CN-116239686-B - CRP camel-derived single domain antibody and preparation method and application thereof

Abstract

The invention discloses a CRP camel-derived single domain antibody, a preparation method and application thereof, and relates to the field of biological medicine. And constructing a camel CRP antibody recombinant plasmid by sequencing structural analysis, cloning and transforming related antibodies in eukaryotic cells, screening to obtain monoclonal antibodies with human CRP resistance, cloning and expressing VHH structural domains of the monoclonal antibodies, and carrying out pairing screening to obtain the monoclonal antibodies with CRP-VHH of antibody activity. The antibody can be applied to ELISA kits, chemiluminescent kits and latex kits, has the same function as the existing kits, and has the application in preparing antitumor drugs.

Inventors

• HAO LIYING
• PENG YONG
• HAN HAOJIE
• Peng Yuelei
• CAO PEIJIE

Assignees

• 北京世纪沃德生物科技有限公司

Dates

Publication Date

20260505

Application Date

20211222

Claims (4)

1. 1. A CRP camel-derived single domain antibody is characterized by comprising a framework region and a complementarity determining region, wherein the complementarity determining region comprises a first complementarity determining region, a second complementarity determining region and a third complementarity determining region, the first complementarity determining region is the 28 th to 34 th amino acid region on the amino acid sequence SEQ ID NO:3, the second complementarity determining region is the 52 th to 58 th amino acid region on the amino acid sequence SEQ ID NO:3, and the third complementarity determining region is the 97 th to 106 th amino acid region on the amino acid sequence SEQ ID NO: 3.
2. 2. An ELISA kit comprising the single domain CRP camelid antibody of claim 1.
3. 3. A chemiluminescent kit comprising the single domain antibody of CRP camelid source of claim 1.
4. 4. A latex kit comprising the single domain CRP camelid antibody of claim 1.

Description

CRP camel-derived single domain antibody and preparation method and application thereof Technical Field The invention relates to the field of biological medicine, in particular to a CRP camel source single domain antibody, and a preparation method and application thereof. Background Immunoglobulin (Ig), an Immunoglobulin with antibody activity or similar chemical structure to an antibody molecule. Antisera can be immunologically bound to the antigen of interest, the primary reason being the presence of immunoglobulins in the antisera, which are the products of immune cells (except monoclonal antibodies), and the distribution of this material over the body fluids, exocrine fluids and the membranes of lymphocytes. Immunoglobulins have a number of components, mainly IgG, accounting for about 70% of the total, about 7-16g/L. Mammalian Ig molecules are symmetrical in structure and are composed of 4 peptide chains, and disulfide bonds are used for linking the peptide chains. As shown in FIG. 1, the heavy chain (HEAVY CHAIN, H chain) is two long chains, the light chain (LIGHT CHAIN, L chain) is two short chains, and the inter-chain disulfide bonds form 4 chains into a classical Y-type. Whether heavy or light, the amino acid sequence of the first domain has a variety, known as the variable region (variable region) of an immunoglobulin molecule that recognizes an antigen. The variable region is followed by a region of relatively fixed sequence, known as the constant region (constant region), each domain consisting of about 110 amino acid residues and having its unique function, except for the hinge region. The variable region of an immunoglobulin comprises four Framework Regions (FR) that provide a framework for the domain, a stable framework structure, and three complementarity determining regions (complementary determining region, CDRs) therebetween that form a complementary relationship with the antigen surface. The amino acid sequence of the CDR regions varies very much, also known as the hypervariable region (hypervariable regions). The constant region sequences are conserved in species compared to the variable regions, with 4 framework regions in each of VH and VL, denoted FR1, FR2, FR3 and FR4, respectively. 1993. In the years Hammers et al isolated a heavy chain antibody with a structure different from that of a conventional antibody from camel peripheral blood lymphocytes, the antibody had a naturally deleted light chain and a heavy chain variable region CH1, and a single domain antibody was obtained by PCR amplification of the variable region of the antibody. The molecular weight of the single domain antibody is only about 15 kDa, the molecular weight of the traditional conventional antibody is generally about 155 kDa, and the single domain antibody is also called a nano antibody (VHH, variable domain of HEAVY CHAIN of heavy-chainantibody) because the molecular weight of the single domain antibody is only about 1/10 of the molecular weight of the single domain antibody. Even though the antibody contains only heavy chains, it still has complete antigen binding properties. The VHH antibody not only has complete antigen affinity, but also has a plurality of advantages, such as that the CDR3 region of the VHH antibody has an exposed large convex ring (kept stable by disulfide bonds) and can penetrate into a slit or a cavity of an antigen, so that the binding capacity of the VHH antibody with the antigen is enhanced, and the VHH antibody has the advantages of small molecular weight, easiness in obtaining and expressing, high water solubility and stability, targeting property, simplicity in humanization and the like. The VHH antibody has good application prospect in the aspects of research tool carrier, disease diagnosis tool, disease treatment strategy and the like. C-reactive protein (CRP) is an acute phase reactive protein produced by the body in the event of inflammation, infection or injury, and when it increases, it suggests that the body is undergoing an inflammatory reaction. Human CRP performs important functions, such as participation in host defenses, antigen clearance, metabolism, etc., by virtue of its interaction with a range of exogenous or autologous factors. The C-reactive protein can interact with DNA and histones, which can scavenge nuclear material released by damaged circulating cells. In addition to the above functions and effects, CRP also has pathogenic effects. For example, an increase in CRP concentration has been shown to be closely related to coronary atherosclerotic disease. CRP is not only a marker of atherosclerotic disease, but is more likely to be involved directly in its formation, and CRP binds to oxidized LDL (low density lipoprotein) to exacerbate tissue damage in coronary infarction and inhibit repair of damaged vascular endothelium. Disclosure of Invention The invention aims to at least solve one of the technical problems in the prior art and provides a CRP camel source sin