CN111504957A - Application of Victoria blue B and its derivatives in the preparation of probes or inhibitors of isomerized proteins - Google Patents

Abstract

The invention provides the application of Victoria blue B and its derivatives in preparing probes or inhibitors of isomerized proteins, belonging to the technical field of biological detection or biological medicine. The research of the present invention confirms that: Victoria blue B and its derivatives Victoria blue R and Victoria blue BO can recognize or inhibit isomerized proteins such as amyloid "lysozyme fiber" or denatured protein "heat-denatured bovine serum albumin". protein binding, thereby activating the fluorescence of Victoria Blue B and its derivatives. Taking advantage of this characteristic of Victoria Blue B and its derivatives, Victoria Blue B and its derivatives can be used in the preparation of probes or inhibitors of isomerized proteins.

Description

A probe or inhibitor of Victoria blue B and its derivatives in the preparation of isomerized proteins application in formulations

technical field

The invention belongs to the technical field of biological detection or biomedicine, and in particular relates to the application of Victoria blue B and its derivatives in the preparation of probes or inhibitors of isomerized proteins.

Background technique

Isomerized proteins are proteins whose secondary structure has been changed by some external conditions, including amyloid proteins, denatured proteins, and assembled proteins. Among them, amyloid (also called protein fibrils) is the result of normal protein misfolding and aggregation. Although different types of amyloid proteins have no homology in structure, they have a common feature: most of the misfolding is to change the secondary structure of the protein, from α-helix to β-sheet, the more severe the fibrosis. , the more β-sheet content.

Many studies have been looking for or synthesizing fluorescent probes to prevent the formation of fibrils. It has been reported that many types of small molecules can be used as probes for the specific recognition of a certain amyloid protein, and the most commonly used dyes for labeling amyloid proteins. There are Congo red, THT derivatives, curcumin derivatives, etc. They basically bind to a certain residue of amyloid and fluoresce, but there are also some common problems, such as easy fluorescence quenching, poor specificity, false positives, and even poor sensitivity. To solve the root cause of amyloid fibrosis, it is necessary to develop and find a variety of inhibitors, which have practical application effects for clinical research. There are many types of inhibitors, such as organic or inorganic nanoparticles, tea polyphenols, indole and other small molecules, but there is still a problem of unclear inhibition mechanism, mainly due to protein fibers The sequence after transformation is not clear, which is also a breakthrough problem to be solved.

SUMMARY OF THE INVENTION

In view of the problems existing in the background art, the purpose of the present invention is to provide the application of Victoria blue B and its derivatives in the preparation of probes or inhibitors of isomerized proteins.

The present invention provides an application of Victoria blue B in preparing probes or inhibitors of isomerized proteins.

Preferably, the isomerized protein includes amyloid or denatured protein.

Preferably, the amyloid includes lysozyme, insulin, α-synuclein, Aβ amyloid, Tau protein, transthyretin, serum amyloid A, amylin, gelsolin, microspheres One or one of protein, prolactin, prion, huntingtin, calcitonin, atrial peptide, apolipoprotein A1, lactagglutinin, transforming growth factor, cystatin and immunoglobulin light chain variety.

Preferably, the denatured protein includes one or more of heat denatured protein, pH denatured protein, mechanical force denatured protein and ultrasonic denatured protein; the secondary structure of the denatured protein is changed.

The present invention also provides the use of derivatives of Victoria Blue B as probes or inhibitors of isomerized proteins.

Preferably, the derivative of Victoria Blue B includes Victoria Blue R or Victoria Blue BO.

Preferably, the isomerized protein includes amyloid.

Beneficial effects: The present invention provides the application of Victoria blue B and its derivatives in preparing probes or inhibitors of isomerized proteins. The research of the present invention confirms that: Victoria blue B and its derivatives, Victoria blue R and Victoria blue BO, can recognize or inhibit isomerized proteins such as amyloid "lysozyme fibers" or denatured proteins "heat-denatured bovine serum albumin". protein binding, thereby activating the fluorescence of Victoria Blue B and its derivatives. Taking advantage of this characteristic of Victoria blue B and its derivatives, Victoria blue B and its derivatives can be used in the preparation of probes or inhibitors of isomerized proteins.

Description of drawings

Fig. 1 is the confocal test chart described in Example 1 of the present invention and the fluorescence monitoring lysozyme growth result;

Fig. 2 is the detection result of circular dichroism described in the embodiment of the present invention 1;

Fig. 3 is the Zeta potential test result and isothermal titration calorimeter test result described in the embodiment of the present invention 1;

Fig. 4 is the test result of transmission electron microscope described in Example 1 of the present invention;

Fig. 5 is the test result of Victoria blue R and Victoria blue BO described in Example 2 of the present invention;

Fig. 6 is the fluorescence situation of the combination of Victoria blue B and heated and unheated bovine serum albumin according to Example 3 of the present invention;

Fig. 7 is the combination of Victoria blue B and lysozyme fiber according to Example 3 of the present invention.

Detailed ways

The present invention provides the application of Victoria blue B in preparing probes or inhibitors of isomerized proteins.

The chemical formula of Victoria Blue B of the present invention is shown in formula I:

The present study confirms that Victoria Blue B can bind to isomerized proteins such as amyloid "lysozyme fibers" or denatured proteins "heat-denatured bovine serum albumin", and activate Victoria Blue B fluorescence. Taking advantage of this property of Victoria Blue B, Victoria Blue B can be applied to the preparation of probes or detection kits for the detection of isomerized proteins. The detection kit includes Victoria Blue B, preferably other available solvents, buffers and other reagents.

The detection method of isomerized protein in this field is mainly fluorescence detection, but the types of related dyes are single, and new dyes need to be developed to make up for the shortcomings of commonly used dyes, such as easy quenching and short emission wavelength. In the present invention, the application of the Victoria Blue B as an isomerized protein probe preferably includes detection of isomerized proteins (including fibrosis, denaturation, changes in assembly form, etc.) in related products on the market (for example, The quality of the product is evaluated by detecting the content of isomerized protein). Using Victoria blue B as a probe for the detection of isomerized proteins has the advantages of low detection limit, high sensitivity, strong fluorescence intensity and long fluorescence wavelength.

At the same time, the present invention also provides the application of Victoria Blue B as an inhibitor of isomerized protein, and the application preferably includes the use of Victoria Blue B to prepare an inhibitor of isomerized protein. It is well known in the art that the production of isomerized proteins in humans is often associated with disease. For example, amyloid, as a result of normal protein misfolding and aggregation, may cause major diseases such as Alzheimer's disease, Parkinson's disease, type 2 diabetes, and prion disease. The combination of Victoria blue B and isomerized protein can effectively inhibit the activity of isomerized protein.

In addition to Victoria Blue B, the present invention also provides the use of derivatives of Victoria Blue B as probes or inhibitors of isomerized proteins. In the present invention, the derivatives of Victoria Blue B include Victoria Blue R or Victoria Blue BO.

The chemical formula of the Victoria Blue R is shown in formula II:

The chemical formula of the Victoria blue BO is shown in formula III:

In the present invention, the derivatives of Victoria Blue B are studied based on the structure of Victoria Blue B. The study found that: Victoria blue R and Victoria blue BO also have a certain inhibitory effect on amyloid lysozyme fibers, similar to Victoria blue B, and the inhibition occurred during the elongation of amyloid lysozyme fibers.

In addition, similar to Victoria Blue B, its derivatives Victoria Blue R, Victoria BO, Victoria Blue FBR, Victoria Blue FGA, Victoria Blue 4R, and dyes similar to Victoria Blue B structural framework such as bromocresol blue, bromocresol green , bromocresol purple, malachite green, etc., and other dyes, such as: Chicago sky blue 6B, quinaldine red, etc., can also be partially used for lysozyme, bovine insulin fiber, CsgA protein and other amyloid or other types of denaturation Protein detection, combined with these isomerized proteins to stimulate fluorescence, and partially inhibit the growth of amyloid fibrils in the isomerized proteins.

The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the protection scope of the present invention.

Example 1

The sources of the raw materials used in this example are: Victoria Blue B, purchased from Shanghai Ruji Biotechnology (Shanghai, China). Lysozyme (egg white), bovine serum albumin, sodium chloride (NaCl), Tris, hydrochloric acid (HCl), glycine, were purchased from Beijing Dingguo Changsheng Biotechnology (Beijing, China).

The lysozyme purchased above was weighed and dissolved in a buffer solution containing 80 mM sodium chloride and 70 mM glycine (pH=2.7), so that the final concentration of lysozyme was 10 μM; heated and stirred with a magnetic stirrer, and the heating temperature was 65 ° C , the stirring revolution is 220rmp, heating for 6h to obtain lysozyme amyloid, which is stored in a refrigerator at 4°C for later use.

The bovine serum albumin purchased above was dissolved in distilled water to a concentration of 100 μM, denatured by heating at 100° C. for 10 minutes in a metal bath, and stored in a 4° C. refrigerator for later use.

Confocal test:

The effects of no addition and addition of the inhibitor Victoria B (50 μM) on the growth of lysozyme into fibers were observed using a laser confocal inverted microscope of model LSM710 purchased from Zeiss, Germany. Take lysozyme samples at different stages incubated at 65 °C and 220 rmp, respectively, to make the final detection concentration 100 μM, and then add a certain amount of dye Victoria B to make the final concentration of both 20 μM. Take 3uL of each sample for preparation, and place the slide gently to ensure that the sample is evenly spread on the slide. Scanning with a 630 nm laser light at a magnification of 20× can obtain the following confocal images. The results are shown in Figure 1-A. Among them, the top and bottom are the original and enlarged images, respectively.

Fluorescence monitoring of lysozyme growth:

The fluorescence intensity of Victoria blue B to identify lysozyme fibers was measured with a fluorescence spectrometer of model RF-5301PC purchased from Shimadzu Corporation of Japan, and quantitative sampling was performed irregularly during the fiber growth process, so that the final concentration of the final detection was 1 mM. After sampling, 1 mM Victoria blue B was added to the samples at each stage to make the final concentration 20 μM, and the fluorescence intensity was measured with a quartz cell with a 3 mm slit purchased from Yehui Glass Instrument Factory. The excitation wavelength and emission wavelength were respectively are 650 nm and 710 nm, and the excitation and emission slits are 5 mm and 10 mm, respectively, during measurement. Repeat the above experiments for three groups, deduct the points with large deviation, and make a curve and a broken line graph. The results are shown in Figure 1-B, where the curve represents the fiber growth.

Circular dichroism detection:

Samples at different stages with and without the addition of the inhibitor Victoria Blue B were tested with a circular dichroism instrument purchased from Biologic. 100 μL of each sample was dissolved in 200 mM Tris-HCl buffer solution to a final concentration of 10 μM. Using a 1 mm quartz cell, the starting wavelength of scanning is 190 nm, the ending wavelength is 260 nm, the scanning time interval is 10 s, and each spectrum is scanned three times on average. The test results are shown in Figure 2. Among them, Figure 2-A and Figure 2-B are the circular digraphs without inhibitor and Victoria blue B (150μM), respectively, Figure 2-C and Figure 2-D are the same as Figure 2-A and Figure 2 -B corresponding α-helix and β-sheet content statistics.

Zeta Potential Test:

The Zeta potential of mature fibers, dyes, and fibers combined with dyes was monitored with a ZEN3600 Malvern particle sizer purchased from Malvern Instruments Ltd., UK. The final concentration of the fibers was 100 μM and the final concentration of the dye was 20 μM. Diluted in 200 mM Tris-HCl buffer pH=7.4. The Zeta potential test results are shown in Figure 3-A.

Isothermal titration calorimeter test:

A series of thermodynamic parameters of Victoria blue B titration of lysozyme were measured with an isothermal titration calorimeter, model MicroCal ITC20, purchased from General Electric Company, USA. Lysozyme was dissolved in a buffer solution containing 70 mM glycine and 80 mM sodium chloride pH=2.7, heated at 65°C, 220 rmp for 4 h, Victoria blue B was dissolved in water to a concentration of 10 μM, and an appropriate amount was injected into the sample pool , take the heated and stirred sample of lysozyme and dilute it to 50 μM with water, draw it with a syringe for use, then stir under the condition of 1000 rmp, and titrate 1 μL of lysozyme fiber into Victoria every 120s to obtain lysozyme fiber titration Victoria Blue B The isothermal titration data, and then according to the fitting curve can calculate the combination of Changshu, reaction heat and other thermodynamic parameters. The test results of the isothermal titration calorimeter are shown in Figure 3-B. Among them, the upper and lower figures represent the binding isotherm and integral heat and the corresponding fitted line, respectively.

Transmission Electron Microscopy Testing:

A JEM model transmission electron microscope produced in Japan was used to observe the above-mentioned samples heated for 4 hours. An appropriate amount of samples were dropped on a 300-mesh copper mesh, and the samples were stained with 0.01 μg/50 μ phosphotungstic acid, and then washed with ultrapure water to clean the background. . Samples were collected at 60, 90, 120, and 240 min of fibrillation, and projection results are shown in Figure 4. Figure 4 represents the morphological changes of protein aggregates in the absence (top half) and presence (bottom half) of Victoria blue B (50 μM) for lysozyme fibrillation.

Example 2

According to the method of Example 1, Victoria Blue R and Victoria Blue BO were tested, and the results are shown in FIG. 5 . Among them, Figure 5-A shows the fiber growth under the condition of fiber growth by adding different concentrations of the inhibitor Victoria Blue BO; Figure 5-B shows the fiber growth by adding different concentrations of the inhibitor Victoria Blue R under the condition of fiber growth situation; Figures 5-C, 5-D and 5-E represent circular dichroism chromatograms without the addition of Victoria Blue BO or Victoria Blue R, Victoria Blue BO (150 μM) and Victoria Blue R (150 μM) additions, respectively; Figure 5- F, 5-G and 5-H represent the α-helix and β-sheet content charts corresponding to Fig. 5-C, 5-D and 5-E respectively.

Figure 5 shows that: Victoria blue R and Victoria blue BO also have inhibitory effects on lysozyme fibers, and can be used as probes for lysozyme fibers.

Example 3

Using the fluorescence detection method, the fluorescence of Victoria blue B binding to heated and unheated bovine serum albumin was detected, and the results are shown in Fig. 6 . Among them, Figure 6-A is the visual display of the fluorescence imager, 1 tube is Victoria Blue B + heated BSA, 2 tubes are Victoria Blue B + BSA, 3 tubes are Victoria Blue B, and 4 tubes are heated Victoria Blue blue B. Figure 6-B shows the fluorescence color development of different treatment groups.

Figure 6 shows that: Victoria blue B does not emit light when combined with the original protein, and emits red fluorescence when combined with heat-denatured bovine serum albumin, which can be used as a heat-denatured bovine serum albumin probe.

The fluorescence intensity of the lysozyme fibers was monitored by detecting Victoria blue B by using the fluorescence spectrum detection method, and the results are shown in Fig. 7 . Among them, Figure 7-A shows the relationship between Victoria Blue B and the concentration of lysozyme fibers added; from Figure 7-A, it can be calculated that the detection limit of Victoria Blue B for detecting lysozyme fibers is 0.3019 μM. Figure 7-B shows the detection of lysozyme fibers by Victoria blue B as a fluorescent probe; from Figure 7-B, it can be concluded that the linear correlation coefficient is R ² =0.9985, so the concentration of lysozyme fibers is in the range of 0-20 μM , Victoria blue B detection is more sensitive.

The above are only the preferred embodiments of the present invention. It should be pointed out that for those skilled in the art, without departing from the principles of the present invention, several improvements and modifications can be made. It should be regarded as the protection scope of the present invention.

Claims (7)

1. Application of Victoria blue B in preparing a probe or an inhibitor of an isomerized protein.

2. The use of claim 1, wherein the isomerized protein comprises an amyloid protein or a denatured protein.

3. The use according to claim 2, wherein the amyloid protein comprises one or more of lysozyme, insulin, α -synuclein, a β amyloid, Tau, transthyretin, serum amyloid a, amylin, gelsolin, microglobulin, prolactin, prion, huntingtin, calcitonin, atrial natriuretic peptide, apolipoprotein a1, lactadherin, transforming growth factor, cystatin and immunoglobulin light chain.

4. The use of claim 2, wherein the denatured protein comprises one or more of heat-denatured protein, pH-denatured protein, mechanical force-denatured protein, and ultrasound-denatured protein; the secondary structure of the denatured protein is changed.

5. Use of derivatives of victoria blue B as probes or inhibitors of isomerised proteins.

6. Use according to claim 5, wherein the derivative of Victoria blue B comprises Victoria blue R or Victoria blue BO.

7. The use of claim 6, wherein the isomerized protein comprises an amyloid protein.

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