MMP-9 nano antibody as well as preparation method and application thereof

A technology of MMP-9 and nano-antibody, which is applied in the field of biomedicine, can solve the problems of poor specific binding ability, weak stability and side effects, etc., and achieve the effect of improving affinity, simple and convenient preparation method, and small molecular weight

Inactive Publication Date: 2021-11-26
SHENZHEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of this application is to provide a MMP-9 nanobody and its preparation method and application, aiming to solve the problems of poor specific binding ability of MMP-9 inhibitors in the prior art, weak stability and relatively large side effects

Method used

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  • MMP-9 nano antibody as well as preparation method and application thereof
  • MMP-9 nano antibody as well as preparation method and application thereof
  • MMP-9 nano antibody as well as preparation method and application thereof

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preparation example Construction

[0069] The second aspect of the embodiment of the present application provides a method for preparing MMP-9 Nanobody, comprising the following steps:

[0070] S01. Design the amino acid mutation sites of CDR1, CDR2 and CDR3 in the CDR region and synthesize the CDRs mutation primers through a large number of sequencing and comparative analysis of multiple natural libraries;

[0071] S02. Provide the synthesized Nanobody template sequence, perform codon optimization on the Nanobody template sequence and introduce a restriction enzyme cleavage site to obtain an optimized Nanobody template sequence;

[0072] S03. Insert the optimized nanobody template sequence into the phagemid carrier to construct a recombinant phagemid, perform phage display on the recombinant phagemid and extract single-stranded DNA, and use the Kunkel mutation method to pair CDRs mutation primers with single-stranded DNA to synthesize heterologous double-stranded DNA, and transformed into Escherichia coli to o...

Embodiment 1

[0111] MMP-9 nanobody and preparation method thereof

[0112] MMP-9 nanobody

[0113] The complete amino acid sequence of the MMP-9 Nanobody G3CC is shown in SEQ.ID No.39, which is:

[0114] MKKTAIAIAVALAGFATVAQAAQVQLQESGGGLVQAGGSLRLSCAASGNIFRYRSMGWYRQAPGKERELVAGISRGTTTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAVYRNQRVYGRGPVLRPLKYWGQGTQVTVSSGQAGQHHHHHHGAYPYDVPDYAS*.

[0115] Preparation method of MMP-9 nanobody

[0116] The preparation method of MMP-9 nanobody comprises the following steps:

[0117] S01. Design the amino acid mutation sites of CDR1, CDR2 and CDR3 in the CDR region and synthesize the CDRs mutation primers through a large number of sequencing comparison analysis of multiple natural libraries, wherein the mutation primers of CDR1, CDR2 and CDR3 are as follows;

[0118]

[0119] S02. Provide the synthesized Nanobody template sequence, perform codon optimization on the Nanobody template sequence and introduce a restriction endonuclease cutting site to obtain an o...

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Abstract

The invention relates to the technical field of biological medicines, and provides an MMP-9 nano antibody. The MMP-9 nano antibody is a single-domain antibody, the single-domain antibody comprises a complementary determining region CDR, the complementary determining region comprises CDR1, CDR2 and CDR3, and a specific amino acid sequence capable of being selected by the CDR1, the CDR2 and the CDR3 is provided, wherein the obtained MMP-9 nano antibody is the single-domain antibody which specifically aims at an active site of an MMP-9 catalytic domain, the CDR3 region is lengthened, the affinity to the MMP-9 catalytic domain can be improved, so that the nano antibody can be specifically and powerfully combined with the active site of the catalytic domain, the molecular weight is small, the penetrating power is high, the stability is high, the side effect is low, the nano antibody can be expressed in a large scale, so that the nano antibody can well inhibit the activity of MMP-9, and then has a good effect of inhibiting breast cancer tumor metastasis.

Description

technical field [0001] The application belongs to the technical field of biomedicine, and in particular relates to a MMP-9 nanobody and its preparation method and application. Background technique [0002] Matrix metalloproteinases (Matrix metalloproteins, MMPs), a class of calcium-dependent zinc-containing endopeptidases composed of 28 family members, can play a key role in the conversion process of ECM by cleaving almost all components in the extracellular matrix effect. [0003] Among them, matrix metalloproteinase 9 (Matrix metalloprotein 9, MMP-9) is one of the important members of the MMPs family, with a molecular weight of 92kDa, and belongs to a kind of gelatinase. Stability-related functions, and the expression of MMP-9 mainly occurs in neutrophils, monocytes, macrophages, microglia, lymphocytes, dendritic cells, fibroblasts, vascular endothelial cells, keratinocytes cells and tumor cells. At present, the role of MMP-9 in angiogenesis, tumor metastasis and invasi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/40C12N15/13C12N15/70A61K39/395A61K45/06A61P35/04A61P35/00
CPCC07K16/40C07K16/005C12N15/70A61K39/3955A61K45/06A61P35/04A61P35/00C07K2317/569C07K2317/565C07K2317/567C07K2317/73C07K2317/76C07K2317/92C12N2800/22C12N2800/101A61K2039/505A61K2300/00
Inventor 叶亮叶其状黄钟吴丹丹郑厚胜
Owner SHENZHEN UNIV
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