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Evolved immunoglobulin binding molecule D-C-G3 and preparation method and application thereof

A technology of D-C-G3 and immunoglobulin, applied in the field of protein, can solve the problems of high production cost, broad spectrum of IBPs and low binding force, and achieve the effect of improving sensitivity and accuracy, low cost and good effect

Inactive Publication Date: 2015-12-16
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the broad spectrum and binding capacity of existing IBPs are still not high, and the production cost remains high

Method used

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  • Evolved immunoglobulin binding molecule D-C-G3 and preparation method and application thereof
  • Evolved immunoglobulin binding molecule D-C-G3 and preparation method and application thereof
  • Evolved immunoglobulin binding molecule D-C-G3 and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 Obtaining D-C-G3 molecules using molecular evolution methods

[0033] That is, a random combinatorial library of 7 single-binding domains including A, B, C, D, and E of SpA and B2 and B3 of SpG was constructed by phage display. Mouse IgG1, 2a, 2b, 3 and 8 kinds of IgG including human IgG, rabbit IgG, bovine IgG, and goat IgG were used as inducing molecules. Through the repeated process of "adsorption"-"elution"-"amplification", the The library undergoes 3-4 rounds of screening, and the positive clones that appear in each round of screening are sequenced to obtain novel combined sequences with advantageous binding properties.

[0034] 1. Construction of phage display library with seven single-domain random combinations of SpA and SpG

[0035]Using pCANTAB5S-SpA phagemid as a template, using uA and dAD, uB and dB, uC and dC, uD and dAD, uE and dE as upstream and downstream primers, PCR amplified A, B, C, D, E of SpA Five-domain fragments; using uG2 and dG2 as ...

Embodiment 2

[0045] For the construction procedure of the prokaryotic expression vector pET32a(+)-D-C-G3, see image 3 .

[0046] 2.1 Primer synthesis

[0047] The primers used for PCR amplification of the cDNA sequence of the evolved immunoglobulin binding molecule D-C-G3 were: upstream 5SNco-u: 5’-GCGCCCATGGGGTCTAGAGCTGATGCGCAAC-3’, downstream 5SSac-d: 5’-GCGCCTCGAGTTATTCTAGACTGCTGGTGTTCGG-3’. The primer DNA sequence was synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd.

[0048] 2.2 PCR amplification of the expression sequence of D-C-G3

[0049] Using pCANTAB5S-D-C-G3 as a template, use the synthetic upstream and downstream primers to amplify, the reaction volume of PCR is 50 μl, add 5 ng of plasmid template, 1 μmol of upstream and downstream primers, 100 μmol of dNTP, Mg 2+ 3mmol, Taq enzyme 1U, add ddH 2 0 to 50 μl. Reaction conditions: 94°C for 30s-60°C for 30s-72°C for 45s; 35 cycles, 72°C extension for 5 minutes before the end of the reaction, the produ...

Embodiment 3

[0065] Example 3 Analysis of the binding characteristics of the novel immunoglobulin evolution molecule D-C-G3 to the four subclasses of mouse IgG, human IgG, rabbit IgG, bovine IgG and goat IgG

[0066] 1. Binding to the four subclasses of mouse IgG:

[0067] 1.1 Four subclasses of mouse IgG were purchased from Sigma, and SpA and SpG were purchased from Sigma.

[0068] 1.21 mg / ml human polyclonal IgG was dialyzed against PBS (pH 7.2). Take 50 μL of 3 mg / ml long-arm activated biotin (purchased from PIERCE company) and add 1 mL IgG (1 mg / mL), shake slightly at room temperature for 4 hours, then add a dialysis bag and dialyze overnight in PBS at 4 ° C. After the dialysis, add an equal amount of glycerol to - Store at 20°C.

[0069] 1. Adjust the concentration of 3D-C-G3, SpA and SpG to 1 mg / ml, and then dilute it with carbonate buffer (pH9.6) 1:200 to coat a 96-well plate. Each protein is coated with 3 rows of duplicate wells, at 4°C After 24 hours, wash with PBST 4-5 times, ...

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PUM

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Abstract

The invention provides an evolved immunoglobulin binding molecule D-C-G3 characterized by having the amino acid sequence shown as SEQ ID NO:1. The invention also provides a cDNA sequence of the evolved immunoglobulin binding molecule D-C-G3 the code of which is shown as claim 1. The cDNA sequence is characterized in that the cDNA sequence is shown as SEQ ID NO:2. Moreover, the invention provides a preparation method of the evolved immunoglobulin binding molecule D-C-G3 as shown in claim 1. The evolved immunoglobulin binding molecule disclosed by the invention can bind total IgG of human and a plurality of animals and IgG of different subclasses in a broad-spectrum manner, and the bonding force of the evolved immunoglobulin binding molecule is higher than that of the immunoglobulin binding molecule compared with the prior art.

Description

technical field [0001] The invention relates to an evolved immunoglobulin binding molecule D-C-G3, which belongs to the protein field. Background technique [0002] Bacterial immunoglobulin (Ig)-binding proteins (IBPs) are a class of bacterial immunoglobulin binding proteins produced by bacteria that specifically bind to host antibodies, and are one of the important pathogenic factors of bacteria. The most studied IBPs are Staphylococcus aureus protein A (StaphylococcalproteinA, SpA), Streptococcus (group C and G) protein G (StreptococcalproteinG, SpG) and part of large Peptostreptococcus surface protein L (PeptostreptococcusmagnusproteinL, PpL). Studies have shown that bacterial IBPs all contain an antibody-binding region consisting of multiple highly homologous binding domains head-to-tail repeated. Each single domain has exactly the same binding properties as the whole molecule, forming the basic Ig binding function. unit. [0003] SpA contains five highly homologous re...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/70G01N33/68C07K16/00C07K1/14
Inventor 潘卫祁培培曹洁王锦红陈秋莉廖文婷张华群何婷丁莹莹
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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