A method for inhibiting ApoB100 thermal aggregation using ApoJ and a method for quantitatively detecting ApoJ activity

Abstract

The present invention provides a method for inhibiting the thermal aggregation of ApoB100 using ApoJ and a method for quantitatively detecting the activity of ApoJ. The activity of ApoJ protein cannot be directly measured, but by utilizing the protein characteristics of ApoJ protein with molecular chaperone function, adding it to ApoB100 protein can reduce the misfolding and aggregation of ApoB100 protein caused by high temperature, thereby protecting ApoB100 protein. The aggregation degree of ApoB100 protein can be obtained by measuring the absorbance value, so that the activity of ApoJ protein can be known. After the ApoJ protein is added, when the protection quantitative ApoB100 protein reduces the denaturation incidence by 50%, the amount of ApoJ protein added is defined as an activity unit, and the activity of ApoJ under different batch conditions can be quantitatively detected, reflecting the differences of ApoJ protein under different batches and different reaction conditions.

Description

Technical Field

[0001] The present invention relates to the field of ApoJ protection and detection technology, and in particular to a method for inhibiting ApoB100 thermal aggregation by using ApoJ and a method for quantitatively detecting ApoJ activity. Background Art

[0002] Apolipoprotein J (ApoJ, also known as clusterin) is a multifunctional glycoprotein newly isolated from human plasma HDL in 1990. It is widely present in various tissues and body fluids of the human body. ApoJ protein is a heterodimeric protein with a relative molecular mass of 75-80KDa, which can bind to human plasma high-density lipoprotein (HDL) and very high-density lipoprotein (VHDL). The functions of ApoJ protein are complex, and it participates in many physiological processes in the body, including the regulation of human functions, inhibition of apoptosis and inflammation, and participation in lipid transport. It has been found that ApoJ protein is highly expressed in many disease processes, such as neurodegenerative diseases, atherosclerosis, myocardial infarction, tumors, etc.

[0003] Molecular chaperones are proteins that bind to unstable conformations of other proteins and stabilize them. They help the bound polypeptides fold, assemble, transport or degrade in vivo by controlling binding and release. In 1997, it was reported that a 14bp element in the promoter of the ApoJ protein, whose sequence is strictly conserved in vertebrates, is specifically recognized by the transcription factor HSF1 and can mediate heat shock-induced transcription in transient expression analysis. This shows that the ApoJ protein is a heat shock protein (heat shock protein, when an organism is exposed to high temperature, it will be stimulated by heat to synthesize this protein to protect the organism itself). ApoJ protein and small heat shock proteins (sHsps) are a class of molecular chaperone proteins with similar functions, although they lack obvious sequence similarity. Both molecular chaperone proteins can effectively prevent the target protein from forming aggregation and precipitation under stress conditions, such as elevated temperature, reduction and oxidation. Studies have shown that ApoJ protein can inhibit the denaturation and aggregation of proteins in starch metabolism-related pathways, including glutathione transferase (GST), creatine phosphokinase (CPK), catalase (CAT) and other proteins. For example, at physiological concentrations, ApoJ protein can effectively protect glutathione s-transferase and catalase from heat precipitation, and can effectively protect α-lactoprotein and bovine serum albumin from dithiothreitol reduction precipitation. In addition, ApoJ protein can also inhibit stress-induced protein precipitation in undiluted human serum. ApoJ protein plays a molecular chaperone role through an ATP-independent mechanism, irreversibly binding to stress proteins to form soluble high-molecular-weight complexes, maintaining the spatial stability of receptor proteins and reducing the misfolding of receptor proteins due to environmental factors. In doing so, they act on slow, unfolded protein pathways. The conformational dynamics and aggregation state of these two proteins may play a crucial role in their chaperone function. Subunit exchange may play an important role in regulating chaperone action; the free form of the protein may be the main form that reflects the chaperone activity, rather than the aggregated state.

[0004] Apolipoprotein (Apo) is a protein in plasma lipoprotein, and its basic function is to transport lipids. Apolipoprotein B (ApoB) can be divided into the following subclasses due to differences in amino acid composition: apolipoprotein B48 (ApoB48) and apolipoprotein B100 (ApoB100). ApoB48 is one of the apolipoproteins of chylomicrons (CM); ApoB100 is one of the apolipoproteins of very low density lipoprotein (VLDL) and low density lipoprotein (LDL). Summary of the invention

[0005] In view of this, the present invention aims to propose a method for inhibiting the thermal aggregation of ApoB100 using ApoJ and a method for quantitatively detecting the activity of ApoJ. The ApoJ can be a natural apolipoprotein or a recombinant apolipoprotein. ApoJ is a protein with a molecular chaperone function, which can reduce the misfolding of ApoB100 protein caused by high temperature, and ultimately protect ApoB100 protein. In view of the problem that there is currently no method for measuring the activity of ApoJ protein, the ApoJ protein has a molecular chaperone function similar to that of heat shock protein, which can inhibit the aggregation and precipitation of proteins after heating. The activity of ApoJ protein is defined and quantified by detecting the changes in the activity of ApoB100 during the heating process.

[0006] To achieve the above object, the technical solution of the present invention is achieved as follows:

[0007] In one aspect of the present invention, a method for inhibiting thermal aggregation of ApoB100 using ApoJ is proposed. ApoJ protein is added to ApoB100 protein, and the ApoJ protein is used to reduce the misfolding of ApoB100 protein caused by high temperature, thereby protecting ApoB100 protein.

[0008] In another aspect of the present invention, a method for quantitatively detecting the activity of ApoJ is provided, which utilizes the method that ApoJ protein can inhibit the thermal aggregation of ApoB100 protein, and the activity of ApoJ protein can be determined by measuring the absorbance of ApoB100 protein.

[0009] Furthermore, the activity of ApoJ was detected by measuring the change in absorbance of ApoB100 protein before and after heating.

[0010] Furthermore, the method for quantitatively detecting ApoJ activity comprises the following steps:

[0011] S1. Take ApoJ protein and ApoB100 protein out of the refrigerator and place them in a 4°C environment to thaw for subsequent use;

[0012] S2. Prepare different groups of solutions, where the blank group is the PB buffer solution of ApoB100 protein, and no heating is required, and the control group is the PB buffer solution of ApoB100 protein, and heating treatment is performed; experimental group: prepare a series of concentration ratios of ApoJ protein and ApoB100 protein, and place them in PB buffer solution, mix well, and then heat treatment;

[0013] S3. Then use a UV spectrophotometer to detect the absorbance values ​​of the blank group, control group and experimental group, and record them as A1, B1, and C1 respectively;

[0014] An ApoJ protein activity meets

[0015] Furthermore, the temperature of the heating treatment in step S2 is 60-200°C.

[0016] Furthermore, in step S2, the mass ratio of ApoB100 protein to ApoJ protein in the experimental group is 1:0.1 to 1:0.7.

[0017] Furthermore, in step S3, an ultraviolet spectrophotometer is used to detect the absorbance values ​​of the blank group, the control group and the experimental group at 360 nm.

[0018] Furthermore, (C1-A1) / (B1-A1)×100% represents the rate at which ApoJ protein protects ApoB100 protein and reduces the occurrence of denaturation.

[0019] Compared with the prior art, the method of using ApoJ to inhibit ApoB100 thermal aggregation and the method of quantitatively detecting ApoJ activity described in the present invention have the following advantages:

[0020] (1) The present invention utilizes the property of ApoJ as a molecular chaperone to protect ApoB100 protein under heating conditions and reduce misfolding caused by high temperature;

[0021] (2) Since the activity of ApoJ protein cannot be measured directly, the activity of ApoJ protein can be defined and measured by the change in the absorbance of ApoB100 protein before and after heating.

[0022] (3) Quantitative detection of the activity of ApoJ protein can reflect the quality differences of ApoJ protein in different batches and under different reaction conditions, and the method is simple, accurate and reliable. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The accompanying drawings constituting a part of the present invention are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the accompanying drawings:

[0024] Figure 1 It is the fitting curve of Example 2 of the present invention. DETAILED DESCRIPTION

[0025] The present invention will be further described below in conjunction with specific embodiments. It should be noted that the data in the following experimental examples are obtained by the inventor through a large number of experiments. Due to space limitations, only a part of them is shown in the specification, and those of ordinary skill in the art can understand and implement the present invention under these data. These embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these changes or modifications also fall within the scope protected by the present application.

[0026] A method for quantitatively detecting ApoJ activity of the present application comprises the following steps:

[0027] S1. Take out ApoJ protein and ApoB100 protein from the -20°C freezer and place them in a 4°C environment to thaw for subsequent use;

[0028] S2. Prepare different groups of solutions, where the blank group is the PB buffer solution of ApoB100 protein, and no heating is required, and the control group is the PB buffer solution of ApoB100 protein, and heating treatment is performed; experimental group: prepare a series of concentration ratios of ApoJ protein and ApoB100 protein, and place them in PB buffer solution, mix well, and then heat treatment;

[0029] S3. Then use a UV spectrophotometer to detect the absorbance values ​​of the blank group, control group and experimental group at 360nm, and record them as A1, B1, and C1 respectively;

[0030] The degree of protein aggregation has a maximum light absorption value at 360nm, so the absorbance value at 360nm can reflect the degree of protein aggregation, folding or thermal denaturation. (B1-A1) represents the 100% denaturation degree of the ApoB100 protein in the control group, and (C1-A1) represents the degree to which the ApoJ protein protects the ApoB100 protein and reduces the occurrence of denaturation. (C1-A1) / (B1-A1)×100% represents the degree to which the ApoJ protein protects the ApoB100 protein and reduces the incidence of denaturation. When the rate of inhibiting the ApoB100 protein to reduce the incidence of denaturation reaches 50%, the amount of ApoJ protein required is defined as one ApoJ protein activity unit, i.e. 1U. The activity of one ApoJ protein satisfies

[0031] The temperature of the heating treatment in step S2 is 60-200° C., and the mass ratio of ApoB100 protein and ApoJ protein added in the experimental group is 1:0.1-1:0.7.

[0032] The method of the present application can provide a method for quantitatively detecting the activity of ApoJ protein, which is convenient for comparing the quality and activity differences of ApoJ protein in different batches and under different reaction conditions, and is simple to operate.

[0033] Example 1

[0034] Experimental equipment and consumables

[0035] Instruments: UV spectrophotometer (Shanghai Meipuda, model UV-1200), metal bath (Changzhou Guohua, model HH-4), electronic balance (Youke), water purifier (SMART-Q15), and a set of pipettes (eppendorf).

[0036] Consumables: ApoB100 (Biotech Biotechnology, concentration 2 mg / ml); ApoJ protein (GenScript, concentration 0.75 mg / ml), 0.2 M phosphate buffer (PB buffer prepared by ourselves)

[0037] The specific experimental steps are as follows:

[0038] (1) After taking out ApoJ protein and ApoB100 protein from the -20°C freezer, place them in a 4°C environment to thaw for subsequent use.

[0039] (2) Turn on the UV spectrophotometer and preheat for 20 minutes. After the preheating is completed, adjust the detection wavelength to 360 nm and use 0.2 M phosphate buffer as a blank control and adjust it to zero.

[0040] (3) The detection reaction system was prepared according to the amount of each component added in Table 1, and the final volume of each system was 70 μL. For both the blank group and the control group, PB buffer was first added to a 1.5 ml EP tube, and then ApoB100 solution was added. The reaction system of experimental groups 1 to 5 was added to a 1.5 ml EP tube in sequence with PB buffer, ApoB100 solution and ApoJ solution. The solvent used in the preparation of ApoB100 solution and ApoJ solution was PB solution.

[0041] Table 1

[0042]

[0043] (4) Use a pipette to evenly blow up each tube several times, and place the reaction system of the control group and experimental groups 1-5 in an 80°C metal bath for heating for 5 minutes. After heating, let it stand at room temperature for 10 minutes, and then add 70 μL of PB buffer, so that the final system of each reaction is 140 μL. The reaction system of the blank group does not need to be heated, but only needs to be left at room temperature for 10 minutes, and then add 70 μL of PB buffer.

[0044] (5) After mixing the reaction solutions in the above tubes, use an ultraviolet spectrophotometer to detect the absorbance of each group at 360 nm. The absorbance of the blank group is recorded as A1, the absorbance of the control group is recorded as B1, and the absorbance of the experimental group is recorded as C1. The specific results are shown in Table 2.

[0045] Table 2

[0046] serial number 0D360 Value (C1-A1) / (B1-A1)×100% Blank group 0.168 / Control group 0.294 / Experimental Group 1 0.231 50％ Experimental Group 2 0.199 24.60％ Experimental Group 3 0.182 11.11％ Experimental Group 4 0.177 7.14％ Experimental Group 5 0.164 -3.17％

[0047] Among them, the reaction system of the control group only contained ApoB100 protein. Under the condition of heating at 80℃ for 5 minutes, its OD360 absorbance value increased from value A1 to value B1, so the OD difference was (B1-A1), which represented the 100% denaturation degree of the control group. The reaction systems of experimental groups 1 to 5 included ApoB100 protein and a certain amount of ApoJ protein. Under the condition of heating at 80℃ for 5 minutes, its OD360 absorbance value increased from value A1 to value C1, so the OD difference was (C1-A1), which represented the degree to which ApoJ protein protected ApoB100 protein and reduced the occurrence of denaturation. (C1-A1) / (B1-A1)×100% represents that ApoJ protein protected ApoB100 protein and reduced the occurrence rate of denaturation.

[0048] By analyzing the data in Table 2, the OD360 value of the control group system after heating increased compared with the blank group, indicating that the ApoB100 protein was denatured during the heating process. Compared with the control group, the OD360 value of experimental groups 1 to 5 decreased, indicating that the degree of denaturation of the ApoB100 protein was inhibited. During the heating process, the addition of ApoJ protein can effectively protect the ApoB100 protein and reduce incorrect folding. And as the amount of ApoJ protein added increases, the OD360 value gradually decreases, and the protective effect on the ApoB100 protein also increases.

[0049] Take experimental group 1 as an example:

[0050] The increase in OD360 value of the control group (B1-A1) was 0.294-0.168=0.126;

[0051] The OD360 value increase value (C1-A1) of experimental group 1 was 0.231-0.168=0.063;

[0052] ApoJ protein protects ApoB100 protein and reduces the incidence of degeneration

[0053] Under this reaction condition setting, when the incidence of quantitative ApoB100 protein denaturation is inhibited by 50%, the amount of ApoJ protein added is defined as one ApoJ protein activity unit, i.e. 1U. For experimental group 1, the amount of ApoJ protein added can protect the ApoB100 protein under heating conditions of 80°C for 5 minutes, and the OD360 value increase (C1-A1) is only 50% of the control group (B1-A1). Then the amount of ApoJ protein used is defined as one ApoJ protein activity unit, i.e. 1.5μg ApoJ protein is equal to 1 ApoJ protein activity unit, i.e. 1U = 1.5μg ApoJ. The specific activity of this batch of ApoJ protein is 1U / 1.5μg = 0.667U / mg.

[0054] Example 2

[0055] Another batch of ApoJ protein and ApoB100 protein was taken out of the -20°C refrigerator and placed in a 4°C environment for thawing for subsequent use, wherein the concentration of ApoB100 protein was 2 mg / mL and the concentration of ApoJ protein was 0.75 mg / mL. The other operating methods were the same as in Example 1, and the detection reaction system was configured according to the amount of each component added in Table 3, and the final volume of each system was 70 μL. The absorbance value of the blank group was recorded as A1, the absorbance value of the control group was recorded as B1, and the absorbance value of the experimental group was recorded as C1. The specific results are shown in Table 4.

[0056] Table 3

[0057]

[0058] Table 4

[0059]

[0060]

[0061] The data in Table 4 were fitted to calculate the specific activity. The fitting curve is shown in Figure 1 .according to Figure 1 It can be seen that when the incidence of quantitative ApoB100 protein reduction and denaturation is 50%, the amount of ApoJ protein used in this batch is 5.152 μg. That is, 5.152 μg ApoJ protein is equal to 1 ApoJ protein activity unit. The specific activity of ApoJ protein in this batch is 1U / 5.152μg=0.194U / mg.

[0062] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for quantitatively detecting ApoJ activity, characterized in that: ApoJ protein is added to ApoB100 protein, and the ApoJ protein is used to reduce the misfolding of ApoB100 protein caused by high temperature, thereby protecting ApoB100 protein and inhibiting thermal aggregation of ApoB100, and the activity of ApoJ is detected by measuring the change in the absorbance value of ApoB100 protein before and after heating; The method for quantitatively detecting ApoJ activity specifically comprises the following steps: S1. Take ApoJ protein and ApoB100 protein out of the refrigerator and place them in a 4°C environment to thaw for subsequent use; S2. Prepare different groups of solutions, where the blank group is the PB buffer solution of ApoB100 protein and does not need to be heated, and the control group is the PB buffer solution of ApoB100 protein and is heated; experimental group: prepare a series of concentration ratios of ApoJ protein and ApoB100 protein, place them in PB buffer solution, mix well and heat them, and the heating temperature is 60-200°C; In the experimental group, the mass ratio of ApoB100 protein and ApoJ protein added was 1:0.1 to 1:0.7; S3. Then use a UV spectrophotometer to detect the absorbance values ​​of the blank group, control group and experimental group, and record them as A1, B1, and C1 respectively; An ApoJ protein activity meets 2. A method for quantitatively detecting ApoJ activity according to claim 1, characterized in that: In step S3, an ultraviolet spectrophotometer is used to detect the absorbance values ​​of the blank group, the control group and the experimental group at 360 nm.

3. A method for quantitatively detecting ApoJ activity according to claim 1, characterized in that: (C1-A1) / (B1-A1)×100% represents the rate at which ApoJ protein protects ApoB100 protein and reduces the occurrence of denaturation.

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