WO2021093787A1

WO2021093787A1 - Luciferase-complementation-based biosensor, preparation method therefor and use thereof

Info

Publication number: WO2021093787A1
Application number: PCT/CN2020/128197
Authority: WO
Inventors: 金宗文; 卫小元; 罗擎颖; 赵江林; 金虹; 朱海; 严义勇; 付辉
Original assignee: 深圳先进技术研究院
Priority date: 2019-11-14
Filing date: 2020-11-11
Publication date: 2021-05-20
Also published as: CN112798786B; CN112798786A

Abstract

Disclosed is a luciferase-complementation-based biosensor, comprising a luciferase amino terminal fragment and a luciferase carboxyl terminal fragment, wherein one of the amino terminal of the luciferase amino terminal fragment and the carboxyl terminal of the luciferase carboxyl terminal fragment is connected to a G protein, and the other terminal is connected to a first marker, and the luciferase amino terminal fragment and the luciferase carboxyl terminal fragment are two complementary fragments of the same luciferase; an analyte antigen connected to the second marker, wherein the second marker is complementary to the first marker; an analyte antibody; and a luciferase substrate. By using a quaternary complementary system of a complementary luciferase amino terminal fragment and carboxyl terminal fragment, a complementary first marker and second marker, a complementary analyte/analyte antigen and analyte antibody and a complementary G protein and analyte antibody, the rapid detection of the analyte can be realized, wherein the detection sensitivity is high, the specificity is strong, and the background noise is low, and same can be used for the quantitative analysis of the analyte in complex samples.

Description

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

Technical field

The invention relates to the technical field of biochemistry, in particular to a biosensor based on luciferase complementation, and a preparation method and application thereof.

Background technique

Among the current methods for detecting small chemical molecules, enzyme-linked immunosorbent assay (ELISA) is widely used. Taking the common chemical small molecule pollutant chloramphenicol as an example, the structure of dichloroamide alcohol and nitrobenzene combined with the macromolecular protein can be used as a complete antigen to effectively prepare corresponding antibodies. Coat the sample to be tested on the ELISA plate, block it with BSA, incubate with chloramphenicol antibody as the primary antibody, and then incubate with the corresponding HRP-labeled secondary antibody. Add HRP substrate TMB color developing solution, then stop the reaction with _{2M H 2} SO ₄ and measure the absorbance of _{OD 450.} Draw a standard curve with standard products, and then analyze the concentration of chloramphenicol contained in the sample according to the standard curve. The ELISA detection method is the most widely used, and the detection limit can reach 0.02μg/kg. There are also ELISA kits for the determination of chloramphenicol on the market. The ELISA detection method has high sensitivity and strong specificity, but it also has some shortcomings. For example, due to the different batches of antibodies, the detection results will be biased. At the same time, it needs to pack the plate and other operations, the operation is complicated, and the equipment used in the detection process It is expensive, and it is impossible to achieve rapid sample detection in a short time (2 to 3 hours), the detection time is long, and there may be false positives.

Summary of the invention

In view of this, the present invention provides a biosensor based on luciferase complementation, by using luciferase amino-terminal fragment and carboxy-terminal fragment complementation, the first label and the second label are complementary, and the analyte/analyte Antigen and analyte antibody complementation, as well as G protein and analyte antibody complementation quaternary complementation system, to achieve rapid detection of the analyte, high detection sensitivity, strong specificity, low background noise, can be used in complex samples to be tested Quantitative analysis of analytes.

In the first aspect, the present invention provides a biosensor based on luciferase complementation, including:

A luciferase amino-terminal fragment and a luciferase carboxy-terminal fragment, one of the amino-terminal of the luciferase amino-terminal fragment and the carboxy-terminal of the luciferase carboxy-terminal fragment is connected to the G protein, and the other is connected to the first A label is connected, and the luciferase amino-terminal fragment and the luciferase carboxy-terminal fragment are two complementary fragments of the same luciferase;

A analyte antigen, the analyte antigen is connected with a second label, and the second label is complementary to the first label;

The antibody to be tested; and

Luciferase substrate.

In this application, by using the luciferase complementary technology, that is, luciferase is separated at a specific site to form amino-terminal (N-terminal) fragments and carboxy-terminal (C-terminal) fragments that cannot catalyze luminescence or can only catalyze very weak fluorescence. ) Fragment. When these two fragments are co-expressed in vivo or mixed in vitro, they cannot spontaneously assemble into a complete luciferase, and thus cannot emit obvious fluorescence; and these two fragments are close to each other under exogenous interaction, forming non-covalent complementation , Reassemble into a complete protein, restore the activity of luciferase, that is, it can catalyze the corresponding substrate to emit light.

In this application, G protein (reference: Purification and some properties of streptococcal protein G, a novel IgG-binding reagent. L

G Kronvall. The Journal of Immunology August 1, 1984, 133(2) 969-974) is a cell surface protein derived from Streptococcus G family. It is a type III Fc receptor that can bind to the Fc segment of antibodies. For example, it can bind to IgG, IgG Fc, IgG Fab, IgG Fab', and IgG F(ab')2.

In the biosensor provided in the present application, the luciferase amino-terminal fragment and the carboxy-terminal fragment can be complementary, the first label and the second label can be complementary, the analyte antigen and the analyte antibody can be complementary, and the G protein and The analyte antibodies can complement each other to form a complementary closed loop, which then pulls in the spatial distance between the luciferase amino-terminal fragment and the carboxy-terminal fragment, so that the two can be reassembled, and the luciferase activity is restored, so that it can interact with the sensor. The luciferase substrate in the analyte is catalyzed to generate luminescence; at the same time, the addition of the analyte competes with the analyte antigen to bind to the analyte antibody, so that the analyte, the analyte antibody, the G protein and the luciferase amino-terminal fragment Linked to one of the carboxy-terminal fragments, the analyte antigen, the second label, the first label, and the other of the luciferase amino-terminal fragment and the carboxy-terminal fragment are connected together to cut the complementary closed loop, That is, the amino-terminal fragment and the carboxy-terminal fragment of luciferase cannot be reassembled in space to restore luciferase activity, and thus cannot catalyze the substrate to emit light. Therefore, according to the change of luminous intensity before and after the addition of the analyte, qualitative detection and analysis of whether the sample contains the analyte can be carried out. At the same time, the change of luminous intensity has a linear relationship with the content of the analyte. The analyte is detected and analyzed to obtain a standard curve, and then the content of the analyte in the sample containing the analyte can be qualitatively detected and analyzed, and the result is more accurate.

In this application, luciferase can be any luciferase, as long as luciferase can be divided into amino-terminal fragments and carboxy-terminal fragments. If the two fragments exist alone, they will not emit fluorescence or have very weak fluorescence, and the two fragments When they are close in space, they can be reassembled together to restore the activity of luciferase.

Optionally, the luciferase includes at least one of Gaussia luciferase, Renilla luciferase, sea luciferase, firefly luciferase, and NanoLuc luciferase. In the present application, the use of the above-mentioned luciferase can generate strong fluorescence after the luciferase amino-terminal fragment and the luciferase carboxy-terminal fragment are spaced closer, which is more conducive to detection and reduces detection errors.

Further, the luciferase is Gaussia luciferase, and the luciferase amino-terminal fragment and the luciferase carboxy-terminal fragment are two separate parts formed by the Gaussia luciferase between G93 and E94. Complementary fragments.

Further, the luciferase is Renilla luciferase, the luciferase amino-terminal fragment and the luciferase carboxy-terminal fragment are the Renilla luciferase between the L110 and P111 positions or G229 and Two complementary fragments formed separately between the K230 site.

Optionally, the luciferase amino-terminal fragment and the luciferase carboxy-terminal fragment are two complementary fragments divided by the same luciferase at loop points. In this application, the two complementary fragments divided into the luciferase amino-terminal fragment and the luciferase carboxy-terminal fragment at the loop point basically do not have the ability to emit light. They need to be close to each other under exogenous action and complement each other to restore luciferase activity and catalyze The substrate glows.

Optionally, one of the amino terminal of the luciferase amino terminal fragment and the carboxy terminal of the luciferase carboxy terminal fragment is connected to the G protein through a first connecting peptide, and the other is connected to the G protein through a first connecting peptide. The second connecting peptide is connected to the first label, and the first connecting peptide and the second connecting peptide are flexible chains.

In this application, two proteins are connected by a connecting peptide of a flexible chain, so that the two proteins spatially maintain their original spatial structure and activity. Optionally, the first connecting peptide and the second connecting peptide are selected from (GGGGS) _n , 2≤n≤20, and n is an integer. In this application, the sequence of the first connecting peptide and the second connecting peptide may be the same or different.

In this application, the first label and the second label can complement each other. Optionally, one of the first label and the second label is biotin, and the other is avidin. Further, the avidin is streptavidin.

In this application, G protein is linked to luciferase amino acid fragment, luciferase carboxy-terminal fragment is linked to biotin, and the analyte antigen is linked to avidin; or biotin is linked to luciferase amino acid fragment, luciferase The carboxy-terminal fragment is connected to the G protein, and the analyte antigen is connected to avidin; or the G protein is connected to the luciferase amino acid fragment, the luciferase carboxy-terminal fragment is connected to avidin, and the analyte antigen is connected to biotin; or Avidin is connected to the amino acid fragment of luciferase, the carboxy-terminal fragment of luciferase is connected to protein G, and the antigen of the analyte is connected to biotin.

Optionally, the analyte antibody titers of 10 ⁵ or more.

In this application, the luciferase substrate is a substance that can be catalyzed by the corresponding luciferase to produce fluorescence. Optionally, the luciferase substrate includes at least one of luciferin, luciferin, coelenterazine and isomers thereof. Specifically, but not limited to, Gaussia luciferase can catalyze the luminescence of the substrate coelenterazine (emission wavelength 480 nm) without ATP.

Optionally, the concentration of the G protein is 25 nM-1000 nM. Further, the concentration of the G protein is 50 nM-500 nM.

Optionally, the concentration of the analyte antigen is 50 nM-2000 nM. Further, the concentration of the analyte antigen is 100 nM-1000 nM.

Optionally, the concentration of the analyte antibody is 25 nM-1000 nM, and the analyte antigen is connected with the second label. Further, the concentration of the analyte antibody is 50 nM-500 nM.

Optionally, the concentration of the first marker is 25 nM-1000 nM. Further, the concentration of the first marker is 100 nM-1000 nM.

Optionally, the concentration ratio of the analyte antibody and the analyte antigen is 1: (1.2-3). Further, the concentration ratio of the analyte antibody and the analyte antigen is 1: (1.5-2). Specifically, the concentration ratio of the analyte antibody and the analyte antigen may be, but not limited to, 1:2.

In the second aspect, the present invention provides a method for preparing a biosensor based on luciferase complementation, including:

Construct a first expression vector containing the luciferase amino-terminal fragment gene, and construct a second expression vector containing the luciferase carboxy-terminal fragment gene, wherein the first expression vector contains the luciferase amino-terminal fragment gene 5'end, and one of the 3'ends of the luciferase carboxyl-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;

The first expression vector and the second expression vector are transformed, expressed and purified to obtain a luciferase amino-terminal fragment and a luciferase carboxy-terminal fragment, the amino-terminal of the luciferase amino-terminal fragment and the One of the carboxy-terminal fragments of the luciferase carboxy-terminal fragment is connected to the G protein, and the other is connected to the first label. The luciferase amino-terminal fragment and the luciferase carboxy-terminal fragment are of the same luciferase Two complementary fragments;

Provide a analyte antigen, the analyte antibody and a luciferase substrate, the analyte antigen is connected with a second label, the second label is complementary to the first label, and the The analyte antigen is combined with the analyte antibody, and the analyte antibody is combined with the G protein to obtain a biosensor based on luciferase complementation.

Optionally, the first expression vector and the second expression vector contain a His tag gene.

In the third aspect, the present invention provides the application of the biosensor according to the first aspect or the biosensor prepared by the preparation method according to the second aspect in substance content detection.

In this application, by adding the analyte, the analyte and the analyte antigen in the biosensor based on luciferase complementation compete for binding to the analyte antibody, so that the luciferase amino-terminal fragment and the carboxy-terminal fragment are The spatial position changes, which in turn affects the luminescence intensity of the luciferase-catalyzed substrate, and the test object is analyzed according to the change of the luminescence intensity. At the same time, since the change of the luminescence intensity is related to the content of the test object, the test object can be quantitatively analyzed. The entire detection process only needs to detect the luminescence intensity, and simple detection instruments such as a microplate reader can be used to reduce the detection cost, and the detection process is convenient and fast, which is more conducive to its application.

Optionally, the application of the biosensor based on luciferase complementation in quantitative detection.

In this application, the test substance can be any substance that can provide corresponding antigens and antibodies, and specifically can be but not limited to small chemical molecules.

Optionally, the application includes:

Mix the luciferase amino-terminal fragment, the luciferase carboxy-terminal fragment, the analyte antigen, the analyte antibody, and the analyte with a known concentration, and then add the fluorescein After the substrates are mixed, the luminescence intensity is detected, and the standard curve of the relationship between the concentration of the analyte and the luminescence intensity is drawn;

After mixing the luciferase amino-terminal fragment, the luciferase carboxy-terminal fragment, the analyte antigen, the analyte antibody, and the luciferase substrate, the detected luminescence intensity is the first luminescence strength;

Mix the luciferase amino-terminal fragment, the luciferase carboxy-terminal fragment, the analyte antigen, the analyte antibody, and the analyte at different concentrations, and then add the luciferin After the enzyme substrates are mixed, the detected luminescence intensity is the second luminescence intensity;

According to the standard curve, the first luminous intensity and the second luminous intensity, the content of the analyte is calculated.

The beneficial effects of this application:

The biosensor based on luciferase complementation and the preparation method thereof provided by the present invention utilize luciferase amino-terminal fragment and carboxy-terminal fragment complementation, first marker and second marker complementation, test substance/test substance antigen A quaternary complementation system that complements the antibody to the test object and the G protein and the antibody to the test object complement each other. By comparing the changes in luminescence intensity before and after the addition of the test object, the rapid detection of the test object is achieved. At the same time, the entire process only requires simple detection equipment. The detection is convenient, the cost is low, and the detection sensitivity is high, the specificity is strong, and the background noise is small. It can be used for the quantitative analysis of the analyte in complex samples.

Description of the drawings

In order to explain the embodiments of the present invention or the technical solutions in the prior art more clearly, the following will briefly introduce the drawings that need to be used in the description of the embodiments or the prior art. The specific embodiments described here are only used to explain the present invention, but not used to limit the present invention.

Fig. 1 is an SDS-PAGE chart of the protein prepared in Example 1 of the present invention. In Fig. 1 (a) is the SDS-PAGE chart of the fusion protein of G protein and the amino-terminal fragment of luciferase, and Fig. 1 (b) SDS-PAGE image of the fusion protein of luciferase carboxy-terminal fragment and monomeric streptavidin.

Fig. 2 is an SDS-PAGE chart of the binding of protein G to the antibody to be tested in Example 1 of the effect of the present invention.

Fig. 3 is a graph of the ELISA test results of Example 2 of the effect of the present invention.

Fig. 4 is a graph showing the detection result of the luminescence signal intensity of the biosensor based on luciferase complementation provided in Example 3 of the effect of the present invention.

FIG. 5 is a schematic diagram of the principle of a biosensor based on luciferase complementation provided in Example 3 of the effect of the present invention.

Detailed ways

The following are preferred implementations of the embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the embodiments of the present invention, several improvements and modifications can be made. These improvements And retouching is also regarded as the protection scope of the embodiments of the present invention.

The present invention provides a biosensor based on luciferase complementation, including:

One of the luciferase amino-terminal fragment and the luciferase carboxy-terminal fragment, the amino-terminal of the luciferase amino-terminal fragment and the carboxy-terminal of the luciferase carboxy-terminal fragment is connected to protein G, and the other is connected to the first label , The luciferase amino-terminal fragment and the luciferase carboxy-terminal fragment are two complementary fragments of the same luciferase; the analyte antigen, the analyte antigen is connected with a second label, and the second label is connected to the first The label is complementary; the analyte antibody; and the luciferase substrate.

In the biosensor provided in the present application, the luciferase amino-terminal fragment and the carboxy-terminal fragment can be complementary, the first label and the second label can be complementary, the analyte antigen and the analyte antibody can complement each other, and the G protein and the analyte antibody can complement each other. The analyte antibodies can complement each other to form a complementary closed loop, which then pulls in the spatial distance between the luciferase amino-terminal fragment and the carboxy-terminal fragment, so that the two can be reassembled, and the luciferase activity is restored, so that it can interact with the sensor. The luciferase substrate in the analyte is catalyzed to generate luminescence; at the same time, the addition of the analyte competes with the analyte antigen to bind to the analyte antibody, so that the analyte, the analyte antibody, the G protein and the luciferase amino-terminal fragment Linked to one of the carboxy-terminal fragments, the analyte antigen, the second label, the first label, and the other of the luciferase amino-terminal fragment and the carboxy-terminal fragment are connected together to cut the complementary closed loop, That is, the amino-terminal fragment and the carboxy-terminal fragment of luciferase cannot be reassembled in space to restore luciferase activity, and thus cannot catalyze the substrate to emit light. Therefore, according to the change of luminous intensity before and after the addition of the analyte, qualitative detection and analysis of whether the sample contains the analyte can be carried out. At the same time, the change of luminous intensity has a linear relationship with the content of the analyte. The analyte is detected and analyzed to obtain a standard curve, and then the content of the analyte in the sample containing the analyte can be qualitatively detected and analyzed, and the result is more accurate.

Compared with ELISA detection, in the biosensor based on luciferase complementation provided by this application, the sensitivity and specificity of detection are greatly improved through the quaternary complementation system, the problem of false positives is avoided, the background noise is small, and it can be used. For the detection of analytes in complex samples, the detection line is low; at the same time, only the luminescence intensity is required to detect, the operation is simple, and the detection cost is low; the luminescence process is fast, and the detection time is short; it can but is not limited to facilitate the detection by the microplate reader. Simultaneous detection of multiple samples, high detection efficiency.

In one embodiment of the present application, the luciferase includes at least one of Gaussia luciferase, Renilla luciferase, sea luciferase, firefly luciferase, and NanoLuc luciferase. In this application, the use of the above-mentioned luciferase can generate strong fluorescence after the luciferase amino-terminal fragment and the luciferase carboxy-terminal fragment are spaced closer, which is more conducive to detection and reduces detection errors.

In one embodiment of the present application, the luciferase is Gaussia luciferase, and the amino-terminal fragment of luciferase and the carboxy-terminal fragment of luciferase are two complementary fragments formed by Gaussia luciferase separated between G93 and E94. .

In one embodiment of the present application, the luciferase is Renilla luciferase, and the amino-terminal fragment of luciferase and the carboxy-terminal fragment of luciferase are Renilla luciferase between L110 and P111 or G229 and K230. Two complementary fragments formed by the separation between.

In one embodiment of the present application, the amino-terminal fragment of luciferase and the carboxy-terminal fragment of luciferase are two complementary fragments of the same luciferase divided into loop points. In this application, the two complementary fragments divided into the luciferase amino-terminal fragment and the luciferase carboxy-terminal fragment at the loop point basically do not have the ability to emit light. They need to be close to each other under exogenous action and complement each other to restore luciferase activity and catalyze The substrate glows.

In one embodiment of the present application, the amino terminal of the luciferase amino terminal fragment and the carboxy terminal of the luciferase carboxy terminal fragment, one of which is connected to the G protein through the first connecting peptide, and the other through the second connection The peptide is connected to the first label, and the first connecting peptide and the second connecting peptide are flexible chains.

In this application, two proteins are connected by a connecting peptide of a flexible chain, so that the two proteins spatially maintain their original spatial structure and activity. Optionally, the first connecting peptide and the second connecting peptide are selected from (GGGGS) _n , 2≤n≤20, and n is an integer. In this application, the sequences of the first connecting peptide and the second connecting peptide may be the same or different. Specifically, n can be, but is not limited to, 2, 3, 4, 5, 6, 10, 15, 18, or 20.

In this application, the first label and the second label can complement each other. Specifically, but not limited to, one of the first label and the second label is biotin, and the other is avidin. Further, the avidin is streptavidin.

In one embodiment, the G protein is connected to the luciferase amino acid fragment, the carboxy-terminal fragment of luciferase is connected to biotin, and the analyte antigen is connected to avidin. In another embodiment, biotin is connected to the luciferase amino acid fragment, the carboxyl terminal fragment of luciferase is connected to the G protein, and the analyte antigen is connected to avidin. In another embodiment, the G protein is connected to the luciferase amino acid fragment, the carboxy-terminal fragment of the luciferase is connected to avidin, and the analyte antigen is connected to biotin. In another embodiment, the avidin is linked to the luciferase amino acid fragment, the carboxy-terminal fragment of luciferase is linked to the G protein, and the analyte antigen is linked to biotin.

In one embodiment of the present application, the analyte antibody titers of 10 ⁵ or more.

In one embodiment of the present application, the concentration of G protein is 25 nM-1000 nM. Further, the concentration of G protein is 50 nM-500 nM.

In one embodiment of the present application, the concentration of the analyte antigen is 50 nM-2000 nM. Further, the concentration of the test substance antigen is 100 nM-1000 nM.

In one embodiment of the present application, the concentration of the analyte antibody is 25 nM-1000 nM, and the second label is attached to the analyte antigen at this time. Further, the concentration of the antibody to be tested is 50 nM-500 nM.

In one embodiment of the present application, the concentration of the first marker is 25 nM-1000 nM. Further, the concentration of the first marker is 100 nM-1000 nM. It is understandable that the luciferase amino-terminal fragment and the luciferase carboxy-terminal fragment are one connected to the G protein and the other to the first label. The concentration of the luciferase amino-terminal fragment and the luciferase carboxy-terminal fragment is based on the connection The G protein is related to the first marker. In one embodiment, the concentrations of the luciferase amino-terminal fragment and the luciferase carboxy-terminal fragment are equal to the concentrations of the protein G and the first label to which they are attached.

In one embodiment of the present application, the concentration ratio of the analyte antibody and the analyte antigen is 1: (1.2-3). Further, the concentration ratio of the test substance antibody and the test substance antigen is 1: (1.5-2). Specifically, the concentration ratio of the analyte antibody and the analyte antigen may be but not limited to 1:2, which is more conducive to the detection and analysis of the biosensor.

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

Construct a first expression vector containing the luciferase amino-terminal fragment gene, and construct a 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 is 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;

The first expression vector and the second expression vector are transformed, expressed and purified to obtain the luciferase amino-terminal fragment and luciferase carboxy-terminal fragment, the amino-terminal of the luciferase amino-terminal fragment and the carboxyl group of the luciferase carboxy-terminal fragment One of the ends is connected to the G protein, and the other is connected to the first label. The luciferase amino-terminal fragment and the luciferase carboxy-terminal fragment are two complementary fragments of the same luciferase;

Provide the analyte antigen, the analyte antibody and the luciferase substrate, the analyte antigen is connected with a second label, the second label is complementary to the first label, and the analyte antigen is combined with the analyte antibody, The analyte antibody is combined with protein G to obtain a biosensor based on luciferase complementation.

In one embodiment of the present application, the first expression vector and the second expression vector contain a His tag gene.

In the present application, the antigen of the analyte to which the second label is attached can be, but not limited to, the method of chemical synthesis to make the antigen of the analyte connect to the second label.

In this application, the antibody to be tested can be commercially available existing antibodies, and can be prepared by existing antibody preparation methods, which is not limited.

The invention also provides the application of the above-mentioned biosensor based on luciferase complementation in substance content detection.

In one embodiment of the present application, the application of a biosensor based on luciferase complementation in quantitative detection. Specifically, it can, but is not limited to, quantitative detection of small chemical molecules, such as chloramphenicol, proteins, and polypeptides.

In an embodiment of this application, the application includes:

Mix the luciferase amino-terminal fragment, luciferase carboxyl-terminal fragment, analyte antigen, analyte antibody, and analyte with a known concentration, and then mix with luciferase substrate to detect the luminous intensity and draw The standard curve of the relationship between the concentration of the analyte and the luminous intensity;

After mixing the luciferase amino-terminal fragment, the luciferase carboxy-terminal fragment, the analyte antigen, the analyte antibody and the luciferase substrate, the detected luminescence intensity is the first luminescence intensity;

Mix the luciferase amino-terminal fragment, luciferase carboxy-terminal fragment, analyte antigen, analyte antibody, and different concentrations of analyte uniformly, and then add luciferase substrate to mix, and the detected luminous intensity is the second light intensity;

In one embodiment, the standard curve is the relationship curve between the concentration of the analyte and the luminous intensity. The concentration of the analyte can be calculated by substituting the difference between the first luminous intensity and the second luminous intensity into the standard curve.

In one embodiment, the application of the biosensor based on luciferase complementation is carried out by the following steps: construct a biosensor based on luciferase complementation; use the biosensor based on luciferase complementation to plot the known concentration of the analyte and Standard curve of luminous intensity relationship; add the sample containing the analyte to a biosensor based on luciferase complementation to detect the luminous intensity; calculate the content of the analyte in the sample according to the standard curve and the luminous intensity.

In this application, the biosensor based on luciferase complementation can be prepared in advance and stored at low temperature for later use. In the application of a biosensor based on luciferase complementation, it is only necessary to add the analyte to detect the change of the luminous intensity, which is simple and fast to operate and takes a short time. Since the luminescence signal comes from the luminescence of the luciferase-catalyzed substrate, there is no other light source, so the background noise is small and the sensitivity is better.

Example 1

Preparation method of biosensor based on luciferase complementation

(1) Expression and purification of fusion protein

Two complementary N-terminal fragments and C-terminal fragments are formed by separating Gaussia luciferase (Gluc) between the G93 and E94 sites, and the coding sequences of the N-terminal fragment and the C-terminal fragment are synthesized. 'Coding sequence and the coding sequence of His-tag side through (GGGGS) coding sequence ₂ is connected to the G protein; the coding sequence of the C-terminal fragment of the 3' to 5 coding sequence of the N-terminal fragment ends by a (GGGGS) encoding ₂ The sequence connects the coding sequence of monomeric streptavidin (mSA) and the coding sequence of His tag, and inserts them into the pET21a expression vector respectively.

The above-mentioned pET21a expression vector was heat-treated at 42°C for 45s and transformed into competent cells BL21, cultured at 37°C and resuscitated and spread on an LB plate containing ampicillin for selection, and cultured at 37°C overnight. On the second day, a single colony was picked for colony PCR and double enzyme digestion to identify positive clones. Add 4mL of LB medium containing ampicillin from positive clones at 1:1000, culture overnight at 37°C, 200rpm, and add 1:100 to 400mL of medium at 37°C, 200rpm the next day, expand culture at 37°C, 200rpm, and detect OD _{When 600} is 0.4-0.6, add 0.5mg/mL IPTG, after inducing expression at 15℃ for 24h, centrifuge at 10000rpm, 2min to collect the bacteria.

The bacteria were resuspended in PBS, and then the bacteria were disrupted using an ultrasonic disintegrator (power 600W*37%) to release the soluble protein in the bacteria, until the solution was clear, centrifuged at 11000 rpm, 4°C for 15 min, and then took the supernatant. Use AKTA protein purifier and GE health Ni column for purification. Using the principle that the filler nickel sulfate and His tag in the Ni column can specifically bind to remove the impurity protein, and then competitively bind to the Ni column filler by the imidazole solution, the histidine fusion protein is eluted from the column. Rinse the column with water first, flush out the ethanol in the column, and then equilibrate the column with PBS. Wait for the UV line (indicating protein) and cout line (indicating the concentration of salt particles) on the instrument to reach the level, and then load the sample (the supernatant collected in the previous step). After loading the sample, rinse the column with PBS to wash away the protein that is not bound to the column. When the UV light level is reached, perform gradient elution with imidazole (25mM, 75mM, 100mM, 150mM, 200mM, 300mM), and monitor the elution in real time. When UV rays change, collect the eluent.

The collected eluate was electrophoresed through the SDS-PAGE manifold and stained with Coomassie brilliant blue. Observe the bands corresponding to the molecular weight position, select the eluent with high expression and high purity, use a 10kD ultrafiltration tube to remove the imidazole, replace it with PBS as the storage system, and store at -80°C. The SDS-PAGE electrophoresis characterization of the purified protein is shown in Figure 1. In Figure 1, (a) is the size of the luciferase amino-terminal fragment, Figure 1 (b) is the size of the luciferase carboxy-terminal fragment, and lane M is Protein molecular weight standard (M). It can be seen that the luciferase amino-terminal fragment (potein G-NGluc) is about 38kDa, the luciferase carboxy-terminal fragment (CGluc-mSA) is about 25kDa, and the luciferase amino-terminal fragment is the G protein connected to the N-terminal fragment through a connecting peptide. The carboxy-terminal fragment of luciferase is a C-terminal fragment that connects monomeric streptavidin through a connecting peptide.

(2) Biotin-labeled analyte antigen

Under N ₂ atmosphere, add R, R-aminoglycol (62 mg, 0.29 mmol), triethylamine (0.12 mL, 0.87 mmol) and dry DMF (2 mL) into a round bottom flask and mix well. Then a solution of biotin (100 mg, 0.29 mmol) in dry DMF (3 mL) was added. Stir at room temperature overnight, then add 15 mL of water, and stir at 4°C for 15 min at low temperature. Filter to remove the precipitated white solid, wash with 50% isopropanol solution, and dry to obtain a crude product. Recrystallization obtains biotin-labeled R,R-aminodiol, where R,R-aminodiol is a chloramphenicol antigen. The reaction process is as follows:

Provide chloramphenicol antibody and coelenterazine (CTZ) to obtain a biosensor based on luciferase complementation.

Example 2

Preparation method of biosensor based on luciferase complementation

Two complementary N-terminal fragments and C-terminal fragments are formed by separating Renilla luciferase (Rluc) between the L110 and P111 sites, and the coding sequences of the N-terminal fragment and the C-terminal fragment are synthesized. The N-terminal fragment of the coding sequence of the 5 'end by a (GGGGS) _coding sequence linked to a coding sequence of the monomeric chain of streptavidin (mSA); and the 3 C-terminal fragment of the coding sequence' end by a (GGGGS) ₃ The coding sequence is connected to the coding sequence of the G protein. Connect the 3'end of the above gene sequence to the encoding gene of the His tag, and insert them into the pET21a expression vector respectively. The luciferase amino-terminal fragment (mSA-NRluc) and the luciferase carboxy-terminal fragment (CRluc-potein G) were prepared according to the same method as in Example 1. The luciferase amino-terminal fragment is a monomeric streptavidin by ligation The peptide is connected to the N-terminal fragment, and the luciferase carboxy-terminal fragment is the C-terminal fragment connected to the G protein through the connecting peptide.

According to the same method as in Example 1, biotin-labeled R,R-aminoglycol was prepared, and chloramphenicol antibody and coelenterazine were provided to obtain a biosensor based on luciferase complementation.

Effect Example 1

Pull-down method to detect the binding ability of anti-chloramphenicol antibody and luciferase amino-terminal fragment

Prepare two 1.5mL centrifuge tubes A and B, add 50μL Ni column material, wash off the preservation solution and equilibrate with PBS, and then use PBS to make the volume to 200μL. Add 1 μL of anti-chloramphenicol antibody (anti-chloramphenicol) at a concentration of 1.8 mg/ml to tube A, and add 1 μL of the luciferase amino-terminal fragment prepared in Example 1 at a concentration of 2 mg/ml to tube B ( protein G-NGluc), incubate at room temperature for 30 minutes. Wash off the unattached protein with PBS, and collect two samples numbered 3 and 4. Add excess anti-chloramphenicol to each tube and incubate at room temperature for 10 minutes. The excess antibody was washed away with PBS, and two samples numbered 5 and 6 were collected. Add 100μL of PBS eluent containing 200mM imidazole to each tube, and collect two samples numbered 7 and 8. The collected samples were detected by SDS-PAGE electrophoresis. The results are shown in Figure 2. Lane M is the protein molecular weight standard (M), Lane 1 is anti-chloramphenicol, and Lane 2 is the luciferase amino group prepared in Example 1. End fragment; Lane 3 is the washing solution after incubation of anti-chloramphenicol with Ni column in tube A, and lane 4 is the washing solution after incubation of protein G-NGluc with Ni column in tube B. Anti-chloramphenicol does not hang on the column. All eluted, protein G-NGluc is bound to the column with His tag and Ni, and it is eluted with the eluent after saturation; Lane 5 is the washing solution after anti-chloramphenicol in tube A passes through the column, Lane 6 is B After the anti-chloramphenicol in the tube passes through the column, the antibody in the A tube is completely eluted without hanging the column, and the antibody in the B tube binds to the G protein in the protein G-NGluc, and only the unbound part is eluted; lanes 7 is the eluate after adding 200 mM imidazole to tube A, lane 8 is the eluate after adding 200 mM imidazole to tube B, the antibody in tube A is not hung on the column, and all the antibodies are washed in the previous step, so there is no band, B Because the protein G-NGluc hangs on the column, the antibody binds to the G protein in the protein G-NGluc and hangs on the column, so the eluate contains two proteins, and the concentration is high, which also indicates that the antibody binds to the protein G-NGluc. Instead of directly binding to the column, it also shows that protein G-NGluc maintains the property of protein G binding to antibodies, ensuring that this part of the binding is effective in the subsequent complementary system.

Effect Example 2

ELISA method to detect the binding ability of anti-chloramphenicol antibody and R, R-aminodiol

Chemically couple bovine serum albumin (BSA) to R,R aminodiol to obtain BSA-R,R-aminodiol. BSA (negative control) and BSA-R, R-aminoglycol were plated on an ELISA adsorption plate at 1 μg/ml, and coated overnight at 4°C. The next day, the coating solution was aspirated, the plate was washed, patted dry, 200 μL of blocking solution (1% BSA) was added, and blocked at 37°C for 1 hour. Aspirate and discard the blocking solution, wash the plate and pat dry, add 100 μL of primary antibody (anti-chloramphenicol, mouse antibody, 1:5000 dilution), and incubate at 37°C for 30 min. Aspirate and discard the primary antibody, wash the plate, pat dry, add 100 μL of the primary antibody (goat anti-mouse, 1:1000 dilution), and incubate at 37°C for 30 min. Aspirate and discard the secondary antibody, wash the plate, pat dry, add 100μL TMB color developing solution, incubate at 37°C for 15min, add 50μL 2M H ₂ SO _{4 to} each well to stop the reaction, and measure the OD ₄₅₀ value on the microplate reader for three groups In parallel experiments, the results are shown in Figure 3. The OD _{450 of the} BSA-R, R-aminoglycol group was significantly higher than that of the negative control group, confirming that anti-chloramphenicol can bind to R, R-aminoglycol.

Effect Example 3

Quantitative detection of chloramphenicol based on luciferase complementary biosensor

Add 1μM potein G-NGluc and 1μM anti-chloramphenicol to the A1 tube, the final volume is 100μL. Add 1μM potein G-NGluc, 1μM anti-chloramphenicol and 20μM chloramphenicol to the A2 tube, and the final volume is 100μL. Add 2μM CGluc-mSA, 2μM biotin-labeled R,R-aminoglycol (chloramphenicol analogue) into the B tube, the final volume is 200μL. After incubating tubes A1, A2 and B at 37°C for 30 minutes, take 100 μL of A1 and 100 μL of A2 and mix with 100 μL of B to form a C1 tube and a C2 tube, and incubate them at 37°C for 1 hour. The final concentration of potein G-NGluc and anti-chloramphenicol in the C1 tube is 0.5 μM, the final concentration of CGluc-mSA and biotin-labeled R, R-aminoglycol in the C2 tube is 1 μM, and the final concentration of chloramphenicol is 10 μM.

Take 200μL of the mixed solution of the C1 tube and the C2 tube after incubation, add 0.5μg coelenterazine (stock solution 0.5mg/ml) respectively, and immediately measure the bioluminescence intensity of the 480±20nm channel in the microplate reader. At the same time, it will contain potein G -NGluc and CGluc-mSA mixture is used as the blank control group C0. The results are shown in Figure 4. According to the luminescence signal intensity difference a between C2 and C0 and the luminescence signal intensity difference b between C1 and C0, you can get a and The difference between b is related to the concentration of chloramphenicol, which enables quantitative analysis of chloramphenicol in samples containing chloramphenicol. At the same time, please refer to Figure 5, which is a schematic diagram of a biosensor based on luciferase complementation. It can be seen that A1 and B are mixed to form a C1 tube, in which CGluc-mSA, biotin-labeled R, R-aminoglycol, anti -Chloramphenicol and potein G-NGluc complement each other to form a loop, which shortens the spatial distance between NGluc and CGluc, recombines them to restore luciferase activity, and can catalyze the substrate CTZ to generate luminescent signal intensity; A2 and B are mixed to form C2 tube, where the addition of chloramphenicol breaks the above closed loop, so that both the above closed loop appears in the C2 tube, and there is a complementation between chloramphenicol, anti-chloramphenicol, and potein G-NGluc. And the single potein G-NGluc, NGluc and CGluc have a spatial distance and cannot be reassembled to restore luciferase activity. Therefore, the intensity of the luminescent signal produced by the catalytic substrate CTZ is reduced, and it is linearly related to the amount of chloramphenicol added. Chloramphenicol is quantitatively detected.

Therefore, the biosensor based on luciferase complementation and the preparation method thereof provided by the present invention utilize the complementation of the amino-terminal fragment and the carboxy-terminal fragment of luciferase, the complementarity of the first marker and the second marker, and the test substance/to-be-tested substance. The quaternary complementation system of the complementation of the antigen and the antibody of the analyte and the complementation of the protein G and the antibody of the analyte can realize the rapid detection of the analyte by comparing the changes of the luminescence intensity before and after the addition of the analyte. At the same time, the whole process only needs simple detection. The instrument is convenient for detection, low cost, high detection sensitivity, strong specificity, and low background noise. It can be used for quantitative analysis of analytes in complex samples.

It should be noted that, based on the disclosure and explanation of the foregoing specification, those skilled in the art to which the present invention belongs can also change and modify the foregoing embodiments. Therefore, the present invention is not limited to the specific embodiments disclosed and described above, and some equivalent modifications and changes to the present invention should also fall within the protection scope of the claims of the present invention. In addition, although some specific terms are used in this specification, these terms are only for convenience of description and do not constitute any limitation to the present invention.

Claims

A biosensor based on luciferase complementation, which is characterized in that it comprises:

A luciferase amino-terminal fragment and a luciferase carboxy-terminal fragment, one of the amino-terminal of the luciferase amino-terminal fragment and the carboxy-terminal of the luciferase carboxy-terminal fragment is connected to the G protein, and the other is connected to the first A label is connected, and the luciferase amino-terminal fragment and the luciferase carboxy-terminal fragment are two complementary fragments of the same luciferase;

A analyte antigen, the analyte antigen is connected with a second label, and the second label is complementary to the first label;

Analyte antibody; and

Luciferase substrate.
The biosensor according to claim 1, wherein one of the first label and the second label is biotin, and the other is avidin.
The biosensor of claim 1, wherein the amino terminal of the luciferase amino terminal fragment and the carboxy terminal of the luciferase carboxy terminal fragment, one of the two is connected to the luciferase carboxy terminal fragment via a first connecting peptide. The G protein is connected, and the other is connected to the first label through a second connecting peptide, and the first connecting peptide and the second connecting peptide are flexible chains.
The biosensor according to claim 1, wherein the luciferase substrate comprises at least one of luciferin, sea luciferin, coelenterazine and isomers thereof.
The biosensor of claim 1, wherein the luciferase comprises at least one of Gaussia luciferase, Renilla luciferase, sea luciferase, firefly luciferase, and NanoLuc luciferase .
The biosensor of claim 5, wherein the luciferase is Gaussia luciferase, and the luciferase amino-terminal fragment and the luciferase carboxy-terminal fragment are the Gaussia luciferase Two complementary fragments formed separately between the G93 and E94 sites.
A method for preparing a biosensor based on luciferase complementation, which is characterized in that it comprises:

Construct a first expression vector containing the luciferase amino-terminal fragment gene, and construct a second expression vector containing the luciferase carboxy-terminal fragment gene, wherein the first expression vector contains the luciferase amino-terminal fragment gene 5'end, and one of the 3'ends of the luciferase carboxyl-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;

The first expression vector and the second expression vector are transformed, expressed and purified to obtain a luciferase amino-terminal fragment and a luciferase carboxy-terminal fragment, the amino-terminal of the luciferase amino-terminal fragment and the One of the carboxy-terminal fragments of the luciferase carboxy-terminal fragment is connected to the G protein, and the other is connected to the first label. The luciferase amino-terminal fragment and the luciferase carboxy-terminal fragment are of the same luciferase Two complementary fragments;

Provide a analyte antigen, the analyte antibody and a luciferase substrate, the analyte antigen is connected with a second label, the second label is complementary to the first label, and the The analyte antigen is combined with the analyte antibody, and the analyte antibody is combined with the G protein to obtain a biosensor based on luciferase complementation.
8. The preparation method of claim 7, wherein the first expression vector and the second expression vector contain a His tag gene.
The application of the biosensor according to any one of claims 1 to 6, or the preparation method according to any one of claims 7-8 in the detection of substance content.
The application according to claim 9, characterized in that it comprises:

Mix the luciferase amino-terminal fragment, the luciferase carboxy-terminal fragment, the analyte antigen, the analyte antibody, and the analyte with a known concentration, and then add the fluorescein After the substrates are mixed, the luminescence intensity is detected, and the standard curve of the relationship between the concentration of the analyte and the luminescence intensity is drawn;

After mixing the luciferase amino-terminal fragment, the luciferase carboxy-terminal fragment, the analyte antigen, the analyte antibody, and the luciferase substrate, the detected luminescence intensity is the first luminescence strength;

Mix the luciferase amino-terminal fragment, the luciferase carboxy-terminal fragment, the analyte antigen, the analyte antibody, and the analyte at different concentrations, and then add the luciferin After the enzyme substrates are mixed, the detected luminescence intensity is the second luminescence intensity;

According to the standard curve, the first luminous intensity and the second luminous intensity, the content of the analyte is calculated.