Construction and application of a DKK3 nanobody and its expression vector

Abstract

The present invention belongs to the field of bioengineering technology, and specifically relates to a DKK3 nanobody, a detection kit and an application thereof. The nucleotide and amino acid sequences of the DKK3 nanobody provided by the present invention are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively. The DKK3 nanobody has a molecular weight of about 16kD, can specifically bind to the DKK3 protein, and has a high affinity, and can be used for accurately detecting the expression of DKK3 in tissues by immunohistochemistry, providing a new method for studying the function of the DKK3 gene.

Description

Technical Field

[0001] The present invention belongs to the field of bioengineering technology, and specifically relates to the construction and application of a DKK3 nano antibody and an expression vector thereof. Background Art

[0002] A specific antibody that is naturally missing heavy chains but still has biological activity in camelids (alpacas, camels) and cartilaginous fish is called a single-domain antibody. The antigen-binding site (VHH) of a single-domain antibody has independent antigen recognition capabilities, and the independently expressed VHH is also called a nanobody. Nanobodies have the advantages of small molecular weight, simple structure, and stable physical and chemical properties, which enable them to pass through some protective barriers in the animal body and enter the diseased site to exert their effects, and can bind to some hidden antigen epitopes and other characteristics.

[0003] DKK3 is a secreted glycoprotein of the Dickkopf (DKK) family, but unlike other members, it does not bind to its common receptors Lrp5 / 6 and Kremen proteins to oppose Wnt signals. DKK3 may play a role in myogenesis by activating the transcription of Myf5, but its role in muscle quality control is not yet clear. High levels of DKK3 in the blood may be a marker of sarcopenia. The muscle content of beef cattle is closely related to its economic value. However, there is currently a lack of specific antibodies for detecting the expression of DKK3 in cattle, resulting in false positives or false negatives. However, nanoantibodies for detecting the expression level of DKK3 have not yet been reported.

[0004] Therefore, it is urgent to develop an antibody that can accurately detect the expression of DKK3 protein in cells or tissues. Summary of the invention

[0005] The purpose of the present invention is to provide a DKK3 nanobody, which has a high specific binding ability with DKK3, providing a basis for accurately detecting the expression level of DKK3 in cells. In order to achieve the above purpose, the present invention provides the following technical solutions:

[0006] The present invention provides a DKK3 nanobody, the amino acid sequence of the DKK3 nanobody is shown as SEQ ID NO.1.

[0007] Preferably, the DKK3 nanobody can specifically bind to the DKK3 protein.

[0008] The nucleotide sequence encoding the above-mentioned DKK3 nanobody is shown in SEQ ID NO.2.

[0009] The present invention also provides a kit for detecting the expression amount of DKK3 protein, wherein the kit comprises the above-mentioned DKK3 nanobody.

[0010] The present invention also provides a recombinant plasmid, which contains the above nucleotide sequence.

[0011] Preferably, the plasmid comprises the above nucleotide sequence, PET28a(+) prokaryotic expression vector and BamH I and ECORI restriction sites.

[0012] The present invention also provides an expression strain, which comprises the above-mentioned recombinant plasmid.

[0013] The present invention also provides the use of the above-mentioned DKK3 nanobody or the above-mentioned recombinant plasmid or the above-mentioned expression strain in the preparation of a reagent or a kit for binding to the DKK3 protein.

[0014] The present invention also provides the use of the above-mentioned DKK3 nanobody in detecting the expression of DKK3 protein in cells or tissues.

[0015] The present invention also provides the use of the above-mentioned DKK3 nanobody in detecting the localization of DKK3 protein in cells or tissues.

[0016] Beneficial effects of the present invention:

[0017] The DKK3 nanobody of the present invention has the characteristics of small molecular weight, stable physicochemical characteristics, high affinity and simple structure, and is easy to express and purify. It has a higher ability to specifically bind to DKK3 than nanobodies screened in the same batch, and can be used to detect the expression of DKK3 in tissues by immunohistochemistry, further providing a new method for the study of the function of the DKK3 gene. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0019] Figure 1 The results of Western Blotting on the purified DKK3 nanobody provided by the present invention;

[0020] Figure 2 The immunohistochemistry method provided by the present invention uses DKK3 nanoantibodies to detect the expression and distribution results of DKK3 in skeletal muscle tissues of Mongolian cattle and the like. DETAILED DESCRIPTION

[0021] The present invention provides a DKK3 nanobody, the amino acid sequence of the DKK3 nanobody is shown in SEQ ID NO.1; the nucleotide sequence of the gene encoding the DKK3 nanobody is shown in SEQ ID NO.2. The present invention utilizes phage display technology to display the expressed exogenous polypeptide or protein on the surface of the phage in the form of a fusion protein, and then screens the phage expressing the specific protein by affinity enrichment method to obtain the DKK3 nanobody.

[0022] In the present invention, the amino acid sequence encoding the DKK3 nanobody (DKK3-AA) is shown in SEQ ID NO.1:

[0023] LQLVESGGGSVQPGESLRLSCVTSGFNVNDYTLGWFRQAPGKEREGVACMS

[0024] PDGSYLHYGDSVNGRFAISRDNAKNSVYLQMNSLKPEDTAIYYCAAHTLFS

[0025] LCVSLGFGSWGQGTQVTVSSAHHSEDPSS

[0026] In the present invention, the nucleotide sequence encoding the DKK3 nanobody is shown in SEQ ID NO.2: TTGCAGCTCGTGGAGTCTGGCGGAGGCTCGGTGCAACCTGGGGAGTCTCTGAGACTCTCCTGTGTAACTTCTGGCTTCAATGTGAATGATTATACGTTGGGCTGGTTCCGCCAGGCCCCGGGAAAGGAGCGTGAGGGGGTCGCATGTATGAGTCCTGATGGGAGTTACTTACACTATGGAGACTCCGTGAATGGCCGATTCGCCATTTCCAGAGACAATGCCAAAAATTCGGTGTATCTGCAGATGAACAGCCTGAAACCGGAGGACACAGCCATCTATTATTGCGCAGCGCACACATTATTTAGCCTGTGTGTCTCCCTTGGCTTTGGTTCCTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCGGCGCACCACAGCGAAGACCCCAGCTCA.

[0027] The DKK3 nanobody with the best affinity was selected and his identification was performed using Western blotting. The results showed that the molecular weight of the DKK3 nanobody of the present invention was about 16KD, and the size of the composite nanobody was about 16KD.

[0028] The purified DKK3 nanobody was used to detect the expression of DKK3 protein in brain tissue by western blotting. The results showed that the purified DKK3 nanobody can be used to detect the expression of DKK3 protein in different structures in tissues by western-blot method.

[0029] The purified DKK3 nanoantibody was used to detect the expression and distribution of DKK3 in the triceps brachii of Mongolian cattle using immunohistochemistry. The results showed that the purified nanoantibody can be used to detect the localization of DKK3 in tissues using immunohistochemistry.

[0030] In the present invention, the DKK3 nanoantibody is selected to be related to the detection target KDD3 protein. When the detection target changes, it needs to be adaptively changed according to the target; the detection sample includes but is not limited to animal cells or tissues; preferably Mongolian cattle, and can be further applied to any animal cells or tissues.

[0031] Given that the DKK3 nanobody has a significantly higher specific binding ability with the DKK3 protein than other types of DKK3 nanobodies screened in the same batch.

[0032] In some embodiments, the present invention also provides the use of the DKK3 nanobody in the preparation of a reagent or kit for binding to the DKK3 protein, and the detection technology of the reagent or kit of the DKK3 nanobody preferably includes one or more of the following methods: immunohistochemistry, Western Blotting, ELISA, immunofluorescence staining and colloidal gold immunoaffinity chromatography.

[0033] In some embodiments, the present invention also provides a kit for detecting DKK3 expression based on immunohistochemistry, comprising the following components: the DKK3 nanobody, HRPAnti His-Tag Mouse, and detection reagents; the detection reagents include a fixative, a buffer, and a color developer.

[0034] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0035] The production processes, experimental methods or detection methods involved in the embodiments of the present invention, unless otherwise specified, are all conventional methods in the prior art, and their names and / or abbreviations are all conventional names in the field, and are very clear and unambiguous in the relevant application fields. Technical personnel in the field 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.

[0036] The various instruments, equipment, raw materials or reagents used in the embodiments of the present invention are not particularly limited in terms of their sources, and are all conventional products that can be purchased through regular commercial channels, or can be prepared according to conventional methods well known to those skilled in the art.

[0037] Example 1 Screening of DKK3 Nanobodies

[0038] 1) The melanoma nanolibrary (prepared according to the method disclosed in Chinese patent CN201910058785.0) is subjected to the first round of panning to obtain B16-DKK3-VHH1;

[0039] The coating concentration of DKK3 protein in the first round of panning was 20 μg / ml;

[0040] 2) performing the second, third, and fourth rounds of panning on the B16-DKK3-VHH1 obtained in step 1) to obtain a phage solution;

[0041] The coating concentration of DKK3 protein in the second round of panning was 10 μg / ml;

[0042] The coating concentration of DKK3 protein in the third round of panning was 10 μg / ml;

[0043] The coating concentration of DKK3 protein in the fourth round of panning was 5 μg / ml;

[0044] 3) mixing the phage solution obtained in step 2) with the TG1 bacterial solution, infecting and culturing the mixture to obtain a strain;

[0045] 4) mixing and infecting the strain obtained in step 3) with KM13 helper phage, performing a first shaking culture on the obtained infection, and then performing a first centrifugation, resuspending the obtained first precipitate in a liquid culture medium, performing a second shaking culture, and then performing a second centrifugation, mixing the obtained second supernatant with a blocking solution, incubating, and then performing an indirect ELISA test to detect the reactivity of the second supernatant with the DKK3 protein to determine that the strain is reactive with the DKK3 protein;

[0046] The temperature of the first oscillation was 37°C, and the temperature of the second oscillation was 30°C;

[0047] The centrifugal force of the first centrifugation is 8000g, and the centrifugal force of the second centrifugation is 2000g;

[0048] 5) extracting plasmid from the strain reactive with DKK3 protein in step 4), using the plasmid as a template and performing PCR amplification with a plasmid primer pair to obtain a nanobody VHH fragment, and connecting the nanobody VHH fragment to an expression vector to obtain a recombinant plasmid;

[0049] The plasmid primers include a plasmid upstream primer F-1: (SEQ ID NO.3) (AATGGATCCTTGCAGCTCGTGGAGTCTG) and a plasmid downstream primer R-1: (SEQ ID NO.4) (TATGAATTCTGAGCTGGGGTCTTCGCT);

[0050] 6) The recombinant plasmid and pBAD18 obtained in step 5) are transformed into Escherichia coli to obtain a nanobody expression strain, and the nanobody expression strain is induced by IPTG, and the protein of the induced nanobody expression strain is extracted, and the protein is identified by SDS-PAGE and Western Blotting, and is identified as DKK3 nanobody according to the molecular weight and his-tag label.

[0051] The first round of panning was performed from the prepared melanoma nanobank to obtain B16-DKK3-VHH1, which was aliquoted and frozen at -80°C.

[0052] During the panning, 50 mM sodium carbonate / sodium bicarbonate buffer was used as the coating buffer, the coating concentration was 20 μg / ml, the coating volume was 2 ml, and the immunotube was coated with the DKK3 protein.

[0053] The washing method is as follows:

[0054] 1) Inoculate 500 μl of melanoma nanobank into 100 ml of 2×YTAG medium and culture at 37°C with shaking at 200 rpm for 1 hour until OD 600 is 0.4;

[0055] 2) Add KM13 helper phage, add 100 μl KM13 helper phage to 100 ml bacterial solution, incubate at 37°C for 30 min, and then shake culture for 30 min;

[0056] 3) Centrifuge at 4000×g for 10 min, remove the culture supernatant, resuspend the bacterial pellet in 100 ml 2×YTAK medium, and culture at 30°C and 200 rpm overnight;

[0057] 4) The next morning, centrifuge the overnight culture at 11,000 × g, 4°C for 10 min, transfer the supernatant to a new centrifuge bottle, add 20 ml of PEG / NaCl solution, mix well, and place on ice for 90 min;

[0058] 5) Centrifuge at 11,000 × g, 4°C for 30 min, discard the supernatant, and then centrifuge again for 2 min to completely aspirate the supernatant;

[0059] 6) Resuspend the pellet with 1.3 ml PBS buffer, divide it into two 1.5 ml centrifuge tubes, and centrifuge at 11600 × g for 10 min;

[0060] 7) Recover the supernatant, named SR-B16-DKK3-VHH1, take 100 μl for titer determination, mix the rest with 1.2 ml MPBS solution, and incubate at room temperature for 1 hour to obtain a mixed solution (DKK3-VHH1 treated with MPBS solution) for standby use.

[0061] Coating protein treatment:

[0062] 1) The day after coating, pour out the liquid in the immune tube and wash the tube three times with PBS buffer.

[0063] 2) Fill each tube with MPBS, block for 2 hours at room temperature, and then wash the tube three times with PBS buffer.

[0064] 3) Add 2 ml of the mixed solution obtained in the above panning step 7) to the immunotube, incubate at room temperature for 2 hours, wash the tube 10 times with PBST solution, and then wash the tube 10 times with PBS buffer.

[0065] 4) Add 2 ml of 100 mM TEA solution to each tube, shake gently at room temperature for 15 min to elute the bound phages, and then add 2 ml of Tris-HCl solution to neutralize.

[0066] 5) Transfer the eluted phage (named SC-B16-DKK3-VHH1) to a 50 ml centrifuge tube and add 16 ml OD 600 TG1 bacterial solution with an OD of 0.4 was placed in a 37°C water bath for 30 minutes to allow the eluted phage to infect the TG1 bacterial solution. (Add 4 ml of OD 600 The TG1 bacterial solution with a dilution of 0.4 was used for infection and finally combined to a total volume of 24 ml).

[0067] 6) Take 100 μl of bacterial solution for titer determination, and centrifuge the remaining bacterial solution at 4000 g for 10 min.

[0068] 7) Use 1 ml of 2×YT medium to resuspend the bacterial pellet, spread the resuspended bacterial solution on 5 2×YTAG solid culture plates (150 mm plates), and place them in an incubator at 30°C for overnight culture.

[0069] 8) The next day, 2×YT medium was used to collect the colonies grown on the plate, and 60% glycerol was added to a final concentration of 15%. This was the primary library bacteria, named B16-DKK3-VHH1, and was aliquoted and frozen at -80°C.

[0070] Determine the titer of rescued phage: SR-B16-DKK3-VHH1 was serially diluted from 10 -7 ~10 -13; Take 10 μl of phage from each dilution to infect 190 μl OD 600 The dilution of TG1 was 0.4; 100 μl of the bacterial solution was taken from each dilution and coated on a 2×YTAG solid culture plate, which was then placed in a 30°C incubator for overnight culture; the colonies on the assay plate were counted and the titer of SR-B16-DKK3-VHH1 was calculated.

[0071] Determine the titer of eluted phage: dilute the bacterial solution used for titer determination in a gradient manner, with the dilution ranging from 10 -1 ~10 -5 ; Take 100 μl of bacterial solution from each dilution and spread it on a 2×YTAG solid culture plate, and culture it in a 30°C incubator overnight; count the colonies on the assay plate and calculate the SC-B16-DKK3-VHH1 titer; and then calculate the input-output ratio I / O of the first round of panning.

[0072] Based on the first round of panning, two to four rounds of panning were performed in sequence: the DKK3 protein coating concentrations were 10 μg / ml, 10 μg / ml, and 5 μg / ml, respectively; the dilutions for the titer determination of rescued phages were 10 -7 ~10 -12 , 10 -8 ~10 -11 , 10 -8 ~10 -11 , 10 -8 ~10 -11 The titer of eluted phage M13-DKK3 was determined by dilutions of 10 -1 ~10 -6 , 10 -1 ~10 -6 , 10 -8 ~10 -11 , 10 -8 ~10 -11 After the phages eluted in the fourth round of panning were neutralized with Tris-HCl solution (1 M, pH 7.4), 200 μl of phages were taken to infect 800 μl of OD 600 TG1 bacterial solution of 0.4 (100 μl was taken for gradient dilution and the rest was used for bacterial preservation), and then 10 -3 ~10 -6 There are 4 dilutions in total. Each dilution is coated on 3 2×YTAG solid culture plates (150 mm plates), 100 μl of bacterial solution is added to each plate, and culture is carried out at 30°C overnight. The colonies on the culture plates are counted, the titers are calculated, and the culture plates are marked as plates and placed in a 4°C refrigerator for use.

[0073] Example 2 Screening of specific nanobodies

[0074] Preparation of monoclonal phage supernatant: 96 monoclonal strains were picked from each plate and inoculated into a 96-well deep-well culture plate. Each well contained 1 ml of 2×YTAG medium. The culture plates were marked as DKK3 library strains and cultured at 30°C with shaking. After 8 hours, 50 μl of bacterial solution was taken from each well and inoculated into 500 μl of 2×YTAG medium, and cultured at 37°C with shaking. 60 μl of 60% glycerol was added to the remaining bacterial solution on the original plate to a final concentration of 15%, and then frozen at -80°C.

[0075] After the transfer plate was shaken and cultured at 37°C for 1 hour, 50μl KM13 (60μl KM13 + 12ml 2×YTAG medium) was added to each well to help the phage, and the infection was allowed to stand at 37°C for 30 minutes, and then shaken and cultured at 37°C for 40 minutes. The deep-well plate was centrifuged at 1800×g for 10 minutes, the supernatant was discarded, and 400μl 2×YTAK medium was added to each well to resuspend the precipitate, and shaken and cultured at 30°C overnight. The next day, centrifuged at a maximum speed of 2020g for 20 minutes, 250μl of phage supernatant was aspirated from each well and transferred to a new deep-well plate, and 250μl of blocking solution (PBS buffer solution containing 3% BSA) was added to each well and incubated at room temperature for 1 hour, ready for indirect ELISA detection.

[0076] Identification of specific monoclonal phage: The reactivity of phage supernatant with DKK3 protein was detected by indirect ELISA test. The specific method is as follows:

[0077] The experimental group, negative control group and BSA control group were designed. The experimental group and negative control group used DKK3 protein to coat the 96-well ELISA plate at a coating concentration of 2μg / ml. The BSA control group used BSA protein to coat the 96-well ELISA plate at a coating concentration of 2μg / ml, 100μl per well, and placed at 4℃ overnight. The next day, the coating liquid in the well was discarded, and 100μl of blocking solution was added to each well to block at 37℃ for 1h. The blocking solution in the well was discarded, and 100μl of phage supernatant obtained from four rounds of screening treated with blocking solution was added to each well of the experimental group and BSA control group as the primary antibody. The negative control added an equal amount of PBS and incubated at 37℃ for 1h. Wash the plate 6 times with PBST washing solution. Add 100μl of secondary antibody (HRP-M13 Antibody, dilution 1:6000) to each well and incubate at 37℃ for 1h. Wash the plate 8 times with PBST washing solution. Add 100 μl of color substrate to each well, react in the dark for 5-15 min, and then add 50 μl of stop solution to each well to terminate the reaction. Place the 96-well ELISA plate on a plate reader to read the OD 450 Absorption value. Analyze the ELISA results and determine the positive strains.

[0078] The glycerol bacteria corresponding to the positive wells were inoculated into 5 ml of 2×YTAG medium, and the culture was shaken at 37°C before the bacterial solution was sent to the sequencing company for sequencing. After the sequencing results were returned, the sequencing results were analyzed, and the strains with correct sequencing were selected to repeat the above experiment again to verify the positive strains. According to the ELISA identification results of the DKK3 monoclonal positive strains as shown in Table 1, the recombinant plasmid construction strain was determined.

[0079] Sequencing was performed by a sequencing company, and strains that could correctly express the VHH fragment clone were selected for indirect ELISA to detect the reactivity of the phage supernatant corresponding to the monoclone with the DKK3 protein. These monoclones all had varying degrees of reactivity with the DKK3 protein.

[0080] Table 1DKK3 monoclonal ELISA screening results

[0081] Positive clone number Experimental Group PBS negative control DKK3-VHH-1G5 2.6481 0.1293 DKK3-VHH-2G3 2.4359 0.5657 DKK3-VHH-1G7 1.6755 0.1293

[0082] The amino acid sequence (DKK3-VHH-1G5) is shown in SEQ ID NO.5: LQLVESGGGSVQPGESLRLSCVTSGFNVNDYTLGWFRQAPGKEREGVACMSPDGSYLHYGDSVNGRFAISRDNAKNSVYLQMNSLKPEDTAIYYCAAHTLFSLCVSLGFGSWGQGTQVTVSSAHHSEDPSS

[0083] The amino acid sequence (DKK3-VHH-2G3) is shown in SEQ ID NO.6: VQLVESGGALVQPGGSLRLSCVASGFGWDYNTIGWFRQAPGKEREAVSCISSSDDSTYYSNSVKGRFSISKDNAKKTVFLQMDSLKPDDTAVYFCAAGAGIRTTVENLCKVLSKDYDKWGQGTQVTVSSAHHRKTPAP

[0084] The amino acid sequence (DKK3-VHH-1G7) is shown in SEQ ID NO.7: VAARGVWGRLGAPAGSLRLSCTNPEAIVTPRAMTWVRQAPRESRSFVALLFANGVSLYNESIKDRFSISGDGAKNRVYLPMSDLNSDDTGVYYCYFRLDGRPDLWGQGTGVTVSTAHHSEHPSS

[0085] Example 3DKK3 Nanobody Activity and Affinity

[0086] Construction of prokaryotic expression recombinant plasmid: The above DKK3-VHH-1G5 glycerol bacteria were inoculated into 5 ml 2×YTAG medium for culture, and the plasmid was extracted using a plasmid small-scale extraction kit as a template plasmid for prokaryotic expression. Primers for prokaryotic expression were designed, and BamH I and ECORI restriction sites were introduced at the 5' and 3' ends of the primers, respectively. The nanobody VHH sequence was amplified using the designed primers, and connected to the PET28a (+) prokaryotic expression vector through the above restriction sites to construct a nanobody prokaryotic expression recombinant plasmid for DKK3-specific identification of the nanobody.

[0087] The screening steps are as follows:

[0088] The recombinant plasmid and PET28a(+) were transformed into BL21(DE3) strain to obtain the corresponding nanobody expression strain. Then the nanobody was induced to express, and the specific method was as follows:

[0089] The transformed bacterial solution was cultured overnight, and the next day, a single clone colony on the culture plate was picked and cultured overnight. The bacterial solution cultured the next day was preserved.

[0090] Pipette 10 μl of glycerol bacteria into 5 ml of Kana-resistant LB medium and culture at 37°C with shaking overnight;

[0091] The next day, 50 μl of bacterial solution was inoculated into 5 ml of Kana-resistant LB culture. Two tubes were inoculated each and cultured at 37°C with shaking until OD 600 is 0.6;

[0092] IPTG was added to one tube of bacterial solution for induction (final concentration 0.6 mM), and no IPTG was added to the other tube as a non-induced control. The cells were cultured overnight at 28°C with shaking for 6 h.

[0093] At the same time, a BL21(DE3) empty strain control was made, and the empty strain control was cultured using LB medium without resistance.

[0094] SDS-PAGE identification of nanobodies:

[0095] The expression of nanobodies will be identified by SDS-PAGE, the specific method is as follows:

[0096] Pipette 1 ml of bacterial solution into a 1.5 ml centrifuge tube and centrifuge at 13000 rpm for 2 min;

[0097] Discard the supernatant and wash the bacterial pellet twice with PBS buffer;

[0098] Resuspend the bacterial pellet with 20 μl PBS buffer, then add 5 μl 5× protein loading buffer and boil the sample in boiling water for 5 minutes. Perform electrophoresis on the sample using a 10% polyacrylamide gel. After the electrophoresis is completed, stain the gel with Coomassie Brilliant Blue for 1 hour, and then decolorize it with a decolorizing solution.

[0099] Screening of nanobodies with anti-DKK3 neutralizing activity: The glycerol strain corresponding to the screened nanobodies was inoculated into 5 ml of Kana-resistant LB medium, cultured at 37°C with shaking for 10 h, and then transferred to 500 ml of Kana-resistant LB medium and cultured at 37°C with shaking until OD 600 When the pH value was 0.6, IPTG (final concentration 0.6 mM) was added to induce expression, and the cells were cultured with shaking at 16° C. for 24 h. The next day, the nanoantibody was purified in small amounts.

[0100] Affinity of DKK3 nanobody: ELISA plate was coated with 2μg / ml DKK3; after BSA blocking, the purified and diluted DKK3 nanobody was used as the primary antibody and diluted 2-fold to 421μg / ml, 210.5μg / ml, 105.25μg / ml, 52.625μg / ml, 26.3125μg / ml, 13.15625μg / ml, 6.578125μg / ml, and 3.289023μg / ml for ELISA identification.

[0101] Identification of the purified product: Identified by ELISA, and the antibody was identified by His tag using Western Blotting: After SDS-PAGE electrophoresis, transferred to NC membrane, directly labeled with His secondary antibody, and the antibody was displayed by development.

[0102] DKK3 nanobody affinity test results:

[0103] After ELISA detection, the antibody concentration was 3.289023 μg / ml, and it could still react specifically with the antigen. The results are shown in Table 2.

[0104] Table 2DKK3 nanobody ELISA screening results

[0105] Concentration (μg / ml) 421 210.5 105.25 52.625 26.313 13.156 6.578 3.289 1.645 OD value of positive group 1.726 1.169 0.724 0.334 0.172 0.072 0.038 0.0236 0.0185 OD value of negative group 0.0084 0.0129 0.0103 0.0097 0.0084 0.0129 0.0103 0.0107 0.0115

[0106] His tag detection: The purified antibody was tested for His tag by Western Blotting, and the molecular weight was found to be about 16KD, which is consistent with the size of nano antibodies. Figure 1 The strain finally obtained is the nanobody protected in the claims of this application, and the sequence of the nanobody with the highest sensitivity is shown in SEQ ID NO.1.

[0107] Example 4 Immunohistochemical Detection

[0108] The specific steps of immunohistochemistry detection are as follows:

[0109] The prepared paraffin sections were placed in xylene and gradient alcohol for dewaxing, and immunohistochemical analysis was performed according to the following steps: PBS washing, 3 min × 3 times, distilled water was added to the wet box, the tissue sections were placed in the box, TritionX-100 was added, and the reaction was carried out for 15 min. After the reaction, PBS washing, 3 min × 3 times, sodium citrate antigen repair solution was used for high temperature repair for 10 min, PBS washing for 3 min × 3 times, 3% H 2 O 2 Solution was added dropwise to the tissue, reacted at 37°C for 30 min, rinsed with PBS for 3 min×3 times, blocking solution was prepared, added dropwise to the tissue, blocked at 37°C for 1 h, the blocking solution was discarded, DKK3 single domain antibody was added to the experimental group, and PBS was added to the control group, incubated at 4°C overnight, the next day, after rewarming for 30 min, rinsed with PBS for 3 min×3 times, anti-His tag antibody containing HRP was added, incubated at 37°C for 1 h, rinsed with PBS solution for 3 min×3 times, DAB color developing solution was used for color development, and observation was performed at any time. After the color development was completed, wash with water to stop the color development, counterstain with hematoxylin, dehydrate and make transparent, and seal the slides.

[0110] Figure 2 The DKK3 nanoantibody was used to detect the expression and distribution of DKK3 in the triceps brachii of Mongolian cattle by immunohistochemistry. Protein samples in tissues were selected, and the DKK3 nanoantibody was used as the primary antibody. The control group was replaced with PBS, and HRPAnti His-Tag Mouse was used as the secondary antibody. The experimental results fully demonstrated that the purified nanoantibody can be used for immunohistochemistry to detect the localization of DKK3 in tissues, and the experimental effect is ideal.

[0111] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A DKK3 nanobody, characterized in that: The amino acid sequence of the DKK3 nanobody is shown in SEQ ID NO.

1.

2. A nucleotide molecule encoding the DKK3 nanobody according to claim 1, characterized in that: The nucleic acid sequence of the nucleotide molecule is shown in SEQ ID NO.

2.

3. A kit for detecting the expression of DKK3 protein, characterized in that: The kit comprises the Nanobody according to claim 1.

4. A recombinant plasmid, characterized in that: The recombinant plasmid comprises the nucleotide molecule according to claim 2.

5. The recombinant plasmid according to claim 4, characterized in that: The plasmid comprises the nucleotide molecule according to claim 2, a PET28a(+) prokaryotic expression vector and BamH I and ECORI restriction sites.

6. An expression strain, characterized in that The expression strain comprises the recombinant plasmid according to claim 4.

7. Use of the DKK3 nanobody according to claim 1, the recombinant plasmid according to any one of claims 4-5, or the expression strain according to claim 6 in the preparation of a reagent or kit for binding to DKK3 protein.

8. Use of the DKK3 nanobody according to claim 1 in detecting the localization of DKK3 protein in cells or tissues for non-diagnostic purposes.

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