A protein or enzyme encapsulation method

Abstract

The present invention discloses a method for encapsulating a protein or enzyme, comprising the following steps: 1) adding a protein or enzyme to be encapsulated into a container, and then adding a wax material, heating the container to 70-80°C, stirring evenly after the wax material melts, wherein the mass ratio of the wax material to the protein or enzyme is between 5-50:1; 2) weighing an inert regulator and adding it to the container in step 1) to continue the enzyme addition mass ratio between 5-50:1; 3) taking another flask to add an emulsifier, and adding a dispersant, stirring until uniform, the inert regulator and the protein or in a constant temperature oil bath, heating to 70-80°C, and stirring; 4) adding the material in step 2 to the flask in step 3), stirring and mixing for 10-30min; 5) turning off the heating, cooling to room temperature, collecting samples, and sieving. This encapsulation method can completely prevent the taq enzyme from participating in the system reaction at low temperature, and release the enzyme in the system to participate in the reaction at a certain temperature, ensuring the activity of the enzyme without introducing new proteins.

Description

Technical Field

[0001] The invention relates to the fields of biopharmaceuticals and medicine, and in particular to a method for encapsulating proteins or enzymes. Background Art

[0002] When DNA polymerase performs PCR reaction, it still has residual enzyme activity during the process of rising from low temperature (20℃~37℃) to high temperature (94℃~95℃). Low-level nonspecific annealing and extension occur at the beginning of the reaction and during thermal cycling, which in turn leads to the formation of nonspecific amplification products in subsequent PCR cycles, and ultimately reduces the sensitivity and yield of the reaction by reducing the desired amplification signal or the signal being masked by high background values. Hot-start PCR starts the PCR enzymatic reaction after the initial denaturation stage, and reduces the formation of primer dimers, primer mismatches and nonspecific amplification through hot start, thereby increasing the specificity and sensitivity of PCR and improving PCR yield. Since hot start was used as a means to block DNA polymerase extension at lower temperatures, many methods have also been developed for the basic components of the reaction such as Mg2+, DNA polymerase, primers and dNTP. There are several technologies now, such as chemical modification, ligand modification, antibody binding method, etc. However, chemical modification will greatly reduce the activity of the enzyme, and the enzyme activation time is long, resulting in a decrease in the yield of PCR products; the ligand modification is not stable enough and the specificity is not strong enough; the antibody preparation cycle in the antibody binding method is long and the cost is high; and at a given temperature, the antibodies may not be completely denatured, which will introduce other reactions into the reaction system. Summary of the invention

[0003] The object of the present invention is to provide a method for encapsulating a protein or an enzyme.

[0004] To achieve this object, the present invention adopts the following technical solutions:

[0005] A method for encapsulating a protein or enzyme, comprising the following steps:

[0006] 1) Add the protein or enzyme to be encapsulated into the container, and then add the wax material, heat the container to 70-80°C, and stir evenly after the wax material melts, wherein the mass ratio of the wax material to the protein or enzyme is between 5-50:1;

[0007] 2) Weigh an inert regulator and add it to the container in step 1) and continue stirring until it is uniform. The mass ratio of the inert regulator to the protein or enzyme is between 5-50:1;

[0008] 3) Take another flask, add emulsifier and dispersant, place it in a constant temperature oil bath, heat it to 70-80℃, and stir;

[0009] 4) Add the materials in step 2 into the flask in step 3) and stir to mix for 10-30 minutes;

[0010] 5) Turn off the heat, cool to room temperature, collect the sample, and sieve.

[0011] As a specific implementation, the waxy material is selected from a mixture of one or more of paraffin wax, beeswax, palm wax, and rice bran wax.

[0012] As a specific implementation manner, in step 3), the stirring speed in the constant temperature oil bath is between 150-500 rpm / min.

[0013] As a specific implementation manner, the stirring speed in step 4) is between 200-2000 rpm / min.

[0014] As a specific implementation, the emulsifier used in step 3) is selected from a mixture of one or more of sodium dodecyl sulfonate, sodium dodecylbenzene sulfonate, polyethylene glycol octylphenyl ether, and sorbitan oleate.

[0015] As a specific implementation, the dispersant used in step 3) is selected from a mixture of one or more of water, ethanol, and ethylene glycol.

[0016] As a specific implementation, the inert regulator used in step 2) is selected from a mixture of one or more of silicon oxide powder, tin oxide powder or titanium oxide powder. The purpose of using the inert regulator is to adjust the density of the final product.

[0017] As a specific implementation, the particle size of the sieved particles in step 5) is between 10-1000 μm. More preferably, the particle size of the sieved particles in step 5) is between 15-100 μm.

[0018] Beneficial effects of the present invention: The encapsulation method of the present invention has a simple process, does not require chemical modification, and does not require low-temperature blocking of DNA polymerase. Under this encapsulation method, the taq enzyme can be completely prevented from participating in the system reaction at low temperature, and the enzyme can be released into the system to participate in the reaction at a certain temperature. The activity of the enzyme is ensured without introducing new proteins. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 is an image of the paraffin microsphere solution obtained in Example 1 under a 400x optical microscope;

[0020] Figure 2 is an image of the beeswax microsphere solution obtained in Example 2 under a 400x optical microscope;

[0021] Figure 3This is an image of the palm wax microsphere solution obtained in Example 3 under a 400x optical microscope. Implementation

[0022] The technical solution of the present invention is further described below with reference to the accompanying drawings and through specific embodiments. Example 1

[0023] This example provides a taq enzyme packaging method, comprising the following steps:

[0024] 1. Take a beaker, add 20 mg of Taq enzyme and 0.1 g of paraffin, heat the container to 70°C, and stir evenly after the paraffin is completely melted;

[0025] 2. Weigh 0.1g of silicon oxide powder, add it to the above paraffin, and continue stirring until uniform;

[0026] 3. Take another 100mL three-necked flask, add 0.6g sodium dodecyl sulfate, then add 50mL ethanol, put it in a constant temperature oil bath, and install a mechanical stirring device at a stirring speed of 300rpm / min, and heat it to 70℃;

[0027] 4. Quickly add the contents of the beaker in step 2 to the flask in step 3, stir, increase the speed to 1000 rpm / min, and time for 15 minutes;

[0028] 5. Turn off the heating and cool to room temperature. Collect the sample in the flask and use a standard sieve to wet screen the particles with a particle size of 10-100μm, that is, the paraffin microsphere solution is finally prepared. Take a drop of the paraffin microsphere solution with a pipette and place it under a 400x optical microscope. The image is attached. Figure 1 .

[0029] The particle size is set to no more than 100 μm to prevent large particle solutions from clogging the pipette.

[0030] The purpose of using an inert regulator here is to prevent the paraffin microspheres from floating on the surface of the liquid and failing to mix evenly with the liquid during use. Example 2

[0031] This example provides a taq enzyme packaging method, comprising the following steps:

[0032] 1. Take a beaker, add 20 mg of Taq enzyme and 0.5 g of beeswax, heat the container to 75°C, and stir evenly after the beeswax is completely melted;

[0033] 2. Weigh 0.3g of tin oxide powder, add it to the above beeswax, and continue stirring until it is uniform;

[0034] 3. Take another 100mL three-necked flask, add 0.8g sodium dodecylbenzene sulfonate, then add 70mL ethanol, put it in a constant temperature oil bath, and install a mechanical stirring device with a stirring speed of 500rpm / min, and heat it to 75℃;

[0035] 4. Quickly add the contents of the beaker in step 2 to the flask in step 3, stir, increase the speed to 500 rpm / min, and time for 30 min;

[0036] 5. Turn off the heating and cool to room temperature. Collect the sample in the flask and use a standard sieve to wet screen the particles with a particle size of 15-100μm. The beeswax microsphere solution is finally prepared. Take a drop of the beeswax microsphere solution with a pipette and place it under a 400x optical microscope. The image is attached. Figure 2 . Example 3

[0037] This example provides a taq enzyme packaging method, comprising the following steps:

[0038] 1. Take a beaker, add 20 mg of Taq enzyme and 1 g of palm wax, heat the container to 80°C, and stir evenly after the palm wax is completely melted;

[0039] 2. Weigh 1g of titanium oxide powder and add it to the palm wax, and continue stirring until it is uniform;

[0040] 3. Take another 100mL three-necked flask, add 0.6g triton 100, then add 50mL ethanol, put it in a constant temperature oil bath, and install a mechanical stirring device with a stirring speed of 150rpm / min, and heat it to 80℃;

[0041] 4. Quickly add the contents of the beaker in step 2 to the flask in step 3, stir, increase the speed to 2000 rpm / min, and time for 10 min;

[0042] 5. Turn off the heating and cool to room temperature. Collect the sample in the flask and use a standard sieve to wet screen the particles with a particle size of 15-100μm. The palm wax microsphere solution is finally prepared. Take a drop of palm wax microsphere solution with a pipette and place it under a 400x optical microscope. The image is attached. Figure 3 .

[0043] The encapsulation method of the present invention can completely prevent the taq enzyme from participating in the system reaction at low temperature, and release the enzyme into the system to participate in the reaction at a certain temperature, thereby ensuring the activity of the enzyme without introducing new proteins.

[0044] The above contents are only preferred embodiments of the present invention. For ordinary technicians in this field, according to the concept of the present invention, there will be changes in the specific implementation methods and application scopes. The content of this specification should not be understood as limiting the present invention.

Claims

1. A method for packaging Taq enzyme, characterized in that: The following steps are involved: 1) Add the Taq enzyme to be encapsulated into the container, and then add the wax material, heat the container to 70-80°C, and stir evenly after the wax material melts, wherein the mass ratio of the wax material to the Taq enzyme is between 5-50:1; 2) Weighing an inert regulator and adding it to the container in step 1) and continuing to stir until uniform, the mass ratio of the inert regulator to the Taq enzyme is between 5-50:1, and the inert regulator is selected from a mixture of one or more of silicon oxide powder, tin oxide powder or titanium oxide powder; 3) Take another flask, add emulsifier and dispersant, place it in a constant temperature oil bath, heat it to 70-80℃, and stir; 4) Add the materials in step 2 into the flask in step 3) and stir to mix for 10-30 minutes; 5) Turn off the heat, cool to room temperature, collect the sample, and sieve.

2. The method for encapsulating Taq enzyme according to claim 1, characterized in that: The waxy material is selected from a mixture of one or more of paraffin wax, beeswax, palm wax and rice bran wax.

3. The method for encapsulating Taq enzyme according to claim 1, characterized in that: In step 3), the stirring speed in the constant temperature oil bath is between 150-500 rpm / min.

4. The method for encapsulating Taq enzyme according to claim 1, characterized in that: The stirring speed in step 4) is between 200-2000 rpm / min.

5. The method for encapsulating Taq enzyme according to claim 1, characterized in that: The emulsifier used in step 3) is selected from a mixture of one or more of sodium dodecyl sulfonate, sodium dodecylbenzene sulfonate, polyethylene glycol octylphenyl ether, and sorbitan oleate.

6. The method for encapsulating Taq enzyme according to claim 1, characterized in that: The dispersant used in step 3) is selected from a mixture of one or more of water, ethanol and ethylene glycol.

7. The method for encapsulating Taq enzyme according to claim 1, characterized in that: Step 5) The particle size of the sieved particles is between 10-1000 μm.

8. The method for encapsulating Taq enzyme according to claim 1, characterized in that: Step 5) The particle size of the sieved particles is between 15-100 μm.

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