otherwise specified, the production processes, experimental methods, or testing methods involved in the embodiments of this invention are all conventional methods in the prior art, and their names and / or abbreviations are all conventional names in the field, which are very clear and distinct in the relevant application areas. Those skilled in the art can understand the conventional process steps based on the names and apply the corresponding equipment, and implement them according to conventional conditions or the conditions recommended by the manufacturer.

[0049] The various instruments, equipment, raw materials or reagents used in the embodiments of this invention are not subject to any special restrictions on their source. They are all conventional products that can be purchased through regular commercial channels and can be prepared according to conventional methods known to those skilled in the art.

[0050] Example 1

[0051] 1.1 Screening of AMH nanobodies

[0052] A non-immune nanobody natural library was prepared according to the method disclosed in Chinese patent CN201910058785.0. The AMH protein expressed in prokaryotes was used to perform the first round of washing on the prepared nanobody natural library to obtain AMH-VHH1, which was then aliquoted and frozen at -80℃.

[0053] During washing, 50 mM sodium carbonate / sodium bicarbonate buffer was used as the coating buffer, with a coating concentration of 20 μg / mL and a coating volume of 2 mL. The AMH protein was used to coat the immunotubes.

[0054] The rinsing method is as follows:

[0055] 1) Inoculate 500 μL of the native nanobody library into 100 mL of 2×YTAG medium and incubate at 37°C with shaking at 200 rpm for 1 hour until OD. 600 It is 0.4;

[0056] 2) Add KM13 helper phage: Add 100 μL of KM13 helper phage to 100 mL of bacterial culture, incubate at 37℃ for 30 min, and then shake and incubate for 30 min.

[0057] 3) Centrifuge at 4000×g for 10 min, remove the supernatant of the culture medium, resuspend the bacterial pellet in 100 mL of 2×YTAK medium, and incubate overnight at 30℃ with shaking at 200 rpm.

[0058] 4) The next morning, centrifuge at 11000×g and 4℃ overnight for 10 min. Transfer the supernatant to a new centrifuge bottle and add 20 mL of PEG / NaCl solution. Mix well and incubate on ice for 90 min.

[0059] 5) Centrifuge at 11000×g, 4℃ for 30 minutes, discard the supernatant, and then centrifuge again for 2 minutes to completely remove the supernatant;

[0060] 6) Resuspend the precipitate in 2.6 mL of PBS buffer, then aliquot it into two 1.5 mL centrifuge tubes and centrifuge at 11600×g for 10 min.

[0061] 7) Recover the supernatant and name it SR-AMH-VHH1. Take 100 μL for titer determination, and mix the remainder with 1.4 mL MPBS solution. Incubate at room temperature for 1 h to obtain the mixture (AMH-VHH1 treated with MPBS solution), which is ready for use.

[0062] The process for treating the coated protein is as follows:

[0063] 1) The day after coating the protein, pour out the liquid in the immunotherapy tube and wash the tube three times with PBS buffer.

[0064] 2) Fill each tube with MPBS, block at room temperature for 2 h, and then wash the tubes 3 times with PBS buffer.

[0065] 3) Add 2 mL of the mixture obtained in step 7) above to the immunotherapy tube, incubate at room temperature for 2 h, wash the tube 10 times with PBST solution, and then wash the tube 10 times with PBS buffer.

[0066] 4) Add 2 mL of 100 mM TEA solution to each tube, shake gently at room temperature for 15 min to elute the bound phage, and then add 2 mL of Tris-HCl solution to neutralize.

[0067] 5) Transfer the eluted phage (named SC-AMH-VHH1) to a 50 mL centrifuge tube and add 16 mL OD. 600The TG1 bacterial suspension was prepared at 0.4 g / L and incubated at 37°C for 30 minutes to infect the eluted phage with the TG1 bacterial suspension. (4 mL of OD solution was added to the immunotherapy tube.) 600 Infection was performed with 0.4 g of TG1 bacterial solution, and the samples were finally combined, for a total volume of 24 mL.

[0068] 6) Take 100 μL of bacterial solution for titer determination, and centrifuge the remaining bacterial solution at 4000 g for 10 min.

[0069] 7) Resuspend the bacterial pellet in 1 mL of 2×YT medium, spread the resuspended bacterial solution on 5 2×YTAG solid culture plates (150 mm plates), and incubate overnight at 30℃.

[0070] 8) The next day, collect the colonies that grow on the plate using 2×YT medium, add 60% glycerol to a final concentration of 15%, which is the primary library of bacteria, named AMH-VHH1, and aliquot and freeze at -80℃.

[0071] Determination of rescue phage titers: SR-AMH-VHH1 was serially diluted, starting from 10... -7 ~10 -13 For each dilution, 10 μL of phage was used to infect 190 μL of OD. 600 TG1 bacterial suspension at 0.4% was prepared; 100 μL of bacterial suspension for each dilution was spread onto 2×YTAG solid culture plates and incubated overnight at 30℃; the colonies on the plates were counted and the SR-AMH-VHH1 titer was calculated.

[0072] Determining the titer of eluted phages: The bacterial suspension used for titer determination is serially diluted, starting from 10⁻⁶. -1 ~10 -5 For each dilution, 100 μL of bacterial culture was spread onto a 2×YTAG solid culture plate and incubated overnight at 30℃. The colonies on the plate were counted, and the SC-AMH-VHH1 titer was calculated. Then, the input / output ratio (I / O) of the first round of washing was calculated.

[0073] Based on the first round of washing, two to four rounds of washing were performed sequentially: AMH protein coating concentrations of 20 μg / mL, 10 μg / mL, and 5 μg / mL, respectively; and the phage rescue titer assay dilutions were 10 μg / mL, 10 μg / mL, and 10 μg / mL, respectively. -7 ~10 -12 10 -8 ~10 -11 10 -8 ~10 -11 The titer of eluted phage M13-AMH was determined at dilutions of 10-1. -1 ~10 -6 10 -1~10 -6 10 -8 ~10 -11 The eluted phage was neutralized with Tris-HCl solution (1 M, pH 7.4), and 200 μL of phage was used to infect 800 μL of OD. 600 Prepare a 0.4 g TG1 bacterial culture (100 μL for serial dilution, the remainder for preservation), then perform 10... -3 ~10 -6 Four dilutions were performed, and each dilution was spread onto three 2×YTAG solid culture plates (150 mm plates). Each plate contained 100 μL of bacterial culture and was incubated overnight at 30°C. Colonies were counted on the culture plates, titers were calculated, and the culture plates were marked as plates and stored at 4°C for later use.

[0074] 1.2 Screening of specific nanobodies

[0075] Preparation of monoclonal phage supernatant: 96 monoclonal strains were picked from each plate and inoculated into a 96-well deep-well plate, each well containing 200 μL of 2×YTAG medium. The plates were labeled with the AMH library strains and incubated at 30°C with shaking. After 8 h, 20 μL of bacterial culture from each well was inoculated into 180 μL of 2×YTAG medium and incubated at 37°C with shaking. The remaining bacterial culture from the original plate was diluted with 60 μL of 60% glycerol to a final concentration of 15% and stored at -80°C. After transferring the plates and incubating at 37°C with shaking for 1 h, 50 μL of KM13 (60 μL KM13 + 12 mL 2×YTAG medium) helper phage was added to each well. The plates were incubated statically at 37°C for 30 min, followed by incubation at 37°C with shaking for 40 min. Centrifuge the deep-well plate at 1800×g for 10 min, discard the supernatant, and resuspend the precipitate in 400 μL of 2×YTAK medium in each well. Incubate overnight at 30°C with shaking. The next day, centrifuge at the maximum speed of 2020×g for 20 min, aspirate 250 μL of phage supernatant from each well and transfer it to a new deep-well plate. Add 250 μL of blocking buffer (PBS buffer containing 3% BSA) to each well and incubate at room temperature for 1 hour, ready for indirect ELISA detection.

[0076] Identification of specific monoclonal phages: The reactivity of phage supernatant with AMH protein was detected by indirect ELISA. The specific method is as follows: An experimental group, a negative control group, and a BSA control group were designed. The experimental and negative control groups used AMH protein to coat 96-well ELISA plates at a concentration of 2 μg / mL. The BSA control group used BSA protein to coat 96-well ELISA plates at a concentration of 2 μg / mL, 100 μL per well, and incubated overnight at 4°C. The next day, the coating liquid was discarded, and 100 μL of blocking buffer was added to each well, and the plates were blocked at 37°C for 1 h. The blocking buffer was then discarded. For the experimental and BSA control groups, 100 μL of phage supernatant obtained from four rounds of screening and treated with blocking buffer was added to each well as primary antibody. The negative control group was incubated with an equal volume of PBS at 37°C for 1 h. The plates were washed 10 times with PBST wash buffer. 100 μL of secondary antibody (HRP-M13 Antibody, dilution 1:6000) was added to each well, and the plates were incubated at 37°C for 1 h. Wash the plate 10 times with PBST washing buffer. Add 100 μL of chromogenic substrate to each well and incubate in the dark for 5–15 min. Then, stop the reaction by adding 50 μL of stop solution to each well. Place the 96-well microplate on a plate reader to read the OD. 450 Absorbance value. Analyze the ELISA results and identify positive strains.

[0077] The glycerol bacteria corresponding to the positive wells were inoculated into 5 mL of 2×YTAG medium and cultured with shaking at 37°C. The bacterial culture was then sent to a sequencing company for sequencing. After the sequencing results were returned, they were analyzed, and the correctly sequenced strains were selected to repeat the above experiment to verify the positive strains.

[0078] 1.3 Sequencing and Specific Monoclonal Phage ELISA Screening Results

[0079] Sequencing was performed using a sequencing company. Cloning strains correctly expressing the VHH fragment were selected for indirect ELISA to detect the reactivity of the phage supernatant corresponding to each clone with AMH protein. AMH protein was coated onto 96-well microplates at a concentration of 2 μg / mL, 50 μL per well, and incubated overnight at 4°C. The next day, the coating liquid was discarded, and 100 μL of blocking buffer was added to each well, blocking at 37°C for 1 hour. The blocking buffer was discarded, and the plates were washed 10 times with PBST. The last column of the 96-well plate was selected as the control group, with 100 μL of PBS added. 100 μL of phage supernatant treated with blocking buffer was added to each of the remaining wells as primary antibody, and incubated at 37°C for 1 hour. The plates were washed 10 times with PBST. 100 μL of secondary antibody (HRP-M13 Antibody, dilution 1:10000) was added to each well, and the plates were incubated at 37°C for 1 hour. The plates were washed 10 times with PBST. Add 50 μl of chromogenic substrate to each well and incubate at 37°C in the dark for 5 minutes. Then, add 50 μL of stop solution to each well to terminate the reaction. Place the 96-well microplate on a plate reader to read the OD. 450 Absorbance values. ELISA results were analyzed to determine the positive well numbers. Each monoclonal antibody showed varying degrees of reactivity to the AMH protein (Table 2).

[0080] Table 2. Screening results of AMH monoclonal ELISA

[0081]

[0082] The correctly sequenced and predicted amino acid sequences are shown in SEQ ID NO.1 (AMH-VHH-A1) and SEQ ID NO.2 (AMH-VHH-A10).

[0083] Example 2: Construction of Pichia pastoris expression vector and expression of AMH nanobodies

[0084] Two clones with strong positive results, AMH-VHH1-A1 (SEQ ID NO.1) and AMH-VHH1-A10 (SEQ ID NO.2), were selected as specific monoclonal positive strains for subsequent experiments.

[0085] The specific method for constructing the Pichia pastoris expression vector for the specific single clone is as follows: the plasmid of the AMH-VHH1 strain was extracted and ligated into the PMD19-T vector. After blue-white screening, it was sent to a sequencing company for sequencing. The plasmids of the successfully sequenced strain and the pMCO-AOXα vector were extracted, double digested, and ligated. Then, the two were digested with Sal I enzyme and transformed into pMCO-AOXα and sent to a sequencing company for sequencing. The successfully sequenced strain was transformed into GS115 to successfully construct the AMH-VHH1-A1 protein expression strain, which was then induced to express and purified.

[0086] Example 3: Detection of AMH Nanobodies

[0087] Purification: After ultrasonic disruption and centrifugation, the supernatant of the induced bacterial culture was collected and purified using a His-tagged nickel column. A 500 mM imidazole elution buffer was prepared, and after elution in a gradient of 20%-100%, a relatively single AMH-VHH-His target band was found in the 20% imidazole elution gradient.

[0088] AMH-VHH-His detection. The purified nanobodies were used as the test proteins for Western blotting identification: a 15% separating gel and a 5% stacking gel were prepared. The original supernatant, supernatant elution buffer, binding buffer, and 20% imidazole elution buffer were added to the lanes respectively. After electrophoresis, wet transfer was performed, followed by blocking at 37°C for 1 h. HRP Anti-His-TagMouse antibody (1:20000) was prepared and incubated at 37°C for 1 h. The membrane was then washed with TBST and exposed to the atmosphere. The purified nanobodies were detected and identified. The bands in the 20% imidazole elution buffer were compared with the blank control in the binding buffer. Based on the position of the protein bands in the original supernatant, and by molecular weight and specific binding, they were identified as AMH nanobodies. Results are as follows: Figure 1 As shown, the results revealed that the molecular weight was approximately 15 kDa, consistent with the size of nanobodies.

[0089] Coomassie Brilliant Blue Detection. The purified nanobodies were used as the test proteins for Coomassie Brilliant Blue identification to determine if the purified nanobodies were monounsaturated. A 15% separating gel and a 5% stacking gel were prepared. The supernatant stock solution, supernatant column buffer, binding buffer, and 20% imidazole elution buffer were added to the respective lanes. After electrophoresis, the colloids were stained with Coomassie Brilliant Blue staining solution. The results are shown below. Figure 2 As shown, the target band in the 20% imidazole eluent is relatively simple.

[0090] Example 4: Preparation of Colloidal Gold Immunochromatography Based on AMH Nanobodies

[0091] Prepare colloidal gold immunochromatographic test strips using AMH-VHH-A1 and AMH-VHH-A10 nanobodies: Add 4 µL of potassium carbonate solution to 1 mL of colloidal gold and mix well. Then add 10 μg of AMH-VHH-A10 and 10 μg of chicken IgG (immunoglobulin) to the colloidal gold and mix well. After standing for 20 min, add 100 µL of 10% BSA and let stand for 20 min. Centrifuge at 12000×g for 10 min and discard the supernatant. Resuspend the colloidal gold containing chicken IgG in 50 µL of gold reconstitution solution and transfer the entire amount to the colloidal gold containing AMH-VHH-A10 nanobodies for resuspending. Use a streak sprayer to extract 25 µL of the nanobodies resuspended and spray it onto the gold pad. Dry overnight at 37°C.

[0092] Add 10 μg AMH-VHH-A1 and 10 μg goat anti-chicken lgY to centrifuge tubes, respectively, and add 10 µL of coating buffer. Mix well and let stand for 1 h. Draw 20 µL of the mixture containing AMH-VHH-A1 using a streak coater to create a T-line, and draw 20 µL of the mixture containing goat anti-chicken lgY using a streak coater to create a C-line. Dry overnight at 37°C.

[0093] Assemble the absorbent pad, NC membrane, gold label pad, and reaction pad onto the base plate in that order, overlapping the pads by 2 mm. Cut the strips into 4 mm wide test strips using a cutter. See the assembly diagram below. Figure 3 As shown.

[0094] Serum from sheep, horses, donkeys, and camels was dropped into the reaction pad of a colloidal gold test strip, and the results were as follows: Figure 4 As shown, we can see that lines C and T have appeared.

[0095] As can be seen from the above embodiments, the AMH nanoantibody provided by the present invention has a molecular weight of approximately 15 kDa, which is consistent with the size of nanobodies. The purified nanobody can be used in the preparation of colloidal gold immunochromatographic test strips.

[0096] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. An AMH Nanobody, characterized in that, The amino acid sequence of the AMH nanobody is shown in SEQ ID NO.1 or SEQ ID NO.

2.

2. The AMH nanobody according to claim 1, characterized in that, The AMH nanobody can specifically bind to the AMH protein.

3. A nucleic acid molecule encoding the AMH nanobody of claim 1, characterized in that, The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO.3 or SEQ ID NO.

4.

4. A kit for detecting AMH protein expression levels, characterized in that, The kit includes the AMH nanobody of claim 1.

5. A recombinant plasmid, characterized in that, The recombinant plasmid comprises the nucleic acid molecule of claim 3.

6. The recombinant plasmid according to claim 5, characterized in that, The recombinant plasmid also includes the pMCO-AOXα vector.

7. An expression strain, characterized in that, The expression strain includes the recombinant plasmid of claim 5.

8. The use of the AMH nanobody of claim 1, the recombinant plasmid of any one of claims 5-6, or the expression strain of claim 7 in the preparation of a reagent for detecting AMH levels in animal serum, characterized in that, The animals mentioned are sheep, donkeys, horses, and camels.

9. The use of the AMH nanobody of claim 1, the recombinant plasmid of any one of claims 5-6, or the expression strain of claim 7 in the preparation of colloidal gold immunochromatographic test strips or kits for detecting AMH levels in animal serum, characterized in that, The animals mentioned are sheep, donkeys, horses, and camels.

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