An anti-VEGFR2 nanobody and its application

Abstract

The present invention provides an anti-VEGFR2 nanobody and its application. The amino acid sequence and nucleotide sequence of the anti-VEGFR2 nanobody of the present invention are shown in SEQ ID No.1 and SEQ ID No.2 respectively. The anti-VEGFR2 nanobody provided by the present invention can specifically bind to the VEGFR2 protein, and the sensitivity to the VEGFR2 protein is 0.625 μg / mL. The application of the anti-VEGFR2 nanobody can be used for immunohistochemistry and Western Blotting to detect the expression of VEGFR2 in tissues or cells, providing a method for the study of the function of the VEGFR2 gene.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering technology, and particularly to an anti-VEGFR2 nanobody and its application. Background Art

[0002] A specific antibody that is naturally lacking in 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 the single-domain antibody has an independent antigen recognition ability, and the independently expressed VHH is also called a nanobody. Compared with traditional tetrameric antibodies, the main characteristics of nanobodies are: small molecular weight, simple structure, stable physical and chemical properties, etc. The excellent properties of nanobodies make them have advantages in many aspects: in terms of antibody entry into the body, nanobodies can cross some protective barriers in the animal body and enter the diseased site to play a role, such as the blood-brain barrier, blood-testis barrier, etc.; in terms of antigen-antibody binding, they can bind to some hidden antigenic epitopes, especially suitable for targets where it is difficult to obtain antibodies, such as GPCRs, ion channels, and enzyme active centers; in terms of reducing production costs, nanobodies have a simple structure and are easy to express in vitro, and at the same time, inclusion bodies are not easily produced during in vitro expression, and the production process is simple.

[0003] The vascular endothelial growth factor (VEGF) family can promote the formation of new blood vessels. Its family members include VEGF-A, VEGF-B, VEGF-C, VEGF-D, and placental growth factor (PLGF), and its receptors include VEGFR1, VEGFR2, and VEGFR3, which are transmembrane tyrosine kinase receptors. Vascular endothelial growth factor receptor 2 (VEGFR2) is one of the most important cytokines in the process of tumor angiogenesis. After binding to vascular endothelial growth factor, it can release a variety of cytokines, not only promoting the formation of new blood vessels, but also participating in mediating changes in vascular permeability. High expression of VEGFRE2 is an important cause of tumor invasion and metastasis. Preparing VEGFR2 nanobodies can be used as a primary antibody in Western Blotting and immunohistochemistry on the one hand, and can become a drug for targeted therapy or inhibiting tumor invasion and metastasis on the other hand. Summary of the Invention

[0004] The object of the present invention is to provide an anti-VEGFR2 nanobody and its applications. The anti-VEGFR2 nanobody of the present invention has a small inherent molecular weight, stable physicochemical characteristics, high affinity, and a simple structure that is easy to recombinantly express and prepare. It can cross the blood-brain barrier and has great application prospects in the diagnosis and treatment of diseases. Using the anti-VEGFR2 nanobody provided by the present invention can specifically bind to the VEGFR2 protein, with a sensitivity of 0.625 μg / mL, and can be used for immunohistochemistry and Western Blotting to detect the expression of VEGFR2 in tissues.

[0005] The first object of the present invention provides an anti-VEGFR2 nanobody, and the amino acid sequence of the anti-VEGFR2 nanobody is shown as SEQ ID No.1; the anti-VEGFR2 nanobody includes a framework region FR and an antigen recognition region CDR.

[0006] The second object of the present invention provides a nucleic acid encoding the above anti-VEGFR2 nanobody, and the nucleotide sequence of the nucleic acid is shown as SEQ ID No.2.

[0007] The third object of the present invention provides a detection reagent or kit, and both the detection reagent and the kit include the above anti-VEGFR-2 nanobody.

[0008] The fourth object of the present invention provides the application of the above anti-VEGFR2 nanobody in the preparation of a drug that binds to the VEGFR2 protein.

[0009] The fifth object of the present invention provides the application of the above anti-VEGFR2 nanobody in the preparation of an immunohistochemical reagent that binds to the VEGFR2 protein.

[0010] The present invention also provides the application of the above anti-VEGFR2 nanobody in the preparation of a Western Blotting detection reagent that binds to the VEGFR2 protein.

[0011] The anti-VEGFR2 nanobody provided by the present invention is screened by phage display technology. Phage display technology can display the expressed foreign polypeptide or protein on the surface of the phage in the form of a fusion protein, and then screen the phage expressing the specific protein by affinity enrichment method.

[0012] Beneficial effects

[0013] Compared with the prior art, the anti-VEGFR2 nanobody developed by the phage display experimental technique in the present invention has a small inherent molecular weight (molecular weight of 15 KD), stable physicochemical characteristics, high affinity, and a simple structure that is easy to recombinantly express and prepare. It can cross the blood-brain barrier and has great application prospects in the diagnosis and treatment of diseases. The anti-VEGFR2 nanobody provided by the present invention can specifically bind to the VEGFR2 protein with a sensitivity of 0.625 μg / mL and can be used for immunohistochemistry and Western Blotting to detect the expression of VEGFR2 in tissues. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 For SDS-PAGE to detect the purification effect of the anti-VEGFR2 nanobody; among them, lanes 1, 2, 3, and 4 are respectively four repeated experiments of VHH1;

[0015] Figure 2 For His-tag to detect the anti-VEGFR2 nanobody; among them, lanes 1, 2, and 3 are respectively three repeated experiments of VHH1;

[0016] Figure 3 For the FR and CDR regions of the anti-VEGFR2 antibody;

[0017] Figure 4 For VEGFR2 applied to Western Blotting to detect the expression of VEGFR2 in tissue proteins; among them, lanes 1, 2, and 3 are respectively three repeated experiments of VEGFR2;

[0018] Figure 5 For VEGFR2 applied to immunohistochemistry to detect the localization of VEGFR2 in tissues. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0019] The present invention will be described in detail below with reference to specific embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but do not limit the present invention in any form. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several adjustments and improvements can still be made. These all belong to the protection scope of the present invention.

[0020] Unless otherwise specified, the raw materials used in the embodiments can be obtained commercially.

[0021] Example 1: Screening of anti-VEGFR2 nanobody using phage display technology

[0022] 1) The melanoma nanobody library (prepared according to the method disclosed in Chinese Patent CN201910058785.0) was subjected to the first round of panning to obtain B16-VEGFR2-VHH1;

[0023] The coating concentration of VEGFR2 protein in the first round of panning was 20 μg / mL;

[0024] 2) The B16-VEGFR2-VHH1 obtained in step 1) was subjected to the second, third, and fourth rounds of panning in sequence to obtain a phage solution;

[0025] The coating concentration of VEGFR2 protein in the second round of panning was 10 μg / mL;

[0026] The coating concentration of VEGFR2 protein in the third round of panning was 10 μg / mL;

[0027] The coating concentration of VEGFR2 protein in the fourth round of panning was 5 μg / mL;

[0028] 3) The phage solution obtained in step 2) was mixed with TG1 bacterial solution, infected, and then cultured to obtain strains;

[0029] 4) The strains obtained in step 3) were mixed with KM13 helper phage and infected. The obtained infectant was subjected to the first shaking culture and then the first centrifugation. The obtained first precipitate was resuspended in a liquid medium and then subjected to the second shaking culture, followed by the second centrifugation. The obtained second supernatant was mixed with a blocking solution, incubated, and then subjected to indirect ELISA to detect the reactivity of the second supernatant with VEGFR2 protein to determine that the strain has reactivity with VEGFR2 protein;

[0030] The temperature of the first shaking was 35 - 42 °C, and the temperature of the second shaking was 28 - 32 °C;

[0031] The centrifugal force of the first centrifugation was 7500 - 8500 g, and the centrifugal force of the second centrifugation was 2000 - 2100 g;

[0032] 5) The strains with reactivity with VEGFR2 protein obtained in step 4) were subjected to plasmid extraction. Using the plasmid as a template, PCR amplification was performed with plasmid primers to obtain the nanobody VHH fragment. The nanobody VHH fragment was ligated with an expression vector to obtain a recombinant plasmid;

[0033] The plasmid primers include a plasmid upstream primer and a plasmid downstream primer. The nucleotide sequence of the plasmid upstream primer is as shown in SEQ ID No.3, specifically as follows:

[0034] CGAGCTC GAGTCGGGGGGAGGTTTGGTGC;

[0035] The nucleotide sequence of the plasmid downstream primer is as shown in SEQ ID No.4, specifically as follows:

[0036] CCTCGAG TGAGGAGACGGTGACCTGGATC;

[0037] 6) Transfer the recombinant plasmid obtained in step 5) and pColdⅠ into Escherichia coli to obtain a nanobody expression strain. After inducing the nanobody expression strain with IPTG, extract the protein of the induced nanobody expression strain, and identify the protein by SDS-PAGE and Western Blotting. The nanobody against VEGFR2 is identified according to the molecular weight and his-tag label.

[0038] Perform the first round of panning on the prepared melanoma nanobody library to obtain B16-VEGFR2-VHH1, aliquot and store it frozen at -80 °C.

[0039] When panning, use 50 mM sodium carbonate / sodium bicarbonate buffer as the coating buffer, the coating concentration is 20 μg / mL, the coating volume is 2 mL, and coat the immunization tube with VEGFR2 protein.

[0040] The panning method is as follows:

[0041] 1) Inoculate 500 μL of the melanoma nanobody library into 100 mL of 2×YTAG medium, and culture it with shaking at 37 °C and 200 rmp for 1 hour until the OD600 is 0.4;

[0042] 2) Add KM13 helper phage, add 100 μL of KM13 helper phage to 100 mL of the bacterial solution, let it stand and infect at 37 °C for 30 min, and then culture it with shaking for 30 min;

[0043] 3) Centrifuge at 4000×g for 10 min, remove the culture medium supernatant, resuspend the bacterial cell pellet with 100 mL of 2×YTAK medium, and culture it with shaking at 30 °C and 200 rmp overnight;

[0044] 4) The next morning, centrifuge the overnight culture at 11000×g and 4 °C 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;

[0045] 5) Centrifuge at 11000×g and 4 °C for 30 minutes, discard the supernatant, and then centrifuge again for 2 min to completely aspirate the supernatant;

[0046] 6) Resuspend the pellet with 1.3 mL of PBS buffer, and then aliquot it into 2 1.5 mL centrifuge tubes, and centrifuge at 11600×g for 10 min;

[0047] 7) Recover the supernatant, name it ZJ-B16-VEGFR2-VHH1, take 100 μL for titer determination, and mix the remaining with 1.2 mL of MPBS solution, incubate together at room temperature for 1 h to obtain a mixture (VEGFR2-VHH1 treated with MPBS solution), for later use.

[0048] Coating protein treatment:

[0049] 1) On the day after coating the protein, pour out the liquid in the immunization tube and wash the tube 3 times with PBS buffer.

[0050] 2) Fill each tube with MPBS, close it at room temperature for 2 h, and then wash the tube 3 times with PBS buffer.

[0051] 3) Add 2 mL of the mixture obtained in step 7) of the panning above to the immunization tube, incubate at room temperature for 2 h, then wash the tube 10 times with PBST solution, and then wash the tube 10 times with PBS buffer.

[0052] 4) Add 2 mL of 100 mM TEA solution to each tube, gently shake at room temperature for 15 min to elute the bound phage, and then add 2 mL of Tris-HCl solution to neutralize.

[0053] 5) Transfer the eluted phage (named XT-B16-VEGFR2-VHH1) to a 50 mL centrifuge tube, add 16 mL of TG1 bacterial solution with an OD600 of 0.4, and incubate in a 37 °C water bath for 30 minutes to allow the eluted phage to infect the TG1 bacterial solution. (And add 4 mL of TG1 bacterial solution with an OD600 of 0.4 to the immunization tube for infection, and finally combine them, with a total volume of 24 mL).

[0054] 6) Take 100 μL of the bacterial solution for titer determination, and centrifuge the remaining bacterial solution at 4000 g for 10 min.

[0055] 7) Resuspend the bacterial cell pellet with 1 mL of 2×YT medium, spread the resuspended bacterial solution on 5 2×YTAG solid culture plates (150 mm plates), and incubate in a 30 °C incubator overnight.

[0056] 8) The next day, collect the colonies grown on the plates with 2×YT medium, add glycerol to a final concentration of 15% at 60%, which is the primary library bacteria, named B16-VEGFR2-VHH1, aliquot and store frozen at -80 °C.

[0057] Determination of the titer of rescued phages: ZJ-B16-VEGFR2-VHH1 was serially diluted at dilutions ranging from 10-7 to 10-13; 10 μL of phages at each dilution was used to infect 190 μL of TG1 bacterial solution with an OD600 of 0.4; 100 μL of the bacterial solution at each dilution was spread on a 2×YTAG solid culture plate and incubated overnight in a 30 °C incubator; the colonies on the assay plate were counted to calculate the titer of ZJ-B16-VEGFR2-VHH1.

[0058] Determination of the titer of eluted phages: The bacterial solution used for titer determination was serially diluted at dilutions ranging from 10-1 to 10-5; 100 μL of the bacterial solution at each dilution was spread on a 2×YTAG solid culture plate and incubated overnight in a 30 °C incubator; the colonies on the assay plate were counted to calculate the titer of XT-B16-VEGFR2-VHH1; and then the input / output ratio I / O of the first round of panning was calculated.

[0059] Based on the first round of panning, the second to fourth rounds of panning were carried out successively: the coating concentrations of VEGFR2 protein were 10 μg / mL, 10 μg / mL, and 5 μg / mL respectively; the dilution ranges for the determination of the titer of rescued phages were 10-7 to 10-12, 10-8 to 10-11, and 10-8 to 10-11 respectively; the dilution ranges for the determination of the titer of eluted phage XT-VEGFR2 were 10-1 to 10-6, 10-1 to 10-6, and 10-1 to 10-6 respectively; after the eluted phages were neutralized with Tris-HCl solution (1 M, pH 7.4), 200 μL of phages was used to infect 800 μL of TG1 bacterial solution with an OD600 of 0.4 (100 μL was taken for serial dilution and the remaining was used for bacterial preservation), and then 4 dilutions from 10-3 to 10-6 were made. 100 μL of the bacterial solution at each dilution was spread on 3 2×YTAG solid culture plates (150 mm plates), and each plate was incubated overnight at 30 °C; the colonies on the culture plates were counted to calculate the titer, and the culture plates were labeled as plates and stored in a 4 °C refrigerator for later use.

[0060] Example 2: Screening of specific nanobodies

[0061] Preparation of monoclonal phage supernatant: 192 monoclonal strains were picked from the plate and inoculated into 2 96-well deep-well culture plates. Each well contained 1 mL of 2×YTAG medium. The culture plates were respectively labeled as VEGFR2-1 and VEGFR2-2 library strains and cultured with shaking at 30 °C. After 8 h, 50 μL of the bacterial liquid was pipetted from each well and inoculated into 500 μL of 2×YTAG medium and cultured with shaking at 37 °C. 60 μL of 60% glycerol was added to the remaining bacterial liquid in the original plate to a final concentration of 15% and stored at -80 °C. After the transferred plate was cultured with shaking at 37 °C for 1 h, 50 μL of KM13 helper phage was added to each well, and the cells were allowed to stand and infect at 37 °C for 30 min, and then cultured with shaking at 37 °C for 40 min. The deep-well plate was centrifuged at 1800×g for 10 min, the supernatant was discarded, and the precipitate was resuspended in 400 μL of 2×YTAK medium in each well and cultured with shaking at 30 °C overnight. The next day, it was centrifuged at a maximum speed of 2020xg for 20 min. 250 μL of phage supernatant was pipetted from each well and transferred to a new deep-well plate, and 250 μL of blocking solution (PBS buffer solution containing 3% BSA) was added to each well and incubated at room temperature for 1 h, to be used for indirect ELISA detection.

[0062] Identification of specific monoclonal phages: The reactivity of the phage supernatant with VEGFR2 protein was detected by indirect ELISA. The specific method was as follows: The experimental group, negative control group and BSA control group were designed. The experimental group and the negative control group used VEGFR2 protein to coat a 96-well ELISA plate at a coating concentration of 2 μg / mL. The BSA control group used BSA protein to coat a 96-well ELISA plate at a coating concentration of 2 μg / mL, 100 μL per well, and placed at 4 °C overnight. The next day, the coating liquid in the wells was discarded, and 100 μL of blocking solution was added to each well and blocked at 37 °C for 1 h. The blocking solution in the wells was discarded. In the experimental group and the BSA control group, 100 μL of the phage supernatant obtained by four rounds of screening treated with the blocking solution was added to each well as the primary antibody, and an equal amount of PBS was added to the negative control. Incubate at 37 °C for 1 h. Wash the plate 6 times with PBST washing solution. 100 μL of secondary antibody (HRP-M13 Antibody, dilution 1:6000) was added to each well and incubated at 37 °C for 1 h. Wash the plate 8 times with PBST washing solution. 100 μL of chromogenic substrate was added to each well and reacted in the dark for 5 - 15 min, and then 50 μL of stop solution was added to each well to terminate the reaction. The 96-well ELISA plate was placed on a microplate reader to read the OD450 absorbance value. Analyze the ELISA results and determine the positive strains.

[0063] Inoculate the glycerol bacteria corresponding to the positive wells into 5 mL of 2×YTAG medium, and after culturing with shaking at 37°C, send the bacterial solution to a sequencing company for sequencing. After the sequencing results are returned, analyze the sequencing results, select the strains with correct sequencing and repeat the above experiment again to verify the positive strains, and determine the recombinant plasmid construction strains according to the ELISA identification results of VEGFR2 monoclonal positive strains (see Table 1).

[0064] Example 3: Activity and Affinity of Anti-VEGFR2 Nanobody

[0065] Construction of prokaryotic expression recombinant plasmid: Inoculate the glycerol bacteria of the above-mentioned clones with correct sequencing results into 5 mL of 2×YTAG medium for culture, and use a plasmid miniprep kit to extract the plasmid as the template plasmid for prokaryotic expression. Then design primers for prokaryotic expression, and introduce SacⅠ and XhoⅠ restriction enzyme sites at the 5' end and 3' end of the primers respectively. Amplify the nanobody VHH sequence using the designed primers, and ligate it into the pColdⅠ prokaryotic expression vector through the above-mentioned restriction enzyme sites to construct a nanobody prokaryotic expression recombinant plasmid for the VEGFR2 specificity identification of the nanobody.

[0066] Primers for prokaryotic expression:

[0067] F (SEQ ID No.3): CGAGCTC GAGTCGGGGGGAGGTTTGGTGC;

[0068] R (SEQ ID No.4): CCTCGAG TGAGGAGACGGTGACCTGGATC;

[0069] The screening steps are as follows:

[0070] Transform the recombinant plasmid and the pColdⅠ empty vector into BL21(DE3) strains to obtain the corresponding nanobody expression strains. Then induce the expression of the nanobody. The specific method is as follows:

[0071] Cultivate the bacterial solution after transformation on the plate overnight, and pick the monoclonal colonies on the culture plate for overnight culture the next day. Preserve the bacterial solution cultured the next day.

[0072] Pipette 10 μL of glycerol bacteria and inoculate them into 5 mL of LB medium with Amp resistance, and culture with shaking at 37°C overnight;

[0073] The next day, pipette 50 μL of the bacterial solution and inoculate it into 5 mL of LB medium with Amp resistance, inoculate 2 tubes each, and culture with shaking at 37°C until the OD600 is 0.6;

[0074] Add IPTG to induce (final concentration 0.4 mM) to one tube of the bacterial solution, and do not add IPTG to the other tube as an uninduced control, and culture with shaking at 15°C overnight;

[0075] At the same time, use BL21(DE3) empty strain as a control. The empty strain control is cultured in LB medium without resistance.

[0076] SDS-PAGE identification of nanobody:

[0077] The expression of the nanobody will be identified by SDS-PAGE. The specific method is as follows:

[0078] Take 1 mL of the bacterial solution and transfer it to a 1.5 mL centrifuge tube. Centrifuge at 13000 rpm for 2 min;

[0079] Discard the supernatant and wash the bacterial cell pellet 2 times with PBS buffer;

[0080] Resuspend the bacterial cell pellet with 20 μL of PBS buffer, then add 5 μL of 5× protein loading buffer, and boil the sample in boiling water for 5 minutes. Electrophorese the sample on a 10% polyacrylamide gel. After the electrophoresis is completed, stain the gel with Coomassie Brilliant Blue staining solution for 1 h, and then decolorize it with decolorizing solution.

[0081] Screening of nanobody with anti-VEGFR2 neutralizing activity: Inoculate the glycerol strain corresponding to the screened nanobody into 5 mL of LB medium with Amp resistance. After culturing with shaking at 37 °C for 10 h, transfer it to 500 mL of LB medium with Amp resistance and culture with shaking at 37 °C until the OD600 reaches 0.6, then add IPTG (final concentration 0.4 mM) for induction of expression, and culture with shaking at 15 °C overnight. The next day, perform a small-scale purification of the above nanobody.

[0082] Affinity of VEGFR2 nanobody: Coat an ELISA plate with 5 μg / mL of VEGFR2; after blocking with BSA, use the purified and diluted VEGFR2 nanobody as the primary antibody and dilute it to 5 μg / mL, 2.5 μg / mL, 1.25 μg / mL, 0.625 μg / mL, 0.3125 μg / mL respectively for ELISA identification.

[0083] Identification of the purified product: Through ELISA identification, screen out the VEGFR2 nanobody with the best affinity, and identify the his tag of this antibody by Western Blotting method: after SDS-PAGE electrophoresis, transfer it to the NC membrane, directly label it with his secondary antibody, and display the antibody by developing technique.

[0084] Example 4: Detection and application of anti-VEGFR2 nanobody

[0085] By techniques such as immunohistochemistry and Western Blotting, using the purified anti-VEGFR2 nanobody as the primary antibody and the HRP-His antibody as the secondary antibody, the expression of VEGFR2 in different tissues was detected.

[0086] Results: ELISA screening results

[0087] The reactivity of the phage supernatants corresponding to 192 monoclonal antibodies with VEGFR2 protein was detected by the indirect ELISA method. Based on the results of the indirect ELISA test, 15 monoclonal antibodies were selected, and these monoclonal antibodies all had good reactivity with VEGFR2 protein (Table 1). The culture broth of the 15 monoclonal antibodies was sent to a sequencing company for sequencing.

[0088] Table 1 ELISA screening results of anti-VEGFR2 monoclonal antibodies

[0089]

[0090]

[0091] The correctly sequenced and predicted amino acid sequences are as follows:

[0092] VEGFR2-VHH1 (shown in SEQ ID No.1):

[0093] ESGGGLVQPGGSLRLSCAAPGFSLSSYQMSWVRQSPGKGPEWVSTIAASSGNTWYADSVKGRFTISKDNAKNTLYLQMNTLKPEDTALYYCAKRNRAGLSAYDYWGQGIQVTVSS;

[0094] VEGFR2-VHH2 (shown in SEQ ID No.5):

[0095] ESGGGLVQPGGSLRLSCAASGFTLGGWNIGWFRQAPGKEREGVLCISDSGESVYYLDSV

[0096] KGRFTISSDYAENTVYLQMNSLKPEDTAIYFCAATYYRCSDYAPEFSSWGQGTQVTVSS;

[0097] VEGFR2-VHH3 (shown in SEQ ID No.6):

[0098] VQPGGSLRLSCAAPGFSLSSYQMSWVRQSPGKGPEWVSTIADNIFNTGYADSVKGRFTISKDNAKNTLYLQMNTLKPEDTALYYCAKRNRAGLSAYDYWGQGIQVTVSS;

[0099] 1. Affinity detection of anti-VEGFR2 nanobody 1:

[0100] After expressing the above 3 clones, the affinity detection results were as follows: The sensitivity of VEGFR2-VHH1 to VEGFR2 protein was 0.625 ug / ml; the sensitivity of VEGFR2-VHH2 to VEGFR2 protein was 1.25 ug / ml; the sensitivity of VEGFR2-VHH3 to VEGFR2 protein was weak, only 5 ug / ml. The most sensitive VHH1 was selected for purification. The purification effect was as Figure 1 shown, and the His tag was detected by Western Blotting method. The result showed a positive His blot band, and the molecular weight was about 15 kDa, as Figure 2 shown, which was consistent with the size of the nanobody. The FR of this antibody is the framework region and the CDR is the antigen recognition region, as Figure 3 shown.

[0101] 2. Application description of anti-VEGFR2 nanobody:

[0102] The content of VEGFR2 in tissue proteins was detected by anti-VEGFR2 nanobody through Western blotting, and the band was clear and single, as Figure 4 shown; the localization of VEGFR2 in tissues could be clearly shown by immunohistochemistry, as Figure 5 shown; In summary, the VEGFR2 nanobody can be well applied to immunohistochemistry and Western blotting detection.

[0103] The above are only the preferred embodiments of the present invention, and do not impose any formal restrictions on the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. Use of an anti-VEGFR2 nanobody or its encoding nucleic acid in the preparation of an immunohistochemical reagent for detecting VEGFR2 protein, characterized in that, The amino acid sequence of the anti-VEGFR2 nanobody is shown in SEQ ID No.

1.

2. The application according to claim 1, characterized in that The nucleotide sequence of the nucleic acid encoding the anti-VEGFR2 nanobody is shown in SEQ ID No.

2.

3. Use of an anti-VEGFR2 nanobody or its encoding nucleic acid in the preparation of a Western Blotting detection reagent for detecting VEGFR2 protein, characterized in that, The amino acid sequence of the anti-VEGFR2 nanobody is shown in SEQ ID No.

1.

4. The application according to claim 3, characterized in that The nucleotide sequence of the nucleic acid encoding the anti-VEGFR2 nanobody is shown in SEQ ID No.2.

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