Low-concentration enzyme preservation solution and application thereof

Abstract

The invention discloses a low-concentration enzyme preservation solution and application thereof. The preservation solution is prepared from the following components in percentage by mass: 25 to 30 percent of sucrose, 0.02 to 0.1 percent of glycine betaine and 0.02 to 0.1 percent of polylysine. Under the synergistic effect of sucrose, glycine betaine and polylysine, the preservation solution realizes long-term stable preservation of the enzyme at 2-8 DEG C under the low concentration of 2-20 [mu] g / mL, and the enzyme still has excellent tissue or cell digestion ability after being preserved for 1 year, so that convenience is brought to the use of the enzyme.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a low-concentration enzyme preservation solution and application thereof. Background Art

[0002] Enzymes play an indispensable and core role in molecular biology and genetic engineering, serving as tools for precise and efficient molecular manipulation. Different enzymes have distinct roles. For example, proteinase K is primarily used in nucleic acid extraction, proteomics analysis, and forensic identification, degrading proteins to release nucleic acids or study protein function. Pancreatic enzymes primarily focus on cellular digestion, such as breaking down extracellular proteins in cell culture, reducing cell membrane permeability, and promoting cell loosening and drainage, or degrading heterologous proteins.

[0003] The activity and stability of enzymes are often affected by factors such as temperature, pH, and storage time. For example, proteinase K typically needs to be stored as a dry powder at 0-4°C with a shelf life of one year, or as a 10 mg / mL solution at -20°C, with no repeated freezing and thawing, for a shelf life of 12 months. The working concentration of an enzyme is typically 2-20 μg / mL, and its storage as a dry powder or at high concentrations makes it inconvenient to use the enzyme, requiring freeze-thaw, preparation, or gradient dilution to the working concentration for each use. It is crucial to enhance the stability of low-concentration enzymes and extend their shelf life at working concentrations. Summary of the Invention

[0004] In order to solve at least one of the above problems, the present invention provides a low-concentration enzyme preservation solution and application thereof.

[0005] In order to achieve the above object, the present invention adopts the following technical means: A first aspect of the present invention provides a low-concentration enzyme preservation solution, which comprises sucrose, glycine betaine and polylysine.

[0006] In some embodiments of the present invention, the mass volume fraction of sucrose in the preservation solution is 25-30%, the mass volume fraction of glycine betaine is 0.02-0.1%, and the mass volume fraction of polylysine is 0.02-0.1%.

[0007] In some embodiments of the present invention, the preservation solution is prepared with PBS buffer or Tris-HCl buffer, pH 7.8-8.2.

[0008] The second aspect of the present invention provides a kit containing the preservation solution of the first aspect, which is used for nucleic acid extraction, fluorescence in situ hybridization or immunohistochemistry detection.

[0009] The third aspect of the present invention provides use of the preservation solution described in the first aspect for preserving low-concentration enzyme reagents.

[0010] In some embodiments of the present invention, the storage solution is filtered through a filter membrane after being prepared and is ready for use. In some embodiments of the present invention, the storage solution is filtered through a 0.22 μm filter membrane after being prepared and is ready for use.

[0011] In some embodiments of the present invention, the enzyme is diluted to a working concentration with a preservation solution and stored at 2-8°C.

[0012] In some embodiments of the invention, the working concentration of the enzyme is 2-20 μg / mL.

[0013] In some embodiments of the present invention, the enzyme includes but is not limited to proteinase K, trypsin.

[0014] Beneficial effects of the present invention Compared with the prior art, the present invention has the following beneficial effects: the present invention provides a low-concentration enzyme preservation solution, which does not affect the immunohistochemistry results, and under the synergistic effect of sucrose, glycine betaine, and polylysine, the preservation solution can achieve long-term stable storage of the enzyme at a low concentration of 2-20 μg / mL at 2-8°C, and still has excellent tissue or cell digestion ability after one year of storage; it solves the cumbersome operation of conventional enzymes such as preparing and using low concentrations immediately, and brings convenience to the use of fluorescence in situ hybridization or immunohistochemistry. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The results of immunohistochemical detection of the antibody target CD10 using tonsil tissue are shown after the proteinase K working solution in Examples 1 to 5 of the present invention has been stored for 1 month, 3 months, 6 months, and 12 months; Figure 2 The results of immunohistochemical detection of the antibody target AMACR using kidney tissue are shown after the proteinase K working solution in Examples 1 to 5 of the present invention has been stored for 1 month, 3 months, 6 months, and 12 months; Figure 3 The graph shows the effect of immunohistochemical detection of the antibody target AMACR using kidney tissue after pancreatic enzyme was stored for 0 days, 6 months, and 12 months in Example 6 of the present invention. DETAILED DESCRIPTION

[0016] The following examples are provided to illustrate preferred embodiments of the present invention. Those skilled in the art will appreciate that the techniques disclosed in the following examples represent techniques discovered by the inventors that can be used to practice the present invention and, therefore, can be considered preferred embodiments of the present invention. However, those skilled in the art will appreciate from this disclosure that many modifications may be made to the specific embodiments disclosed herein while still achieving the same or similar results without departing from the spirit or scope of the present invention.

[0017] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art to which this invention belongs, and the disclosures herein and the materials they cite are hereby incorporated by reference. Those skilled in the art will recognize or be able to ascertain, through routine experimentation, many technical equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the claims.

[0018] A low-concentration enzyme preservation solution comprises sucrose, glycine betaine and polylysine; wherein the mass volume fraction of sucrose is 25-30%, the mass volume fraction of glycine betaine is 0.02-0.1%, and the mass volume fraction of polylysine is 0.02-0.1%; and the preservation solution is prepared in PBS buffer or Tris-HCl buffer, with a pH of 7.8-8.2.

[0019] Accurately weigh or measure each reagent component into a clean sterile container, add the corresponding buffer, stir thoroughly, filter with a 0.22 μm filter membrane, and store for later use.

[0020] The enzyme was diluted to a working concentration of 2-20 μg / mL using the above-mentioned storage solution and stored at 2-8°C until use.

[0021] The technical solution of the present application will be further described in detail below in conjunction with specific implementation methods.

[0022] Example 1 Preparation of enzyme preservation solution: Weigh 30 g sucrose, 0.5 g glycine betaine, and 0.5 g polylysine, add Tris-HCl buffer to make up the volume to 1 L, stir thoroughly, and filter through a 0.22 μm filter membrane to obtain enzyme preservation solution 1 for later use.

[0023] The obtained enzyme preservation solution 1 was used to prepare a proteinase K working solution with a final concentration of 2.5 μg / mL. The proteinase K working solution was divided into 5 tubes, labeled tube 1, tube 2, tube 3, tube 4, and tube 5. (1) Immunohistochemistry (antibody) testing was performed immediately after tube 1 was aliquoted; (2) Tube 2 was stored at 2-8°C and immunohistochemistry (antibody) was performed after 1 month; (3) Tube 3 was stored at 2-8°C and subjected to immunohistochemistry (antibody) testing 3 months later; (4) Tube 4 was stored at 2-8°C and subjected to immunohistochemistry (antibody) testing after 6 months; (5) Tube 5 was stored at 2-8°C and subjected to immunohistochemistry (antibody) testing 12 months later; The antibody catalog number, clone number, dilution, and other information used for immunohistochemistry (antibody) testing are as follows:

[0024] Immunohistochemistry (antibody) testing methods are as follows: The experiment was performed using the LYNX480 fully automatic immunohistochemical staining machine from Tuling (Hangzhou) Biotechnology Co., Ltd. Other reagents used, such as secondary antibodies and auxiliary reagents, were all from commercial kits from Tuling (Hangzhou) Biotechnology Co., Ltd. After heat repair, a low-concentration proteinase K working solution was added, 150 μL / section, and incubated at room temperature for 10 minutes. Other steps were consistent with the conventional immunohistochemistry experimental process.

[0025] Example 2 The other steps were the same as those in Example 1, except for the preparation of the enzyme preservation solution: 30 g sucrose and 0.5 g glycine betaine were weighed, the volume was made up to 1 L with Tris-HCl buffer, the mixture was thoroughly stirred, and the mixture was filtered through a 0.22 μm filter membrane before storage to obtain enzyme preservation solution 2.

[0026] Example 3 The other steps were the same as those in Example 1, except for the preparation of the enzyme preservation solution: 30 g of sucrose and 0.5 g of polylysine were weighed, the volume was made up to 1 L with Tris-HCl buffer, the mixture was thoroughly stirred, and the solution was filtered through a 0.22 μm filter membrane before storage to obtain enzyme preservation solution 3.

[0027] Example 4 The other steps were the same as those in Example 1, except for the preparation of the enzyme preservation solution: 0.5 g of glycine betaine and 0.5 g of polylysine were weighed, the volume was made up to 1 L with Tris-HCl buffer, the mixture was thoroughly stirred, and the solution was filtered through a 0.22 μm filter membrane before storage to obtain enzyme preservation solution 4.

[0028] Example 5 The other steps were the same as those in Example 1, except for the preparation of the enzyme preservation solution: 0.555 g of CaCl2 and 0.125 mL of glycerol were weighed, the volume was made up to 1 L with Tris-HCl buffer, the mixture was thoroughly stirred, and the solution was filtered through a 0.22 μm filter membrane before storage to obtain enzyme preservation solution 5.

[0029] The results of immunohistochemistry (antibody) detection using proteinase K working solutions prepared using different formulations in Examples 1 to 5 are shown in Tables 1 and Figure 1 and Figure 2 shown.

[0030] Table 1 Immunohistochemical test results of Examples 1 to 5

[0031] Figure 1 The results of proteinase K working solutions from Examples 1 to 5 after storage for 1 month, 3 months, 6 months, and 12 months, respectively, were analyzed using tonsil tissue for testing against the antibody target CD10. The results demonstrate that long-term storage of low-concentration protease working solutions for at least one year is only possible when sucrose, glycine betaine, and polylysine are present in the storage solution simultaneously. None of these three elements can be missing.

[0032] Figure 2 The results of AMACR testing of the proteinase K working solutions in Examples 1 to 5 for the antibody target site using kidney tissue are shown after storage for 1 month, 3 months, 6 months, and 12 months, respectively. These results are consistent with those of testing of the antibody target site CD10 using tonsil tissue. Long-term storage of the low-concentration proteinase K working solution for at least one year can only be achieved when sucrose, glycine betaine, and polylysine are co-present in the storage solution.

[0033] Example 6 Use the enzyme preservation solution in Example 1 to dilute pancreatic enzyme to a working concentration of 20 μg / mL, and divide into 3 tubes, labeled tube 1, tube 2, and tube 3, wherein: (1) Immunohistochemistry (antibody) testing was performed immediately after tube 1 was aliquoted (day 0); (2) Tube 2 was stored at 2-8°C and subjected to immunohistochemistry (antibody) testing after 6 months; (3) Tube 3 was stored at 2-8°C and immunohistochemistry (antibody) testing was performed after 12 months.

[0034] The detection method is the same as in Example 1, the primary antibody is AMACR antibody reagent, and the detection tissue is kidney. Figure 3 shown.

[0035] The results showed that the ingredients added to the preservation solution did not affect the test results. At the same time, the immunohistochemistry (antibody) test results did not change after the pancreatic enzyme was preserved in the preservation solution for 12 months. The results were very good, indicating that the preservation solution can also achieve long-term preservation of pancreatic enzyme working solution for at least 1 year.

[0036] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the present application.

Claims

1. A low-concentration enzyme preservation solution, characterized in that: The preservation solution comprises sucrose, glycine betaine and polylysine.

2. A low-concentration enzyme preservation solution according to claim 1, characterized in that: The mass volume fraction of sucrose in the preservation solution is 25-30%, the mass volume fraction of glycine betaine is 0.02-0.1%, and the mass volume fraction of polylysine is 0.02-0.1%.

3. A low-concentration enzyme preservation solution according to claim 2, characterized in that: The preservation solution is prepared with PBS buffer or Tris-HCl buffer, pH 7.8-8.

2.

4. A kit comprising the preservation solution according to any one of claims 1 to 3.

5. Use of the preservation solution according to any one of claims 1 to 3 for preserving low-concentration enzyme reagents.

6. The use according to claim 5, characterized in that: After the storage solution is prepared, it is filtered through a filter membrane for later use.

7. The use according to claim 5 or 6, characterized in that: Dilute the enzyme to the working concentration with preservation solution and store at 2-8°C.

8. The use according to claim 7, characterized in that: The working concentration of the enzyme was 2-20 μg / mL.

9. The use according to claim 7, characterized in that: The enzymes include proteinase K and trypsin.

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