CN116836292A - Protein bioengineering sensor and application thereof - Google Patents

Abstract

The invention discloses a protein bioengineering sensor and application thereof, wherein the protein bioengineering sensor comprises a first fusion protein fragment of an immunogenic protein and a nano luciferase small subunit, and a second fusion protein fragment of the immunogenic protein and a nano luciferase large subunit, wherein the immunogenic protein is connected with the nano luciferase small subunit through a first connector, the immunogenic protein is connected with the nano luciferase large subunit through a second connector, and the first connector and the second connector are flexible peptide chains. The protein bioengineering sensor can be used for detecting antibodies targeting the immunogenic protein, and also can be used for screening small molecule drugs or antibody drugs targeting the immunogenic protein. The invention also discloses an HA protein bioengineering sensor and application thereof in detection of H7N9 influenza virus antibodies and screening of antiviral drugs. The sensor has the advantages of specificity, sensitivity, rapidness, simplicity, easiness in operation and the like in application.

Description

Protein bioengineering sensor and application thereof

Technical Field

The invention belongs to the technical field of medicine and health, in particular to the field of H7N9 influenza virus antibody detection and antiviral drug screening, and more particularly relates to a protein bioengineering sensor and application thereof.

Background

Influenza is a common acute respiratory infectious disease, can cause respiratory infection, and has remarkable morbidity and mortality in high-risk groups with low immunity, such as old people, children and the like. In 2013, a new recombinant type H7N9 avian influenza A virus appears in China, which causes human infection, and then H7N9 is popular in China for 5 times. Because of the mutation of part of amino acids of H7N9 of the novel recombinant avian influenza virus, the recombinant avian influenza virus can infect mammalian host cells, and although the pathogenicity of the recombinant H7N9 virus to an avian host is reduced, asymptomatic or mild avian diseases can be caused, most of the diseases of patients infected with the recombinant H7N9 virus can develop pneumonia and acute respiratory distress syndrome, and the death rate is as high as 40%. By 7 months 2019, a total of 1568 cases were diagnosed by the laboratory as human infection cases, of which 613 were death cases. The current clinical treatment strategy for H7N9 is supportive care and antiviral drug application within 48 hours after symptoms appear.

The current diagnosis method of human influenza virus mainly comprises serological detection, virus culture, real-time reverse transcription polymerase chain reaction (rRT-PCR), antigen detection analysis, immunofluorescence and the like. While rRT-PCR is the gold standard for current laboratory virus identification, it has high sensitivity and specificity, but has high requirements in terms of time, instrumentation and technical expertise, which greatly limits its application in this field. In addition, some laboratory methods for detecting influenza viruses are based on antigen-antibody reactions, such as lateral immunochromatographic assays and enzyme-linked immunosorbent assays (ELISA), hemagglutination inhibition assays and neuraminidase inhibition assays, and are also commonly used for classification of infectious viruses. However, these techniques often take a long time and have complicated operation procedures, and some operators who need specific equipment and a certain professional background are difficult to implement widely in primary hospitals. Therefore, the development of a rapid, specific and sensitive detection method has important significance.

Disclosure of Invention

In order to solve the defects in the prior art, the invention aims to provide a protein bioengineering sensor and application thereof. Influenza virus HA protein is another very important glycoprotein on the viral envelope besides Neuraminidase (NA), is immunogenic, and its neutralizing antibodies can effectively inhibit viral replication and growth, and HA protein-mediated host receptor recognition and subsequent membrane fusion play an irreplaceable role in the viral infection process, so HA protein is attracting increasing attention as a promising drug target. The invention clones the large subunit and the small subunit of the nano luciferase gene onto the HA protein of the H7N9 influenza virus respectively, successfully constructs a group of HA protein bioengineering sensors fused with the luciferase reporter gene, and is successfully applied to H7N9 influenza virus antibody detection and high-flux drug screening. The specific scheme is as follows:

the first aspect of the invention provides a protein bioengineering sensor comprising a first fusion protein fragment of an immunogenic protein and a small subunit of a nano-luciferase, and a second fusion protein fragment of the immunogenic protein and a large subunit of the nano-luciferase, wherein the immunogenic protein is linked to the small subunit of the nano-luciferase through a first linker, the immunogenic protein is linked to the large subunit of the nano-luciferase through a second linker, and the first linker and the second linker are flexible peptide chains.

Further, the first fusion protein fragment and the second fusion protein fragment comprise a 6 x HIS tag sequence.

Further, the amino acid sequence of the nano luciferase small subunit is shown as SEQ ID NO. 1;

the amino acid sequence of the large subunit of the nano luciferase is shown as SEQ ID NO. 2;

preferably, the amino acid sequences of the first linker and the second linker are shown in SEQ ID NO. 3.

Further, the first fusion protein fragment comprises an immunogenic protein, a 6×his tag sequence, a first linker and a nano-luciferase small subunit in order from N-terminus to C-terminus;

the second fusion protein fragment comprises an immunogenic protein, a 6×his tag sequence, a second linker and a nano-luciferase large subunit in sequence from the N-terminus to the C-terminus.

Further, the immunogenic protein is HA protein;

the amino acid sequence of the HA protein is shown as SEQ ID NO. 4.

Further, the amino acid sequence of the first fusion protein fragment is shown as SEQ ID NO. 5;

the amino acid sequence of the second fusion protein fragment is shown as SEQ ID NO. 6;

preferably, the nucleotide sequence of the first fusion protein fragment is shown in SEQ ID NO. 7;

preferably, the nucleotide sequence of the second fusion protein fragment is shown in SEQ ID NO. 8.

The second aspect of the invention provides the use of the protein bioengineering sensor in the detection of antibodies targeting immunogenic proteins.

Further, the method for detecting antibodies targeting immunogenic proteins by the protein bioengineered sensor comprises:

the first fusion protein fragment and the second fusion protein fragment are mixed according to a volume ratio of 1:1, mixing to obtain a protein bioengineering sensor;

mixing a protein bioengineering sensor, a sample to be detected, a substrate and a reaction buffer solution to prepare a reaction system;

detecting the fluorescence intensity by an enzyme-labeled instrument;

preferably, the concentration of the protein bioengineered sensor is 10-100nM;

preferably, the reaction buffer is: 25mM NaH ₂ PO ₄ ,pH7.5,150mM NaCl,0.05％Tween 20,0.2％BSA；

Preferably, the reaction system comprises 20 mu l of a protein bioengineering sensor, 20 mu l of a sample to be detected, 4 mu l of a substrate and 26 mu l of a reaction buffer solution;

preferably, the protein bioengineered sensor is an HA protein bioengineered sensor.

The third aspect of the invention provides application of the protein bioengineering sensor in screening small molecule drugs or antibody drugs targeting immunogenic proteins and application of the protein bioengineering sensor in analysis of action epitopes of the small molecule drugs or antibody drugs targeting immunogenic proteins.

Further, the method for screening the small molecule drugs or the antibody drugs targeting the immunogenic proteins by using the protein bioengineering sensor comprises the following steps:

the first fusion protein fragment and the second fusion protein fragment are mixed according to a volume ratio of 1:1, mixing to obtain a protein bioengineering sensor;

mixing a protein bioengineering sensor, a humanized antibody premix of targeted immunogenic proteins, a drug to be screened, a substrate and a reaction buffer solution to prepare a reaction system;

detecting the fluorescence intensity by an enzyme-labeled instrument;

preferably, the concentration of the protein bioengineered sensor is 2-100nM;

preferably, the concentration of the humanized antibody targeting the immunogenic protein is 1-100nM;

preferably, the reaction buffer is: 25mM NaH ₂ PO ₄ ,pH7.5,150mM NaCl,0.05％Tween 20,0.2％BSA；

Preferably, the reaction system is 20 μl of a premix of the protein bioengineering sensor and the humanized antibody targeting the immunogenic protein, 0.5 μl of the drug to be screened, 4 μl of the substrate, and 45.5 μl of the reaction buffer solution;

preferably, the protein bioengineered sensor is an HA protein bioengineered sensor.

The beneficial effects of the invention are as follows:

the invention discloses a protein bioengineering sensor based on fusion of an immunogenic protein and a nano-luciferase (NanoLuc Luciferase), which comprises a first fusion protein fragment of the immunogenic protein and a nano-luciferase small subunit and a second fusion protein fragment of the immunogenic protein and a nano-luciferase large subunit, wherein the immunogenic protein is connected with the nano-luciferase small subunit through a first connector, the immunogenic protein is connected with the nano-luciferase large subunit through a second connector, and the first connector and the second connector are flexible peptide chains. The protein bioengineering sensor can be used for detecting antibodies targeting the immunogenic protein, can also be used for screening small molecular drugs or antibody drugs targeting the immunogenic protein, and is beneficial to greatly simplifying and shortening working procedures of antibody detection and drug screening.

The invention designs the HA protein bioengineering sensor based on the HA protein of the H7N9 influenza virus and the nano luciferase, can be used for detecting the H7N9 influenza virus and developing medicaments, expands the application range of the HA protein, and particularly provides a brand new, quick and reliable detection method for detecting the influenza virus antibody and screening novel medicaments under the conditions of lacking a quick and simple detection method and generating wide medicament resistance of the influenza virus. The experimental result shows that the detection result of the HA protein bioengineering sensor HAs strong correlation with ELISA signal in the mouse in-vitro serological antibody detection (figure 8-9;R =0.76-0.85). The humanized antibody MED8852 targeting the neck region of the HA protein is used for competitive high-throughput drug screening, so that the compound library can be subjected to high-efficiency drug screening, and meanwhile, other competitive experiments targeting antibodies of other epitopes of the HA protein can be used for completing drug action epitope analysis. In addition, analysis of experimental results shows that the optimal analytical concentration of the sensor for antibody detection and high throughput drug screening is 2-10nM, and rapid detection of picomolar levels of antibody can be accomplished. Therefore, the bioengineering sensor has the characteristics of rapidness, sensitivity, specificity, simplicity, low dosage and the like, and can be used as a novel technology for H7N9 influenza virus antibody detection and high-flux drug screening.

Drawings

Fig. 1 is a schematic diagram of the HA protein bioengineering sensor provided in example 1 of the present invention in detection of H7N9 influenza virus antibodies and screening of antiviral drugs.

Fig. 2 is a preparation result of the HA protein bioengineering sensor protein provided in example 2 of the present invention.

FIG. 3 shows the result of optimizing the substrate of nano-luciferase (NanoLuc Luciferase) provided in example 3 of the present invention.

Fig. 4 shows the interaction result of the HA protein bioengineering sensor provided in example 3 of the invention with humanized antibody MED 8852.

Fig. 5 is a result of drug screening based on HA protein bioengineering sensor targeting H7N9 influenza virus HA protein provided in example 3 of the present invention.

Fig. 6 is a detection result of the mouse immune antibody of H7N9 influenza virus based on the HA protein bioengineering sensor and ELISA method provided in example 4 of the present invention.

Fig. 7 shows the detection results of the HA protein-based bioengineering sensor and ELISA method based on the H7N9 influenza virus mouse immune antibody provided in example 4 of the present invention.

Fig. 8 is a detection result of the humanized antibody of H7N9 influenza virus based on the HA protein bioengineering sensor and ELISA method provided in example 4 of the present invention.

FIG. 9 is a comparison result of the detection of H7N9 influenza virus mouse immune antibody using HA protein bioengineering sensor and ELISA method provided in example 4 of the present invention.

FIG. 10 shows the comparison result of the detection of H7N9 influenza virus humanized antibody using HA protein bioengineering sensor and ELISA method according to example 4 of this invention.

Detailed Description

For a better understanding and appreciation of the invention, reference will be made to the accompanying drawings and the detailed description of the invention, which are to be taken in connection with the accompanying drawings, but are not to be construed as limiting the invention.

Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

The HA protein bioengineering sensor in the embodiment of the invention is mainly based on a protein bioengineering sensor designed by fusion of H7N9 influenza virus HA protein and nano luciferase (NanoLuc Luciferase). The HA protein bioengineering sensor is expressed by a mature stable mammalian cell expression system, and is efficiently purified by methods such as affinity chromatography and the like to obtain a high-quality sensor. Subsequently, its application in vitro antibody serological detection of H7N9 influenza virus and high throughput drug screening was evaluated.

The HA bioengineering sensor and related antibodies displayed by the invention are expressed by high density suspension mammalian cells (HEK 293F) of the laboratory, and Union 293 medium is purchased from Shanghai Yonggang Biotechnology Co.

The HA protein bioengineering sensor plasmid in the embodiment of the invention is constructed by gene cloning in the laboratory, and plasmid sequence identification is completed by the Optic and biological technology Co.Ltd.

Affinity chromatography packing Ni Sepharose excel and ion exchange packing Q Sepharose High Performance used in the protein purification technique of the present invention are available from Cytiva, inc. and full-automatic protein purifiers are available from Shanghai Yonggang Biotech, inc.

The purity of the HA protein bioengineering sensor in the embodiment of the invention is finally identified by SDS-PAGE, and the purity is more than 90%.

In the embodiment of the invention, H7N9 influenza virus antibody in-vitro serological detection is carried out, serum is obtained from HA protein immunized mice, and 8 weeks of BALB/c female mice are adopted.

Example 1

The protein bioengineering sensor is mainly based on fusion design of an immunogenic protein and nano luciferase (NanoLuc Luciferase), and comprises a first fusion protein fragment of the immunogenic protein and the nano luciferase small subunit and a second fusion protein fragment of the immunogenic protein and the nano luciferase large subunit, wherein the immunogenic protein is connected with the nano luciferase small subunit through a first connector, the immunogenic protein is connected with the nano luciferase large subunit through a second connector, and the first connector and the second connector are flexible peptide chains. In a preferred embodiment the first fusion protein fragment and the second fusion protein fragment comprise a 6 x HIS tag sequence. In a preferred embodiment, the first fusion protein fragment comprises, in order from N-terminus to C-terminus, an immunogenic protein, a 6 x HIS tag sequence, a first linker, and a nano-luciferase small subunit; the second fusion protein fragment comprises an immunogenic protein, a 6×his tag sequence, a second linker and a nano-luciferase large subunit in sequence from the N-terminus to the C-terminus.

In a specific embodiment, the amino acid sequence of the small subunit of nano-luciferase is shown in SEQ ID NO. 1; the amino acid sequence of the large subunit of the nano luciferase is shown as SEQ ID NO. 2; the amino acid sequences of the first connector and the second connector are shown in SEQ ID NO. 3.

The protein bioengineering sensor provided by the invention can be used for detecting antibodies targeting the immunogenic protein and also can be used for screening small molecule drugs or antibody drugs targeting the immunogenic protein.

In this example, the HA protein is taken as an example to specifically illustrate the protein bioengineering sensor of the invention. The H7N9 influenza virus HA protein is another very important glycoprotein on the virus envelope besides Neuraminidase (NA), HAs immunogenicity, and can effectively inhibit the replication and growth of viruses, and the HA protein-mediated host receptor recognition and subsequent membrane fusion play an irreplaceable role in the virus infection process.

The design of the HA protein bioengineering sensor and the application thereof in H7N9 influenza virus antibody detection and antiviral drug screening (the pattern diagram is shown in figure 1).

The HA protein bioengineering sensor comprises a first fusion protein fragment HA-SB of HA protein and nano luciferase small subunit SB and a second fusion protein fragment HA-LB of HA protein and nano luciferase large subunit LB, wherein the HA protein is connected with SB through a first connector, and the HA protein is connected with LB through a second connector. HA-SB and HA-LB also included 6 XHIS tag sequences.

Specifically, the HA-SB comprises, in order from N-terminus to C-terminus, an HA protein, a 6 XHIS tag sequence, a first linker, and SB; the HA-LB comprises an HA protein, a 6×HIS tag sequence, a second linker and LB from the N-terminal to the C-terminal. The amino acid sequence of the small subunit of the nano luciferase is shown as SEQ ID NO. 1; the amino acid sequence of the large subunit of the nano luciferase is shown as SEQ ID NO. 2; the amino acid sequences of the first connector and the second connector are shown in SEQ ID NO. 3; the HA protein is amino acids at positions HA1-524 of H7N9 influenza virus, and the amino acid sequence of the HA protein is shown as SEQ ID NO. 4; the HA-SB amino acid sequence is shown in SEQ ID NO. 5; the HA-LB amino acid sequence is shown as SEQ ID NO. 6.

The HA protein bioengineering sensor provided by the embodiment can realize H7N9 influenza virus antibody detection and antiviral drug (including small molecule drugs and antibody drugs) screening. As shown in the schematic diagram in fig. 1, under the specific binding of the Fab end of the antibody, the subunits of the nano luciferase fused on the HA protein are close to each other and recombined into active nano luciferase, and after a reaction substrate is added, the substrate is catalyzed to emit light, and then a fluorescence signal can be detected by a multifunctional enzyme-labeled instrument. While there is only very weak binding between the size subunits of the nanoluciferase, its ability to combine into an active nanoluciferase depends on the interaction between the immunogenic material to which it is attached and the antibody. As shown in the schematic diagram in FIG. 1, the pre-mixture of the HA bioengineering sensor and the antibody can generate the same luminous effect as that shown in the detection of the pre-antibody, and after the small molecule drug and the antibody drug are added, the active drug can compete for the binding epitope of the antibody and the HA protein, so that the fluorescent signal is weakened. Further by setting a competition experiment of the compound with gradient concentration, the dose-dependent competition analysis of the compound can be completed.

Example 2

Expression and purification of HA protein bioengineered sensors (see fig. 2).

1. Mammalian cell (HEK 293F) expression of HA protein bioengineered sensor:

first, by feedingThe mammalian cell codons were optimized to obtain the HA-SB nucleotide sequence (shown as SEQ ID NO. 7) and the HA-LB nucleotide sequence (shown as SEQ ID NO. 8) of example 1, two genes, HA-SB and HA-LB, were successfully constructed by gene cloning, and the gene sequences were verified by gene sequencing. Genes synthesized by mammalian cell codon optimization were inserted into pCMV3 expression vectors containing CMV promoter and fused with IgK signal peptide at the N-terminus for secretory expression. The mature high-density suspension HEK293 cell expression system is adopted for expression, and the mammalian cell expression system is adopted for expression, so that the correct folding of the protein and the formation of a natural spatial conformation can be ensured; protein expression media mature commercial Union 293 media was selected. The expression conditions are optimized to obtain the optimal expression environment, and the expression parameters including the expression time, the temperature, the transfection reagent and the like are optimized in the small bottle. Subsequently, the expansion culture was performed according to the optimal expression conditions. The density of the invention is 4-8 multiplied by 10 ⁶ 293F cells with a viability of 98% or more were transiently transfected with plasmid, and were transfected at a mass ratio of plasmid to PEI of transfection reagent of 1:3 on day one, followed by downstream protein purification on day seven.

2. Purification of HA protein bioengineering sensor:

first, cell viability was measured and cell culture supernatants were collected by centrifugation, the cell viability was 80% or more to the optimum protein expression conditions, and then the supernatants were collected by centrifugation at 4000 Xg for 10 minutes. Simultaneously, ni Sepharose excel of the packing was equilibrated with Binding buffer (20 mM Tris-HCl, pH8.0,250mM NaCl) and the equilibrated packing was incubated with the cell culture supernatant for 1 hour. And then separating and purifying the target protein, firstly separating the incubated filler from the supernatant, and then performing gradient elution. The first step, flushing with a Binding buffer of at least 20 times of column volume; a second step of washing sequentially with a Bindingbuffer containing 20mM and 30mM imidazole, 10 column volumes per fraction; in the third step, 10 column volumes were eluted with 250mM imidazole. Finally, the purity of the protein of interest was analyzed on the basis of SDS-PAGE of each component sample, or further ion exchange purification was considered. Ion exchange purification was performed by an automated purification apparatus, bufferA:20mM Tris-HCl, pH8.0,50mM NaCl,Buffer B:20mM Tris-HCl, pH8.0,1M NaCl, elution was performed in a gradient of 20 column volumes (0-100% B), and finally, the target protein was analyzed based on the chromatographic peak and SDS-PAGE results, concentrated, split-packed and stored at-80 ℃.

3. Results: as shown in fig. 2.

Example 3

High throughput drug screening based on HA protein bioengineered sensors targeting H7N9 influenza virus (as in figures 3-5).

Bioengineering sensor based on fusion design of immunogenic protein and nano luciferase can complete competitive drug screening of antibody under the action of antibody targeting immunogenic protein. Furthermore, a drug action epitope assay may be performed in a competitive screen employing synchronization of two or more antibodies. And the screening method is easy to realize a high-throughput and automatic screening mode due to the rapid, simple and stable flow. In this example, taking HA protein as an example, a high-throughput drug screening based on HA protein bioengineering sensor targeting H7N9 influenza virus was performed.

1. Establishment of a high throughput screening method:

1) Analysis of optimal concentration of HA protein bioengineering sensor and antibody:

the concentration of the following HA protein bioengineering sensor refers to the final concentration of a system obtained by mixing HA-SB and HA-LB according to a ratio of 1:1, the concentrations are all the final concentration of the system, a 70 μl reaction system is adopted to react in a 96-well plate, and a reaction buffer solution is as follows: 25mM NaH ₂ PO ₄ pH7.5,150mM NaCl,0.05%Tween 20,0.2%BSA. Humanized antibody MED8852 targets the HA protein neck region. The reaction system was 20. Mu.l of sensor, 20. Mu.l of sample, 4. Mu.l of substrate, and buffer solution was added to 70. Mu.l, and the substrate was diluted 1:50 according to Promega instruction, and the present invention showed that 4. Mu.l was the optimum amount by the substrate amount optimization analysis (see FIG. 3). First, a series of sensor concentrations of 0.1nM, 0.5nM, 1nM, 2nM, 10nM, 20nM and corresponding antibody concentrations were set, and final analysis found that the sensor optimum concentration was 2nM and the antibody MED8852 optimum concentration was 2nM (left side of fig. 4). The fluorescence intensity generated at the optimal concentration sensor showed an antibody concentration dependence, which was also based on this sensor for H7N9 flowBasis for the detection of serological antibodies in vitro.

2) Epitope competition assay of antibody Fab fragments:

the gradient concentrations of MED8852Fab were set to compete with MED8852 for binding to the HA neck binding site. Analysis found that fluorescence intensity decreased with increasing competition for Fab concentration, exhibiting concentration dependence (right side of fig. 4). Subsequent high throughput drug screening is supported by this epitope competition binding principle.

2. High throughput drug screening:

high throughput drug screening employs competition assays of MED8852 to obtain reliable screening results and drug binding epitope assays. The reaction system is as follows: a final concentration of 20. Mu.l of a premix of 2nM HA sensor and 2nM MED8852, 0.5. Mu.l of compound, 45.5. Mu.l of buffer solution and 4. Mu.l of substrate. Screening of 7766 compounds in total was completed for both FDA-approved Drug Library (apedbio) and Custom Compound Library (Target MOI) libraries. The screening procedure is as follows: firstly, adding a buffer solution and a compound into a 96-well plate, secondly, adding a mixture of an HA sensor and an antibody mixed according to a ratio of 1:1, thirdly, incubating for 10-20 minutes under low-speed vibration, fourthly, adding a substrate, and fifthly, measuring fluorescence intensity by an enzyme-labeled instrument (measuring mode is Luminescence, integration Time:1000ms, attention: none). The whole flow is quick and simple, the automatic running water operation is convenient, and the result is stable and reliable.

3. Results: as shown in fig. 5;

all compound screening results are expressed as z-score and percentage ofeffect (%), with z-score less than-2 and Percentage ofeffect less than-90 being selected as positive screening results in this example.

Example 4

In vitro serological detection of H7N9 influenza virus antibodies based on HA protein bioengineered sensors (see FIGS. 6-10).

1. HA protein immunized mice obtained antibodies:

1) Mouse selection:

healthy BALB/c female mice were selected and bred to the eighth week for immunization.

2) Immunization strategies:

the three-needle immunization strategy is adopted, the immunization part is subcutaneously injected at the back of the mouse in a multipoint way, the immune protein is selected from H7N9 influenza virus HA protein subtype HA2013 and SD12, and the protein and Freund's adjuvant are mixed into a water-in-oil state according to the volume ratio of 1:1. The first needle (d 0), 100ug of protein was mixed with 250. Mu.l Freund's complete adjuvant, the second needle (d 21), 100ug of protein was mixed with 250. Mu.l Freund's incomplete adjuvant, and the third needle (d 42), 100ug of protein was mixed with 250. Mu.l Freund's incomplete adjuvant. Blood was collected around the eyes 7 days after the third immunization and serum was isolated and frozen at-80 ℃.

2. Conventional ELISA detects antibodies (left side of fig. 6-8):

first, protein HA2013 was diluted to 5ug/ml with a coating buffer solution (0.05M sodium carbonate) at pH9.6,

then in 50. Mu.l/Kong Jiazhi 96-well plates, at 4℃overnight. The following day, first step, washing the plate 3 times with PBS-T (PBS+0.05% Tween 20), adding 180 μl/well of PBS-B (PBS+5% BSA) at 37deg.C, blocking for 1 hr, and washing the plate 3 times with PBS-T; secondly, the initial dilution multiple of the serum to be tested is 1000 times, then the serum is diluted according to a 5-time gradient, PBS is used as a blank control, 50 μl/hole is incubated for 1 hour at 37 ℃, and then PBS-T is used for washing the plate for 3 times; thirdly, 50 μl of HRP-labeled goat anti-mouse IgG diluted 1:5000 with PBS-b (PBS+2.5% BSA) was added, incubated at 37deg.C for 1 hr, and the secondary antibody incubation was completed with PBS-T wash plate 6 times; fourthly, adding 50 μl TMB color development solution, incubating for 2-5 minutes at room temperature in a dark place, and adding 50 μl 1M sulfuric acid solution to terminate the reaction; fifthly, detecting by an enzyme-labeled instrument, wherein the reference wavelength is 570nm, and the detection wavelength is 450nm.

3. HA sensor detection antibody (right side of fig. 6-8):

the detection of the HA sensor antibody adopts a sensor with the concentration of 2nM, the reaction buffer solution and the high flux drug screening buffer solution are adopted, and the reaction system is 70 μl:20 μl of the sensor at a concentration of 2nM, 20 μl of the gradient diluted serum sample to be tested, 26 μl of the buffer solution, and 4 μl of the substrate. The method comprises the steps of adding a buffer solution into a 96-well plate, adding a serum sample to be detected, adding a sensor, performing low-speed vibration incubation for 20 minutes, adding a substrate, and performing detection fluorescence by an enzyme-labeled instrument, wherein the detection by the enzyme-labeled instrument is set in a high-flux medicine screening mode.

4. Results: as shown in fig. 9-10;

compared with the traditional ELISA method and the HA sensor for synchronously detecting the mouse immune antibody and the humanized antibody MED8852, the results show that the detection results of the two methods have better correlation (R=0.76-0.85), and the detection of the HA sensor HAs the obvious advantages of high sensitivity, rapidness, simplicity and the like.

The foregoing is merely a preferred embodiment of the present invention and not all embodiments thereof, and any equivalent modifications of the technical solution of the present invention that will be obvious to those skilled in the art upon reading the present specification are intended to be encompassed by the claims of the present invention.

SEQUENCE LISTING

<110> university of medical science in south China

<120> a protein bioengineering sensor and application thereof

<130> CP122010119C

<160> 8

<170> PatentIn version 3.3

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His Lys Cys Asp Asp Asp Cys Met Ala Ser Ile Arg Asn Asn Thr Tyr

485 490 495

Asp His Ser Lys Tyr Arg Glu Glu Ala Met Gln Asn Arg Ile Gln Ile

500 505 510

Asp Pro Val Lys Leu Ser Ser Gly Tyr Lys Asp Val

515 520

<210> 5

<211> 556

<212> PRT

<213> artificial sequence

<400> 5

Met Asn Thr Gln Ile Leu Val Phe Ala Leu Ile Ala Ile Ile Pro Thr

1 5 10 15

Asn Ala Asp Lys Ile Cys Leu Gly His His Ala Val Ser Asn Gly Thr

20 25 30

Lys Val Asn Thr Leu Thr Glu Arg Gly Val Glu Val Val Asn Ala Thr

35 40 45

Glu Thr Val Glu Arg Thr Asn Ile Pro Arg Ile Cys Ser Lys Gly Lys

50 55 60

Arg Thr Val Asp Leu Gly Gln Cys Gly Leu Leu Gly Thr Ile Thr Gly

65 70 75 80

Pro Pro Gln Cys Asp Gln Phe Leu Glu Phe Ser Ala Asp Leu Ile Ile

85 90 95

Glu Arg Arg Glu Gly Ser Asp Val Cys Tyr Pro Gly Lys Phe Val Asn

100 105 110

Glu Glu Ala Leu Arg Gln Ile Leu Arg Glu Ser Gly Gly Ile Asp Lys

115 120 125

Glu Ala Met Gly Phe Thr Tyr Ser Gly Ile Arg Thr Asn Gly Ala Thr

130 135 140

Ser Ala Cys Arg Arg Ser Gly Ser Ser Phe Tyr Ala Glu Met Lys Trp

145 150 155 160

Leu Leu Ser Asn Thr Asp Asn Ala Ala Phe Pro Gln Met Thr Lys Ser

165 170 175

Tyr Lys Asn Thr Arg Lys Ser Pro Ala Leu Ile Val Trp Gly Ile His

180 185 190

His Ser Val Ser Thr Ala Glu Gln Thr Lys Leu Tyr Gly Ser Gly Asn

195 200 205

Lys Leu Val Thr Val Gly Ser Ser Asn Tyr Gln Gln Ser Phe Val Pro

210 215 220

Ser Pro Gly Ala Arg Pro Gln Val Asn Gly Leu Ser Gly Arg Ile Asp

225 230 235 240

Phe His Trp Leu Met Leu Asn Pro Asn Asp Thr Val Thr Phe Ser Phe

245 250 255

Asn Gly Ala Phe Ile Ala Pro Asp Arg Ala Ser Phe Leu Arg Gly Lys

260 265 270

Ser Met Gly Ile Gln Ser Gly Val Gln Val Asp Ala Asn Cys Glu Gly

275 280 285

Asp Cys Tyr His Ser Gly Gly Thr Ile Ile Ser Asn Leu Pro Phe Gln

290 295 300

Asn Ile Asp Ser Arg Ala Val Gly Lys Cys Pro Arg Tyr Val Lys Gln

305 310 315 320

Arg Ser Leu Leu Leu Ala Thr Gly Met Lys Asn Val Pro Glu Ile Pro

325 330 335

Lys Gly Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu Asn Gly

340 345 350

Trp Glu Gly Leu Ile Asp Gly Trp Tyr Gly Phe Arg His Gln Asn Ala

355 360 365

Gln Gly Glu Gly Thr Ala Ala Asp Tyr Lys Ser Thr Gln Ser Ala Ile

370 375 380

Asp Gln Ile Thr Gly Lys Leu Asn Arg Leu Ile Glu Lys Thr Asn Gln

385 390 395 400

Gln Phe Glu Leu Ile Asp Asn Glu Phe Asn Glu Val Glu Lys Gln Ile

405 410 415

Gly Asn Val Ile Asn Trp Thr Arg Asp Ser Ile Thr Glu Val Trp Ser

420 425 430

Tyr Asn Ala Glu Leu Leu Val Ala Met Glu Asn Gln His Thr Ile Asp

435 440 445

Leu Ala Asp Ser Glu Met Asp Lys Leu Tyr Glu Arg Val Lys Arg Gln

450 455 460

Leu Arg Glu Asn Ala Glu Glu Asp Gly Thr Gly Cys Phe Glu Ile Phe

465 470 475 480

His Lys Cys Asp Asp Asp Cys Met Ala Ser Ile Arg Asn Asn Thr Tyr

485 490 495

Asp His Ser Lys Tyr Arg Glu Glu Ala Met Gln Asn Arg Ile Gln Ile

500 505 510

Asp Pro Val Lys Leu Ser Ser Gly Tyr Lys Asp Val His His His His

515 520 525

His His Gly Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser

530 535 540

Gly Val Thr Gly Tyr Arg Leu Phe Glu Glu Ile Leu

545 550 555

<210> 6

<211> 703

<212> PRT

<213> artificial sequence

<400> 6

Met Asn Thr Gln Ile Leu Val Phe Ala Leu Ile Ala Ile Ile Pro Thr

1 5 10 15

Asn Ala Asp Lys Ile Cys Leu Gly His His Ala Val Ser Asn Gly Thr

20 25 30

Lys Val Asn Thr Leu Thr Glu Arg Gly Val Glu Val Val Asn Ala Thr

35 40 45

Glu Thr Val Glu Arg Thr Asn Ile Pro Arg Ile Cys Ser Lys Gly Lys

50 55 60

Arg Thr Val Asp Leu Gly Gln Cys Gly Leu Leu Gly Thr Ile Thr Gly

65 70 75 80

Pro Pro Gln Cys Asp Gln Phe Leu Glu Phe Ser Ala Asp Leu Ile Ile

85 90 95

Glu Arg Arg Glu Gly Ser Asp Val Cys Tyr Pro Gly Lys Phe Val Asn

100 105 110

Glu Glu Ala Leu Arg Gln Ile Leu Arg Glu Ser Gly Gly Ile Asp Lys

115 120 125

Glu Ala Met Gly Phe Thr Tyr Ser Gly Ile Arg Thr Asn Gly Ala Thr

130 135 140

Ser Ala Cys Arg Arg Ser Gly Ser Ser Phe Tyr Ala Glu Met Lys Trp

145 150 155 160

Leu Leu Ser Asn Thr Asp Asn Ala Ala Phe Pro Gln Met Thr Lys Ser

165 170 175

Tyr Lys Asn Thr Arg Lys Ser Pro Ala Leu Ile Val Trp Gly Ile His

180 185 190

His Ser Val Ser Thr Ala Glu Gln Thr Lys Leu Tyr Gly Ser Gly Asn

195 200 205

Lys Leu Val Thr Val Gly Ser Ser Asn Tyr Gln Gln Ser Phe Val Pro

210 215 220

Ser Pro Gly Ala Arg Pro Gln Val Asn Gly Leu Ser Gly Arg Ile Asp

225 230 235 240

Phe His Trp Leu Met Leu Asn Pro Asn Asp Thr Val Thr Phe Ser Phe

245 250 255

Asn Gly Ala Phe Ile Ala Pro Asp Arg Ala Ser Phe Leu Arg Gly Lys

260 265 270

Ser Met Gly Ile Gln Ser Gly Val Gln Val Asp Ala Asn Cys Glu Gly

275 280 285

Asp Cys Tyr His Ser Gly Gly Thr Ile Ile Ser Asn Leu Pro Phe Gln

290 295 300

Asn Ile Asp Ser Arg Ala Val Gly Lys Cys Pro Arg Tyr Val Lys Gln

305 310 315 320

Arg Ser Leu Leu Leu Ala Thr Gly Met Lys Asn Val Pro Glu Ile Pro

325 330 335

Lys Gly Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu Asn Gly

340 345 350

Trp Glu Gly Leu Ile Asp Gly Trp Tyr Gly Phe Arg His Gln Asn Ala

355 360 365

Gln Gly Glu Gly Thr Ala Ala Asp Tyr Lys Ser Thr Gln Ser Ala Ile

370 375 380

Asp Gln Ile Thr Gly Lys Leu Asn Arg Leu Ile Glu Lys Thr Asn Gln

385 390 395 400

Gln Phe Glu Leu Ile Asp Asn Glu Phe Asn Glu Val Glu Lys Gln Ile

405 410 415

Gly Asn Val Ile Asn Trp Thr Arg Asp Ser Ile Thr Glu Val Trp Ser

420 425 430

Tyr Asn Ala Glu Leu Leu Val Ala Met Glu Asn Gln His Thr Ile Asp

435 440 445

Leu Ala Asp Ser Glu Met Asp Lys Leu Tyr Glu Arg Val Lys Arg Gln

450 455 460

Leu Arg Glu Asn Ala Glu Glu Asp Gly Thr Gly Cys Phe Glu Ile Phe

465 470 475 480

His Lys Cys Asp Asp Asp Cys Met Ala Ser Ile Arg Asn Asn Thr Tyr

485 490 495

Asp His Ser Lys Tyr Arg Glu Glu Ala Met Gln Asn Arg Ile Gln Ile

500 505 510

Asp Pro Val Lys Leu Ser Ser Gly Tyr Lys Asp Val His His His His

515 520 525

His His Gly Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser

530 535 540

Gly Val Phe Thr Leu Glu Asp Phe Val Gly Asp Trp Glu Gln Thr Ala

545 550 555 560

Ala Tyr Asn Leu Asp Gln Val Leu Glu Gln Gly Gly Val Ser Ser Leu

565 570 575

Leu Gln Asn Leu Ala Val Ser Val Thr Pro Ile Gln Arg Ile Val Arg

580 585 590

Ser Gly Glu Asn Ala Leu Lys Ile Asp Ile His Val Ile Ile Pro Tyr

595 600 605

Glu Gly Leu Ser Ala Asp Gln Met Ala Gln Ile Glu Glu Val Phe Lys

610 615 620

Val Val Tyr Pro Val Asp Asp His His Phe Lys Val Ile Leu Pro Tyr

625 630 635 640

Gly Thr Leu Val Ile Asp Gly Val Thr Pro Asn Met Leu Asn Tyr Phe

645 650 655

Gly Arg Pro Tyr Glu Gly Ile Ala Val Phe Asp Gly Lys Lys Ile Thr

660 665 670

Val Thr Gly Thr Leu Trp Asn Gly Asn Lys Ile Ile Asp Glu Arg Leu

675 680 685

Ile Thr Pro Asp Gly Ser Met Leu Phe Arg Val Thr Ile Asn Ser

690 695 700

<210> 7

<211> 1671

<212> DNA

<213> artificial sequence

<400> 7

atgaacactc aaatcctggt attcgctctg attgcgatca ttccaacaaa tgcagacaaa 60

atctgcctcg gacatcatgc cgtgtcaaac ggaaccaaag taaacacatt aactgaaaga 120

ggagtggaag tcgtcaatgc aactgaaaca gtggaacgaa caaacatccc caggatctgc 180

tcaaaaggga aaaggacagt tgacctcggt caatgtggac tcctggggac aatcactgga 240

ccacctcaat gtgaccaatt cctagaattt tcagccgatt taattattga gaggcgagaa 300

ggaagtgatg tctgttatcc tgggaaattc gtgaatgaag aagctctgag gcaaattctc 360

agagaatcag gcggaattga caaggaagca atgggattca catacagtgg aataagaact 420

aatggagcaa ccagtgcatg taggagatca ggatcttcat tctatgcaga aatgaaatgg 480

ctcctgtcaa acacagataa tgctgcattc ccgcagatga ctaagtcata taaaaataca 540

agaaaaagcc cagctctaat agtatggggg atccatcatt ccgtatcaac tgcagagcaa 600

accaagctat atgggagtgg aaacaaactg gtgacagttg ggagttctaa ttatcaacaa 660

tcttttgtac cgagtccagg agcgagacca caagttaatg gtctatctgg aagaattgac 720

tttcattggc taatgctaaa tcccaatgat acagtcactt tcagtttcaa tggggctttc 780

atagctccag accgtgcaag cttcctgaga ggaaaatcta tgggaatcca gagtggagta 840

caggttgatg ccaattgtga aggggactgc tatcatagtg gagggacaat aataagtaac 900

ttgccatttc agaacataga tagcagggca gttggaaaat gtccgagata tgttaagcaa 960

aggagtctgc tgctagcaac agggatgaag aatgttcctg agattccaaa gggaagaggc 1020

ctatttggtg ctatagcggg tttcattgaa aatggatggg aaggcctaat tgatggttgg 1080

tatggtttca gacaccagaa tgcacaggga gagggaactg ctgcagatta caaaagcact 1140

caatcggcaa ttgatcaaat aacaggaaaa ttaaaccggc ttatagaaaa aaccaaccaa 1200

caatttgagt tgatagacaa tgaattcaat gaggtagaga agcaaatcgg taatgtgata 1260

aattggacca gagattctat aacagaagtg tggtcataca atgctgaact cttggtagca 1320

atggagaacc agcatacaat tgatctggct gattcagaaa tggacaaact gtacgaacga 1380

gtgaaaagac agctgagaga gaatgctgaa gaagatggca ctggttgctt tgaaatattt 1440

cacaagtgtg atgatgactg tatggccagt attagaaata acacctatga tcacagcaaa 1500

tacagggaag aggcaatgca aaatagaata cagattgacc cagtcaaact aagcagcggc 1560

tacaaagatg tgcaccatca tcaccatcac ggcagttcag gtggtggcgg gagcggaggt 1620

ggaggctcga gcggtgtgac cggctaccgg ctgttcgagg agattctgta a 1671

<210> 8

<211> 2112

<212> DNA

<213> artificial sequence

<400> 8

atgaacactc aaatcctggt attcgctctg attgcgatca ttccaacaaa tgcagacaaa 60

atctgcctcg gacatcatgc cgtgtcaaac ggaaccaaag taaacacatt aactgaaaga 120

ggagtggaag tcgtcaatgc aactgaaaca gtggaacgaa caaacatccc caggatctgc 180

tcaaaaggga aaaggacagt tgacctcggt caatgtggac tcctggggac aatcactgga 240

ccacctcaat gtgaccaatt cctagaattt tcagccgatt taattattga gaggcgagaa 300

ggaagtgatg tctgttatcc tgggaaattc gtgaatgaag aagctctgag gcaaattctc 360

agagaatcag gcggaattga caaggaagca atgggattca catacagtgg aataagaact 420

aatggagcaa ccagtgcatg taggagatca ggatcttcat tctatgcaga aatgaaatgg 480

ctcctgtcaa acacagataa tgctgcattc ccgcagatga ctaagtcata taaaaataca 540

agaaaaagcc cagctctaat agtatggggg atccatcatt ccgtatcaac tgcagagcaa 600

accaagctat atgggagtgg aaacaaactg gtgacagttg ggagttctaa ttatcaacaa 660

tcttttgtac cgagtccagg agcgagacca caagttaatg gtctatctgg aagaattgac 720

tttcattggc taatgctaaa tcccaatgat acagtcactt tcagtttcaa tggggctttc 780

atagctccag accgtgcaag cttcctgaga ggaaaatcta tgggaatcca gagtggagta 840

caggttgatg ccaattgtga aggggactgc tatcatagtg gagggacaat aataagtaac 900

ttgccatttc agaacataga tagcagggca gttggaaaat gtccgagata tgttaagcaa 960

aggagtctgc tgctagcaac agggatgaag aatgttcctg agattccaaa gggaagaggc 1020

ctatttggtg ctatagcggg tttcattgaa aatggatggg aaggcctaat tgatggttgg 1080

tatggtttca gacaccagaa tgcacaggga gagggaactg ctgcagatta caaaagcact 1140

caatcggcaa ttgatcaaat aacaggaaaa ttaaaccggc ttatagaaaa aaccaaccaa 1200

caatttgagt tgatagacaa tgaattcaat gaggtagaga agcaaatcgg taatgtgata 1260

aattggacca gagattctat aacagaagtg tggtcataca atgctgaact cttggtagca 1320

atggagaacc agcatacaat tgatctggct gattcagaaa tggacaaact gtacgaacga 1380

gtgaaaagac agctgagaga gaatgctgaa gaagatggca ctggttgctt tgaaatattt 1440

cacaagtgtg atgatgactg tatggccagt attagaaata acacctatga tcacagcaaa 1500

tacagggaag aggcaatgca aaatagaata cagattgacc cagtcaaact aagcagcggc 1560

tacaaagatg tgcaccatca tcaccatcac ggcagttcag gtggtggcgg gagcggaggt 1620

ggaggctcga gcggtgtctt cacactcgaa gatttcgttg gggactggga acagacagcc 1680

gcctacaacc tggaccaagt ccttgaacag ggaggtgtgt ccagtttgct gcagaatctc 1740

gccgtgtccg taactccgat ccaaaggatt gtccggagcg gtgaaaatgc cctgaagatc 1800

gacatccatg tcatcatccc gtatgaaggt ctgagcgccg accaaatggc ccagatcgaa 1860

gaggtgttta aggtggtgta ccctgtggat gatcatcact ttaaggtgat cctgccctat 1920

ggcacactgg taatcgacgg ggttacgccg aacatgctga actatttcgg acggccgtat 1980

gaaggcatcg ccgtgttcga cggcaaaaag atcactgtaa cagggaccct gtggaacggc 2040

aacaaaatta tcgacgagcg cctgatcacc cccgacggct ccatgctgtt ccgagtaacc 2100

atcaacagct aa 2112

Claims (10)

1. The protein bioengineering sensor is characterized by comprising a first fusion protein fragment of an immunogenic protein and a nano luciferase small subunit, and a second fusion protein fragment of the immunogenic protein and a nano luciferase large subunit, wherein the immunogenic protein is connected with the nano luciferase small subunit through a first connector, the immunogenic protein is connected with the nano luciferase large subunit through a second connector, and the first connector and the second connector are flexible peptide chains.

2. The protein bioengineered sensor of claim 1, wherein the first and second fusion protein fragments comprise a 6 x HIS tag sequence.

3. The protein bioengineering sensor according to claim 1, wherein the amino acid sequence of the nano-luciferase small subunit is shown in SEQ ID No. 1;

the amino acid sequence of the large subunit of the nano luciferase is shown as SEQ ID NO. 2;

preferably, the amino acid sequences of the first linker and the second linker are shown in SEQ ID NO. 3.

4. The protein bioengineered sensor of claim 1, wherein the first fusion protein fragment comprises in order from N-terminus to C-terminus an immunogenic protein, a 6 x HIS tag sequence, a first linker and a nano-luciferase small subunit;

the second fusion protein fragment comprises an immunogenic protein, a 6×his tag sequence, a second linker and a nano-luciferase large subunit in sequence from the N-terminus to the C-terminus.

5. The protein bioengineered sensor of claim 1, wherein the immunogenic protein is HA protein;

the amino acid sequence of the HA protein is shown as SEQ ID NO. 4.

6. The protein bioengineering sensor of claim 5, wherein the amino acid sequence of the first fusion protein fragment is shown in SEQ ID No. 5;

the amino acid sequence of the second fusion protein fragment is shown as SEQ ID NO. 6;

preferably, the nucleotide sequence of the first fusion protein fragment is shown in SEQ ID NO. 7;

preferably, the nucleotide sequence of the second fusion protein fragment is shown in SEQ ID NO. 8.

7. Use of the protein bioengineered sensor according to claim 1 for detection of antibodies targeting immunogenic proteins.

8. The use of claim 7, wherein the protein bioengineering sensor detects antibodies targeting an immunogenic protein comprising:

the first fusion protein fragment and the second fusion protein fragment are mixed according to a volume ratio of 1:1, mixing to obtain a protein bioengineering sensor;

mixing a protein bioengineering sensor, a sample to be detected, a substrate and a reaction buffer solution to prepare a reaction system;

detecting the fluorescence intensity by an enzyme-labeled instrument;

preferably, the concentration of the protein bioengineered sensor is 10-100nM;

preferably, the reaction buffer is: 25mM NaH ₂ PO ₄ ,pH7.5,150mM NaCl,0.05％Tween 20,0.2％BSA；

Preferably, the reaction system comprises 20 mu l of a protein bioengineering sensor, 20 mu l of a sample to be detected, 4 mu l of a substrate and 26 mu l of a reaction buffer solution;

preferably, the protein bioengineered sensor is an HA protein bioengineered sensor.

9. The use of the protein bioengineering sensor of claim 1 for screening small molecule drugs or antibodies targeting immunogenic proteins for epitope analysis.

10. The use according to claim 9, wherein the method of screening for small molecule drugs or antibodies targeting immunogenic proteins by the protein bioengineering sensor comprises:

the first fusion protein fragment and the second fusion protein fragment are mixed according to a volume ratio of 1:1, mixing to obtain a protein bioengineering sensor;

mixing a protein bioengineering sensor, a humanized antibody premix of targeted immunogenic proteins, a drug to be screened, a substrate and a reaction buffer solution to prepare a reaction system;

detecting the fluorescence intensity by an enzyme-labeled instrument;

preferably, the concentration of the protein bioengineered sensor is 2-100nM;

preferably, the concentration of the humanized antibody targeting the immunogenic protein is 1-100nM;

preferably, the reaction buffer is: 25mM NaH ₂ PO ₄ ,pH7.5,150mM NaCl,0.05％Tween 20,0.2％BSA；

Preferably, the reaction system is 20 μl of a premix of the protein bioengineering sensor and the humanized antibody targeting the immunogenic protein, 0.5 μl of the drug to be screened, 4 μl of the substrate, and 45.5 μl of the reaction buffer solution;

preferably, the protein bioengineered sensor is an HA protein bioengineered sensor.