CN113637078B - A nanobody against protein kinase p38δ, nucleic acid, expression vector, host cell and application thereof - Google Patents

Abstract

The invention provides a nanobody, nucleic acid, an expression vector, a host cell and application thereof aiming at protein kinase p38 delta, wherein the nanobody is provided with at least one complementarity determining region shown as the following: CDR1 of the amino acid sequence shown in SEQ ID No. 1; CDR2 of the amino acid sequence shown in SEQ ID No. 2; and CDR3, which is the complementary determining region of the amino acid sequence shown in SEQ ID NO. 3. The nano antibody has a unique antigen complementarity determining region aiming at the protein kinase p38 delta, shows high specific binding activity to the protein kinase p38 delta and can effectively inhibit the activity of the p38 delta protein kinase. And does not cross-react with other family proteins p38 alpha, p38 gamma and p38 delta of p38 MAPK.

Description

A nanobody, nucleic acid, expression vector, host cell against protein kinase p38δ cells and their applications

technical field

The present invention relates to the field of biotechnology, and particularly relates to a nanobody against protein kinase p38δ, nucleic acid, expression vector, host cell and application thereof.

Background technique

p38δ is a serine/threonine-specific protein kinase in the p38 MAPK family, which is mainly involved in the regulation of signal transduction between extracellular stimuli and intracellular responses. There are four members of the p38 MAPK family, namely p38α, p38β, p38γ and p38δ. Although all p38 MAPKs are highly conserved in sequence and structure, they function differently in physiological and pathological settings. Among them, p38α and p38β are widely expressed in various cells and tissues, regulating the basic life activities of cells. For example: p38α has high expression levels in leukocytes, liver, spleen, cerebellum, bone marrow, thyroid and placenta; p38β is mainly expressed in brain and heart. The expression of p38γ and p38δ is histiocyte-specific and strictly regulated, p38γ is mainly expressed in skeletal muscle; p38δ is mainly expressed in epidermal cells of lung, kidney, intestine, salivary gland, testis, ovary, adrenal gland and pituitary. In recent years, studies have revealed that p38δ plays an important regulatory function in the occurrence and development of various diseases, such as diabetes, immune diseases, neurodegenerative diseases, and cancer. p38δ is a potential disease diagnostic biomarker and therapeutic target. In terms of drug development, due to the high degree of conservation among p38 MAPKs, it is difficult to develop p38δ-specific inhibitors that do not cross-react with other p38 MAPK proteins, so there is no effective p38δ inhibitor yet.

Nanobodies are small-molecule antibodies derived from the variable region of heavy chain antibodies, with a molecular weight of about 15kDa, with high affinity, strong stability, good histocompatibility, and easy screening and preparation. Antibodies, clinical detection antibodies, and scientific research antibodies have been extensively researched and developed. Nanobodies are mainly formed by 4 conserved framework regions (FR) and 3 antigenic complementarity determining regions (CDRs) in series. Among them, the antigenic complementarity determining regions (CDRs) are the main execution sites of antigen recognition and antigen binding. Therefore, the key to the screening of nanobodies is to obtain the complementarity determining regions that can mediate specific binding to antigens. The complementarity determining regions in Nanobodies can be divided into three independent regions according to their different positions in the whole antibody, namely complementarity determining region 1 (CDR1), complementarity determining region 2 (CDR2) and complementarity determining region 3 (CDR3) . Compared with traditional antibodies, the amino acid sequence of the complementarity-determining regions of nanobodies is longer, which enables them to interact with antigens in the more hidden structures. Therefore, in addition to binding antigens, nanobodies can also be used as inhibitors and agonists to change the activity and function of antigens. Therefore, screening p38δ-specific nanobodies will provide a new strategy for the development of p38δ-targeted drugs.

SUMMARY OF THE INVENTION

The first object of the present invention is to provide a nanobody against protein kinase p38δ, the nanobody has a unique antigen complementarity determining region against protein kinase p38δ, shows highly specific binding activity to protein kinase p38δ, and can effectively inhibit Activity of p38delta protein kinase. And it does not cross-react with other family proteins p38α, p38β and p38δ of p38MAPK.

The second object of the present invention is to provide nucleic acid encoding the nanobody against protein kinase p38δ.

The third object of the present invention is to provide an expression vector containing the nucleic acid of the nanobody against protein kinase p38δ.

The fourth object of the present invention is to provide a host cell containing the expression vector.

The fifth object of the present invention is to provide the application of a nanobody against protein kinase p38δ in the preparation of a kit for detecting protein kinase p38δ.

The sixth object of the present invention is to provide the application of a nanobody against protein kinase p38δ in the preparation of a drug for inhibiting the activity of protein kinase p38δ.

The present invention solves its technical problems by adopting the following technical solutions.

The present invention provides a nanobody against protein kinase p38δ, the nanobody has at least one complementarity determining region shown in the following: the complementarity determining region CDR1 of the amino acid sequence shown in SEQ ID NO.1; as shown in SEQ ID NO. The complementarity determining region CDR2 of the amino acid sequence shown in 2; and the complementarity determining region CDR3 of the amino acid sequence shown in SEQ ID NO.3.

According to a preferred embodiment, the Nanobody has the amino acid sequence shown in SEQ ID NO.7.

The present invention also provides a nucleic acid encoding the nanobody against protein kinase p38δ, wherein the nucleic acid can encode at least one of the following amino acid sequences: the amino acid sequence shown in SEQ ID NO.1; the amino acid sequence shown in SEQ ID NO. .2 the amino acid sequence shown; and the amino acid sequence shown in SEQ ID NO.3.

According to a preferred embodiment, the nucleic acid has at least one of the following coding sequences: the coding sequence shown in SEQ ID NO.4; the coding sequence shown in SEQ ID NO.5; and the coding sequence shown in SEQ ID NO. 6 shows the coding sequence.

According to a preferred embodiment, the nucleic acid has the coding sequence shown in SEQ ID NO.8.

The present invention also provides an expression vector containing the nucleic acid of the nanobody against protein kinase p38δ, and the expression vector includes a prokaryotic expression vector, a eukaryotic expression vector or an in vitro expression vector system.

According to a preferred embodiment, the expression vector is pET22b.

The present invention also provides a host cell containing the expression vector, and the host cell includes prokaryotes or eukaryotes.

The present invention also provides the application of the nanobody against protein kinase p38δ in preparing a kit for detecting protein kinase p38δ.

The present invention also provides the application of the nanobody against protein kinase p38δ in the preparation of a medicament for inhibiting the activity of protein kinase p38δ, and the medicament includes an inhibitor or agonist of protein kinase p38δ activity.

Based on the above technical solutions, the nanobody, nucleic acid, expression vector, host cell and application of the present invention against protein kinase p38δ have at least the following technical effects:

The nanobody against protein kinase p38δ of the present invention has at least one of the following complementarity determining regions: the complementarity determining region CDR1 of the amino acid sequence shown in SEQ ID NO.1; the complementarity determining region of the amino acid sequence shown in SEQ ID NO.2 region CDR2; and the complementarity determining region CDR3 of the amino acid sequence shown in SEQ ID NO.3. The nanobody has a unique antigen complementarity determining region for protein kinase p38δ, shows highly specific binding activity to protein kinase p38δ, and does not cross-react with other family proteins p38α, p38β and p38γ of p38 MAPK. At the same time, the nanobody of the present application has outstanding thermal stability and acid-base stability. It provides a new direction for the detection of protein kinase p38δ and the treatment of diseases related to the regulation of protein kinase p38δ.

Description of drawings

In order to explain the embodiments of the present invention or the technical solutions in the prior art more clearly, the following briefly introduces the accompanying drawings that need to be used in the description of the embodiments or the prior art. Obviously, the accompanying drawings in the following description are only These are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can also be obtained according to these drawings without creative efforts.

Figure 1 is a schematic diagram of the expression and purification of p38γ and p38δ in Example 1 of the present invention, wherein Figure 1A shows the composition of the main elements of the expression vector pET28a-p38γ/p38δ-2×Strep; The expression strain of p38γ/p38δ-2×Strep was induced to express the protein and purified with MagStrep "type3" XT magnetic beads, and the expression and purified products were analyzed by SDS-PAGE protein electrophoresis;

Figure 2 is a schematic diagram of the stable binding of Nb13-6 to p38δ in Example 2 of the present invention, wherein Figure 2A shows the composition of the main components of the Nanobody expression vector pET22b-NB-FLAG; Control Nb is a control Nanobody containing the same backbone sequence; Figure 2 2B shows ELISA detection of the binding of Escherichia coli cellulite extract containing Nanobody expression vector to p38δ and control antigen BSA; Figure 2C shows co-immunoprecipitation detection of Escherichia coli cellulite extract containing Nanobody expression vector and The binding of p38δ; Figure 2D shows the indirect ELISA method to determine the dissociation constant K _D of Nanobody Nb13-6 and p38δ;

Fig. 3 shows the situation that Nb13-6 does not cross-react with other proteins of the p38 MAPK family in Example 3 of the present invention, wherein Fig. 3A shows the cross-reaction between Nb13-6 and p38 MAPK family proteins detected by ELISA; Fig. 3B shows the immunization Cross-reaction between Nb13-6 and p38 MAPK family proteins detected by co-precipitation;

Figure 4 shows the inhibitory ability of Nb13-6 on p38δ activity in Example 4 of the present invention, wherein Figure 4A shows that Nb13-6 inhibits the phosphorylation of p38δ on SQSTM1 protein; Figure 4B shows that Nb13-6 inhibits MDA-MB-231 , MCF7 cell proliferation.

Detailed ways

In order to make the objectives, technical solutions and advantages of the present invention clearer, the technical solutions of the present invention will be described in detail below. Obviously, the described embodiments are only some, but not all, embodiments of the present invention. Based on the embodiments of the present invention, all other implementations obtained by those of ordinary skill in the art without creative work fall within the protection scope of the present invention.

The nanobody against protein kinase p38δ of the present invention and its application will be specifically described below with reference to specific examples. The examples are only used to explain and understand the present invention, and are not used to limit the scope of the present invention.

The present invention proposes a nanobody against protein kinase p38δ, the nanobody has at least one complementarity determining region shown in the following: the complementarity determining region CDR1 of the amino acid sequence shown in SEQ ID NO.1; as shown in SEQ ID NO. .2 the complementarity determining region CDR2 of the amino acid sequence shown in SEQ ID NO.3; and the complementarity determining region CDR3 of the amino acid sequence shown in SEQ ID NO.3. It can be understood that the Nanobody of the present invention has three complementarity determining regions CDR1, CDR2 and CDR3, and at least one of the three complementarity determining regions satisfies: the complementarity determining region CDR1 has the amino acid shown in SEQ ID NO.1 sequence; the complementarity determining region CDR2 has the amino acid sequence shown in SEQ ID NO.2; or the complementarity determining region CDR3 has the amino acid sequence shown in SEQ ID NO.3. As a result, the nanobody has a unique antigenic complementarity determining region for the protein kinase p38δ, shows a highly specific binding activity to the protein kinase p38δ, can effectively inhibit the activity of the p38δ protein kinase, and does not interact with other family proteins of p38 MAPK p38α, p38α, p38β and p38γ cross-react.

Further preferably, the present invention also provides a nucleic acid encoding the above-mentioned Nanobody against protein kinase p38δ, the nucleic acid can encode at least one of the following amino acid sequences: the amino acid sequence shown in SEQ ID NO. The amino acid sequence shown in SEQ ID NO.2; and the amino acid sequence shown in SEQ ID NO.3. Preferably, the nucleic acid has at least one of the following coding sequences: the coding sequence shown in SEQ ID NO.4; the coding sequence shown in SEQ ID NO.5; and the coding sequence shown in SEQ ID NO.6 the coding sequence. In order to be able to encode at least one complementarity determining region of the Nanobody against the protein kinase p38δ of the present invention.

Further preferably, the present invention also provides an expression vector containing the nucleic acid of the nanobody against protein kinase p38δ, and the expression vector includes a prokaryotic expression vector, a eukaryotic expression vector or an in vitro expression vector system. Preferably, the expression vector is pET22b.

Further preferably, the present invention also provides a host cell containing the expression vector, and the host cell includes prokaryotes or eukaryotes. Other in vitro expression systems comprising the expression vector may also be employed.

Further preferably, the present invention also provides the application of the nanobody against protein kinase p38δ in the preparation of a detection kit for detecting protein kinase p38δ.

Further preferably, the present invention also provides the application of the nanobody against protein kinase p38δ in the preparation of a drug for inhibiting the activity of protein kinase p38δ.

The features and performances of the present invention will be further described in detail below in conjunction with the embodiments.

Example 1

This Example 1 provides the screening of Nanobodies against the protein kinase p38δ. Specific steps are as follows:

蛋白纯化系统构建p38δ、p38γ表达载体，如图1A。其中p38δ氨基酸序列如SEQ ID NO.9所示，p38γ氨基酸序列如SEQ ID NO.10所示；p38δDNA编码序列如SEQ ID NO.11所示，p38γDNA编码序列如SEQ ID NO.12所示；2×Strep标签氨基酸序列如SEQ ID NO.13所示，2×Strep标签DNA编码序列如SEQ ID NO.14所示。将构建好的表达载体转化至大肠杆菌表达细菌BL21(DE3)中，通过IPTG诱导其表达，利用MagStrep“type3”XT磁珠进行蛋白纯化，利用SDS-PAGE及考马斯亮蓝染色检测表达及纯化产物，最终获得高纯度p38δ和p38γ蛋白，如图1B。(1) Based on the third generation developed by IBA

The protein purification system constructs p38δ and p38γ expression vectors, as shown in Figure 1A. The p38δ amino acid sequence is shown in SEQ ID NO.9, the p38γ amino acid sequence is shown in SEQ ID NO.10; the p38δ DNA coding sequence is shown in SEQ ID NO.11, and the p38γ DNA coding sequence is shown in SEQ ID NO.12; 2 The amino acid sequence of the ×Strep tag is shown in SEQ ID NO.13, and the DNA coding sequence of the 2×Strep tag is shown in SEQ ID NO.14. The constructed expression vector was transformed into E. coli expressing bacteria BL21(DE3), its expression was induced by IPTG, protein purification was carried out using MagStrep "type3" XT magnetic beads, and the expression and purified products were detected by SDS-PAGE and Coomassie brilliant blue staining. , and finally obtained high-purity p38δ and p38γ proteins, as shown in Figure 1B.

(2) The purified p38δ and p38γ proteins were coated on magnetic beads respectively; the nanobody phage library was diluted into a solution containing 2.5% BSA TBST; ."Identification of nanobodies against hepatocellular carcinoma markerglypican-3." Molecular Immunology 131(2021):13-22.) Incubate with p38γ-coated magnetic beads for 1 hour at room temperature to remove non-specifically bound phage; The phage library solution was again incubated with p38δ-coated magnetic beads for 2 hours at room temperature, then the phage solution was discarded, the magnetic beads were washed 20 times with TBST and 10 times with PBS, and then the magnetic beads were incubated with 100 μL of eluent (Triethylamine, 0.1 M). The beads were incubated for 10 min, and finally the eluate was neutralized with 100 μL of 1 M Tris-HCl solution. The eluate was added to 1 ml of E. coli SS320 and incubated at 37°C for 30 min. The phage was amplified by the helper phage M13K07. Finally, the purified phage was collected and used for the next round of screening.

(3) After completing the third round of screening phage infection, E. coli SS320 was plated, and 40 single clones containing phage plasmids were picked for sequencing. According to the sequencing results, monoclonal clones with higher repeat rate were selected for nanobody expression and identification.

Example 2

This example provides methods for the identification of Nanobodies.

(1) The clone Nb13-6 obtained in Example 1 was selected, its DNA sequence is shown in SEQ ID NO.8, and its encoded amino acid sequence is shown in SEQ ID NO.7. The nucleotide sequence encoding the Nanobody was subcloned into the expression vector pET22b by molecular cloning, and the FLAG tag sequence was fused to its C-terminus, as shown in Figure 2A. The control Nanobody expression vector was constructed in the same way.

(2) The constructed nanobody expression vector was transformed into Escherichia coli expression strain BL21 (DE3), the expression was induced by IPTG, and then the bacterial cell peritoneal protein was extracted by hypotonicity.

(3) ELISA to detect the binding of peritope protein and p38δ in bacterial cells expressing nanobody:

The p38δ and BSA proteins were coated on a 96-well microtiter plate. After blocking with the blocking solution, the nanobody bacterial cells were incubated at 37°C for 2 hours, and then the anti-FALG mouse monoclonal antibody was incubated at 37°C for 1 hour. The secondary antibody was incubated at 37°C for 1 hour, and the color reaction and termination reaction were carried out according to the ELISA kit (Solebo), and the absorbance value (OD450) at 450 nM of the reaction solution was finally detected, as shown in Figure 2B. The result analysis showed that Nb13-6 Binds specifically to p38δ.

(4) Co-immunoprecipitation to detect the binding of nanobody Nb13-6 to p38δ:

Nanobody bacterial cell periplasmic extract was incubated with p38δ expressing bacterial lysate for 2 hours at 4°C, then 10 μL anti-FLAG magnetic beads were added and incubated at 4°C for another 1 hour. After washing the magnetic beads with TBST for 3 times, SDS-PAGE and Coomassie brilliant blue Co-immunoprecipitation results were detected by staining. As shown in Figure 2C, the results showed that Nb13-6 co-precipitated p38δ.

(5) Indirect ELISA method to measure the dissociation constant K _D of nanobody Nb13-6 and p38δ:

The p38δ and BSA proteins were coated on a 96-well microtiter plate, and after blocking with the blocking solution, the nanobodies were prepared at concentrations of 0 μM, 0.1 μM, 0.2 μM, 0.4 μM, 0.8 μM, 1.6 μM, 3.2 μM, and 6.4 μM at 37°C. The antigen was incubated for 2 hours, then the anti-FALG mouse monoclonal antibody was incubated at 37°C for 1 hour, and then the HRP-labeled mouse secondary antibody was incubated at 37°C for 1 hour. The 450nM absorbance (OD450) of the reaction solution was detected, a 4-parameter curve was fitted by Graphpad Prism software, and the dissociation constant K _D was calculated. As shown in Fig. 2D, the result analysis shows that the dissociation constant K _D =(4.99±0.10)×10 ^-7 M of Nb13-6 and p38δ.

Example 3

In this Example 3, the cross-reaction of Nb13-6 with other p38 MAPK family proteins was detected.

(1) ELISA to detect the cross-reaction of Nb13-6 with other family proteins of p38 MAPK: p38α, p38β, p38γ and p38δ proteins were coated on a 96-well microtiter plate, and after blocking with the blocking solution, the nanobody bacterial cells were peri-extracted to 37°C. Incubate for 2 hours, then incubate with anti-FALG mouse monoclonal antibody at 37°C for 1 hour, and then incubate with HRP-labeled mouse secondary antibody at 37°C for 1 hour, carry out color reaction and termination reaction according to ELISA kit (Solebo), and finally detect the reaction Absorbance at 450 nM (OD450) of the solution. As shown in Figure 3A, the analysis of the results showed that Nb13-6 specifically bound to p38δ, and did not cross-react with p38α, p38β, and p38γ.

(2) Co-immunoprecipitation to detect the cross-reaction of Nb13-6 with other p38 MAPK family proteins: Nanobody Nb13-6 was incubated with p38α, p38β, p38γ and p38δ expressing bacterial lysates for 2 hours at 4°C, and then 10 μL of MagStrep “type3 "XT magnetic beads were incubated at 4°C for another 1 hour. After washing the beads with TBST three times, the co-immunoprecipitation results were detected by SDS-PAGE and Coomassie brilliant blue staining. As shown in Figure 3B, the results showed that Nb13-6 specifically co-precipitated p38δ, and did not cross-react with p38α, p38β, and p38γ.

Example 4

It has been confirmed that the activation of p38δ drives the proliferation of breast cancer cells. This example uses this biological phenomenon to evaluate the effect of Nb13-6 on p38δ protein kinase activity.

First, the above nanobody expression plasmid was expressed in breast cancer cells MDA-MB-231 cells by transfection method. After 48 hours of transfection, the cell viability was detected by CCK-8 method, and then the cell growth was evaluated. As shown in Figure 4A, the results showed that Nb13-6 significantly inhibited the growth of MDA-MB-231 cells compared with the control Nanobody.

Second, the clonogenic ability of the cells was assayed using plate clonogenicity. MDA-MB-231 cells and MCF7 cells were seeded into 6-well plates at 5000 cells per well, and then the control Nanobody or Nb13-1 was overexpressed by transient transfection. After 7 days of cell growth, cells were fixed with methanol and stained with crystal violet. As shown in Figure 4B, the results showed that Nb13-6 significantly reduced the clonogenic ability of MDA-MB-231 and MCF7 cells compared with the control Nanobody.

In summary, the nanobody against protein kinase p38δ of the present invention has a unique antigenic complementarity determining region against protein kinase p38δ, shows highly specific binding activity to protein kinase p38δ, can effectively inhibit the activity of p38δ, and does not interact with the protein kinase p38δ. The other family proteins p38α, p38β and p38δ of p38 MAPK cross-react.

The above are only specific embodiments of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art can easily think of changes or substitutions within the technical scope disclosed by the present invention. should be included within the protection scope of the present invention.

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catgagaacg tcattgggct cctggatgtc ttcaccccag cctcctccct gcgcaacttc 300

tatgacttct acctggtgat gcccttcatg cagacggatc tgcagaagat catggggatg 360

gagttcagtg aggagaagat ccagtacctg gtgtatcaga tgctcaaagg ccttaagtac 420

atccactctg ctggggtcgt gcacagggac ctgaagccag gcaacctggc tgtgaatgag 480

gactgtgaac tgaagattct ggattttggg ctggcgcgac atgcagacgc cgagatgact 540

ggctacgtgg tgacccgctg gtaccgagcc cccgaggtga tcctcagctg gatgcactac 600

aaccagacag tggacatctg gtctgtgggc tgtatcatgg cagagatgct gacagggaaa 660

actctgttca aggggaaaga ttacctggac cagctgaccc agatcctgaa agtgaccggg 720

gtgcctggca cggagttttgt gcagaagctg aacgacaaag cggccaaatc ctacatccag 780

tccctgccac agacccccag gaaggatttc actcagctgt tcccacgggc cagcccccag 840

gctgcggacc tgctggagaa gatgctggag ctagacgtgg acaagcgcct gacggccgcg 900

caggccctca cccatccctt ctttgaaccc ttccgggacc ctgaggaaga gacggaggcc 960

cagcagccgt ttgatgattc cttagaacac gagaaactca cagtggatga atggaagcag 1020

cacatctaca aggagattgt gaacttcagc cccattgccc ggaaggactc acggcgccgg 1080

agtggcatga agctg 1095

<210> 12

<211> 1101

<212> DNA

<213> Homo sapiens

<400> 12

atgagctctc cgccgcccgc ccgcagtggc ttttaccgcc aggaggtgac caagacggcc 60

tgggaggtgc gcgccgtgta ccgggacctg cagcccgtgg gctcgggcgc ctacggcgcg 120

gtgtgctcgg ccgtggacgg ccgcaccggc gctaaggtgg ccatcaagaa gctgtatcgg 180

cctttccagt ccgagctgtt cgccaagcgc gcctaccgcg agctgcgcct gctcaagcac 240

atgcgccacg agaacgtgat cgggctgctg gacgtattca ctcctgatga gaccctggat 300

gacttcacgg acttttacct ggtgatgccg ttcatgggca ccgacctggg caagctcatg 360

aaacatgaga agctaggcga ggaccggatc cagttcctcg tgtaccagat gctgaagggg 420

ctgaggtata tccacgctgc cggcatcatc cacagagacc tgaagcccgg caacctggct 480

gtgaacgaag actgtgagct gaagatcctg gacttcggcc tggccaggca ggcagacagt 540

gagatgactg ggtacgtggt gacccggtgg taccgggctc ccgaggtcat cttgaattgg 600

atgcgctaca cgcagacggt ggacatctgg tctgtgggct gcatcatggc ggagatgatc 660

acaggcaaga cgctgttcaa gggcagcgac cacctggacc agctgaagga gatcatgaag 720

gtgacgggga cgcctccggc tgagtttgtg cagcggctgc agagcgatga ggccaagaac 780

tacatgaagg gcctccccga attggagaag aaggattttg cctctatcct gaccaatgca 840

agccctctgg ctgtgaacct cctggagaag atgctggtgc tggacgcgga gcagcgggtg 900

acggcaggcg aggcgctggc ccatccctac ttcgagtccc tgcacgacac ggaagatgag 960

ccccaggtcc agaagtatga tgactccttt gacgacgttg accgcacact ggatgaatgg 1020

aagcgtgtta cttacaaaga ggtgctcagc ttcaagcctc cccggcagct gggggccagg 1080

gtctccaagg agacgcctct g 1101

<210> 13

<211> 30

<212> PRT

<213> Artificial Sequence

<400> 13

Gly Ser Trp Ser His Pro Gln Phe Glu Lys Gly Gly Gly Gly Ser Gly Gly

1 5 10 15

Gly Ser Gly Gly Ser Ala Trp Ser His Pro Gln Phe Glu Lys

20 25 30

<210> 14

<211> 93

<212> DNA

<213> Artificial Sequence

<400> 14

ggatcctgga gccacccgca gttcgagaaa ggtggaggtt ccggaggtgg atcgggaggt 60

tcggcgtgga gccacccgca gttcgaaaaa tga 93

Claims (10)

1. A nanobody that specifically binds to protein kinase p38δ, wherein the nanobody has the complementarity determining region CDR1 of the amino acid sequence shown in SEQ ID NO.1, the complementarity of the amino acid sequence shown in SEQ ID NO.2 The determining region CDR2 and the complementarity determining region CDR3 of the amino acid sequence shown in SEQ ID NO.3. 2 . The nanobody specifically binding to protein kinase p38δ according to claim 1 , wherein the nanobody has the amino acid sequence shown in SEQ ID NO.7. 3 . 3. A nucleic acid encoding the nanobody of claim 1 or 2 that specifically binds to protein kinase p38δ, characterized in that the nucleic acid can encode the amino acid sequence shown in SEQ ID NO.1, such as SEQ ID NO. The amino acid sequence shown in 2 and the amino acid sequence shown in SEQ ID NO.3. 4. The nucleic acid according to claim 3, wherein the nucleic acid has the coding sequence shown in SEQ ID NO.4, the coding sequence shown in SEQ ID NO.5 and the coding sequence shown in SEQ ID NO.6 the coding sequence shown. 5. The nucleic acid according to claim 4, wherein the nucleic acid has the coding sequence shown in SEQ ID NO.8. 6. An expression vector containing the nucleic acid encoding the Nanobody specifically binding to protein kinase p38δ according to any one of claims 3 to 5, wherein the expression vector comprises a prokaryotic expression vector, a eukaryotic expression vector vector or in vitro expression vector system. 7. The expression vector according to claim 6, wherein the expression vector is pET22b. 8. A host cell containing the expression vector of claim 6 or 7, wherein the host cell comprises a prokaryotic cell or a eukaryotic cell. 9 . The application of the nanobody specifically binding to protein kinase p38δ according to claim 1 in the preparation of a kit for detecting protein kinase p38δ. 10 . 10 . The application of the nanobody specifically binding to protein kinase p38δ according to claim 1 in the preparation of a medicament for inhibiting the activity of protein kinase p38δ, wherein the medicament comprises an inhibitor or agonist of protein kinase p38δ activity. 11 .

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