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Nanometer antibody specifically binding to protein kinase p38delta, nucleic acid, expression vector, host cell and application thereof

A technology for binding proteins and nanobodies, applied in the fields of antibodies, applications, immunoglobulins, etc., can solve the problems of no p38δ inhibitors and difficulties, and achieve outstanding thermal stability, acid-base stability, and high specific binding activity Effect

Active Publication Date: 2021-10-15
WEST CHINA HOSPITAL SICHUAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In terms of drug development, due to the high degree of conservation among p38 MAPKs proteins, 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 so far

Method used

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  • Nanometer antibody specifically binding to protein kinase p38delta, nucleic acid, expression vector, host cell and application thereof
  • Nanometer antibody specifically binding to protein kinase p38delta, nucleic acid, expression vector, host cell and application thereof
  • Nanometer antibody specifically binding to protein kinase p38delta, nucleic acid, expression vector, host cell and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] This Example 1 provides a screen for Nanobodies that specifically bind the protein kinase p38δ. Specific steps are as follows:

[0039] (1) According to the third generation developed by IBA company Protein purification system to construct p38δ, p38γ expression vectors, such as figure 1 a. The amino acid sequence of p38δ is shown in SEQ ID NO.9, the amino acid sequence of p38γ is shown in SEQ ID NO.10; the coding sequence of p38δDNA is shown in SEQ ID NO.11, and the coding sequence of p38γDNA is shown in SEQ ID NO.12. The amino acid sequence of the 2×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 Escherichia coli expression bacteria BL21(DE3), its expression was induced by IPTG, the protein was purified using MagStrep "type3" XT magnetic beads, and the expressed and purified product was detected by SDS-PAGE and Coomassie brilliant blue staining , and...

Embodiment 2

[0043] This example provides methods for the identification of Nanobodies.

[0044] (1) Select the clone Nb13-1 obtained in Example 1, 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 at its C-terminus, such as figure 2 As shown in A, the control Nanobody expression vector was constructed in the same way.

[0045] (2) The constructed Nanobody expression vector was transformed into Escherichia coli expression strain BL21(DE3), the expression was induced by IPTG, and then the pericellular protein was extracted by low-osmotic hair.

[0046] (3) ELISA detection of the binding of bacterial cell periplasmic protein expressing Nanobody to p38δ:

[0047] Coat p38δ and BSA proteins to 96-well ELISA plates, and after blocking with blocking solution, incubate the nanobody bacterial ...

Embodiment 3

[0053] In Example 3, the cross-reactivity between Nb13-1 and other p38 MAPK family proteins was detected.

[0054] (1) ELISA detection of cross-reaction between Nb13-1 and other p38 MAPK family proteins: Coat p38α, p38β, p38γ and p38δ proteins to 96-well microtiter plates, after blocking with blocking solution, nanobody bacterial cells were surrounded by extracts at 37°C Incubate for 2 hours, then incubate with anti-FALG mouse monoclonal antibody at 37°C for 1 hour, then incubate with HRP-labeled mouse secondary antibody at 37°C for 1 hour, carry out color reaction and stop reaction according to ELISA kit (Solebo), and finally detect the reaction The 450nM absorbance value (OD450) of the solution, such as image 3 As shown in A, the result analysis shows that Nb13-1 specifically binds to p38δ, and does not cross-react with p38α, p38β, and p38γ.

[0055] (2) Co-immunoprecipitation detection of cross-reactivity between Nb13-1 and other p38 MAPK family proteins: Nanobody bacteri...

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Abstract

The invention provides a nanometer antibody specifically binding to protein kinase p38delta, nucleic acid, an expression vector, a host cell and application thereof. The nanometer antibody has at least one of the following complementarity determining regions: a complementarity determining region CDR1 of an amino acid sequence as shown in SEQ ID NO.1; a complementarity determining region CDR2 of an amino acid sequence as shown in SEQ ID NO.2; and a complementarity determining region CDR3 of an amino acid sequence as shown in SEQ ID NO.3. The nanometer antibody has a unique antigen complementarity determining region aiming at the protein kinase p38delta, shows high specific binding activity to the protein kinase p38delta, and does not have cross reaction with other family proteins p38alpha, p38gamma and p38delta of p38MAPK. Meanwhile, the nanometer antibody provided by the invention has outstanding thermal stability and acid-base stability.

Description

technical field [0001] The invention relates to the field of biotechnology, and in particular to a nanobody specifically binding to protein kinase p38δ, nucleic acid, expression vector, host cell and application thereof. Background technique [0002] 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. The p38 MAPK family has four members, namely p38α, p38β, p38γ and p38δ. Although all p38 MAPKs are highly conserved in sequence and structure, their functions in physiological and pathological environments are not the same. Among them, p38α and p38β are widely expressed in various cells and tissues, and regulate the basic life activities of cells. For example: p38α has a higher expression level in leukocytes, liver, spleen, cerebellum, bone marrow, thyroid and placenta; p38β is mainly expressed in the brain and heart. The express...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/40C12N15/13C12N15/70C12N1/21G01N33/573A61K39/395A61P3/10A61P37/00A61P25/00A61P35/00
CPCC07K16/40C12N15/70G01N33/573A61P3/10A61P37/00A61P25/00A61P35/00C07K2317/569C07K2317/565C07K2317/33C07K2317/94G01N2333/912A61K2039/505
Inventor 张臣良毕锋
Owner WEST CHINA HOSPITAL SICHUAN UNIV
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