A hepatitis B virus particle lysate and its preparation and application

Abstract

The present invention belongs to the technical field of medical detection, and specifically discloses a hepatitis B virus particle lysate and its preparation and application. The lysate comprises the following components in parts by weight: 10-20 parts of thiourea, 70-90 parts of urea, 8-12 parts of sodium dodecyl sarcosinate, 3-5 parts of Tween, 4-8 parts of CHAPS, 1-3 parts of TCEP, 3-5 parts of Triton X-100, 8-12 parts of glycerol, 1-3 parts of Tirs-HCl, and 0-100 parts of deionized water. The lysate provided by the present invention can expose HBcAg in hepatitis B virus particles and has little influence on detection antibodies.

Description

Technical Field

[0001] The present invention relates to the technical field of medical detection, and particularly relates to a hepatitis B virus particle lysate and its preparation and application. Background Art

[0002] Currently, in clinical practice, the antiviral efficacy evaluation and treatment endpoint judgment of chronic hepatitis B patients are mainly carried out by detecting hepatitis B five-item and hepatitis B virus DNA (HBV DNA). However, due to nucleoside analogue treatment, hepatitis B virus gene integration, and the expression termination mutation of hepatitis B e antigen G1896A, these serological markers cannot be directly used as the basis for judging the treatment endpoint.

[0003] In the existing literature reports, the hepatitis B core-related antigen (HBcrAg) including hepatitis B core antigen (HBcAg), hepatitis B e antigen (HBeAg), and P22cr has important value for making a correct judgment on the clinical treatment endpoint of chronic hepatitis B patients. However, HBcAg is a component unit of HBV core particles and is wrapped by the outer membrane composed of hepatitis B virus S protein in the mature state. It is difficult to directly detect HBcAg. It is necessary to remove the virus outer membrane and break the core particles into monomers to achieve the detection of HBcAg. Currently, at room temperature, there is no special reagent for exposing and lysing core particles. In laboratory-level research, heating at 70 °C for 15 to 30 minutes is generally used to destroy HBV core particles. However, this treatment method is not applicable to clinical serum samples (coagulation will occur after heating). Therefore, a suitable hepatitis B virus particle lysate is particularly important. Summary of the Invention

[0004] In view of the above-mentioned disadvantages of the prior art, the purpose of the present invention is to provide a hepatitis B virus particle lysate and its preparation and application, which can expose HBcAg in hepatitis B virus particles and have little influence on the detection antibody.

[0005] To achieve the above object and other related objects, the first aspect of the present invention provides a hepatitis B virus particle lysate, which comprises the following components in parts by weight: 10 - 20 parts of thiourea, 70 - 90 parts of urea, 8 - 12 parts of sodium lauroyl sarcosinate, 3 - 5 parts of Tween, 4 - 8 parts of CHAPS (3 - [3 - (cholamidopropyl) dimethylammonio] propanesulfonate), 1 - 3 parts of TCEP (Tris (2 - carboxyethyl) phosphine), 3 - 5 parts of Triton X - 100, 8 - 12 parts of glycerol, 1 - 3 parts of Tirs - HCl, and 0 - 100 parts of deionized water.

[0006] Furthermore, the hepatitis B virus particle lysate includes the following components in proportions by weight: 10-20 parts of thiourea, 70-90 parts of urea, 8-12 parts of sodium lauryl sarcosinate, 3-5 parts of Tween, 4-8 parts of CHAPS, 1-3 parts of TCEP, 3-5 parts of Triton X-100, 8-12 parts of glycerol, 1-3 parts of Tirs-HCl, and 10-100 parts of deionized water.

[0007] Furthermore, the pH of the hepatitis B virus particle lysate is between 7.2 and 7.8.

[0008] Furthermore, the Tween is selected from at least one of tween 20, tween 40, tween 60, and tween 80. The second aspect of the present invention provides a method for preparing the hepatitis B virus particle lysate according to the first aspect, comprising the following steps:

[0009] (1) Add thiourea, urea, sodium lauryl sarcosinate (SLS), CHAPS, and TCEP into deionized water in sequence and stir evenly;

[0010] (2) adjusting the pH of the solution obtained in step (1) to 7.2 to 7.8;

[0011] (3) Add Triton X-100, glycerol, and Tirs-HCl to the solution obtained in step (2) and stir evenly;

[0012] (4) Tween 60 is added to the solution obtained in step (3), and the mixture is stirred evenly to obtain the hepatitis B virus particle lysate.

[0013] Furthermore, in step (1), the stirring speed is 300-400 rpm, and the stirring time is 30-40 min.

[0014] Furthermore, in step (2), the stirring speed is 300-400 rpm, and the stirring time is 30-40 min.

[0015] Furthermore, in step (4), the stirring speed is 100-120 rpm, and the stirring time is 20-30 min.

[0016] The third aspect of the present invention provides the use of the hepatitis B virus particle lysis solution according to the first aspect or the hepatitis B virus particle lysis solution prepared according to the method described in the second aspect in lysing hepatitis B virus particles.

[0017] A fourth aspect of the present invention provides a method for lysing hepatitis B virus particles, comprising lysing hepatitis B virus particles using the hepatitis B virus particle lysing solution according to the first aspect or the hepatitis B virus particle lysing solution prepared according to the method described in the second aspect.

[0018] As described above, the hepatitis B virus particle lysate of the present invention, its preparation and application have the following beneficial effects:

[0019] Sodium lauryl sarcosinate (SLS) and CHAPS in the lysate provided by the present invention can effectively lyse the outer membrane of hepatitis B virus particles, and thiourea, urea and TCEP can effectively lyse the core particles, making HBcAg present in a monomer state and being detected in the largest amount. It is suitable for lysing HBcAg in hepatitis B virus particles and has little influence on the detection of antibodies. Specific embodiments

[0020] The following specific examples illustrate the embodiments of the present invention. Those skilled in the art can easily understand the other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0021] The present invention provides a hepatitis B virus particle lysate, which comprises the following components in parts by weight: 10 - 20 parts of thiourea, 70 - 90 parts of urea, 8 - 12 parts of sodium lauryl sarcosinate (SLS), 3 - 5 parts of Tween, 4 - 8 parts of CHAPS, 1 - 3 parts of TCEP, 3 - 5 parts of Triton X-100, 8 - 12 parts of glycerol, 1 - 3 parts of Tirs-HCl, and 0 - 100 parts of deionized water.

[0022] Among them, the Tween is selected from at least one of tween20, tween40, tween60, and tween80.

[0023] The preparation method of the hepatitis B virus particle lysate provided by the present invention comprises the following steps:

[0024] (1) Place deionized water on a magnetic stirrer with a stirring speed of 300 - 400 rpm, and sequentially add thiourea, urea, sodium lauryl sarcosinate (SLS), CHAPS, and TCEP, and stir for 30 - 40 min;

[0025] (2) Adjust the pH of the solution obtained in step (1) to 7.2 - 7.8;

[0026] (3) Add Triton X-100, glycerol, and Tirs-HCl to the solution obtained in step (2), stir evenly with a stirring speed of 300 - 400 rpm, and stir for 30 - 40 min;

[0027] (4) Add tween 60 to the solution obtained in step (3), with a stirring speed of 100 - 120 rpm, and stir for 20 - 30 min to obtain the hepatitis B virus particle lysate.

[0028] The lysate provided by the present invention is used for lysing hepatitis B virus particles, can expose HBcAg inside the hepatitis B virus particles, and has little influence on the detection antibody.

[0029] The following specifically lists examples to illustrate the present invention in detail. It should also be understood that the following examples are only used to specifically illustrate the present invention and cannot be construed as limiting the protection scope of the present invention. Some non-essential improvements and adjustments made by those skilled in the art based on the above content of the present invention all fall within the protection scope of the present invention. The specific process parameters and the like in the following examples are also only an example within a suitable range, that is, those skilled in the art can make selections within a suitable range through the description in this article, rather than being limited to the specific values in the following examples.

[0030] Example 1

[0031] The hepatitis B virus particle lysate in this example is composed of the following components in parts by weight: 15 parts of thiourea, 80 parts of urea, 10 parts of sodium lauroyl sarcosinate (SLS), 4 parts of tween 60, 6 parts of CHAPS, 2 parts of TCEP, 4 parts of Triton X-100, 10 parts of glycerol, 3 parts of Tirs-HCl, and 66 parts of deionized water.

[0032] The preparation method of the hepatitis B virus particle lysate in this example includes the following steps:

[0033] (1) Place deionized water on a magnetic stirrer with a stirring speed of 300 rpm, and sequentially add thiourea, urea, sodium lauroyl sarcosinate (SLS), CHAPS, and TCEP, and stir for 30 min.

[0034] (2) Adjust the pH of the solution obtained in step (1) to 7.2;

[0035] (3) Add Triton X-100, glycerol, and Tirs-HCl to the solution obtained in step (2), with a stirring speed of 300 rpm, and stir for 30 min;

[0036] (4) Add tween 60 to the solution obtained in step (3), with a stirring speed of 100 rpm, and stir for 20 min to obtain the hepatitis B virus particle lysate.

[0037] Example 2

[0038] The hepatitis B virus particle lysate in this example is composed of the following components in parts by weight: 10 parts of thiourea, 70 parts of urea, 8 parts of sodium lauroyl sarcosinate (SLS), 3 parts of tween 60, 4 parts of CHAPS, 1 part of TCEP, 3 parts of Triton X-100, 8 parts of glycerol, 1 part of Tirs-HCl, and 92 parts of deionized water.

[0039] The preparation method of the hepatitis B virus particle lysate in this example comprises the following steps:

[0040] (1) Place deionized water on a magnetic stirrer with a stirring speed of 350 rpm, and sequentially add thiourea, urea, sodium lauroyl sarcosinate (SLS), CHAPS, and TCEP, and stir for 35 minutes.

[0041] (2) Adjust the pH of the solution obtained in step (1) to 7.5;

[0042] (3) Add Triton X-100, glycerol, and Tirs-HCl to the solution obtained in step (2), with a stirring speed of 350 rpm, and stir for 35 minutes;

[0043] (4) Add tween 60 to the solution obtained in step (3), with a stirring speed of 110 rpm, and stir for 25 minutes to obtain the hepatitis B virus particle lysate.

[0044] Example 3

[0045] The hepatitis B virus particle lysate in this example is composed of the following components in parts by weight: 20 parts of thiourea, 90 parts of urea, 12 parts of sodium lauroyl sarcosinate (SLS), 5 parts of tween 60, 8 parts of CHAPS, 3 parts of TCEP, 5 parts of Triton X-100, 12 parts of glycerol, 3 parts of Tirs-HCl, and 42 parts of deionized water.

[0046] The preparation method of the hepatitis B virus particle lysate in this example comprises the following steps:

[0047] Step (1): Place deionized water on a magnetic stirrer with a stirring speed of 400 rpm, and sequentially add thiourea, urea, sodium lauroyl sarcosinate (SLS), CHAPS, and TCEP, and stir for 40 minutes.

[0048] (2) Adjust the pH of the solution obtained in step (1) to 7.8;

[0049] (3) Add Triton X-100, glycerol, and Tirs-HCl to the solution obtained in step (2), with a stirring speed of 400 rpm, and stir for 40 minutes;

[0050] (4) Add tween 60 into the solution obtained in step (3), with a stirring speed of 120 rpm and stir for 30 min to obtain the hepatitis B virus particle lysate.

[0051] Example 4

[0052] Use the hepatitis B virus particle lysates prepared in Examples 1 to 3 to process the sera of chronic hepatitis B patients, and then perform HBcrAg detection. The results are as follows:

[0053] Table 1

[0054]

[0055]

[0056] Note: An HBcrAg concentration ≥ 5 pg / ml is a positive result, between 1 - 5 pg / ml is a suspected positive, and < 1 pg / ml is a negative result.

[0057] As can be seen from Table 1, Example 1 detected 3 cases out of 6 HBV DNA positive samples, with 1 suspected case, and detected 1 case out of 2 samples with HBV DNA below the detection limit; Example 2 detected 3 cases out of 6 positive samples, with 2 suspected cases, and detected 1 case out of 2 samples with HBV DNA below the detection limit; Example 3 detected 4 cases out of 6 positive samples, with 1 suspected case, and detected 1 case out of 2 samples with HBV DNA below the detection limit. From the above results, it can be seen that the hepatitis B virus particle lysates of Examples 1 to 3 can all lyse HBcAg in hepatitis B virus particles, and among them, the sample detection results after being treated with the hepatitis B virus particle lysate of Example 3 are the most accurate.

[0058] The above examples only illustratively explain the principle and its efficacy of the present invention, rather than being used to limit the present invention. Any person familiar with this technology can modify or change the above examples without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or changes completed by those with ordinary knowledge in the technical field without departing from the spirit and technical idea disclosed by the present invention should still be covered by the claims of the present invention.

Claims

1. A hepatitis B virus particle lysate, characterized in that, It is composed of components with the following weight parts ratio: 10 - 20 parts of thiourea, 70 - 90 parts of urea, 8 - 12 parts of sodium dodecyl sarcosinate, 3 - 5 parts of tween, 4 - 8 parts of CHAPS, 1 - 3 parts of TCEP, 3 - 5 parts of Triton X-100, 8 - 12 parts of glycerol, 1 - 3 parts of Tirs-HCl, and 0 - 100 parts of deionized water; the pH of the hepatitis B virus particle lysate is adjusted to 7.2 - 7.

8.

2. The hepatitis B virus particle lysate according to claim 1, characterized in that, It is composed of components with the following weight parts ratio: 10 - 20 parts of thiourea, 70 - 90 parts of urea, 8 - 12 parts of sodium dodecyl sarcosinate, 3 - 5 parts of tween, 4 - 8 parts of CHAPS, 1 - 3 parts of TCEP, 3 - 5 parts of Triton X-100, 8 - 12 parts of glycerol, 1 - 3 parts of Tirs-HCl, and 10 - 100 parts of deionized water.

3. The preparation method of the hepatitis B virus particle lysate according to any one of claims 1 to 2, characterized in that, It includes the following steps: (1) Add thiourea, urea, sodium dodecyl sarcosinate, CHAPS, and TCEP into deionized water in sequence and stir evenly. (2) Adjust the pH of the solution obtained in step (1) to 7.2 - 7.

8. (3) Add Triton X-100, glycerol, and Tirs-HCl into the solution obtained in step (2) and stir evenly. (4) Add tween 60 into the solution obtained in step (3) and stir evenly to obtain the hepatitis B virus particle lysate.

4. The preparation method of the hepatitis B virus particle lysate according to claim 3, characterized in that: In step (1), the stirring speed is 300 - 400 rpm and stir for 30 - 40 min.

5. The preparation method of the hepatitis B virus particle lysate according to claim 3, characterized in that: In step (2), the stirring speed is 300 - 400 rpm and stir for 30 - 40 min.

6. The preparation method of the hepatitis B virus particle lysate according to claim 3, wherein: In step (4), the stirring speed is 100 - 120 rpm and stir for 20 - 30 min.

7. Use of the hepatitis B virus particle lysate according to any one of claims 1 - 2 and the hepatitis B virus particle lysate prepared by the method according to any one of claims 3 - 6 for lysing hepatitis B virus particles.

8. A method for lysing hepatitis B virus particles, characterized in that: Lyse hepatitis B virus particles with the hepatitis B virus particle lysate according to any one of claims 1 - 2 and the hepatitis B virus particle lysate prepared by the method according to any one of claims 3 - 6.

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