CN120064671B - Composition for detecting content of infant saliva PRB1 and application - Google Patents

Abstract

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

Description

Composition for detecting content of infant saliva PRB1 and application

Technical Field

The invention relates to the field of detection of infant sialyl proline-rich protein content, in particular to a composition for detecting infant saliva PRB1 content and application thereof.

Background

Proline-rich proteins are an important class of proteins in saliva that protect dental health. They maintain saliva as a supersaturated calcium and phosphate solution which prevents enamel demineralization, stimulates demineralization of the demineralized enamel, and progressively remineralizes newly erupted teeth. Human proline-rich proteins are of various types, mainly including proline-rich protein BstNI subfamily 1 (pro line-rich protein BstNI subfamily 1, PRB 1), proline-rich protein BstNI subfamily 3 (pro line-rich protein BstNI subfamily 3, PRB 3), proline-rich protein HaeIII subfamily 1 (proline rich protein HaeIII subfamily 1, PRH 1) and proline-containing protein BstNI subfamily 4 (proline rich protein BstNI subfamily, PRB 4). PRB1 is generally considered to have a relatively high correlation with caries. If the PRB1 content is insufficient or the function is abnormal, the remineralization of the tooth bodies can be influenced, and the occurrence risk of dental caries is increased. Although there are few studies on the correlation of PRB3, PRH1 and PRB4 with caries, they are members of salivary-rich proteins, and may indirectly affect the occurrence and development of caries in terms of maintaining stable oral environment, etc. Therefore, in the intensive study and analysis of caries correlation and risk analysis, it is necessary to individually detect or effectively distinguish between PRB1, PRB3, PRH1 and PRB 4.

However, since PRB1, PRB3, PRH1 and PRB4 have similar physical and chemical properties, and have similar domains or amino acid sequences, cross-reactions may occur in antibodies used in immunodetection methods such as enzyme-linked immunosorbent assay (ELISA) and the like. For example, antibodies directed against PRB1 may bind to certain similar 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 infant saliva and the limitations of the existing detection techniques, it is of great scientific and clinical value to develop a PRB1 detection method that is rapid, sensitive and suitable for infant samples.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention provides a composition for detecting the content of PRB1 in saliva of infants and application thereof.

According to the first aspect of the invention, the composition for detecting the PRB1 content of saliva of infants comprises TFIIB or biotinylation TFIIB, PRB1, carboxymethyl dextran SPR chip, EDC, NHS and ethanolamine hydrochloride, wherein the TFIIB or biotinylation TFIIB is used at 400 nM-1 mM, PRB1 is used as a standard substance, the PRB1 is used at 0.02 nM-1200 nM, EDC and NHS are mixed, the EDC concentration in a mixed solution is 0.2M-0.4M, the NHS concentration in the mixed solution is 0.1M-0.2M, the ethanolamine hydrochloride concentration in the mixed solution is 0.05-5M, and the pH=8.5.

In some embodiments, the dissociation equilibrium constant of the TFIIB and PRB1 is 4.18X10 ^-9 mo1/L.

In certain embodiments, the composition further comprises horseradish peroxidase-labeled avidin, wherein the concentration of horseradish peroxidase-labeled avidin is 0.01-3 μg/mL.

In a second aspect, the invention provides the use of a composition according to the first aspect for the preparation of a kit for detecting the content of PRB1 in saliva of a baby. The preparation method comprises the steps of preparing a gradient standard solution of PRB1, activating carboxyl groups on the surface of a carboxymethyl dextran SPR chip, immersing the activated carboxymethyl dextran SPR chip in a TFIIB solution or a biotinylation TFIIB solution for coupling, introducing the coupled SPR chip into the gradient standard solution or a sample solution to be tested, preparing a standard curve according to the concentration of the gradient standard solution and a response value of the gradient standard solution, fitting to obtain a standard equation, and obtaining the PRB1 content according to the response value of the sample solution to be tested and the standard equation.

In certain embodiments, the second aspect specifically comprises the steps of immersing an activated carboxymethyl dextran SPR chip in a 120 mu M TFIIB solution, coupling until a response value is stable, blocking redundant carboxyl groups of the carboxymethyl dextran SPR chip by adopting 1 mol/L of ethanolamine hydrochloride with pH=8.5, introducing the carboxymethyl dextran SPR chip coupled with the TFIIB into a first gradient standard solution to obtain a first response value, preparing a first standard curve according to the first response value and the concentration of the first gradient standard solution, fitting to obtain a first standard equation, and obtaining the PRB1 content according to the first response value of the sample liquid to be tested and the first standard equation.

In certain embodiments of the second aspect, the first gradient standard solution is a PRB1 solution of 400nM, 200nM, 100nM, 50nM, 25nM, and 10nM, 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.

In certain embodiments, the second aspect specifically comprises the steps of immersing an activated carboxymethyl dextran SPR chip in a 120 mu M biotinylation TFIIB solution to be coupled to a stable response value, adopting 1 mol/L of ethanol hydrochloride with pH=8.5 to seal redundant carboxyl of the carboxymethyl dextran SPR chip, introducing the carboxymethyl dextran SPR chip coupled with the biotinylation TFIIB into a second gradient standard solution to obtain a second response value, washing the carboxymethyl dextran SPR chip coupled with the biotinylation TFIIB, introducing the carboxymethyl dextran SPR chip coupled with the biotinylation TFIIB into a horseradish peroxidase labeled avidin solution containing 0.01-3 mu g/mL to obtain a third response value, preparing a standard curve according to the sum of the second response value of the second gradient standard solution and the third response value of the second gradient standard solution and the concentration of the second gradient standard solution, and obtaining a second standard equation according to the second response value of the sample solution to be tested, and the PRB standard solution to be tested and the PRB standard solution to obtain a standard equation of the sample to be tested.

In some embodiments, the second gradient standard solution is a PRB1 solution of 400nM, 200nM, 100nM, 50nM, 25nM and 10nM, and the second standard equation is y=21.68 x≡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 tested.

In certain embodiments, during activation of the carboxymethyl dextran saccharification SPR chip, 300. Mu.L of EDC/NHS mixture is introduced to the surface of the SPR chip at a flow rate of 20. Mu.L/min, the EDC concentration in the DC/NHS mixture is 0.3M and the NHS concentration is 0.15M.

In an embodiment, in the process of coupling the activated carboxymethyl dextran SPR chip with the TFIIB or the biotinylated TFIIB, the target response value reaches 2000-3000 after coupling, the pH value of the solution is 4.5-5.5, and after the residual carboxyl is blocked by ethanolamine hydrochloride, the carboxymethyl dextran SPR chip is washed and dried.

In certain embodiments, the preparation of the biotinylated TFIIB comprises the steps of constructing a recombinant expression vector of the birA sequence and a recombinant expression vector carrying the Avi sequence and the TFIIB sequence, mixing and transfecting eukaryotic cells with the recombinant expression vector of the birA sequence and the recombinant expression vector carrying the Avi sequence and the TFIIB sequence, culturing transformed cells, collecting culture solution thereof, collecting cells from the culture solution, lysing the cells by using the lysate, centrifuging to collect supernatant, and collecting the biotinylated TFIIB from the supernatant.

The kit has the beneficial effects that the content of PRB1, PRB3, PRH1 and PRB4 in infant saliva is detected by a general ELISA kit, and CAT in caries-free groups, low caries groups and high caries groups is detected by a resazurin paper method, and the PRB1 is closely related to caries symptoms of infants detected by association analysis, while PRB3, PRH1 and PRB4 cannot be closely related to caries symptoms of infants.

In addition, the composition provided by the invention comprises TFIIB or biotin-marked TFIIB fusion protein, and the detection method of the surface plasmon resonance sensor can be used for effectively detecting the PRB1 content in infant saliva, effectively distinguishing the PRB1 from other three saliva proline-rich proteins, and providing firm technical support for predicting and diagnosing caries symptoms of the infant compared with the existing kit.

Drawings

Fig. 1 is a linear fit curve of apparent rate constants versus input concentrations of PRB1, PRB3, PRH1, and PRB 4.

Fig. 2 is a first standard curve and a first standard equation provided in example 3.

FIG. 3 is a SDS-PAGE map of a solution containing biotin-labeled TFIIB fusion protein.

FIG. 4 is a second standard curve and a second standard equation provided in example 5.

Fig. 5 is a bar graph of standard deviation of sample solutions tested on day 1 using the methods provided in example 1, example 3, and example 5.

Fig. 6 is a bar graph of standard deviation of sample solutions tested on day 3 using the methods provided in example 1, example 3, and example 5.

Fig. 7 is a bar graph of standard deviation of sample fluid at day 7 tested by the methods provided in example 1, example 3, and example 5.

FIG. 8 is a graph showing the results of analysis of caries symptoms in infants using the methods of example 1, example 3 and example 5 to detect sample solutions on days 1, 3 and 7.

Fig. 9 is the ROC curve of example 1.

Fig. 10 is the ROC curve of example 3.

Fig. 11 is the ROC curve of example 5.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail with reference to the following examples. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention. The reagents which are not specifically described in the present invention are conventional reagents and are commercially available, and the methods which are not specifically described in the present invention are conventional experimental methods and are known from the prior art.

Example 1 PRB1 is closely associated with caries symptoms in infants

1. Preparation of sample solution I

100 Children 3-5 years old in kindergarten in the city are randomly extracted, caries conditions of all children are checked by referring to caries diagnosis standards prescribed by World Health Organization (WHO) basic method for oral health investigation, and caries Index (DMF Index) is expressed. 100 children were divided into 3 groups according to caries conditions, ① caries-free groups (DMF index=0), ② low caries groups (DMF index=1 to 4), ③ high caries groups (DMF Index. Gtoreq.5). Then, 10 persons of the same sex and age were randomly extracted from each group, and 30 infants were subjected to the experiment. Wherein, the caries-free group, the low caries group and the high caries group are complete dentition, have no systemic and hereditary diseases, have no history of using fluorine-containing mouthwash and antibacterial drug-containing mouthwash for a long time, and have no drug administration in the last 1 month.

The Cariogenic Activity (CAT) test was carried out on 30 extracted children with different cariogenic conditions by means of the Resazurin paper sheet method. As a result, the average CAT values of the caries-free group, the low caries group and the high caries group were 0.011, 1.29 and 2.86, respectively.

Collecting 3-5 mL of non-irritating saliva of tested infants in each of the caries-free group, the low caries group and the high caries group by adopting a spitting method, mixing saliva of each group, centrifuging 10min at 10000 r/min, diluting the supernatant with PBS buffer solution with pH=7 for 1000 times, and respectively serving as sample liquid I of each of the caries-free group, the low caries group and the high caries group.

2. PRB1, PRB3, PRH1 and PRB4 content detection in sample liquid

And detecting the content of PRB1 in each group of sample liquid by using a detection kit (NDC-KSJ-4 TCZHN-96,Nordic BioSite) of PRB 1. The PRB3 content of each set of sample solutions was detected using an ELISA kit for PRB3 (EKL 54894, biomatik). The PRH1 content of each group of sample solutions was tested using an ELISA kit for PRH1 (ZY-E64803H, zea mays). The PRB4 content of each set of sample fluid was detected using ELISA kit for PRB4 (LMAI Bio, LM-PRB 4-Hu).

As a result, the average value of PRB1 content in each of the sample solutions of the caries-free group, the low caries group and the high caries group was 4.78mg/mL, 1.49mg/mL and 1.20 mg/mL, respectively. The average PRB3 content in each sample solution of the caries-free group, the low caries group and the high caries group is 1.48mg/mL, 1.46mg/mL and 1.47mg/mL respectively. The average value of PRH1 content in each group of sample solutions of the caries-free group, the low caries group and the high caries group is 0.51mg/mL, 0.39mg/mL and 0.48mg/mL respectively. The average value of PRB4 content in each group of sample liquids of the caries-free group, the low caries group and the high caries group is 0.13mg/mL, 0.16mg/mL and 0.09mg/mL respectively.

TABLE 1

	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 set	1.29	1.49	1.46	0.39	0.16
						High caries set	2.86	1.20	1.47	0.48	0.09
Pearson correlation coefficient		-0.87	-0.45	-0.18	0.62

Table 1 shows that the PRB1 content, PRB3 content, PRH1 content and PRB4 content of each group were respectively analyzed by pearson correlation coefficient with CAT, and as a result, the pearson correlation coefficient of PRB1 content and CAT detected by using the commercial kit was-0.87, the pearson correlation coefficient of PRB3 content and CAT detected by using the commercial kit was-0.45, the pearson correlation coefficient of PRH1 content and CAT detected by using the commercial kit was-0.18, and the pearson correlation coefficient of PRB4 content and CAT detected by using the commercial kit was-0.62. That is, commercial kits are used to find that PRB1 in saliva of infants shows a strong correlation with CAT value, which indicates that PRB1 is closely related to caries symptoms of infants, and has application prospect as a marker for detecting caries symptoms of infants.

EXAMPLE 2 affinity studies of TFIIB with saliva-rich protein

1. Experimental materials

1-Ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC), N-hydroxysuccinimide (NHS) and ethanolamine are Sigma products. Human universal Transcription Factor (TFIIB), available from MCE under the accession number HY-P71357.PRB1 was purchased from Shanghai ze leaf organisms. Recombinant protein PRB3 protein is purchased from Ai Pudi under the trade designation PA3000-36003. Recombinant human PRH1 protein, available from abcam under accession number ab167837.PRB4 protein was purchased from Shanghai Zea japonica organisms.

Phosphate buffer (Phosphate buffered saline/Tween-20, PBST) 0.01M, pH=7.4. All chemical reagents were analytically pure and all experimental waters were Mill-Q deionized double distilled water.

Surface plasmon resonance sensors and carboxymethyl dextran (carboxymethyl dextran, CMD) pretreated sample cells were purchased from Labsystems Affinity Sensors company in the uk. Surface plasmon resonance sensors are used to monitor the interactions (binding and dissociation) of biological macromolecules that occur at the surface of a sample cell. The basic principle is based on the phenomenon of Resonance Mirror (RM), by which the change in refractive index of the bio-sensor chip is monitored and reflected, which is proportional to the mass of the biomolecules bound to the surface of the sensor chip. In the analysis of the interaction of biomolecules, one of the molecules to be measured, generally called ligand, is immobilized on the sensitive substrate surface of the sample cell, then another component, ligand, is added to the sample cell, when the ligand binds to (or dissociates from) the ligand immobilized on the substrate surface, the laser entering the light attenuation region at resonance angle will change the resonance angle (refractive index), and the change is continuously monitored and recorded by the instrument and processed by the computer to form a continuous reaction curve, and the interaction and affinity between the ligand on the sensor surface and the ligand molecule can be represented by plotting time in rad/s. After each reaction, the bound ligand is washed away, called regeneration, and a different concentration of ligand or other ligands can be reintroduced.

2. CMD sample cell surface activation and TFIIB immobilization

The surface plasma resonance sensor is started and preheated for 1h, and corresponding parameters are set. The CMD-pretreated cuvette was placed in the instrument and rinsed with 50 μl PBST until baseline equilibrated. A1:1 (V/V) EDC/NHS mixture (0.4M EDC and 0.1M NHS) was added and reacted for about 7min for CDM carboxyl activation. Rinse with PBST and leave 40. Mu.L in the sample well, add 10. Mu.L of TFIIB to a final concentration of 120. Mu.M, and allow to react well. The activated carboxyl groups may be covalently bound to amino groups of the TFIIB such that the TFIIB is immobilized on the CMD surface. Unreacted activation sites were blocked with 1M ethanolamine. The pool was rinsed with PBST to stabilize the baseline.

3. Binding monitoring of human saliva Propofol to TFIIB

TFIIB modified wells were washed 3 times with 40. Mu.L of PBST and 40. Mu.L of PBST was retained in the well, and a 3min baseline was recorded. The reaction was completed to equilibrium by adding 10. Mu.L of PRB1 with final concentrations of 1200nM, 600nM, 300nM, 150nM, 75nM and 35nM, or adding 10. Mu.L of PRB3 with final concentrations of 1200nM, 600nM, 300nM, 150nM, 75nM and 35nM, or adding 10. Mu.L of PRH1 with final concentrations of 1200nM, 600nM, 300nM, 150nM, 75nM and 35nM, or adding 10. Mu.L of PRB4 with final concentrations of 1200nM, 600nM, 300nM, 150nM, 75nM and 35nM, washing 3 times with PBST, and recording the reaction curve.

4. Regeneration of sample cells

Soaking the sample cell with 10mM HCl for 0.5-1 min, thoroughly washing with PBST, washing off the combined human saliva protein rich protein, and obtaining the sample cell coated with TFIIB.

5. Standard curve and binding kinetics

If human saliva-rich protein can generate a binding effect with TFIIB, the human saliva-rich protein can cause the change of the resonance angular velocity, and a binding curve of resonance angular velocity change values at different time points and time is drawn.

And (3) fitting and analyzing the binding curve according with the second-order reaction dynamics, calculating the apparent rate constant of each person at the input concentration of the salivary prolin, and then plotting the apparent rate constant and the input concentration of each person to perform linear fitting. The fitting formula is Kobs =koncx+koff, wherein Kobs is an apparent rate constant, s ^-1, kon is a binding rate constant, L/(mo 1 ^-1s^-1), koff is a dissociation rate constant, s ^-1, and Cx is the input concentration of the human salivary prolin. Equilibrium constant K _D = Koff/Kon.

As shown in fig. 1, the apparent rate constants of PRB1, PRB3, PRH1, and PRB4 binding to TFIIB are plotted against the individual human salivary prolin input concentrations. As can be seen from fig. 1, both PRB1 and PRB4 can bind to TFIIB, but neither PRB3 nor PRH1 can bind to TFIIB, and the dissociation equilibrium constant of PRB1 is 4.18×10 ^-9 mo1/L, and that of PRB4 is 5.67×10 ^-6 mo1/L, thus demonstrating that the highest affinity of TFIIB to PRB1 can be used as a reagent for detecting PRB1 and distinguishing various prolines.

Example 3 detection of PRB1 content Using TFIIB and surface plasmon resonance sensor

In the embodiment, quantitative detection of the content of PRB1 in saliva is further realized by using a TFIIB and a surface plasmon resonance chip. The method comprises the following steps:

1. Preparation of sample solution II

Sample fluid II was prepared according to the same procedure as in the grouping of example 1 except that 10 children were additionally drawn from each of the non-cariogenic, low cariogenic and high cariogenic groups, respectively. The Cariogenic Activity (CAT) test was carried out on 30 extracted children with different cariogenic conditions by means of the Resazurin paper sheet method. As a result, the average CAT values of the caries-free group, the low caries group and the high caries group were 0.039, 1.93 and 2.68, respectively, in this order.

2. SPR chip activation

The carboxymethyl dextran SPR chip (quick test technology Co., ltd.) is placed in an SPR instrument, and the instrument temperature is set to be 20-30 ℃. 300. Mu.L of EDC/NHS mixed solution is introduced to the surface of the SPR chip at a flow rate of 20. Mu.L/min, and carboxyl groups on the surface of the chip are activated. EDC concentration in the EDC/NHS mixture was 0.3M and NHS concentration was 0.15M.

3. Coupling TFIIB

And immersing the SPR chip after the surface carboxyl is activated into a 120 mu M TFIIB solution for coupling, so that the SPR chip is coupled to the activated carboxyl, wherein a stable response value (RU) is required to reach 2000-3000, and the pH value of the solution is 4.5-5.5. 1 mol/L of ethanolamine hydrochloride with pH=8.5 is added dropwise to block the excess carboxyl groups. The chip is washed by absolute ethyl alcohol before each chip treatment, and repeatedly washed by a large amount of deionized water and dried by nitrogen.

4. First standard curve and first standard equation

The treated chip was loaded into an SPR apparatus (with its baseline zeroed, i.e., its existing response value cleared), and PRB1 diluted to 400nM, 200nM, 100nM, 50nM, 25nM and 10nM was introduced at 20. Mu.L/min, or PRB3 was added at a final concentration of 400nM, 200nM, 100nM, 50nM, 25nM and 10nM, or PRH1 was added at a final concentration of 10. Mu.L, 400nM, 200nM, 100nM, 50nM, 25nM and 10nM, or PRB4 solution at a final concentration of 400nM, 200nM, 100nM, 50nM, 25nM and 10nM was added to allow specific binding of the protein to the ligand immobilized on the chip. During protein binding, the SPR instrument monitors the refractive index change of the chip surface in real time, thereby obtaining a response value (RU) reflecting the amount of protein binding. After the standard protein solution with each concentration is injected, the corresponding first response value is recorded after the response value reaches a stable state. And (3) manufacturing a first standard curve according to the first response value and the concentration of which the concentrations reach stability, and fitting to obtain a first standard equation.

As shown in fig. 2, the stable RU values of PRB3, PRH1 and PRB4 are not significantly different from each other and the stable RU values between different concentrations are not significantly changed, and in addition, the stable RU values are significantly lower than PRB1 at different concentrations, which is not beneficial to curve fitting and detection, and the method provided by the invention can effectively distinguish PRB1, PRB3, PRH1 and PRB4.

In addition, fig. 2 also shows a first standard curve and a first standard equation with a higher fitting degree, where the first standard equation is y= 122.14ln (x) -216.92, and r ² = 0.9647, and the standard deviation is 0.027.

Calculated as lod=3.3×σ/S, where σ is the standard deviation and S is the slope of the calibration curve. Y=s= 122.14/x is obtained by deriving y= 122.14ln (x) -216.92 according to the derivative formula (lnx)' =x1. Let x=1, the slope S be 122.14. Knowing the standard deviation σ=0.027, substituting 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, assuming x=1, the detection limit is about 7.29×10 ⁻⁴ nM.

5. Analysis of caries symptoms associated with young children

The levels of PRB1 in the non-caries group, low caries group and high caries group sample fluid I and the levels of PRB1 in the non-caries group, low caries group and high caries group sample fluid II are respectively detected by using TFIIB and a surface plasmon resonance sensor, and the correlation analysis of the caries symptoms of the infants is respectively carried out, and the results are shown in Table 2.

TABLE 2

As can be seen from table 2, whether sample liquid I or sample liquid II, the PRB1 content was detected using TFIIB and the surface plasmon resonance sensor, and the detection sensitivity was higher, and it was able to be highly correlated with caries prevalence, and the correlation effect was superior to that of example 1.

EXAMPLE 4 preparation of Biotin-labeled TFIIB fusion proteins

The biotin ligase is a bifunctional protein encoded by the BirA gene of Escherichia coli, can recognize the specific sequence of a protein molecule, so that biotin is bound to the protein, and can also act as a repressor protein to inhibit the synthesis of biotin from an operon. In the embodiment, the N end or the C end of the target protein TFIIB is added with a small tag (LHILLDAQKMVWNHNR, SEQ ID NO: 1) which can be specifically identified by biotin ligase BirA, the small tag can be specifically identified by biotin ligase BirA, and lysine residues on the small tag are covalently bound with biotin, so that the biotinylation marking of the TFIIB is realized. In addition, the affinity between the target protein TFIIB and PRB1 is further utilized to enrich saliva, and signal amplification and specificity detection are realized through the interaction between streptavidin and biotin.

1. Construction and identification of pQCIH-birA recombinant expression vector

The upstream primer (TTTTGCGGCCGCCATGAAGGATAACACCGTGCCACTG, lower case NotI cleavage site, SEQ ID NO: 2) and downstream primer (GGGCGGATCCTTATTTTTCTGCACTACGCAGG, lower case BamHI cleavage site, SEQ ID NO: 3) were designed based on the BirA sequence reported by GenBank (ID: 914965, CDS sequence NC-002695.2) and the pre-amplified fragment length was about 960 bp. The optimal DNA polymerase is found out by PCR amplification with 3 different DNA polymerases given in the materials section using E.coli DH5 alpha genome DNA as template. The PCR reaction conditions were 94℃pre-denatured 5 min, followed by 32 cycles at 94℃40 s, 56℃40 s, 72℃1 min and 72℃extension for 10min. About 1000 bp of the amplified product was recovered, digested with BamHI and NotI, ligated overnight with SolutionI in a ligation kit, DH 5. Alpha. Competent cells were transformed, and cultured overnight by picking up a monoclonal and shaking, and after plasmid extraction, PCR was performed according to the plasmid extraction kit instructions, and positive clones were sequenced and correctly designated pQCIIH-BirA.

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

The TFIIB sequence (NM_ 001514.5) is used as a template, and an upstream primer (ATAGGATCCACGACTGCGTGGGTGAGTCGTCTATAAAA, lowercase BamHI, SEQ ID NO: 4) and a downstream primer (AACAGCGGCCGCTTTTTTTATCTTGTTAAAATTACAGAGAGTTC, lowercase NotI cleavage site, SEQ ID NO: 5) are designed for PCR amplification to obtain a TFIIB fragment with the cleavage site. The pCMV-N-3X Flag-Avi-Neo (D2973, biyun) was digested with BamHI and NotI to obtain pCMV-N-3X Flag-Avi-Neo linearization fragment. And (3) connecting a TFIIB fragment with an enzyme cutting site with the pCMV-N-3X Flag-Avi-Neo linearization fragment, transferring a connection product into escherichia coli DH5 alpha, screening positive clones, extracting plasmids according to a plasmid extraction kit instruction after identification, and naming the positive clone with correct sequencing as pCMV-Avi-TFIIB to be a TFIIB recombinant expression vector carrying an Avi expression tag.

3. TFIIB biotinylation

HEK293T cells are placed in a complete culture medium of a 6 cm culture dish for growth, namely DMEM, 10% fetal calf serum and 1% green streptomycin, when the cell density is 60% -80%, 65 mu L of 2 mol/L CaCl ₂, pQCIH-BirA and pCMV-Avi-TFIIB are mixed according to the proportion of 1:1 for transfection (5 mu g each), sterilized water is supplemented to 500 mu L, the mixture is fully mixed, 500 mu L of 2 XHBS is added, and the mixture is blown to be slightly milky (about 50 times) and added into HEK293T cells. Simultaneously, pCMV-Avi-TFIIB and pQCIH-BirA were transfected respectively, and negative controls were set. And after transfection for 6-8 hours, changing fresh complete culture medium, and after cell transfection for 36-44 hours, collecting cells. The culture supernatant is discarded, the pre-cooled 1 XPBS solution is used for cleaning 1-2 times, then 1 mL PBS is added, the cells are scraped off rapidly by a cell scraper and transferred to a 1.5 mL centrifuge tube, the supernatant is removed after short centrifugation for 20-30 s, 300 mu L of IP lysate （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), is added for full lysis on ice for 30 min,4 ℃ and 13000 r/min are centrifuged for 10min, the supernatant is transferred to a new centrifuge tube, 200 mu L of the lysate and 100 mu L of streptavidin agarose beads are taken for rotary co-incubation for 1h in a 4 ℃ chromatography cabinet by a rotary mixer, after the biotin and the streptavidin are fully combined, 1000 r/min is centrifuged for 2 min, the precipitate is added to the 1 XPBS solution pre-cooled by 1 mL, and the non-specific binding substance is removed after washing for 4 times. The precipitate was added to 200. Mu.L of 1 Xloading buffer, the protein was denatured by 100℃20 min, centrifuged at 13000 r/min for 1min, and the supernatant was a solution containing biotin-labeled TFIIB fusion protein (FIG. 3), and WB was used to verify that one antibody was a TFIIB antibody, product No. SAB1410591, merck, inc.

Example 5 detection of PRB1 content Using biotinylated TFIIB and surface plasmon resonance sensor

This example further utilizes biotinylated TFIIB and a surface plasmon resonance sensor to detect the proline protein rich content in saliva, and determines caries prevalence based on the proline protein rich content. The method comprises the following steps:

1. SPR chip activation

The SPR chip was activated in the same manner as in example 3.

2. Coupling biotinylated TFIIB

And immersing the SPR chip after the surface carboxyl is activated into 120 mu M of biotinylation TFIIB solution for coupling, so that the SPR chip is coupled to the activated carboxyl, wherein a stable response value (RU) is required to reach 2000-3000, and the pH value of the solution is 4.5-5.5. 1mol/L of pH=8.5 ethanolamine hydrochloride was added dropwise to block the excess carboxyl groups. The chip is washed by absolute ethyl alcohol before each chip treatment, and repeatedly washed by a large amount of deionized water and dried by nitrogen.

3. Second standard curve and second standard equation

The treated chip was loaded into an SPR instrument (with its baseline zeroed, i.e., its existing response cleared), and 20. Mu.L/min was passed into PRB1 solutions diluted to 400nM, 200nM, 100nM, 50nM, 25nM and 10nM, which allowed specific binding to biotinylated TFIIB immobilized on the chip to a second response (RU 1) unchanged. After washing the SPR chip with 20. Mu.L/min of PBST solution, 1.5. Mu.g/mL of horseradish peroxidase-labeled avidin solution was added at 20. Mu.L/min, and incubated at 37℃for 1h, so that the specific binding of avidin to biotinylated antigen to the third response value (RU 2) was no longer changed. And recording the sum of response values of RU1 and RU2, drawing a second standard curve of the response values and the concentration, and fitting to obtain a second standard equation.

Fig. 4 shows a second standard curve and a second standard equation with higher fitting degree, the second standard equation is y=21.68x≡0.6777, and 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.

The limit of detection lod=3.3×σ/S, where σ is the standard deviation and S is the slope of the calibration curve. The function y=21.68x0.6777 is then derived to obtain the slope S: according to the derivative formula (x n) '=nx (n-1), y' =s= 21.68 × 0.6777x (0.6777-1) = 14.692536x (-0.3223). In practical applications, x usually takes a certain value within the linear range of the calibration curve (generally, x corresponding to the middle point of the calibration curve may be taken, where x=1 is assumed for convenience of calculation). When x=1, the slope s= 14.692536 ×1 (-0.3223) = 14.692536. Knowing the standard deviation σ=0.035, substituting σ and S into the detection limit formula lod=3.3×σ/s=3.3×0.035/14.692536 ≡0.0079nM. Therefore, assuming x=1, the detection limit is about 0.0079nM.

4. Analysis of caries symptoms associated with young children

The levels of PRB1 in the non-caries group, low caries group and high caries group sample fluid I and the levels of PRB1 in the non-caries group, low caries group and high caries group sample fluid II are respectively detected by using TFIIB and a surface plasmon resonance sensor, and the correlation analysis of the caries symptoms of the infants is respectively carried out, and the results are shown in Table 3.

TABLE 3 Table 3

As can be seen from table 3, whether sample fluid I or sample fluid II, the PRB1 content therein was detected using TFIIB and a surface plasmon resonance sensor, the detection sensitivity was higher, it was able to be highly correlated with caries prevalence, and the correlation effect was superior to that of example 1.

Example 6 PRB1 content detection reproducibility analysis and correlation analysis

1. Reproducibility analysis

The method provided in example 1, example 3 and example 5 was used to detect the PRB1 content in the samples I of the non-caries group, low-caries group and high-caries group, the samples were stored at low temperature, and the detection was repeated 10 times for each group on days 1, 3 and 7.

As shown in fig. 5, 6 and 7, the reproducibility of the method provided in example 1 was rapidly decreased as the storage time of the sample solution was increased, while examples 3 and 4 still had higher reproducibility and the standard deviation was always significantly lower than that of example 1. Therefore, the invention detects the PRB1 in saliva by utilizing the affinity between the TFIIB and the PRB1, and the reproducibility and the accuracy are higher than those of the existing ELISA detection kit.

2. Correlation analysis of caries symptoms of infants

The method provided in example 1, example 3 and example 5 was used to detect the PRB1 content in the samples of non-caries, low caries and high caries groups, respectively, the samples were stored at low temperature, each group of samples was detected 10 times on days 1, 3 and 7, and the infant caries symptoms associated analysis was performed.

As shown in FIG. 8, as the storage time of the sample fluid increases, the correlation between the detection results of the sample fluid I for the non-caries group, the low caries group and the high caries group and the caries prevalence rate decrease significantly, while the correlation between the detection results provided in examples 3 and 5, respectively, is still higher for infants suffering from caries symptoms. Therefore, the invention utilizes the affinity effect of TFIIB and PRB1, not only can effectively detect the PRB1 content in infant saliva, but also can effectively distinguish PRB1 from other three saliva proline-rich proteins, and has higher detection sensitivity, accuracy and repeatability for the infant saliva PRB1 content than the existing kit, thereby providing firm technical support for predicting and diagnosing infant caries symptoms.

Example 7 reverse validation experiment

The present application further verifies the specificity and sensitivity of PRB1 as a caries marker through a reverse verification test to evaluate the specificity and sensitivity of example 1, example 3 and example 5, respectively, and to exclude false positive/false negative interference.

1. Sample selection

A total of 100 children 35 years old were randomly selected from two kindergartens, and their saliva samples were collected without grouping. Non-irritating saliva was collected as in example 1, and the supernatant was collected by centrifugation and diluted to give a sample solution to be tested.

2. Detection method

PRB1 content in the samples was measured by the methods of example 1 (commercial kit), example 3 (TFIIB+SPR), example 5 (biotinylated TFIIB+SPR), respectively.

3. Caries diagnosis

The samples were initially diagnosed according to PRB1 content into non-carious group, low carious group and high carious group. PRB1 content greater than 4.0 mg/mL is caries-free group. PRB1 content is 1.5-4.0 mg/mL, and the tooth caries is low. PRB1 levels below 1.5 mg/mL are high caries groups.

The samples were tested for Caries Activity (CAT) using the Resazurin paper method to determine actual caries. CAT <0.1 is a caries-free group, CAT <2.0 less than or equal to 0.1 is a low caries group, and CAT <2.0 more than or equal to 2.0 is a high caries group.

4. Data comparison

Comparing the preliminary diagnosis result with the actual caries condition, counting the numbers of True Positives (TP), false Positives (FP), true Negatives (TN) and False Negatives (FN). Wherein true positives refer to low caries or high caries using the resazurin sheet method and the test results of examples 1, 3 or 5. False positives refer to low caries or high caries as measured by the method of example 1, example 3 or example 5, respectively, and no caries as measured by the resazurin paper method. True negative means no caries using the resazurin sheet method and the test results of examples 1, 3 or 5. False negative means that the result of detection by the method of example 1, example 3 or example 5, respectively, is no caries, whereas the result of detection by the resazurin paper method is low caries or high caries. From this, the sensitivity and the specificity were calculated, the sensitivity=true positive number/(true positive number+false negative number), the specificity=true negative number/(true negative number+false positive number), and the ROC curve was generated from the sensitivity and the specificity.

The ROC curve is a graph of sensitivity (vertical axis) versus 1-specificity (horizontal axis) calculated for different PRB1 concentration thresholds by varying the diagnostic threshold. The ROC curves of example 1, example 3 and example 5 are shown in fig. 9, 10 and 11, respectively. The statistical results are shown in Table 4.

TABLE 4 Table 4

From this, the AUC values of example 3 and example 5 are higher than example 1 and the detection accuracy is higher than example 1, indicating that both methods can more accurately predict caries in infants when detecting their saliva PRB1 content. The highest AUC value of example 5 indicates the best overall performance in diagnosing caries, and higher detection sensitivity and specificity. Thus, the effectiveness of PRB1 as a caries marker for children can be confirmed by a reverse validation experiment and ROC curve analysis. The methods of example 3 and example 5 are superior to the existing ELISA kit (example 1) in terms of both detection sensitivity and specificity, providing a more reliable technical support for early diagnosis of caries in young children.

The foregoing is only a preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions easily contemplated by those skilled in the art within the technical scope of the present invention should be covered by the present invention.

Claims (9)

1. A composition for detecting the PRB1 content of saliva of infants is characterized by comprising TFIIB or biotinylation TFIIB, PRB1, carboxymethyl dextran SPR chip, EDC, NHS and ethanolamine hydrochloride, wherein the TFIIB or biotinylation TFIIB is used at 400 nM-1 mM, PRB1 is used as a standard substance, PRB1 is used at 0.02 nM-1200 nM, EDC and NHS are mixed, EDC is used at 0.2M-0.4M, NHS is 0.1M-0.2M, ethanolamine hydrochloride is used at 0.05-5M, and pH=8.5.

2. The composition of claim 1, wherein the TFIIB has a dissociation equilibrium constant for PRB1 of 4.18 x 10 ^-9 mo1/L.

3. The composition of claim 1, further comprising horseradish peroxidase-labeled avidin, wherein the horseradish peroxidase-labeled avidin is used at a concentration of 0.01-3 μg/mL.

4. Use of a composition according to any one of claims 1 to 3 for the preparation of a kit for detecting the PRB1 content of saliva in infants, characterized in that it comprises:

Preparing a gradient standard solution of PRB 1;

activating carboxyl groups on the surface of the carboxymethyl dextran SPR chip;

Immersing the activated carboxymethyl dextran SPR chip in a TFIIB solution or a biotinylation TFIIB solution for coupling;

Introducing the coupled SPR chip into the gradient standard solution or the sample solution to be tested;

a standard curve is manufactured according to the concentration of the gradient standard solution and the response value of the gradient standard solution, and a standard equation is obtained through fitting;

and obtaining the PRB1 content according to the response value of the sample liquid to be detected and the standard equation.

5. The application of the preparation method according to claim 4, wherein the activated carboxymethyl dextran SPR chip is immersed in a TFIIB solution or a biotinylation TFIIB solution for coupling, the coupled SPR chip is introduced into the gradient standard solution or the sample solution to be tested, a standard curve is manufactured according to the concentration of the gradient standard solution and the response value of the gradient standard solution, a standard equation is obtained by fitting, and the PRB1 content is obtained according to the response value of the sample solution to be tested and the standard equation, and the preparation method specifically comprises the following steps:

Immersing the activated carboxymethyl dextran SPR chip in 120 mu M TFIIB solution, coupling to a stable response value, and blocking redundant carboxyl groups of the carboxymethyl dextran SPR chip by adopting 1 mol/L of ethanolamine hydrochloride with pH=8.5;

introducing the carboxymethyl dextran SPR chip coupled with TFIIB into a first gradient standard solution to obtain a first response value;

A first standard curve is manufactured according to the first response value and the concentration of the first gradient standard solution, and a first standard equation is obtained through fitting;

and obtaining the PRB1 content according to the first response value of the sample liquid to be detected and the first standard equation.

6. The use according to claim 5, wherein the first gradient standard solution is a PRB1 solution of 400nM, 200nM, 100nM, 50nM, 25nM and 10nM, 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.

7. The application of the preparation method according to claim 4, wherein the activated carboxymethyl dextran SPR chip is immersed in a TFIIB solution or a biotinylation TFIIB solution for coupling, the coupled SPR chip is introduced into the gradient standard solution or the sample solution to be tested, a standard curve is manufactured according to the concentration of the gradient standard solution and the response value of the gradient standard solution, a standard equation is obtained by fitting, and the PRB1 content is obtained according to the response value of the sample solution to be tested and the standard equation, and the preparation method specifically comprises the following steps:

immersing the activated carboxymethyl dextran SPR chip in 2mg/mL biotinylated TFIIB solution, coupling to a stable response value, and blocking redundant carboxyl groups of the carboxymethyl dextran SPR chip by adopting 1 mol/L of ethanolamine hydrochloride with pH=8.5;

Introducing the carboxymethyl dextran SPR chip coupled with the biotinylated TFIIB into a second gradient standard solution to obtain a second response value;

Washing the carboxymethyl dextran SPR chip coupled with the biotinylated TFIIB, and introducing the chip into a horseradish peroxidase-labeled avidin solution containing 0.01-3 mug/mL until a third response value is obtained;

Making a standard curve according to the sum of the second response value of the second gradient standard solution 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;

and obtaining the PRB1 content of the sample liquid to be tested according to the sum of the second response value of the sample liquid to be tested and the third response value of the sample liquid to be tested and the second standard equation.

8. The use according to claim 7, wherein the second gradient standard solution is a PRB1 solution of 400nM, 200nM, 100nM, 50nM, 25nM and 10nM, the second standard equation is y=21.68 x ζ0.6777, 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 tested.

9. The use according to claim 4, wherein the step of preparing the biotinylated TFIIB comprises:

constructing a recombinant expression vector of the BirA sequence and a recombinant expression vector carrying the Avi sequence and the TFIIB sequence;

mixing and transfecting a recombinant expression vector of the BirA sequence and a recombinant expression vector carrying the Avi sequence and the TFIIB sequence into eukaryotic cells;

Culturing the transformed cells and collecting the culture solution;

collecting cells from the culture broth, lysing the cells with a lysate, centrifuging to collect a supernatant, and collecting the biotinylated TFIIB from the supernatant.