Composition and application for detecting the content of PRB1 in saliva of infants

Abstract

The present invention relates to the field of detection of the content of acidic proline-rich protein in children's saliva, and specifically discloses a composition for detecting the content of PRB1 in children's saliva and its application. The composition provided by the present invention contains TFIIB or a biotin-labeled TFIIB fusion protein. Using the surface plasmon resonance sensor detection method, it can not only effectively detect the content of PRB1 in children's saliva, but also effectively distinguish PRB1 from the other three saliva proline-rich proteins. Moreover, the detection sensitivity, accuracy and reproducibility of the content of PRB1 in children's saliva are higher than those of existing kits, providing solid technical support for predicting and diagnosing the symptoms of dental caries in children.

Description

Technical Field

[0001] The present invention relates to the field of detection of the content of acidic proline-rich protein in the saliva of infants, and particularly relates to a composition and application for detecting the content of PRB1 in the saliva of infants. Background Art

[0002] Proline-rich proteins are an important class of proteins in saliva that play a protective role in dental health. They can maintain saliva as a supersaturated solution of calcium and phosphate, which can prevent enamel demineralization, stimulate the remineralization of demineralized tooth enamel, and gradually remineralize and mature newly erupted teeth. There are multiple types of human proline-rich proteins, mainly including proline-rich protein BstNI subfamily 1 (PRB1), proline-rich protein BstNI subfamily 3 (PRB3), proline-rich protein HaeIII subfamily 1 (PRH1), and proline-rich protein BstNI subfamily 4 (PRB4). It is generally believed that the correlation between PRB1 and dental caries is relatively high. If the content of PRB1 is insufficient or its function is abnormal, it may affect tooth remineralization and increase the risk of dental caries. Although there are few studies on the correlation between PRB3, PRH1, and PRB4 and dental caries, as members of salivary proline-rich proteins, they may indirectly affect the occurrence and development of dental caries in aspects such as maintaining the stability of the oral environment. Therefore, it is particularly necessary to separately detect or effectively distinguish PRB1, PRB3, PRH1, and PRB4 during the in-depth exploration and analysis of the correlation and risk analysis of dental caries.

[0003] However, due to the relatively similar physicochemical properties of PRB1, PRB3, PRH1, and PRB4, with similar domains or amino acid sequences, antibodies used in immunoassay methods such as enzyme-linked immunosorbent assay (ELISA) may cross-react. For example, antibodies against PRB1 may bind to certain similar antigenic epitopes in PRB3, PRB4, or PRH1, resulting in false positive results or inaccurate detection signals, affecting the quantitative analysis of the target protein. Therefore, in view of the potential biological significance of PRB1 in the saliva of infants and the limitations of existing detection techniques, developing a rapid, sensitive, and suitable PRB1 detection method for infant samples has important scientific and clinical value. Summary of the Invention

[0004] In view of the deficiencies of the prior art, the present invention provides a composition for detecting the content of PRB1 in the saliva of infants and its application.

[0005] In the first aspect of the present invention, there is provided a composition for detecting the content of PRB1 in the saliva of infants, comprising TFIIB or biotinylated TFIIB, PRB1, carboxymethyl dextranylated SPR chip, EDC, NHS and ethanolamine hydrochloride; the use concentration of TFIIB or biotinylated TFIIB is 400 nM - 1 mM, PRB1 is used as a standard, the use concentration of PRB1 is 0.02 nM - 1200 nM, EDC and NHS are used in combination, the concentration of EDC in the mixed solution is 0.2 M - 0.4 M, the concentration of NHS is 0.1 M - 0.2 M, and the use concentration of ethanolamine hydrochloride is 0.05 - 5 M, pH = 8.5.

[0006] In certain embodiments, the dissociation equilibrium constant between the TFIIB and PRB1 is 4.18×10 -9 mo1 / L.

[0007] In certain embodiments, the composition further comprises horseradish peroxidase-labeled avidin, and the use concentration of horseradish peroxidase-labeled avidin is 0.01 - 3 μg / mL.

[0008] In the second aspect of the present invention, there is provided the application of the composition of the first aspect in the preparation of a kit for detecting the content of PRB1 in the saliva of infants. This application includes: preparing a gradient standard solution of PRB1; activating the carboxyl groups on the surface of the carboxymethyl dextranylated SPR chip; immersing the activated carboxymethyl dextranylated SPR chip into a TFIIB solution or a biotinylated TFIIB solution for coupling; introducing the coupled SPR chip into the gradient standard solution or the sample solution to be measured; making a standard curve according to the concentration of the gradient standard solution and the response value of the gradient standard solution, and fitting to obtain a standard equation; obtaining the content of PRB1 according to the response value of the sample solution to be measured and the standard equation.

[0009] In certain embodiments, the steps in the second aspect specifically include: immersing the activated carboxymethyl dextranylated SPR chip into a 120 μM TFIIB solution for coupling until the response value is stable, and using 1 mol / L pH = 8.5 ethanolamine hydrochloride to block the excess carboxyl groups on the carboxymethyl dextranylated SPR chip; introducing the carboxymethyl dextranylated SPR chip coupled with TFIIB into the first gradient standard solution to obtain a first response value; making a first standard curve according to the first response value and the concentration of the first gradient standard solution and fitting to obtain a first standard equation; obtaining the content of PRB1 according to the first response value of the sample solution to be measured and the first standard equation.

[0010] In certain embodiments of the second aspect, the first gradient standard solution is a PRB1 solution with concentrations of 400 nM, 200 nM, 100 nM, 50 nM, 25 nM, and 10 nM, and the first standard equation is y = 122.14ln(x) - 216.92, where y is the first response value and x is the PRB1 content.

[0011] In certain embodiments, the steps in the second aspect specifically include: immersing the activated carboxymethyl dextranylated SPR chip in a 120 μM biotinylated TFIIB solution until the response value is stable, and blocking the excess carboxyl groups of the carboxymethyl dextranylated SPR chip with 1 mol / L hydrochloric acid ethanolamine at pH = 8.5; introducing the carboxymethyl dextranylated SPR chip conjugated with biotinylated TFIIB into the second gradient standard solution to obtain a second response value; then washing the carboxymethyl dextranylated SPR chip conjugated with biotinylated TFIIB and introducing it into a horseradish peroxidase-labeled avidin solution containing 0.01 - 3 μg / mL until a third response value is obtained; making a standard curve based on the sum of the second response value and the third response value of the second gradient standard solution, and the concentration of the second gradient standard solution, and fitting to obtain a second standard equation; obtaining the PRB1 content of the test sample solution based on the sum of the second response value and the third response value of the test sample solution, and the second standard equation.

[0012] In certain embodiments, the second gradient standard solution is a PRB1 solution with concentrations of 400 nM, 200 nM, 100 nM, 50 nM, 25 nM, and 10 nM, and the second standard equation is y = 21.68x^0.6777, where y is the sum of the second response value and the third response value, and x is the PRB1 content in the test sample solution.

[0013] In the activation process of the carboxymethyl dextranylated SPR chip, a 300 μL EDC / NHS mixture is introduced onto the surface of the SPR chip at a flow rate of 20 μL / min. The concentration of EDC in the EDC / NHS mixture is 0.3 M, and the concentration of NHS is 0.15 M.

[0014] In a certain embodiment, during the conjugation of the activated carboxymethyl dextranylated SPR chip with TFIIB or biotinylated TFIIB, after conjugation, the target response value reaches 2000 - 3000, the pH value of the solution is 4.5 - 5.5. After blocking the excess carboxyl groups with hydrochloric acid ethanolamine, the carboxymethyl dextranylated SPR chip is rinsed and dried.

[0015] In some embodiments, the steps for preparing the biotinylated TFIIB include: constructing a recombinant expression vector of the BirA sequence and a recombinant expression vector carrying the Avi sequence and the TFIIB sequence; co-transfecting the recombinant expression vector of the BirA sequence and the recombinant expression vector carrying the Avi sequence and the TFIIB sequence into eukaryotic cells; culturing the transformed cells and collecting their culture supernatants; collecting the cells from the culture supernatants, lysing the cells with a lysis solution, centrifuging to collect the supernatant, and collecting the biotinylated TFIIB from the supernatant.

[0016] Advantageous effects: In the present invention, the contents of PRB1, PRB3, PRH1, and PRB4 in the saliva of infants are detected by a general ELISA kit, and the CAT of the caries-free group, the low caries group, and the high caries group is detected by the resazurin paper method. Through correlation analysis, it is found that PRB1 is closely associated with the symptoms of dental caries in infants, while PRB3, PRH1, and PRB4 are not closely associated with the symptoms of dental caries in infants.

[0017] In addition, the composition provided by the present invention contains TFIIB or a biotin-labeled TFIIB fusion protein. Using the surface plasmon resonance sensor detection method, it can not only effectively detect the content of PRB1 in the saliva of infants, but also effectively distinguish PRB1 from the other three salivary proline-rich proteins. Moreover, the detection sensitivity, accuracy, and reproducibility of the content of PRB1 in the saliva of infants are higher than those of the existing kits, providing strong technical support for predicting and diagnosing the symptoms of dental caries in infants. Description of the Drawings

[0018] Figure 1 It is a linear fitting curve of the apparent rate constant and the input concentrations of PRB1, PRB3, PRH1, and PRB4.

[0019] Figure 2 It is the first standard curve and the first standard equation provided in Example 3.

[0020] Figure 3 It is an SDS-PAGE diagram of a solution containing a biotin-labeled TFIIB fusion protein.

[0021] Figure 4 It is the second standard curve and the second standard equation provided in Example 5.

[0022] Figure 5 It is a bar chart of the standard deviation of the sample solutions detected on the first day by the methods provided in Example 1, Example 3, and Example 5.

[0023] Figure 6 It is a bar chart of the standard deviation of the sample solutions detected on the third day by the methods provided in Example 1, Example 3, and Example 5.

[0024] Figure 7 Bar chart of standard deviation of the sample solution detected by the methods provided for Example 1, Example 3 and Example 5 on the 7th day.

[0025] Figure 8 Analysis result chart of the association between the symptoms of dental caries in children and the sample solutions detected by the methods provided for Example 1, Example 3 and Example 5 on the 1st, 3rd and 7th days.

[0026] Figure 9 ROC curve for Example 1.

[0027] Figure 10 ROC curve for Example 3.

[0028] Figure 11 ROC curve for Example 5. Detailed implementation manners

[0029] In order to make the objectives, technical solutions and advantages of the present invention clearer and more understandable, the present invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention. Reagents not specifically described in detail in the present invention are all conventional reagents and can be obtained from commercial channels; methods not specifically described in detail are all conventional experimental methods and can be known from the prior art.

[0030] Example 1. PRB1 is closely associated with the symptoms of dental caries in children

[0031] 1. Preparation of sample solution I

[0032] Randomly select 100 children aged 3 - 5 years from two kindergartens in this city. Refer to the caries diagnosis standard stipulated in the "Basic Methods of Oral Health Survey" of the World Health Organization (WHO) to check the caries conditions of all children, and express them with the decayed - missing - filled index (DMF Index). According to the caries conditions, the 100 children are divided into 3 groups: ① caries - free group (DMF Index = 0); ② low - caries group: (DMF Index = 1 - 4); ③ high - caries group (DMF Index≥5). Then, randomly select 10 children with comparable genders and ages from each group, for a total of 30 children to participate in the experiment. Among them, the caries - free group, the low - caries group and the high - caries group all have complete primary dentition, no systemic or genetic diseases, no history of long - term use of fluoride mouthwash or antibacterial mouthwash, and no medication taken in the past 1 month.

[0033] Use the resazurin paper strip method to conduct a cariogenic activity (CAT) test on the 30 children with different caries conditions selected. As a result, the average CAT values of the caries - free group, the low - caries group and the high - caries group are 0.011, 1.29 and 2.86 respectively.

[0034] The non-stimulated saliva of 3 - 5 mL from the test children in the caries-free group, low caries group and high caries group was collected by the expectoration method. After the saliva of each group was mixed, it was centrifuged at 10,000 r / min for 10 min. The supernatant was diluted 1000 times with PBS buffer solution with pH = 7 and used as the sample solution I of the caries-free group, low caries group and high caries group respectively.

[0035] 2. Detection of the contents of PRB1, PRB3, PRH1 and PRB4 in the sample solution

[0036] The content of PRB1 in the sample solution of each group was detected by the detection kit for PRB1 (NDC-KSJ-4TCZHN-96, Nordic BioSite). The content of PRB3 in the sample solution of each group was detected by the ELISA kit for PRB3 (EKL54894, Biomatik). The content of PRH1 in the sample solution of each group was detected by the ELISA kit for PRH1 (ZY-E64803H, Zeye Bio). The content of PRB4 in the sample solution of each group was detected by the ELISA kit for PRB4 (LMAI Bio, LM-PRB4-Hu).

[0037] As a result, the average values of the PRB1 content in the sample solutions of the caries-free group, low caries group and high caries group were 4.78 mg / mL, 1.49 mg / mL and 1.20 mg / mL respectively. The average values of the PRB3 content in the sample solutions of the caries-free group, low caries group and high caries group were 1.48 mg / mL, 1.46 mg / mL and 1.47 mg / mL respectively. The average values of the PRH1 content in the sample solutions of the caries-free group, low caries group and high caries group were 0.51 mg / mL, 0.39 mg / mL and 0.48 mg / mL respectively. The average values of the PRB4 content in the sample solutions of the caries-free group, low caries group and high caries group were 0.13 mg / mL, 0.16 mg / mL and 0.09 mg / mL respectively.

[0038] Table 1

[0039] CAT PRB1 (mg / mL) PRB3 (mg / mL) PRH1 (mg / mL) PRB4 (mg / mL) Caries-free group 0.011 4.78 1.48 0.51 0.13 Low caries group 1.29 1.49 1.46 0.39 0.16 High caries group 2.86 1.20 1.47 0.48 0.09 Pearson correlation coefficient -0.87 -0.45 -0.18 0.62

[0040] Table 1 shows the Pearson correlation coefficient analysis of the PRB1 content, PRB3 content, PRH1 content, and PRB4 content in each group with CAT. The results show that the Pearson correlation coefficient between the PRB1 content detected by a commercial kit and CAT is -0.87, the Pearson correlation coefficient between the PRB3 content detected by a commercial kit and CAT is -0.45, the Pearson correlation coefficient between the PRH1 content detected by a commercial kit and CAT is -0.18, and the Pearson correlation coefficient between the PRB4 content detected by a commercial kit and CAT is -0.62. That is, using a commercial kit, a strong correlation is found between PRB1 and CAT values in the saliva of these children, indicating that PRB1 is closely associated with the symptoms of dental caries in children and has the application prospect as a biomarker for detecting the symptoms of dental caries in children.

[0041] Example 2. Affinity Study between TFIIB and Salivary Proline-Rich Proteins

[0042] 1. Experimental Materials

[0043] 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), N-Hydroxysuccinimide (NHS), and ethanolamine are Sigma products. Human general transcription factor (TFIIB) is purchased from MCE, catalog number HY-P71357. PRB1 is purchased from Shanghai Zeye Biotechnology. Recombinant protein PRB3 is purchased from Aptibody, product number PA3000-36003. Recombinant human PRH1 protein is purchased from abcam, catalog number ab167837. PRB4 protein is purchased from Shanghai Zeye Biotechnology.

[0044] Phosphate buffered saline / Tween-20 (PBST) 0.01M, pH = 7.4. All chemical reagents are of analytical grade, and all experimental water is Mill-Q deionized double-distilled water.

[0045] The surface plasmon resonance sensor and the sample cell pretreated with carboxymethyl dextran (CMD) were purchased from Labsystems Affinity Sensors in the UK. The surface plasmon resonance sensor is used to monitor the interactions (binding and dissociation) of biomacromolecules occurring on the surface of the sample cell. Its basic principle is based on the resonant mirror (RM) phenomenon, which monitors and reflects the change in the refractive index of the biosensing chip, and this change is proportional to the mass of the biomolecules bound to the surface of the chip. When performing biomolecular interaction analysis, first, one of the molecules to be tested, generally called the ligand, is immobilized on the sensitive substrate surface of the sample cell. Then, another component - the ligand - is added to the sample cell. When the ligand binds (or dissociates) with the ligand immobilized on the substrate surface, the laser that enters the light attenuation region at the resonant angle will undergo a change in the resonant angle (refractive index). This change is continuously monitored and recorded by the instrument and processed by a computer to form a continuous reaction curve, measured in rad / s and plotted against time, which can represent the interaction and affinity between the ligand on the sensor surface and its ligand molecule. After each reaction, the bound ligand is washed away, called regeneration, and then different concentrations of the ligand or other ligands can be introduced again.

[0046] 2. Activation of the CMD sample cell surface and immobilization of TFIIB

[0047] The surface plasmon resonance sensor was turned on and preheated for 1 h, and the corresponding parameters were set. The sample cell pretreated with CMD was placed in the instrument and rinsed with 50 μL of PBST until the baseline was balanced. A 1:1 (V / V) mixture of EDC / NHS (0.4 M EDC and 0.1 M NHS) was added and reacted for about 7 min to activate the carboxyl groups of CDM. It was rinsed with PBST, and 40 μL was retained in the sample cell. 10 μL of TFIIB with a final concentration of 120 μM was added and allowed to react fully. The activated carboxyl groups can covalently bind to the amino groups of TFIIB, thus immobilizing TFIIB on the CMD surface. The unreacted activated sites were blocked with 1 M ethanolamine. The sample cell was rinsed with PBST to stabilize the baseline.

[0048] 3. Monitoring the binding of human salivary proline-rich protein to TFIIB

[0049] Wash the sample cell modified with TFIIB three times with 40 μL of PBST, and retain 40 μL of PBST in the sample cell. Record the 3-minute baseline. Add 10 μL of PRB1 with final concentrations of 1200 nM, 600 nM, 300 nM, 150 nM, 75 nM, and 35 nM, or add 10 μL of PRB3 with final concentrations of 1200 nM, 600 nM, 300 nM, 150 nM, 75 nM, and 35 nM, or add 10 μL of PRH1 with final concentrations of 1200 nM, 600 nM, 300 nM, 150 nM, 75 nM, and 35 nM, or add 10 μL of PRB4 with final concentrations of 1200 nM, 600 nM, 300 nM, 150 nM, 75 nM, and 35 nM. React fully to reach equilibrium, wash three times with PBST, and record the reaction curve.

[0050] 4. Regeneration of the sample cell

[0051] Soak the sample cell in 10 mM HCl for 0.5 - 1 min, and thoroughly rinse with PBST to wash away the bound human salivary proline-rich protein, and re-obtain the TFIIB-coated sample cell.

[0052] 5. Standard curve and binding kinetics

[0053] If human salivary proline-rich protein can bind to TFIIB, it can cause a change in the resonance angular velocity. Plot the binding curve of the change in resonance angular velocity value at different time points versus time.

[0054] Since the binding curve conforms to second-order reaction kinetics, perform fitting analysis on it, calculate the apparent rate constant at each input concentration of human salivary proline-rich protein, and then plot the apparent rate constant against each input concentration of human salivary proline-rich protein and perform linear fitting. The fitting formula is Kobs = Kon Cx + Koff; where Kobs is the apparent rate constant, s -1 ; Kon is the binding rate constant, L / (mol -1 s -1 ); Koff is the dissociation rate constant, s -1 ; Cx is the input concentration of each human salivary proline-rich protein. The equilibrium constant K D = Koff / Kon.

[0055] As Figure 1 shown, the plots of the apparent rate constants of the binding of PRB1, PRB3, PRH1, and PRB4 to TFIIB against the input concentrations of each human salivary proline-rich protein are presented. According to Figure 1 it can be seen that both PRB1 and PRB4 can bind to TFIIB, while neither PRB3 nor PRH1 can bind to TFIIB, and the dissociation equilibrium constant of PRB1 is 4.18×10-9 mol / L, the dissociation equilibrium constant of PRB4 is 5.67×10 -6 mol / L, indicating that TFIIB has the highest affinity for PRB1 and can be used as a reagent for detecting PRB1 and differentiating various proline-rich proteins.

[0056] Example 3: Detection of PRB1 content using TFIIB and surface plasmon resonance sensor

[0057] In this example, TFIIB and a surface plasmon resonance chip were further used to quantitatively detect the PRB1 content in saliva. The specific steps are as follows:

[0058] 1. Preparation of sample solution II

[0059] According to the grouping in Example 1, 10 children were separately selected from each of the caries-free group, low caries group, and high caries group, and sample solution II was prepared by the same method. The caries activity (CAT) test was performed on the 30 children with different caries statuses using the resazurin paper strip method. As a result, the average CAT values of the caries-free group, low caries group, and high caries group were 0.039, 1.93, and 2.68 respectively.

[0060] 2. Activation of SPR chip

[0061] The carboxymethyl dextranylated SPR chip (Swift Test Technology Co., Ltd.) was placed in an SPR instrument, and the instrument temperature was set at 20°C - 30°C. A 300 μL EDC / NHS mixture was passed over the surface of the SPR chip at a flow rate of 20 μL / min to activate the carboxyl groups on the chip surface. The concentration of EDC in the EDC / NHS mixture was 0.3 M, and the concentration of NHS was 0.15 M.

[0062] 3. Coupling of TFIIB

[0063] The SPR chip with activated surface carboxyl groups was immersed in a 120 μM TFIIB solution for coupling, so that it was coupled to the activated carboxyl groups, and the stable response value (RU) was required to reach 2000 - 3000, and the solution pH value was 4.5 - 5.5. 1 mol / L hydrochloric acid ethanolamine with pH = 8.5 was added dropwise to block the excess carboxyl groups. Before each chip treatment, it was rinsed clean with anhydrous ethanol, repeatedly washed with a large amount of deionized water, and dried with nitrogen.

[0064] 4. The first standard curve and the first standard equation

[0065] Load the processed chip into the SPR instrument (zero the baseline, that is, clear the existing response value), and introduce PRB1 diluted to 400 nM, 200 nM, 100 nM, 50 nM, 25 nM, and 10 nM at a flow rate of 20 μL / min, or add 10 μL of PRB3 with a final concentration of 400 nM, 200 nM, 100 nM, 50 nM, 25 nM, and 10 nM, or add 10 μL of PRH1 with a final concentration of 400 nM, 200 nM, 100 nM, 50 nM, 25 nM, and 10 nM, or add 10 μL of PRB4 solution with a final concentration of 400 nM, 200 nM, 100 nM, 50 nM, 25 nM, and 10 nM, so that the protein specifically binds to the ligand immobilized on the chip. During the protein binding process, the SPR instrument will monitor the change in refractive index on the chip surface in real time, thereby obtaining the response value (RU) reflecting the protein binding amount. After injecting the standard protein solution at each concentration and waiting for the response value to reach a stable state, record the corresponding first response value. Make the first standard curve based on the first response value reaching stability at each concentration and the concentration, and fit to obtain the first standard equation.

[0066] As Figure 2 shown, the stable RU values of PRB3, PRH1, and PRB4 have no significant difference from each other and the change in stable RU values between different concentrations is not significant. In addition, their stable RU values are significantly lower than that of PRB1 at different concentrations, which is not conducive to curve fitting and detection, indicating that the method provided by the present invention can effectively distinguish PRB1, PRB3, PRH1, and PRB4.

[0067] In addition, Figure 2 the first standard curve and the first standard equation with a high degree of fitting are also shown. The first standard equation is y = 122.14ln(x) - 216.92, R 2 = 0.9647, and the standard deviation is 0.027.

[0068] According to the calculation of LOD = 3.3×σ / S, where σ is the standard deviation and S is the slope of the calibration curve. According to the derivative formula (lnx)′ = x1, take the derivative of y = 122.14ln(x) - 216.92, and we can get y′ = S = 122.14 / x. Assume x = 1, and the slope S is 122.14. Given that the standard deviation σ = 0.027, substitute σ and S into the detection limit formula: LOD = 3.3×σ / S = 3.3×0.027 / 122.14 ≈ 7.29×10 −4 nM. Therefore, under the assumption of x = 1, the detection limit is about 7.29×10 −4 nM.

[0069] 5. Association analysis with the symptoms of dental caries in young children

[0070] The contents of PRB1 in sample solution I of the caries-free group, low caries group, and high caries group, as well as the contents of PRB1 in sample solution II of the caries-free group, low caries group, and high caries group, were detected using TFIIB and a surface plasmon resonance sensor respectively, and the correlation analysis of the symptoms of dental caries in children was carried out respectively. The results are shown in Table 2.

[0071] Table 2

[0072]

[0073] As can be seen from Table 2, whether it is sample solution I or sample solution II, using TFIIB and a surface plasmon resonance sensor to detect the content of PRB1 in it, the detection sensitivity is higher, and it can be highly correlated with the caries prevalence rate, and the correlation effect is better than that of Example 1.

[0074] Example 4, Preparation of Biotin-Labeled TFIIB Fusion Protein

[0075] Biotin ligase is a bifunctional protein encoded by the Escherichia coli BirA gene. It can recognize the specific sequence of protein molecules, enabling biotin to bind to the protein. At the same time, it can also act as a repressor protein to inhibit the operon that synthesizes biotin. In this example, a small tag (LHILLDAQKMVWNHNR, SEQ ID NO:1) that can be specifically recognized by biotin ligase BirA was added to the N-terminus or C-terminus of the target protein TFIIB. This small tag can be specifically recognized by biotin ligase BirA, and biotin was covalently bound to the lysine residue on this tag, thereby realizing the biotinylation labeling of TFIIB. In addition, the affinity between the target protein TFIIB and PRB1 was further used to enrich from saliva, and signal amplification and specific detection were achieved through the interaction between streptavidin and biotin.

[0076] 1. Construction and Identification of pQCXIH-BirA Recombinant Expression Vector

[0077] Design upstream primer (TTTTgcggccgcCATGAAGGATAACACCGTGCCACTG, lowercase letters represent NotⅠ restriction site, SEQ ID NO:2) and downstream primer (GGGCggatccTTATTTTTCTGCACTACGCAGG, lowercase letters represent BamHⅠ restriction site, SEQ ID NO:3) according to the BirA sequence reported in GenBank (ID: 914965, CDS sequence number NC_002695.2). The length of the pre-amplified fragment is about 960 bp. Using the genomic DNA of Escherichia coli DH5α as a template, perform PCR amplification with the 3 different DNA polymerases given in the materials section to find the best DNA polymerase. PCR reaction conditions: pre-denaturation at 94°C for 5 min, then perform 32 cycles at 94°C for 40 s, 56°C for 40 s, and 72°C for 1 min, and extend at 72°C for 10 min. Recover the amplified product of about 1000 bp. After digestion with BamHⅠ and NotⅠ enzymes respectively for the amplified product and pQCXIH plasmid (Clontech), connect them overnight with SolutionⅠ in the ligation kit, transform DH5α competent cells, pick monoclonal colonies and culture them by shaking overnight. After extracting the plasmid according to the plasmid extraction kit instructions, perform PCR identification, and sequence the positive clones. The correctly identified one is named pQCXIH-BirA.

[0078] 2. Construction and identification of plenti-Avi-TFIIB recombinant expression vector

[0079] Using the TFIIB sequence (NM_001514.5) as a template, design upstream primer (ATAggatccACGACTGCGTGGGTGAGTCGTCTATAAAA, lowercase letters represent BamHI, SEQ ID NO:4) and downstream primer (AACAgcggccgcTTTTTTTATCTTGTTAAAATTACAGAGAGTTC, lowercase letters represent NotⅠ restriction site, SEQ ID NO:5) for PCR amplification to obtain the TFIIB fragment with restriction sites. Digest pCMV-N-3X Flag-Avi-Neo (D2973, Beyotime) with BamHI and NotⅠ enzymes to obtain the linearized fragment of pCMV-N-3X Flag-Avi-Neo. Connect the TFIIB fragment with restriction sites and the linearized fragment of pCMV-N-3X Flag-Avi-Neo. Transfer the ligation product into Escherichia coli DH5α, screen positive clones. After identification, extract the plasmid according to the plasmid extraction kit instructions, and name the positive clone with correct sequencing as pCMV-Avi-TFIIB, which is the TFIIB recombinant expression vector carrying the Avi expression tag.

[0080] 3. Biotinylation of TFIIB

[0081] Grow HEK 293T cells in complete medium in a 6 cm culture dish, namely DMEM, 10% fetal bovine serum, 1% penicillin-streptomycin. When the cell density reaches 60% - 80%, mix 65 μL of 2 mol / L CaCl2, and mix pQCXIH-BirA and pCMV-Avi-TFIIB at a ratio of 1:1 for transfection (5 μg each). Supplement sterile water to 500 μL, mix well, add 500 μL of 2×HBS, pipette until slightly milky (about 50 times), and add to HEK293T cells. At the same time, transfect pCMV-Avi-TFIIB and pQCXIH-BirA separately, and set negative controls. Replace with fresh complete medium 6 - 8 h after transfection, and collect the cells 36 - 44 h after cell transfection. Discard the supernatant of the medium, wash 1 - 2 times with pre-cooled 1×PBS solution, then add 1 mL of PBS, quickly scrape the cells with a cell scraper and transfer them to a 1.5 mL centrifuge tube. After a short centrifugation for 20 - 30 s, remove the supernatant, add 300 μL of IP lysis buffer (Tris-HCl 50 mmol / L, NaCl 100 mmol / L, EDTA 2.5 mmol / L, EGTA 2.5 mmol / L, NP40 0.5%, Glycerol 5%, Sodium Vanadate 0.1 mmol / L, NaF 1 mmol / L, β-glycerophosphate 10 mmol / L, Cocktail 1%, PMSF 1 mmol / L), and lyse fully on ice for 30 min. Centrifuge at 4 ℃ and 13,000 r / min for 10 min, transfer the supernatant to a new centrifuge tube. Take 200 μL of the lysate supernatant and co-incubate with 100 μL of streptavidin agarose beads in a 4 ℃ chromatography cabinet using a rotary mixer for 1 h. After the biotin and streptavidin are fully bound, centrifuge at 1,000 r / min for 2 min, add the precipitate to 1 mL of pre-cooled 1×PBS solution, and wash 4 times to remove non-specific binders. Take the precipitate and add it to 200 μL of 1×loading buffer, denature the protein at 100 ℃ for 20 min, centrifuge at 13,000 r / min for 1 min, and the supernatant is the solution containing the biotin-labeled TFIIB fusion protein (as Figure 3 ), verified by WB. The primary antibody in WB verification is the TFIIB antibody, catalog number SAB1410591, from Merck.

[0082] Example 5 Detection of PRB1 Content Using Biotinylated TFIIB and Surface Plasmon Resonance Sensor

[0083] In this example, biotinylated TFIIB and a surface plasmon resonance sensor were further used to detect the content of proline-rich proteins in saliva, and the prevalence of dental caries was determined based on the content of proline-rich proteins. The specific steps are as follows:

[0084] 1. Activation of SPR Chip

[0085] The SPR chip was activated using the same method as in Example 3.

[0086] 2. Coupling of Biotinylated TFIIB

[0087] The activated SPR chip with surface carboxyl groups was immersed in a 120 μM biotinylated TFIIB solution for coupling, so that it was coupled to the activated carboxyl groups, and the stable response value (RU) was required to reach 2000 - 3000, and the pH value of the solution was 4.5 - 5.5. 1 mol / L pH = 8.5 ethanolamine hydrochloride was added dropwise to block the excess carboxyl groups. Before each chip treatment, it was rinsed thoroughly with absolute ethanol, washed repeatedly with a large amount of deionized water, and dried with nitrogen.

[0088] 3. Second Standard Curve and Second Standard Equation

[0089] The processed chip was placed into the SPR instrument (its baseline was zeroed, that is, its existing response value was cleared), and PRB1 solutions diluted to 400 nM, 200 nM, 100 nM, 50 nM, 25 nM, and 10 nM were passed through at 20 μL / min, so that it specifically bound to the biotinylated TFIIB immobilized on the chip until the second response value (RU1) no longer changed. After the SPR chip was washed with PBST solution at 20 μL / min, a 1.5 μg / mL horseradish peroxidase-labeled avidin solution was passed through at 20 μL / min and incubated at 37 °C for 1 h, so that avidin specifically bound to the biotinylated antigen until the third response value (RU2) no longer changed. The sum of the response values of RU1 and RU2 was recorded, and the second standard curve of it and the concentration was plotted, and the second standard equation was obtained by fitting.

[0090] Figure 4 The second standard curve and the second standard equation with a higher fitting degree are shown. The second standard equation is y = 21.68x^0.6777, R 2 = 0.9905, where y is the sum of the second response value and the third response value, x is the PRB1 content, and the standard deviation is 0.035.

[0091] The limit of detection LOD = 3.3×σ / S, where σ is the standard deviation and S is the slope of the calibration curve. Then, the function y = 21.68x^0.6777 is differentiated to obtain the slope S: According to the differentiation formula (x^n)′ = nx^(n−1), the derivative gives y′ = S = 21.68×0.6777x^(0.6777−1) = 14.692536x^(−0.3223). In practical applications, x usually takes a value within the linear range of the calibration curve (generally, the x value corresponding to the midpoint of the calibration curve can be taken, etc. For the convenience of calculation here, assume x = 1). When x = 1, the slope S = 14.692536×1^(−0.3223) = 14.692536. Given the standard deviation σ = 0.035, substituting σ and S into the limit of detection formula: LOD = 3.3×σ / S = 3.3×0.035 / 14.692536 ≈ 0.0079 nM. Therefore, assuming x = 1, the limit of detection is approximately 0.0079 nM.

[0092] 4. Association analysis with dental caries symptoms in children

[0093] The content of PRB1 in sample solution I of the caries-free group, low caries group, and high caries group, as well as the content of PRB1 in sample solution II of the caries-free group, low caries group, and high caries group, were detected using TFIIB and surface plasmon resonance sensors respectively, and the association analysis with dental caries symptoms in children was carried out separately. The results are shown in Table 3.

[0094] Table 3

[0095]

[0096] As can be seen from Table 3, whether it is sample solution I or sample solution II, when using TFIIB and surface plasmon resonance sensors to detect the content of PRB1, the detection sensitivity is higher, it can be highly associated with the caries prevalence rate, and the association effect is better than that of Example 1.

[0097] Example 6 Reproducibility analysis and association analysis of PRB1 content detection

[0098] 1. Reproducibility analysis

[0099] The methods provided in Example 1, Example 3, and Example 5 were respectively used to detect the content of PRB1 in sample solution I of the caries-free group, low caries group, and high caries group. The sample solution was stored at low temperature and detected and analyzed on the 1st day, the 3rd day, and the 7th day respectively. Each group of sample solutions was repeatedly detected 10 times each time.

[0100] As Figure 5 、 Figure 6 and Figure 7As shown, with the increase in the storage time of the sample solution, the reproducibility of the method provided in Example 1 decreased rapidly, while those of Example 3 and Example 4 still had high reproducibility, and their standard deviations were always significantly lower than that of Example 1. This shows that the method of detecting PRB1 in saliva by using the affinity between TFIIB and PRB1 in the present invention has higher reproducibility and accuracy than the existing ELISA detection kits.

[0101] 2. Correlation analysis of dental caries symptoms in young children

[0102] The methods provided in Example 1, Example 3 and Example 5 were respectively used to detect the content of PRB1 in the sample solution I of the caries-free group, the low caries group and the high caries group. The sample solution I was stored at low temperature and detected and analyzed on the 1st day, the 3rd day and the 7th day respectively. Each group of sample solutions was detected, repeated 10 times, and the correlation analysis of dental caries symptoms in young children was carried out.

[0103] As Figure 8 shown, with the increase in the storage time of the sample solution, the correlation between the detection results of the sample solution I of the caries-free group, the low caries group and the high caries group and the caries prevalence rate decreased significantly, while the correlation between the detection results provided by Example 3 and Example 5 and the dental caries symptoms in young children was still relatively high. This shows that the method of using the affinity between TFIIB and PRB1 in the present invention can not only effectively detect the content of PRB1 in the saliva of young children, but also effectively distinguish PRB1 from the other three salivary proline-rich proteins. Moreover, the detection sensitivity, accuracy and reproducibility of the content of PRB1 in the saliva of young children are higher than those of the existing kits, providing solid technical support for predicting and diagnosing dental caries symptoms in young children.

[0104] Example 7: Reverse verification experiment

[0105] The present application further verified the specificity and sensitivity of PRB1 as a dental caries biomarker through reverse verification tests to evaluate the specificity and sensitivity of Example 1, Example 3 and Example 5 respectively, and exclude false positive / false negative interference.

[0106] 1. Sample selection

[0107] A total of 100 children aged 3 - 5 years from two randomly selected kindergartens were not grouped, and their saliva samples were collected. Non-stimulated saliva was collected according to the method in Example 1, centrifuged to obtain the supernatant, and diluted as the sample solution to be detected.

[0108] 2. Detection method

[0109] The methods of Example 1 (commercial kit), Example 3 (TFIIB + SPR), and Example 5 (biotinylated TFIIB + SPR) were respectively used to detect the content of PRB1 in the samples.

[0110] 3. Dental caries diagnosis

[0111] Samples were initially diagnosed into caries-free group, low caries group and high caries group according to the PRB1 content. Samples with PRB1 content greater than 4.0 mg / mL were in the caries-free group. Samples with PRB1 content between 1.5 and 4.0 mg / mL were in the low caries group. Samples with PRB1 content less than 1.5 mg / mL were in the high caries group.

[0112] The caries activity test (CAT) was performed on the above samples using resazurin paper strip method to determine the actual caries situation. Samples with CAT < 0.1 were in the caries-free group, samples with 0.1 ≤ CAT < 2.0 were in the low caries group, and samples with CAT ≥ 2.0 were in the high caries group.

[0113] 4. Data comparison

[0114] The initial diagnosis results were compared with the actual caries situation, and the numbers of true positive (TP), false positive (FP), true negative (TN) and false negative (FN) were counted. Among them, true positive means that the results of resazurin paper strip method and the detection results of Example 1, Example 3 or Example 5 are both low caries or high caries. False positive means that the results of the methods of Example 1, Example 3 or Example 5 are low caries or high caries respectively, while the result of resazurin paper strip method is caries-free. True negative means that the results of resazurin paper strip method and the detection results of Example 1, Example 3 or Example 5 are both caries-free. False negative means that the results of the methods of Example 1, Example 3 or Example 5 are caries-free respectively, while the result of resazurin paper strip method is low caries or high caries. Then the sensitivity and specificity were calculated. Sensitivity = number of true positives / (number of true positives + number of false negatives), Specificity = number of true negatives / (number of true negatives + number of false positives), and the ROC curve was generated according to the sensitivity and specificity.

[0115] The ROC curve is obtained by changing the diagnostic threshold, calculating the sensitivity and specificity at different PRB1 concentration thresholds, and plotting the relationship curve between sensitivity (vertical axis) and 1 - specificity (horizontal axis). As Figure 9 、 Figure 10 and Figure 11 are the ROC curves of Example 1, Example 3 and Example 5 respectively. The statistical results are shown in Table 4.

[0116] Table 4

[0117]

[0118] It can be seen that the AUC values of Example 3 and Example 5 are higher than that of Example 1, and the detection accuracies are both higher than that of Example 1, indicating that when detecting the PRB1 content in the saliva of young children, these two methods can more accurately predict the dental caries situation of young children. The AUC value of Example 5 is the highest, indicating that its comprehensive performance in diagnosing dental caries is the best, with higher detection sensitivity and specificity. Therefore, through the reverse verification experiment and ROC curve analysis, the effectiveness of PRB1 as a dental caries marker for young children can be clarified. The methods of Example 3 and Example 5 are superior to the existing ELISA kit (Example 1) in terms of detection sensitivity and specificity, providing more reliable technical support for the early diagnosis of dental caries in young children.

[0119] As described above, the above are only the preferred specific embodiments of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed by the present invention should be covered by the protection scope of the present invention.

Claims

1. A composition for detecting the content of PRB1 in the saliva of infants and young children, characterized in that, Comprising: TFIIB or biotinylated TFIIB, PRB1, carboxymethylated dextran SPR chip, EDC, NHS and ethanolamine hydrochloride; The use concentration of TFIIB or biotinylated TFIIB is 400 nM to 1 mM, PRB1 is used as a standard, the use concentration of PRB1 is 0.02 nM to 1200 nM, EDC and NHS are used in combination, the concentration of EDC in the mixed solution is 0.2 M to 0.4 M, the concentration of NHS is 0.1 M to 0.2 M, and the use concentration of ethanolamine hydrochloride is 0.05 to 5 M, pH = 8.

5.

2. The composition according to claim 1, wherein The dissociation equilibrium constant of TFIIB and PRB1 is 4.18×10 -9 mol / L.

3. The composition according to claim 1, wherein It further comprises horseradish peroxidase-labeled avidin, and the use concentration of horseradish peroxidase-labeled avidin is 0.01 to 3 μg / mL.

4. Use of the composition according to any one of claims 1 to 3 in the preparation of a kit for detecting the content of PRB1 in the saliva of infants, characterized in that, The application includes: Preparing a gradient standard solution of PRB1; Activating the carboxyl groups on the surface of the carboxymethylated dextran SPR chip; Immersing the activated carboxymethylated dextran SPR chip into a TFIIB solution or a biotinylated TFIIB solution for coupling; Passing the coupled SPR chip into the gradient standard solution or the sample solution to be tested; Making a standard curve based on the concentration of the gradient standard solution and the response value of the gradient standard solution, and fitting to obtain a standard equation; Obtaining the content of PRB1 according to the response value of the sample solution to be tested and the standard equation.

5. The application according to claim 4, characterized in that, Immersing the activated carboxymethylated dextran SPR chip into a TFIIB solution or a biotinylated TFIIB solution for coupling; passing the coupled SPR chip into the gradient standard solution or the sample solution to be tested; making a standard curve based on the concentration of the gradient standard solution and the response value of the gradient standard solution, and fitting to obtain a standard equation; Obtaining the content of PRB1 according to the response value of the sample solution to be tested and the standard equation, specifically including: Immersing the activated carboxymethylated dextran SPR chip into a 120 μM TFIIB solution for coupling until the response value is stable, and using 1 mol / L pH = 8.5 ethanolamine hydrochloride to block the excess carboxyl groups of the carboxymethylated dextran SPR chip; Passing the carboxymethylated dextran SPR chip coupled with TFIIB into the first gradient standard solution to obtain a first response value; Making a first standard curve based on the first response value and the concentration of the first gradient standard solution and fitting to obtain a first standard equation; Obtaining the content of PRB1 according to the first response value of the sample solution to be tested and the first standard equation.

6. The application according to claim 5, characterized in that The first gradient standard solution is a PRB1 solution of 400 nM, 200 nM, 100 nM, 50 nM, 25 nM and 10 nM, and the first standard equation is y = 122.14ln(x) - 216.92, where y is the first response value and x is the content of PRB1.

7. The application according to claim 4, characterized in that, Immerse the activated carboxymethyl dextranylated SPR chip in TFIIB solution or biotinylated TFIIB solution for coupling; pass the coupled SPR chip into the gradient standard solution or the sample solution to be measured; make a standard curve according to the concentration of the gradient standard solution and the response value of the gradient standard solution, and fit to obtain a standard equation; Obtain the content of PRB1 according to the response value of the sample solution to be measured and the standard equation, specifically including: Immerse the activated carboxymethyl dextranylated SPR chip in 2 mg / mL biotinylated TFIIB solution for coupling until the response value is stable, and block the excess carboxyl groups of the carboxymethyl dextranylated SPR chip with 1 mol / L hydrochloric acid ethanolamine at pH = 8.5; Pass the carboxymethyl dextranylated SPR chip coupled with biotinylated TFIIB into the second gradient standard solution to obtain a second response value; Then wash the carboxymethyl dextranylated SPR chip coupled with biotinylated TFIIB and pass it into a horseradish peroxidase-labeled avidin solution containing 0.01 - 3 μg / mL until a third response value is obtained; Make a standard curve according to the sum of the second response value and the third response value of the second gradient standard solution, and the concentration of the second gradient standard solution, and fit to obtain a second standard equation; Obtain the PRB1 content of the sample solution to be measured according to the sum of the second response value and the third response value of the sample solution to be measured, and the second standard equation.

8. The application according to claim 7, wherein The second gradient standard solution is a PRB1 solution of 400 nM, 200 nM, 100 nM, 50 nM, 25 nM and 10 nM, and the second standard equation is y = 21.68x^0.6777, where y is the sum of the second response value and the third response value, and x is the PRB1 content in the sample solution to be measured.

9. The application according to claim 4, wherein The preparation steps of the biotinylated TFIIB include: Construct a recombinant expression vector of the BirA sequence and a recombinant expression vector carrying the Avi sequence and the TFIIB sequence; Mix and transfect the recombinant expression vector of the BirA sequence and the recombinant expression vector carrying the Avi sequence and the TFIIB sequence into eukaryotic cells; Culture the transformed cells and collect their culture supernatants; Collect cells from the culture supernatant, lyse the cells with a lysis solution, centrifuge to collect the supernatant, and collect the biotinylated TFIIB from the supernatant.

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