Evolved immunoglobulin binding molecule, and its preparation method and uses

A technology of immunoglobulin and binding molecules, which is applied in biochemical equipment and methods, chemical equipment and methods, botanical equipment and methods, etc., can solve the problems of LD5 functional characteristics without further elaboration, and achieve improved sensitivity and accuracy , low cost, good effect

Inactive Publication Date: 2009-05-13
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Specifically, the single Ig-binding domains of SpA and PpL were cloned, the single-domain DNA fragments were randomly spliced ​​in vitro, and then these recombinant molecules were expressed on the surface of phage by phage display technology, and the single-domain random combination of SpA and PpL was constructed. Phage display library, using human Ig as bait to perform affinity screening on the library, the result is a new type of Ig composed of the B3 domain of PpL and the D, A, B, C (mainly D) domains of SpA arranged in intervals Binding molecular form (MDPL-MDPA)n(MDPL, mono-domain of protein L; MDPA, mono-domain of protein A), which does not exist in natural IBP and has not been reported in recombinant IBP molecules, therefore It is a new molecular structure form of IBP. Since it is obtained by means of molecular evolution, we collectively refer to these molecules as evolved Ig-binding molecules, and evolved Ig-binding molecules LD5 (L-D-L-D-L; L is the B3 domain of PpL, and D is the D domain of SpA) is the most representative dominant clone among them. Progress in Biochemistry, Volume 32, Issue 6, 2005, pages 535-543 have a detailed description, but this article does not further elaborate on the various functional properties of LD5

Method used

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  • Evolved immunoglobulin binding molecule, and its preparation method and uses
  • Evolved immunoglobulin binding molecule, and its preparation method and uses
  • Evolved immunoglobulin binding molecule, and its preparation method and uses

Examples

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Embodiment 1

[0139] Example 1 A method for prokaryotic preparation of a novel evolutionary immunoglobulin binding molecule

[0140] 1. cDNA sequence and cloning of new evolved immunoglobulin binding molecules

[0141] Recombinant phagemid vector pCANTAB5S-LD5, cloned with cDNA sequence encoding evolutionary immunoglobulin binding molecule LD5 (LDLDL; L is the B3 domain of PpL, D is the D domain of SpA), was obtained through molecular evolution screening and preparation The specific steps of pCANTAB5S-LD5 are described in "Phage Display SpA and PpL lg Binding Single Domain Random Combinatorial Library and Affinity Screening", Progress in Biophysics and Biochemistry, 2005 Vol. 32, No. 6, pages 535-543 Disclosure, that is, the 27# / 44# in the third round of screening and the 4# / 33# cloning vector in the fourth round of screening in Table 4 in the literature. The cDNA sequence is shown in SEQ ID No. 1, and the amino acid sequence is shown in SEQ ID No. .2 shown.

[0142] Evolutionary immunoglobulin...

Embodiment 2

[0174] Example 2 Binding of novel evolutionary immunoglobulin binding molecules to various antibodies and antibody fragments

[0175] 1. Combination with human polyclonal IgG:

[0176] 1.1 Human polyclonal IgG was purchased from Sigma, and PpL and SpA were purchased from Sigma.

[0177] 1.2 1mg / ml human polyclonal IgG was dialyzed with PBS (pH 7.2). Take 50μL of 3mg / ml long-arm activated biotin (purchased from PIERCE) and add 1mL IgG (1mg / mL). After shaking slightly at room temperature for 4h, add a dialysis bag and dialyze in PBS overnight at 4℃. After dialysis, add the same amount of glycerol to Store at -20°C.

[0178]1.3 After adjusting the concentration of LD5, PpL and SpA to 1mg / ml, the 96-well plate was coated with carbonate buffer (pH9.6) 1:200, and each protein was coated with 3 rows of multiple wells. After 24h at 4℃ Wash the plate with PBST for 4-5 times, then wash the plate with blocking solution (2% BSA 0.05% TWEEN-20) at 37°C for 1 hour. The biotin-labeled IgG starts...

Embodiment 3

[0201] Example 3 Confirmation of the dual-site binding mode of the new evolved immunoglobulin binding molecule κ light chain and VH3 heavy chain

[0202] SpA has a strong affinity with the Fc segment of IgG, and SpA can also bind to the VH region belonging to the VH3 gene family (SassoEH, Silvcrman GJ, Mannik M. Human 1gA and IgG F(ab')2 that bind to staphylococcalSpA belong to the VHIII subgroup. J Immunol. 1991:147:1877-1883). PpL binds to immunoglobulin by interacting with the light chain of Ig, mainly subtypes 1, 3, and 4 of the kappa light chain (NilSon BH, Solomon A, BjorckL, et al. PpL from peptostreptococcus magnus binds to the kappa light chain variabledomain; J Biol Chem. 1992; 267(4): 2234-9). From the binding experiments of LD5 and various Ig molecules, LD5 has a higher affinity for IgGFab IgM IgA than SpA and PpL. It is speculated that LD5 has dual-site binding properties for the variable regions of VH3 heavy chain and kappa light chain. The competitive inhibition tes...

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Abstract

The invention relataes to novel evolutionary immunoglobulin binding molecules, as well as their preparation methods and applications. The invention discloses separated evolutionary immunoglobulin binding molecules, which are proteins with amino acid sequences as shown by SEQ ID NO : 1, or conservative variant proteins with immunoglobulin binding activity. The invention also discloses the gene encoding, genetic engineering preparation methods and applications of immunoglobulin binding molecules. The disclosed immunoglobulin molecule broad-spectrum combined with various immuneglobulin shows high immunoglobulin whole molecule binding activity, and can be used in large-scale purification of genetic engineering antibodies, purification of natural antibodies and monoclonal antibodies, enzyme-linked immunosorbent assay, and immuno-chromatography and immunohistochemical methods for immune antibody detection and diagnosis.

Description

Technical field [0001] The present invention relates to recombinant immunoglobulin binding molecules, in particular to immunoglobulin binding molecules obtained by rearranging the Ig binding domains of Staphylococcus aureus protein and Peptostreptococcus surface protein, and its preparation method and application. Background technique [0002] Ig binding molecules (Ig binding proteins, IBP) are a class of proteins expressed on the surface of bacteria that can bind to specific parts of the host's Ig, regulate the host's immune response to bacteria, and are one of the important pathogenic factors of bacteria. There are three main types of IBP that have been studied the most: Staphylococcus aureus protein A (SpA), human streptococcus (group C and G) protein G (SpG), and part of the surface protein L (PpL) of Streptococcus macrocephalus. [0003] SpA has a molecular weight of about 57KDa and is composed of about 524 amino acids. Its extracellular part contains 5 Ig binding domains wi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/00C12N15/31C12N15/70C12N1/21G01N33/53G01N33/577
Inventor 潘卫蒋少华徐容贾建安沈毅珺杨华王锦红陈秋莉何俊陈璐
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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