CN116855578A

CN116855578A - System for detecting target protein content and detection method using same

Info

Publication number: CN116855578A
Application number: CN202310668482.7A
Authority: CN
Inventors: 毋亚仙; 邢珊; 刘万里
Original assignee: Sun Yat Sen University Cancer Center
Current assignee: Sun Yat Sen University Cancer Center
Priority date: 2023-06-06
Filing date: 2023-06-06
Publication date: 2023-10-10
Anticipated expiration: 2043-06-06
Also published as: CN116855578B

Abstract

The application provides a system for detecting the content of target protein, which comprises: (1) A first antibody that binds to a protein of interest, a second antibody that binds to a protein of interest with a first label, a conjugate that binds to the first label; (2) A single stranded deoxyribonucleic acid concatemer with a second label bound to the conjugate, the single stranded deoxyribonucleic acid concatemer being prepared by a primer exchange reaction; (3) A promoter sequence associated with the single stranded deoxyribonucleic acid tandem, and a ribonucleic acid polymerase associated with the promoter sequence; (4) Cas13a protein, crRNA, and RNA substrate with fluorescent label. The system for detecting the content of the target protein is used for detecting serum trace IL-15, the detection sensitivity is 153 times higher than that of ELISA (enzyme-linked immunosorbent assay) of the traditional detection method, the detection performance is improved, complex thermal cycle instruments such as PCR (polymerase chain reaction) and the like are not needed, and amplicon pollution is avoided.

Description

System for detecting target protein content and detection method using same

Technical Field

The application belongs to the technical field of molecular biology, and particularly relates to a system for detecting target protein content and a detection method using the same.

Background

Cas13a is an iv-a type RNA ribonuclease that degrades the RNA strand targeted by CRISPR RNA (crRNA), wherein after specific binding of the crRNA to the target RNA, the crRNA-target RNA duplex will bind to the positively charged central channel of the LbuCas13a protein Nuclease (NUC) leaf, causing a significant conformational change in the Cas13a protein, triggering the movement of the higher eukaryotic and prokaryotic nucleotide binding (HEPN) 1 domain to the HEPN2 domain, activating the HEPN catalytic site of the Cas13a protein, thereby cleaving single stranded RNA in a non-specific manner.

Cytokines are a series of soluble small molecule proteins with a wide range of biological activities synthesized and secreted by immune cells such as T cells, B cells, NK cells and macrophages, as well as some non-immune cells, which act as a molecular messenger, allowing the immune system cells to communicate with each other to produce a coordinated effect on the target antigen, with immunomodulatory and effector functions in many diseases. At present, researches indicate that the level of the peripheral blood cytokines is closely related to the curative effect of tumor immunotherapy, and the method can be used for predicting the curative effect of tumor immunotherapy, and compared with the current clinical markers for immunotherapy prediction such as tumor tissue PD-L1 expression level, tumor mutation load detection (TMB) and microsatellite instability (MSI), the peripheral blood cytokines detection has the advantages of simple sampling, low detection cost, sustainable monitoring and the like, so that the method is more suitable for clinical immunotherapy curative effect prediction.

However, the content of part of cytokines in peripheral blood is low, and the current clinical methods for detecting peripheral blood proteins mainly comprise enzyme-mediated chemiluminescence systems or chromogenic systems, such as electrochemiluminescence and ELISA, and the sensitivity of the methods is insufficient, the lower limit of the detected proteins is only 0.01-50 ng/mL, and samples with low cytokine content cannot be distinguished.

In order to increase the sensitivity of protein detection, it is necessary to establish a protein detection system having high sensitivity.

Disclosure of Invention

The application aims to provide a system for detecting the content of target protein and a detection method using the system, which remarkably improve the sensitivity of protein detection and expand the applicable concentration range of protein detection.

According to a first aspect of the present application there is provided a system for detecting the content of a protein of interest, the system comprising: (1) A first antibody that binds to a protein of interest, a second antibody that binds to a protein of interest with a first label, a conjugate that binds to the first label; (2) A single stranded deoxyribonucleic acid concatemer with a second label bound to the conjugate, the single stranded deoxyribonucleic acid concatemer being prepared by a primer exchange reaction; (3) A promoter sequence associated with the single stranded deoxyribonucleic acid tandem, and a ribonucleic acid polymerase associated with the promoter sequence; (4) Cas13a protein, crRNA, and RNA substrate with fluorescent label.

The system for detecting the content of the target protein is selected for detection, and the target protein can be captured by an ELISA double-antibody sandwich method by utilizing a first antibody combined with the target protein, a second antibody provided with a first marker and combined with the target protein; the first marker is combined with the second marker through a conjugate, and the conversion from protein signals to nucleic acid signals is completed by utilizing a single-stranded deoxyribonucleic acid concatemer with the second marker; a ribonucleic acid polymerase coupled to the promoter sequence using the promoter sequence coupled to the single stranded deoxyribonucleic acid tandem, thereby generating a corresponding target ribonucleic acid; using Cas13a protein, crRNA and RNA substrate with fluorescent label, using CRISPR/Cas13a as report system, under the guidance of crRNA, cas13a protein specifically targets target RNA, making the activity of the side cutting enzyme of Cas13a activated, cutting the RNA substrate with fluorescent label (i.e. RNA fluorescent report probe) around, releasing fluorescent signal; finally, by analyzing the fluorescence intensity, the target protein concentration (fluorescence signal rise can be detected only in the presence of Cas13a, crRNA and target RNA). According to the system for detecting the content of the target protein, provided by the application, the corresponding first antibody and second antibody can be replaced according to the target protein, so that the universality of the detection system is realized, and the sensitivity of protein detection is obviously improved.

Preferably, the single stranded DNA concatemer is a sequence beginning with 2 bases TT and bounded by base A by a repeat CAACTTAAC, 2500.+ -.500 nt in length.

Preferably, the promoter sequence is a T7 promoter.

Since the pre-synthesized single-stranded DNA concatemer is selected to be a sequence which starts with 2 bases TT and is formed by connecting a repeated sequence CAACTTAAC with a base A, the length is 2500+/-500 nt, and the hybridization can be theoretically carried out with nearly hundred probes with T7 promoter sequences, thereby achieving the first signal amplification. And, utilize the supplies in (3) to make up TMA transcription system, make ribonucleic acid polymerase (namely T7 RNA polymerase) combine with T7 promoter sequence (T7 promoter sequence is a part of AS/S probe sequence), thus start transcription, produce the target RNA, achieve the second signal amplification. And taking the CRISPR/Cas13a as a detection system, combining target RNA specificity with crRNA under the guidance of crRNA, so that the conformation of the Cas13a is changed and activated, thereby efficiently and indiscriminately cutting surrounding ssRNA, and at the moment, the RNA fluorescence reporter probe in the system is also cut, and a fluorescence signal is released, so that the third signal amplification is achieved. The system for detecting the content of the target protein provided by the application adopts three times of signal amplification, and the sensitivity of protein detection is obviously improved.

Preferably, the crRNA is selected from T3crRNA, and the sequence of the T3crRNA is shown as SEQ ID NO. 11.

Aiming at the target RNA sequence, crRNA molecules for identifying the positions of different target RNA sequences are designed at every 3 bases, the length of the selected crRNA molecules for identifying the target RNA sequences is analyzed, and according to the analysis of the detection result, the detection is carried out by adopting T3crRNA with the sequence shown as SEQ ID NO.11, and the detected fluorescence slope is higher than that of other crRNAs.

Preferably, the fluorescently labeled RNA substrate is a laboratory detection kit v2 fluorescent probe from RNAse Alert (hereinafter referred to as v2 fluorescent probe).

The fluorescence probes in the CRISPR/Cas13a system are selected, and three fluorescence probes of v2, UUAUU and UUUU are selected for testing under the same concentration of 2 mu M, so that the fluorescence slope obtained by v2 is found to be the highest (P value is smaller than 0.0001). The laboratory detection kit v2 fluorescent probe of RNAse Alert company adopts a brand new and higher-sensitivity novel RNA substrate, one end of which is marked with a fluorescent reporter group molecule (fluor) and the other end of which is marked with a quenching group.

Preferably, the first label is biotin, the conjugate is streptavidin, and the second label is biotin.

The biotin and the streptavidin can be combined mutually, so that the biotin is selected as the first marker and the streptavidin is selected as the second marker, and the streptavidin is selected as the conjugate, so that the second antibody with the first marker and the single-stranded deoxyribonucleic acid (DNA) concatemer with the second marker can be bridged mutually through the connection of the first marker and the second marker by the conjugate, so that the protein signal of the target protein combined with the second antibody is converted into the nucleic acid signal of the single-stranded DNA concatemer, and the detection means which can be used for detecting the protein signal are enlarged.

Preferably, the system is a kit.

According to a second aspect of the present application, there is provided a protein content detection method based on the above system for detecting a target protein content, comprising the steps of: s1, capturing target proteins: capturing the target protein by adopting a first antibody which is combined with the target protein and a second antibody which is provided with a first marker and combined with the target protein through an ELISA double-antibody sandwich method; s2, protein signal conversion: adding the conjugate into a system, combining the conjugate with a second antibody with a first marker in S1, washing to remove the redundant conjugate, and adding a hybridization complex with a second marker into the system, coupling the first marker with the second marker through the conjugate, so that a protein signal is converted into a nucleic acid signal, wherein the hybridization complex is prepared by hybridization compounding a single-stranded deoxyribonucleic acid concatemer with a promoter sequence in advance; s3, amplifying nucleic acid signals: uniformly mixing the hybridization complex after mixing and incubation in the S2 and ribonucleic acid polymerase, and performing transcription mediated amplification to generate target ribonucleic acid; s4, fluorescence quantitative detection: uniformly mixing the target ribonucleic acid, crRNA, cas13a protein and a fluorescent reporter probe in the S3, and measuring the concentration of the target protein by analyzing the fluorescence intensity; protein content detection methods do not include methods aimed at diagnosis and/or treatment of disease.

In the application, the inventor establishes a novel high-sensitivity micro protein detection system which generates fluorescent signals through a CRISPR/Cas13a system after the PER serial bodies are subjected to multiple activation of TMA, is named as an immune-PER-TME-CRISPR/Cas 13a (iPTC) system, and has higher detection sensitivity than ELISA 153 times when being used for detecting serum micro IL-15, thereby realizing the improvement of detection performance, avoiding complex thermal cycle instruments such as PCR and the like, and avoiding amplicon pollution.

Preferably, in S3, the concentration ratio of single stranded dna concatemers to promoter sequences is 1:80, the final concentration of single stranded deoxyribonucleic acid concatemers was 2.5nM.

When the concentration ratio of single-stranded deoxyribonucleic acid concatemer to promoter sequence is 1: 80. the resulting fluorescence slope was higher at a final concentration of 2.5nM for the single stranded DNA concatemers.

Preferably, in S4, the initial concentration of the fluorescent reporter probe is 2 μm and the final concentration of Cas13a protein is 200nM.

When v2 fluorescent reporter probe and Cas13a protein were selected for testing at the above concentrations, a higher fluorescence slope could be measured.

Drawings

FIG. 1 is a schematic of the fluorescence slope of different single stranded deoxyribonucleic acid concatemers, with the upper left inset showing the fluorescence intensity of the CRISPR/Cas13a system observed under UV-visible reading, resulting from 3a, 6a, 9a and PER concatemers, respectively, from left to right;

FIG. 2 is a graph showing the detection results of different crRNAs targeting recognition target RNAs in a CRISPR/Cas13a system;

FIG. 3 is a detection result of different lengths of crRNA targeting recognition target RNA in a CRISPR/Cas13a system;

FIG. 4 is a schematic representation of fluorescence slopes of different fluorescent probes in a CRISPR/Cas13a system;

FIG. 5 is a graph showing the measured fluorescence slopes of PER concatemers and AS/S probes at different hybridization temperatures;

FIG. 6 is a graph showing the measured fluorescence slope at different ratios of PER concatemers to AS/S probes;

FIG. 7 is a graph showing the slope of fluorescence measured at various concentrations of PER concatemers;

FIG. 8 is a schematic representation of fluorescence slope of fluorescent probes at different concentrations in a CRISPR/Cas13a system;

FIG. 9 is a schematic representation of fluorescence slope of different concentrations of Cas13a protein in a CRISPR/Cas13a system;

FIG. 10 is a plot of IL-15 concentration versus fluorescence value;

FIG. 11 is a schematic diagram showing the results of the protein content detection method (iPTC) for detecting the specificity;

FIG. 12 is a schematic diagram showing the results of IL-2 assay by protein content assay (iPTC);

FIG. 13 is a schematic diagram showing the results of ELISA method for IL-15 test;

FIG. 14 shows the principle of detection of the protein content detection method (iPTC) method.

Detailed Description

In order that the manner in which the above-recited embodiments of the application are attained and can be readily understood by those skilled in the art, a more particular description of the application will be rendered by reference to specific embodiments thereof which are illustrated in the appended drawings. All other embodiments, which can be made by those skilled in the art based on the embodiments of the present application without making any inventive effort, shall fall within the scope of the present application.

Example 1

The materials selected in this embodiment are: the target protein is IL-15, the first marker is biotin, the second marker is biotin, the conjugate is streptavidin, the first antibody is a coated antibody aiming at IL-15, the second antibody is a detection antibody aiming at IL-15, the single-stranded deoxyribonucleic acid concatemer is a sequence which starts with 2 bases TT and is connected with a repeated sequence CAACTTAAC by bases A and has the length of 2500+/-500 nt, the promoter sequence is a T7 promoter, the crRNA is T3crRNA, and the RNA substrate with fluorescent marker is a laboratory detection kit v2 fluorescent probe of RNAse Alert company.

The nucleic acid primer of the application is synthesized by Guangzhou Rui Bo biological company or Shanghai biological company, and purified by PAGE method or HPLC method. The primer sequences and the sequences are shown in Table 1.

TABLE 1 description of the sequences

The detection is carried out according to the following steps:

(1) Expression and purification of Cas13a proteins

(1) Transforming the Cas13a plasmid (containing an Amp resistance sequence) into Rosetta2DE3 competence by adopting a general mode in the field, and culturing;

(2) inducing expression in a manner common in the art;

(3) protein purification was performed in a manner common in the art.

(2) Preparation of crRNA

1. First, ssDNA templates (sense strand and antisense strand) with promoter sequences corresponding to the crRNA of interest were synthesized, and an annealing system comprising 10. Mu.L of sense strand, 10. Mu.L of antisense strand, 10. Mu.L of 10 XPCR buffer, 70. Mu.L of RF water (enzyme-free water) was configured, wherein the sequences of each sense strand and antisense strand are shown in Table 1 above.

2. Placing the annealing system in a metal bath at 95 ℃ for 10min, slowly cooling to room temperature for about 1.5h, and then centrifuging at 8000rpm at 4 ℃ for 2min;

3. adding 10 mu L of sodium acetate and 250 mu L of absolute ethyl alcohol into the system, mixing the mixture evenly in a reversed manner, centrifuging the mixture at 12000rpm and 4 ℃ for 15min, wherein white precipitation is visible at the bottom of the tube, and discarding the supernatant;

4. adding 1mL of 75% ethanol to wash the precipitate, mixing well, centrifuging at 12000rpm and 4 ℃ for 15min, discarding the supernatant, and repeating the steps twice;

5. after the supernatant was aspirated, the EP tube was placed in a fume hood and left to dry upside down until the white precipitate became clear, and an appropriate amount of RF water was added to dissolve, and then the dsDNA concentration was measured on a spectrophotometer.

The T7 in vitro transcription system includes: mu.L of dsDNA, 10. Mu.L of NTP buffer mix, 2. Mu. L T7 RNA polymerase mix, 17. Mu.L of RF water. The T7 in vitro transcription system is placed in a 37 ℃ water bath box for overnight incubation, and a transcription product is prepared.

6. Centrifuging the above transcription product at 8000rpm at 4deg.C for 2min, adding 20 μl RF water, 2 μl DNase (final concentration of 2U/. Mu.L), incubating at 37deg.C for 15min, and removing residual dsDNA in the system;

7. adding 100 μl of RF water and 1mL of phenol-chloroform extraction reagent, shaking vigorously for 15s, standing at room temperature for 10min, centrifuging at 12000rpm and 4deg.C for 15min, transferring the uppermost aqueous phase layer to a new EP tube, adding equal volume of isopropanol, mixing, standing at room temperature for more than 30 min;

8. centrifuge at 12000rpm at 4℃for 10min, discard supernatant and wash twice with pre-chilled 75% ethanol. And finally, placing the precipitate in a fume hood for airing, adding a proper amount of RF water for dissolution, and measuring the concentration by a spectrophotometer for later use. (3) CRISPR/Cas13a system and fluorescence detection

1. The CRISPR/Cas13a system comprises 11 μl RF water, 2 μl crRNA (5 μΜ), 2 μl Thermo pol reaction buffer (10×), 2 μ L V2 fluorescent probe (2 μΜ), 1 μl Cas13a protein (2 μΜ).

2. After the above system is evenly mixed, the mixture is centrifuged briefly, the mixture is transferred into 384-well plates, 18 mu L of target RNA is added into each well, 2 mu L of target RNA is added, the mixture is covered by a cover plate film, and the mixture is placed into a centrifuge for centrifugation for 30 seconds, and the mixture is subjected to on-line detection within 10 minutes. Placing the enzyme label into an enzyme label instrument, and setting the following parameters: excitation wavelength (Excitation wavelength) =485 nM, emission wavelength (Emission wavelength) =535 nM, gain value=80, measurement time (Kinetic duration) =1 h, interval time (Interval time) =2 min;

3. the results of Fluorescence Intensity (FI) were analyzed.

(4) Preparation of primer exchange reaction (Primer Exchange Reaction, PER) sequences

1. Preparing a PER reaction system, wherein the final concentration of each reagent is as follows: 0.8U/. Mu.L Bst DNA polymerase large fragment, 0.5. Mu.M hairpin aa,1.8mM dATP/dTTP/dCTP solution mixture, 0.1. Mu.M dGTP cleaner, 1 XSOH buffer, 10mM MgSO4, RF water to corresponding volumes; wherein the sequence of hairpin aa is shown in Table 1.

2. After being evenly mixed, the mixture is centrifuged for a short time, and is placed in a constant temperature water bath box at 37 ℃ for incubation for 15min, so as to remove dGTP possibly existing in the system;

3. taking out and adding the primer a with the final concentration of 1 mu M, uniformly mixing, centrifuging briefly, and placing in a constant-temperature water bath box at 37 ℃ for incubation for 2 hours; the sequence of primer a is shown in Table 1.

4. Taking out, incubating in a metal bath with a temperature of 80 ℃ in advance for 20min, inactivating the polymerase at a high temperature, and terminating the reaction to obtain a single-stranded deoxyribonucleic acid concatemer, and performing the following purification process.

(5) Purification of primer exchange reaction sequences

1. Protein impurities and DNA fragments of less than 100 bases in the primer exchange reaction were removed using the e.z.n.a.cycle Pure kit, which is the most reliable and rapid kit for purifying PCR products.

2. Add 4-5 volumes of CP buffer to the prepared system, slightly shake and briefly centrifuge and transfer to HiBind's DNA mini-column, put into 2mL receiving tube.

3. Centrifuge at room temperature for 1 minute with a centrifugal force of 13000 Xg.

4. After the waste liquid in the receiving tube obtained by centrifugation is poured out, the receiving tube is put into the DNA mini column of HiBind again.

5. After adding 700. Mu.L of the DNA washing buffer and centrifuging at room temperature for 1 minute by using the same centrifugal force of 13000 Xg, the waste liquid was poured off, and this step was repeated for a total of two washes.

6. The empty tube from which the waste liquid was poured was centrifuged at 13000×g for 2 minutes at room temperature to remove residual alcohol and other solutions.

7. The HiBind DNA mini-column was transferred to a new 1.5mL Ep tube, 50. Mu.L of RF water was added, and the mixture was allowed to stand at room temperature for 2 minutes.

8. The purified single-stranded deoxyribonucleic acid concatemers (hereinafter, each of which is replaced by PER concatemers) were obtained by centrifugation at 13000 Xg for 1 minute at room temperature, and the concentrations thereof were measured by a spectrophotometer.

Wherein the PER concatemer is a sequence which starts with 2 bases TT and is formed by connecting a repeated sequence CAACTTAAC with a base A, and the length of the repeated sequence is 2500nt.

(6) Detection of ssDNA concatamers of different lengths on magnetic beads with TMA-CRISPR/Cas13a System

1. PER concatemer hybridizes to the T7 promoter sequence: total volume was 200 μl,100fM of biotin-bearing 3a, 6a, 9a, PER concatamer and blank (RF water), 10pM of T7-bearing promoter sequence (AS/S probe), 1 xpcr buffer, and finally RF water was added to make up to 200 μl, the system was placed in a metal bath with an early warming, and after 10min at 95 ℃ it was allowed to slowly cool naturally to room temperature in the metal bath for approximately 1h.

Wherein 3a is a sequence starting with 2 bases TT and connecting 3 repeated sequences CAACTTAAC by base A, 6a, 9a and so on; the specific sequences of 3a, 6a, 9a are shown in table 1 above; the PER concatemer is the single-stranded deoxyribonucleic acid concatemer in (5) above.

2. Taking 200 mu L of magnetic beads with streptavidin, washing twice by a magnetic rack, discarding supernatant, adding the hybridization product into the washed magnetic beads, incubating for 1h on a shaking table at room temperature, combining the magnetic beads with streptavidin with DNA concatemers with biotin, washing 4 times by 0.05% PBST washing liquid, removing AS/S probes which are not combined with the DNA concatemers, and discarding washing liquid;

3. TMA transcription system: the final concentration is 5mM ATP/GTP/CTP/UTP mixture, 1 Xreaction buffer, 1. Mu.L of T7 RNA polymerase mixture, finally RF water is added to supplement 20. Mu.L, the prepared TMA transcription system is added into the prepared magnetic beads, incubation is carried out for 40min at 37 ℃, then the supernatant is transferred into a new Ep tube by a magnetic rack, and 2. Mu.L is added into the CRISPR/Cas13a system;

4. CRISPR/Cas13a system: 200nM purified Cas13a protein (LbuCas 13 a), 500nM crRNA,200nM V2 fluorescent reporter probe, 1 Xthermo pol reaction buffer, 2. Mu.L of the product prepared above, and finally make up to 20. Mu.L with RF water. Adding the system into 384-well plates, putting the system into an enzyme-labeled instrument, and setting the parameters as follows: excitation wavelength (Excitation wavelength) =485 nM, emission wavelength (Emission wavelength) =535 nM, gain value=80, measurement time (Kinetic duration) =1 h, interval time (Interval time) =2 min.

(7) Protein detection method (Immuno-TMA-CRISPR/Cas 13a, iPTC) operation procedure (for example, detection of IL-15 content in serum)

S1, capturing target proteins: capturing target proteins by an ELISA double-antibody sandwich method;

1. dilution of standard: the IL-15 standard was diluted with standard dilutions at 6.25pg/mL, 3.12pg/mL, 1.56pg/mL, 1pg/mL, 0.5pg/mL;

2. adding a sample: taking out patient serum from-80 ℃ in advance, thawing, adding the patient serum and diluted standard substances into a commercial 96-well plate coated with a capture antibody, incubating for 1h at 37 ℃ with 100 mu L of each well, and washing with 0.05% PBST washing solution once;

3. adding a detection antibody: dilution with primary antibody in kit 1:100 to biotin secondary antibody dilution, per well of 100u L,37 degrees C1 h incubation, 0.05% PBST washing three times. Wherein the first antibody and the second antibody are both antibodies against IL-15.

S2, protein signal conversion:

1. mixing single-stranded deoxyribonucleic acid concatemers (PER concatemers) with a second marker biotin concentration of 500ng/mL, incubating for 20min at 37 ℃ for 100 μl PER well, and washing with 0.05% PBST wash three times; wherein, the single-stranded deoxyribonucleic acid concatemer is prepared by the primer exchange reaction of the (4) and (5); the length of the single-stranded deoxyribonucleic acid concatemer is 2500+/-500 nt, and the single-stranded deoxyribonucleic acid concatemer has 125 pairs of AS/S probe binding sites.

2. The single-stranded deoxyribonucleic acid concatemer with the second marker is mixed and incubated with the target protein combined with the second antibody with the first marker and the conjugate streptavidin in the S1, and the first marker is combined with the second marker through the conjugate, so that a protein signal is converted into a nucleic acid signal.

S3, amplifying nucleic acid signals:

1. the single-stranded deoxyribonucleic acid concatemer after 10nM of S2 is mixed, the promoter sequence of the AS/S probe of 200nM and 1 XPCR buffer are mixed, the mixture is supplemented to a corresponding volume by RF water, the mixture is slowly and naturally cooled to room temperature in a metal bath after 10min at 95 ℃ for about 1h, and the promoter-concatemer hybridization complex is prepared.

2. After 4-fold dilution of the promoter-concatemer hybridization complex with 5% Bovine Serum Albumin (BSA), 25. Mu.L per well was incubated at 37℃for 20min and washed four times with 0.05% PBST wash.

3. Adding a TMA transcription system: the target RNA is produced by mixing a promoter-concatemer hybridization complex with T7 RNA polymerase capable of binding to a T7 promoter sequence, and performing transcription-mediated amplification (TMA). TMA system was configured as described in (6) above, with 20. Mu.L per well, incubated at 37℃for 40min.

S4, fluorescence quantitative detection:

1. kong Zhongshang clear (containing target RNA) was transferred into new Ep tubes, plus 2 μl in the CRISPR/Cas13a system.

2. The CRISPR/Cas13a system comprises target ribonucleic acid, crRNA, cas13a protein and a fluorescence reporter probe, the specific system configuration is as shown in the (6), the system is added into 384-well plates, fluorescence is measured by an enzyme-labeled instrument, and the result of Fluorescence Intensity (FI) is analyzed to measure the concentration of the target protein.

The test conditions are optimized

Detection was performed using 3a, 6a, 9a, and PER concatamers (single-stranded deoxyribonucleic acid concatamers), designated as 3a, 6a, 9a, and PER groups, respectively. As shown in fig. 1, the test results of these four sets of tests show that under otherwise identical control conditions, as ssDNA length increases from 3a to 6a, 9a, PER concatemers, the resulting fluorescence intensity increases, with the PER concatemers producing target RNAs that target CRISPR/Cas13a with the strongest ability to activate CRISPR/Cas13a, with slopes up to 1327.0, whereas the slopes of short-chain DNA,9a, 6a, and 3a at the same concentration are only 441.4, 255.5, and 40.5.

In order to make the experimental result more visual, the CRISPR/Cas13a system is transferred into an octant tube, and fluorescence generated by the CRISPR/Cas13 system in 3a, 6a and 9a and PER serial body tubes is directly excited by adopting a Dyight channel (488 nm wavelength) in a gel imaging system Tanon-2500, as can be seen from the upper left-hand insert chart of FIG. 1, after enzyme digestion for 30min, the PER serial body group can observe that the solution emits a very bright fluorescence signal, the fluorescence intensity emitted by the 9a group with the same concentration is inferior, the fluorescence of the 6a group is weaker, and the fluorescence signal in the 3a group solution is extremely weak. These results all demonstrate that since the single stranded dna concatemer is ssDNA of about 2500nt in length with a repeat unit of a sequence, the more binding sites for AS/S probes than 3a, 6a and 9a, i.e., the more sites that bind T7 transcriptase, the more transcription efficient the more target RNAs are generated, resulting in enhanced bypass activity of the CRISPR/Cas13a system, thus improving the sensitivity of detection, and that the technique is compatible with solid support ELISA plates.

The parameters and conditions of the iPTC protein detection method are fully optimized. First, parameters of the CRISPR/Cas13a system are optimized, and the recognition sequence of crRNA has a great influence on the parachuting activity of Cas13a protein, so that crRNA molecules for recognizing different target RNA sequence positions are designed at every 3 bases for target RNA sequences, T3crRNA, T4 crRNA, T5 crRNA and T6 crRNA are designed as crrnas, and are tested respectively, and sequences of T3crRNA, T4 crRNA, T5 crRNA and T6 crRNA are shown in table 1. The test results are shown in FIG. 2, and by comparing the fluorescence slopes shown in the graphs, it is found that the fluorescence slopes measured by using T3crRNA are all higher than those of other four crRNAs, and the differences are statistically significant (P values are all less than 0.0001).

Meanwhile, the length of the crRNA recognition target RNA sequence is analyzed, T7 crRNA, T3crRNA, T2 crRNA and T1 crRNA are designed and respectively tested, the specific sequences of the T7 crRNA, the T3crRNA, the T2 crRNA and the T1 crRNA are shown in table 1, the sequence length comprises 23-32 nt, the test result is shown in figure 3, the slope of the T3crRNA under the same condition is the largest, and therefore, the T3crRNA (hereinafter, crRNA is used for replacing) is selected for subsequent exploring experiments.

Then, the fluorescent probes in the CRISPR/Cas13a system are selected, and three types of fluorescent probes, namely a v2 fluorescent probe, a UUUAUU and a UUUU, are selected for testing at the same concentration of 2 mu M, wherein the v2 fluorescent probe is a laboratory detection kit v2 of RNAse Alert company, a novel RNA substrate with brand new and higher sensitivity is adopted, one end of the substrate is marked with a fluorescent reporter group molecule (fluor) and the other end of the substrate is marked with a quenching group. The sequences of UUAUU and UUUUU are shown in table 1, and the test results are shown in fig. 4, and the corresponding fluorescence slope using v2 can be found to be highest.

Next, the parameters of the hybridization system of the PER concatemer and the AS/S probe are optimized, the hybridization temperature is optimized, hybridization conditions of slowly cooling to room temperature after incubation at 95 ℃ for 5min, incubation at 37 ℃ for 1h and incubation at 25 ℃ for 1h are respectively set for detection, the detection results are shown in fig. 5, it can be found that the hybridization conditions of slowly cooling to room temperature after incubation at 95 ℃ for 5min are selected, the obtained hybridization products are detected, and the fluorescence slope finally generated is the highest.

The transcription efficiency of T7 has a close relationship with the hybridization amount of AS/S probes on PER concatamers, on one hand, too many probes on PER concatamers can lead to false positives or prevent the specific binding of T7 transcriptase to promoters due to too high probe density. On the other hand, too little hybridization of PER concatemers with probes results in a decrease in T7 transcription efficiency, resulting in insufficient amplification of fluorescent signals, and thus detection was performed when AS/S probe concentrations were 100 times, 80 times, and 50 times, respectively, and the fluorescent signals generated by the test were shown in FIG. 6, and it was found from FIG. 6 that the fluorescence slope obtained was the highest when the probe concentration was 80 times that of PER concatemers. Further, the concentrations of PER concatemers were examined, and the concentrations of 1.25nM, 2.5nM, 5nM and 10nM were selected and examined, and as a result of the examination, as shown in FIG. 7, 2.5nM was found to be the optimal concentration of PER concatemers.

Finally, final concentrations of v2 fluorescent probe, cas13a protein were investigated. The v2 fluorescent probes with final concentrations of 1. Mu.M, 1.5. Mu.M and 2. Mu.M were selected and tested, and the test results are shown in FIG. 8, wherein the obtained fluorescence slope was found to be highest when the final concentration of the fluorescent reporter probe was 2. Mu.M. Cas13a protein with final concentration of 80nM, 100nM, 200nM and 500nM is selected for testing, and the test results are shown in figure 9, and it can be found that the highest fluorescence slope can be obtained when Cas13a protein with final concentration of 200nM is selected for testing.

IL-15 standard with different concentrations is detected by adopting the most preferred test conditions explored above, and the specific concentrations are 6.25pg/mL, 3.12pg/mL, 1.56pg/mL, 1pg/mL and 0.5pg/mL. The corresponding curve of the measured IL-15 concentration and fluorescence value is shown in FIG. 10, the upper left inset of FIG. 10 is a logarithmic XY plot of the fluorescence value of the iPTC system for detecting IL-15 standard versus the target protein concentration, and LOD is calculated to be 0.032pg/mL.

Repeatability test of protein detection method (iPTC)

To evaluate the reproducibility of the above protein assay (hereinafter, each was replaced with the iPTC assay), the inventors selected 0.5pg/mL and 5pg/mL IL-15 standard for 5-fold parallel assays under the same conditions using the established protein assay, and the test results are shown in Table 2, and the Relative Standard Deviation (RSD) with respect to the average value was 3.69% and 3.07% for 0.5pg/mL and 5pg/mL IL-15, respectively, indicating good reproducibility and stability of the iPTC assay.

TABLE 2 IL-15 repeated measurement results

Detection specificity of protein detection method (iPTC)

IL-12 at 100pg/mL, IL-7 at 100pg/mL, IL-18 at 100pg/mL, and a mixture of BSA at 100pg/mL and IL-15 at 10pg/mL were used as test samples to examine the analytical specificity of IL-15 on the iPTC method. As the test results are shown in fig. 11, it is clear from fig. 11 that targeting only IL-15 can result in a significant increase in fluorescence; despite the use of other interleukins and proteins at a concentration 10 times higher (100 pg/mL) than IL-15, their resulting fluorescence signal values are still low. These results indicate that the iPTC method also has good specificity due to the specificity of the antibody binding to the antigen.

Sensitivity and versatility of protein detection method (iPTC)

For IL-2 standard, the primary antibody (coated antibody) and the secondary antibody (detection antibody) were replaced with antibodies against IL-2, and IL-12 protein detection was performed by the iPTC method, and the results are shown in FIG. 12, wherein the left graph of FIG. 12 shows the trend of absorbance with the concentration of IL-2, and the right graph of FIG. 12 shows the trend of fluorescence intensity with the concentration of IL-2. As can be seen from FIG. 12, the absorbance and fluorescence intensity increased significantly with increasing IL-2 concentration. Fluorescence intensity was linearly related to the logarithm of IL-2 concentration over the range of 62.5fg/mL to 31.2pg/mL, linear regression equation fi=19011×lgc+36767 (R ² =0.974), the LOD was calculated to be 48fg/mL; the sensitivity of this LOD was 103 times that of ELISA (lod=4.96 pg/mL), indicating that the sensitivity of this method was higher than that of the conventional ELISA; meanwhile, the detection of other proteins by the method can be realized by only changing the types of antibodies aiming at antigens, so that the universality of the detection method is realized.

Comparative example 1 ELISA (enzyme-Linked immunosorbent assay) which is a conventional protein detection technique

1. Serum and standard protein are added: adding 100uL of serum sample and multiple diluted standard protein into each hole, incubating for 1h at room temperature, washing the plate 3 times with 0.05% PBST washing solution for 1min each time, and drying;

2. incubation resistance: dilution with primary antibody in the kit was used at 1:100, diluting the primary antibody with biotin to be working concentration, adding 100uL of IL-15Detection Antibody in each hole, incubating for 1h at room temperature, washing the plate 3 times with 0.05% PBST washing solution for 1min each time, and drying;

3. incubation of streptavidin-conjugated horseradish peroxidase (SA-HRP): adding 100uL of SA-HRP with working concentration into each hole, incubating for 20min at room temperature in a dark place, washing the plate 3 times with 0.05% PBST washing solution for 1min each time, and drying;

4. color development: adding 100uL of color development liquid (3, 3', 5' -tetramethyl benzidine, TMB) into each hole, and incubating for 20min at room temperature in a dark place;

5. and (3) terminating: 50uL of stop solution (2N H) was added to each well ₂ SO ₄ ) The yellowing in the holes can be seen;

6. and (5) detecting a light absorption value by a machine: the absorbance was measured using a fully automatic multi-wavelength microplate reader at wavelengths of 450nm and 620nm (calibration wavelength).

The ELISA method and the iPTC method are used for detecting the IL-15 standard substance diluted by the double ratio, the detection result of the ELISA method is shown in figure 13, the detection result of the iPTC method is shown in figure 10, the upper left diagram of figure 13 shows that the absorbance value of the IL-15 standard substance detected by the ELISA method is in linear relation with the concentration of IL-15, the LOD of the IL-15 detected by the ELISA method is calculated to be 4.92pg/mL, the upper left diagram of figure 10 shows that the fluorescence value of the IL-15 standard substance detected by the iPTC is calculated to be an XY diagram of the logarithm of the target protein concentration, the LOD of the IL-15 detected by the iPTC is calculated to be 32fg/mL, and the ELISA sensitivity is improved by 153 times.

Meanwhile, 130 patients with recurrent or metastatic nasal cancer are detected by using an ELISA method and an iPTC method, and ROC analysis results show that the AUC of the predicted NPC immunotherapy effect of sIL-15 detected by iPTC is 0.88, and ELISA is only 0.69. Fig. 14 shows the detection principle of the iPTC method.

The above embodiments are only for illustrating the technical solution of the present application and not for limiting the scope of the present application, and although the present application has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the present application may be modified or substituted without departing from the spirit and scope of the technical solution of the present application.

Claims

1. A system for detecting the content of a protein of interest, the system comprising:

(1) A first antibody that binds to a protein of interest, a second antibody that binds to the protein of interest with a first label, a conjugate that binds to the first label;

(2) A single stranded deoxyribonucleic acid concatemer with a second label bound to the conjugate, the single stranded deoxyribonucleic acid concatemer prepared by primer exchange reaction;

(3) A promoter sequence associated with the single stranded deoxyribonucleic acid tandem, a ribonucleic acid polymerase associated with the promoter sequence;

(4) Cas13a protein, crRNA, and RNA substrate with fluorescent label.

2. The system for detecting the content of a target protein according to claim 1, wherein the single-stranded deoxyribonucleic acid concatemer is a sequence which starts with 2 bases TT and is formed by connecting a repeated sequence CAACTTAAC with bases A, and the length of the repeated sequence is 2500+/-500 nt.

3. The system for detecting the content of a protein of interest according to claim 1, wherein the promoter sequence is a T7 promoter.

4. The system for detecting the content of target proteins according to claim 1, wherein the crRNA is T3crRNA, and the sequence of the T3crRNA is shown as SEQ ID NO. 11.

5. The system for detecting the content of a protein of interest according to claim 1, wherein the RNA substrate with a fluorescent label is a V2 fluorescent probe.

6. The system for detecting the level of a protein of interest according to claim 1, wherein the first label is biotin, the conjugate is streptavidin, and the second label is biotin.

7. The system for detecting the content of a protein of interest according to claim 1, wherein the system is a kit.

8. A protein content detection method based on the system for detecting a protein content of interest according to any one of claims 1 to 7, comprising the steps of:

s1, capturing target proteins: capturing the target protein by adopting a first antibody combined with the target protein and a second antibody which is provided with a first marker and combined with the target protein through an ELISA double-antibody sandwich method;

s2, protein signal conversion: adding the conjugate into a system, combining the conjugate with the second antibody with the first marker in the S1, washing to remove the excessive conjugate, adding a hybridization complex with the second marker into the system, and coupling the first marker with the second marker through the conjugate so as to convert a protein signal into a nucleic acid signal, wherein the hybridization complex is prepared by hybridization compounding the single-stranded deoxyribonucleic acid concatemer with the promoter sequence in advance;

s3, amplifying nucleic acid signals: uniformly mixing the hybridization complex after mixing and incubation in the S2 and the ribonucleic acid polymerase, and performing transcription mediated amplification to generate target ribonucleic acid;

s4, fluorescence quantitative detection: uniformly mixing the target ribonucleic acid, the crRNA, the Cas13a protein and the fluorescent reporter probe in the S3, and measuring the concentration of the target protein by analyzing the fluorescence intensity;

the protein content detection method does not include a method for the purpose of disease diagnosis and/or treatment.

9. The method for detecting the protein content according to claim 8, wherein in S3, the ratio of the final concentration of the single-stranded DNA concatemer to the final concentration of the promoter sequence is 1:80, wherein the final concentration of the single stranded deoxyribonucleic acid concatemer is 2.5nM.

10. The method of claim 8, wherein in S4 the initial concentration of the fluorescent reporter probe is 2 μΜ and the final concentration of the Cas13a protein is 200nM.