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A biosensor based on luciferase complementation and its preparation method and application

A biosensor, luciferase technology, applied in the field of biochemistry, can solve the problems of deviation in detection results, long detection time, complicated operation, etc., and achieve the effects of low background noise, low cost and high detection sensitivity

Active Publication Date: 2022-04-29
SHENZHEN INST OF ADVANCED TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The ELISA detection method has high sensitivity and strong specificity, but there are also some shortcomings. For example, due to different batches of antibodies, the detection results will be biased. Expensive, and it is impossible to achieve rapid detection of samples in a short period of time (2-3 hours), the detection time is long, and there may be the possibility of false positives

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  • A biosensor based on luciferase complementation and its preparation method and application
  • A biosensor based on luciferase complementation and its preparation method and application
  • A biosensor based on luciferase complementation and its preparation method and application

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preparation example Construction

[0073] The present invention also provides a method for preparing the above-mentioned biosensor based on luciferase complementation, including:

[0074] Constructing the first expression vector containing the luciferase amino-terminal fragment gene, and constructing the second expression vector containing the luciferase carboxy-terminal fragment gene, wherein the 5' end of the luciferase amino-terminal fragment gene in the first expression vector, and one of the 3' ends of the luciferase carboxy-terminal fragment gene in the second expression vector is inserted into the G protein gene, and the other is inserted into the gene of the first marker;

[0075] The first expression vector and the second expression vector are transformed, expressed and purified to obtain the luciferase amino-terminal fragment and the luciferase carboxy-terminal fragment, the amino-terminal of the luciferase amino-terminal fragment and the carboxyl group of the luciferase carboxyl-terminal fragment One...

Embodiment 1

[0093] A kind of preparation method of biosensor based on luciferase complementation

[0094] (1) Expression and purification of fusion protein

[0095] Gaussia luciferase (Gluc) was divided between the G93 and E94 sites to form two complementary N-terminal fragments and C-terminal fragments, and the coding sequences of the N-terminal fragments and C-terminal fragments were synthesized. Pass the 5' end of the coding sequence of the N-terminal fragment through (GGGGS) 2 The coding sequence of the G protein is connected to the coding sequence of the His tag; the 3' end of the coding sequence of the C-terminal fragment is passed (GGGGS) 2 The coding sequence of the monomeric streptavidin (mSA) and the coding sequence of the His tag were connected and inserted into the pET21a expression vector respectively.

[0096] The above pET21a expression vector was transformed into competent cells BL21 after heat treatment at 42°C for 45s, revived by culturing at 37°C, spread on LB plates ...

Embodiment 2

[0104] A kind of preparation method of biosensor based on luciferase complementation

[0105] Two complementary N-terminal fragments and C-terminal fragments formed by dividing Renilla luciferase (Rluc) between L110 and P111 sites were synthesized into coding sequences of the N-terminal fragment and the C-terminal fragment. Pass the 5' end of the coding sequence of the N-terminal fragment through (GGGGS) 3 The coding sequence of the monomeric streptavidin (mSA) was joined to the coding sequence; the 3' end of the coding sequence of the C-terminal fragment was passed (GGGGS) 3 The coding sequence of G protein is connected with the coding sequence of G protein. The 3' end of the above gene sequence was connected to the gene encoding the His tag, and inserted into the pET21a expression vector respectively. According to the same method as in Example 1, luciferase amino-terminal fragment (mSA-NRluc) and luciferase carboxyl-terminal fragment (CRluc-potein G) were prepared, and the...

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Abstract

The present invention provides a biosensor based on luciferase complementation, comprising a luciferase amino-terminal fragment and a luciferase carboxyl-terminal fragment, the amino-terminal of the luciferase amino-terminal fragment and the carboxyl-terminal of the luciferase carboxyl-terminal fragment One of them is connected to G protein, and the other is connected to the first marker. The amino-terminal fragment of luciferase and the carboxy-terminal fragment of luciferase are two complementary fragments of the same luciferase; an antigen, a second marker complementary to the first marker; an antibody to the analyte; and a luciferase substrate. By using the four-fold combination of luciferase amino-terminal fragment and carboxy-terminal fragment complementary, first marker and second marker complementary, analyte / analyte antigen and analyte antibody complementary, and G protein and analyte antibody complementary The meta-complementary system realizes the rapid detection of the analyte, has high detection sensitivity, strong specificity, and low background noise, and can be used for quantitative analysis of the analyte in complex samples.

Description

technical field [0001] The invention relates to the technical field of biochemistry, in particular to a biosensor based on luciferase complementation and its preparation method and application. Background technique [0002] Among the current detection methods for chemical small molecules, enzyme-linked immunosorbent assay (ELISA) is widely used. Taking chloramphenicol, a common chemical small molecule pollutant, as an example, its dichloroamide alcohol and nitrobenzene structures can be used as complete antigens to effectively prepare corresponding antibodies after being combined with macromolecular proteins. Coat the sample to be detected on the microtiter plate, block with BSA, incubate with chloramphenicol antibody as the primary antibody, and then incubate with the corresponding HRP-labeled secondary antibody. Add HRP substrate TMB chromogenic solution for color development, and then use 2MH 2 SO 4 Stop the reaction, measure OD 450 of absorbance. Use the standard to...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N33/58G01N33/53G01N21/64
CPCG01N33/6854G01N33/581G01N33/5308G01N21/6486G01N33/53G01N21/64G01N33/68G01N33/58
Inventor 金宗文卫小元罗擎颖赵江林金虹朱海严义勇付辉
Owner SHENZHEN INST OF ADVANCED TECH
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