Method for detecting interaction between proteins in vitro, detection kit and application of detection kit

An in vitro detection and protein technology, which is applied in the direction of biological testing, measuring devices, material inspection products, etc., can solve the problems of high false negative probability and long time-consuming, and achieve simplified operation process, saving time and cost, and detection of protein interaction The effect of action

Pending Publication Date: 2020-05-26
THE HONG KONG POLYTECHNIC UNIV SHENZHEN RES INST
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] The main purpose of the present invention is to provide a method for detecting the interaction between proteins in vitro, which aims to solve the technical problems of the prior art such as long time-consuming and high probability of false negative

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  • Method for detecting interaction between proteins in vitro, detection kit and application of detection kit
  • Method for detecting interaction between proteins in vitro, detection kit and application of detection kit
  • Method for detecting interaction between proteins in vitro, detection kit and application of detection kit

Examples

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Embodiment 1

[0089] This example provides a method for detecting protein-protein interaction in vitro, which realizes the detection of protein-protein interaction outside the cell using bimolecular fluorescence complementation technology. Table 1 is the plasmid vector used in this embodiment, and Table 2 is the primer information used in this embodiment

[0090] The method for detecting the interaction between proteins in vitro in this embodiment specifically includes the following steps:

[0091] 1. Preparation of fusion protein A containing luciferase protein fragment SmBiT and NusB protein

[0092] 1) Construction of plasmid pCU231: bla

[0093] Select pETMCSIII as a plasmid vector for overexpressing N-terminal histidine-tagged recombinant proteins;

[0094] Use primers N smbit_F and N lg / smbit_R (Table 2) to amplify the smBiT-linker fragment containing the luciferase gene fragment smBiT from the plasmid pBiT2.1-N[TK / SmBiT]; The smBiT-linker fragment was inserted into the NdeI-NcoI...

Embodiment 2

[0118] This example provides a method for detecting protein-protein interaction in vitro, which realizes the detection of protein-protein interaction outside the cell using bimolecular fluorescence complementation technology. Table 3 is the plasmid vector used in this embodiment, and Table 4 is the primer information used in this embodiment

[0119] The method for detecting the interaction between proteins in vitro in this embodiment specifically includes the following steps:

[0120] 1. Preparation of fusion protein C containing luciferase protein fragment LgBiT and NusB protein

[0121] 1) Construction of plasmid pCU250: bla

[0122] Select pNG209 (Table 3) as a plasmid vector for overexpressing the C-terminal histidine-tagged recombinant protein; then, prepare the plasmid vectors pCU198 and pCU202 in sequence, the specific process is as described in step 1 of Example 1, wherein pCU202 includes Luciferase gene lgbit-linker fragment.

[0123] Use primers lgbit nusB_F and...

Embodiment 3

[0144] This example provides a method for detecting protein-protein interaction in vitro, which realizes the detection of protein-protein interaction outside the cell using bimolecular fluorescence complementation technology. Table 5 is the plasmid vector used in this embodiment, and Table 6 is the primer information used in this embodiment

[0145] The method for detecting the interaction between proteins in vitro in this embodiment specifically includes the following steps:

[0146] 1. Preparation of fusion protein E containing luciferase protein fragment SmBiT and NusB protein

[0147] Construction of recombinant plasmid vector pCU231: Escherichia coli was used for overexpression and purification to obtain fusion protein E.

[0148] 2. Preparation of fusion protein F containing luciferase protein fragment LgBiT and NusE protein

[0149] Construction of recombinant plasmid vector pCU236: bla Escherichia coli was used for overexpression and purification to obtain fusion...

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Abstract

The invention provides a method for detecting interaction between proteins in vitro, a detection kit and application of the detection kit. A bimolecular fluorescence complementation technology is adopted outside a cell for detection, and the method comprises the following steps: S01, constructing a first recombinant plasmid which comprises a first detection protein gene, a luciferase gene segmentand a protein purification tag gene; then, carrying out overexpression on the first recombinant plasmid vector to obtain a first fusion protein; wherein the first fusion protein contains a first detection protein and a luciferase protein fragment; S02, constructing a second recombinant plasmid, wherein the second recombinant plasmid contains a second detection protein gene, a luciferase complementary gene segment and a protein purification tag gene; performing overexpression on the second recombinant plasmid vector to obtain a second fusion protein; the second fusion protein contains a seconddetection protein and a luciferase complementary protein fragment; and S03, incubating the first fusion protein and the second fusion protein outside the cell, adding a luciferase substrate, observingwhether fluorescence is emitted or not, and detecting the luminous intensity.

Description

technical field [0001] The invention belongs to the technical field of active drug screening, and specifically relates to a method for detecting protein-protein interaction in vitro, a detection kit and applications thereof. Background technique [0002] With the continuous development of life science research, the biological functions through the interaction between proteins are gradually discovered. The interaction between proteins can affect the activity of cells, and the interaction between some proteins is normal for organisms. Function and state have a decisive influence, and can even be used to explain and predict various life processes and phenomena. In recent years, assisting the development of new drugs by detecting the interaction between proteins has become the focus of relevant researchers. [0003] Currently, detection and analysis methods for protein-protein interactions include: yeast two-hybrid system, tandem affinity purification technology, gel filtration...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N33/58
CPCG01N33/6845G01N33/582Y02A50/30
Inventor 马聪
Owner THE HONG KONG POLYTECHNIC UNIV SHENZHEN RES INST
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