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Multifunctional protein molecule switch used for antibody detection

A molecular switch and protein molecular technology, applied in the field of protein detection, can solve the problems that have not yet been reported on Nanoluc luciferase detection antibody

Active Publication Date: 2021-04-23
CHONGQING MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] At present, there is no report on the use of different fragments of Nanoluc luciferase for the detection of antibodies

Method used

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  • Multifunctional protein molecule switch used for antibody detection
  • Multifunctional protein molecule switch used for antibody detection
  • Multifunctional protein molecule switch used for antibody detection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Example 1 A NanoSwitch molecular switch for Flag antibody detection

[0064] 1. Principle

[0065] This example takes Flag antibody as an example to demonstrate the basic design of the present invention. Its design idea and working principle are as follows:

[0066] (1) SmBiT is fused to the N-terminus of LgBiT; (2) A polypeptide capable of binding to an antibody such as 3×flag is inserted between LgBiT and SmBiT ( figure 2 A); (3) When no antibody exists, SmBiT will be able to bind to the corresponding position of LgBiT, and NanoSwitch is in a fully active state; (4) When specific antibodies exist and bind to specific polypeptides in NanoSwitch , causing steric hindrance and allosteric effects, leading to the combination barrier between SmBiT and LgBiT, at this time the NanoSwitch will be in a partially inactive state, which is manifested by the decrease of the optical signal after adding the substrate ( figure 2 A), so this signal change can be used to reflect th...

Embodiment 2

[0087] Improvement of Embodiment 2 NanoSwitch-3×flag

[0088] The purpose of this embodiment is to provide three improvement schemes on the basis of Embodiment 1.

[0089] Improvement 1: Place three 1×flags at the N-terminal of SmBiT, between SmBiT and LgBiT, and at the C-terminal of LgBiT (NanoSwitch-1×flag-3);

[0090] Improvement scheme 2, on the basis of improvement scheme 1, only keep the first two 1×flags; (NanoSwitch-1×flag-2NM), remove the 1×flag at the C-terminal.

[0091] Improvement scheme 3, on the basis of improvement scheme 1, removes the 1×flag at the N-terminal, and retains the last two 1×flags (NanoSwitch-1×flag-2MC).

[0092] 1. Method

[0093] 1. Construction of plasmid NanoSwitch-1×flag-2MC

[0094] Using the plasmid NanoSwitch-3×flag as a template, it was amplified with primers F flag-SV40+R amp. The PCR reaction system is: plasmid C11-3flag-N11S 10ng, primers F flag-SV40 (10μM) and R amp (10μM) 0.4μl each, 2XPrimeSTAR Max Premix 10μl, sterilized ultra...

Embodiment 3

[0126] Example 3 NanoSwitch-1×flag-2 Specificity Test for Detecting Flag Antibody

[0127] Prokaryotic expression and purification of NanoSwitch-1×flag-2NM was performed to further prove the detection specificity of the flag antibody.

[0128] 1. Method

[0129] 1. Plasmid construction and prokaryotic expression and purification

[0130] Using the plasmid PET28a as a template, it was amplified with the primer F pet28a-Chis2+R rop-bsa1. The PCR reaction system was: plasmid PET28a 10 ng, primers F pet28a-Chis2 (10 μM) and R rop-bsa1 (10 μM) 0.4 μl each, 2XPrimeSTAR Max Premix 10 μl, and sterilized ultrapure water to make up the volume to 20 μl. Reaction conditions: pre-denaturation at 95°C for 3 minutes; 35 cycles at 95°C for 15s, 55°C for 15s, and 72°C for 45s. The amplified fragment was recovered with a gel recovery kit, and the recovered fragment was named frag16.

[0131] Using the plasmid PET28a as a template, it was amplified with primers R pet28a-Chis3+F rop-bsal. Th...

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Abstract

The invention discloses a multifunctional protein molecule switch used for antibody detection, and belongs to the field of protein detection. The molecule switch is fusion protein and comprises the following parts connected in sequence from an N terminal to a C terminal: (1) SmBiT, (2) antigen epitope peptide which can be subjected to specific binding by an antibody to be detected, and (3) LgBiT. The molecule switch disclosed by the invention can affect binding of the SmBiT and the LgBiT through the specific binding of the antibody and the antigen epitope peptide, the enzymatic activity of fluorescein is greatly changed to cause change of the enzymatic activity before and after, and the change can reflect the concentration of the antibody. The multifunctional protein molecule switch disclosed by the invention can be used for detecting the 2019-nCoV, accuracy and specificity are almost 100%, and the molecule switch has a good application value.

Description

technical field [0001] The invention belongs to the field of protein detection. Background technique [0002] The protein molecular switch is a biosensor that can be used to detect various target biomolecules. Its principle is to use specially designed protein molecules to "perceive" and reflect the different conformation and functional states of the target molecule before and after binding the target molecule. exists, so as to achieve the detection purpose. Protein molecular switches are widely used in disease diagnosis and basic biomedical research. Recent advances in protein molecular switch design, represented by the work of 2020 Breakthrough Prize winner David Baker. They designed and constructed a synthetic protein molecular switch LOCKR (Naure, 2019), and then used the system for biological signal regulation (Nature, 2019), and recently for the detection of new coronavirus antibodies and antigens (BioRxiv, 2020). [0003] Allosteric regulatory enzymes are often use...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N9/02G01N33/68G01N33/569G01N33/58
CPCC12N9/0069C07K2319/43C07K2319/60G01N33/56983G01N2333/165G01N2469/20G01N33/542Y02A50/30G01N33/569C07K19/00
Inventor 胡接力黄爱龙李杰
Owner CHONGQING MEDICAL UNIVERSITY
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