Method for purifying nano-particle vaccine wrapped by bacterial outer membrane

Abstract

The invention provides a method for purifying a nanoparticle vaccine wrapped by a bacterial outer membrane, which comprises the following steps of: firstly, inducing and expressing a target protein in bacteria, and collecting thalli; secondly, ultrasonically crushing bacterial thalli, centrifuging, and carrying out subsequent purification on centrifuged precipitates; and washing the precipitate obtained in the previous step by using a low-concentration sodium dodecyl sarcosinate solution, dissolving the washed precipitate by using a high-concentration sodium dodecyl sarcosinate solution, destroying bacterial cell membranes, and releasing the virus-like particles, thereby realizing purification of the virus-like particles existing in the inclusion body form. The method is suitable for purifying the virus-like particles existing in the form of inactive inclusion bodies, and solves the purification problem of the virus-like particles existing in the form of inclusion bodies after large proteins are presented in the virus-like particles.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and particularly relates to a method for purifying nanoparticle vaccines wrapped by bacterial outer membranes. Background Art

[0002] Virus-like particles have strong immunogenicity, but even a very short peptide can destroy the structure of the virus-like particles. Generally, the smaller the peptide, the easier it is to be presented on the surface of the virus-like particles, while the longer the sequence, the less likely it is to be correctly presented on the surface of the virus-like particles, and it is usually insoluble after induced expression. Whether the expressed protein is soluble or not is the key link in whether the virus-like particles can be purified.

[0003] The existing technical solutions usually induce the expression of the target protein in bacteria. If the target protein exists in a soluble form, it is purified with the soluble supernatant. Common purification methods include: sulfuric acid precipitation combined with sucrose density gradient centrifugation, iodixanol density gradient centrifugation, etc., or direct purification by molecular sieve.

[0004] Obviously, the existing target protein purification methods are limited to proteins in soluble form. If the target protein exists in the form of inactive inclusion bodies after expression, the above methods are not applicable.

[0005] Therefore, how to achieve the purification of inactive target proteins (insoluble) in the form of inclusion bodies, especially the purification of nanoparticle vaccines (virus-like particles) wrapped by bacterial membranes, is a problem that needs to be solved. Summary of the invention

[0006] In view of the problems existing in the prior art, the purpose of the present invention is to provide a method for purifying bacterial outer membrane-coated nanoparticle vaccines, which is suitable for purifying target proteins in the form of inactive inclusion bodies, and solves the problem of purifying large proteins after presentation in virus-like particles.

[0007] The objective of the present invention is achieved through the following technical solutions:

[0008] A method for purifying a bacterial outer membrane-encapsulated nanoparticle vaccine comprises the following steps:

[0009] (1) Induce the expression of target protein in bacteria and collect bacteria;

[0010] (2) Ultrasonic disruption of bacterial cells, centrifugation, and subsequent purification of the precipitate;

[0011] (3) washing the precipitate obtained in step (2) with a low concentration of sodium lauryl sarcosyl solution, and then dissolving the washed precipitate with a high concentration of sodium lauryl sarcosyl solution to destroy the bacterial cell membrane and release virus-like particles (i.e., nanoparticle vaccine), thereby purifying the target protein in the form of inclusion bodies.

[0012] Furthermore, in order to remove other impurities including the damaged cell membrane, the method further includes performing sucrose density gradient centrifugation on the obtained virus-like particles.

[0013] Furthermore, in step (2), the ultrasonic conditions are: ultrasonic time: 10 minutes, working time: 10 seconds, interval time: 10 seconds, alarm temperature 4°C, power: 25%.

[0014] Furthermore, in step (3), the mass concentration of the low-concentration sodium lauryl sarcosinate solution is 0.1% to 0.3%, and the mass concentration of the high-concentration sodium lauryl sarcosinate solution is 0.3% to 0.9%.

[0015] Furthermore, in step (3), the precipitate is first resuspended with a low concentration sodium lauryl sarcosyl solution and then immediately centrifuged at 12000 rpm / min for 10 minutes; the precipitate treated with the low concentration sodium lauryl sarcosyl solution is then resuspended with a high concentration sodium lauryl sarcosyl solution and shaken on a shaker until the precipitate is completely dissolved.

[0016] In the present invention, sodium lauryl sarcosinate is used to treat the cell membrane based on its surfactant properties. Sodium lauryl sarcosinate is an amphiphilic chemical substance with a hydrophobic lauroyl group at one end and a hydrophilic carboxyl group at the other end. This structure can interact with water and the lipid layer in the cell membrane. The cell membrane is mainly composed of lipids and proteins, wherein the lipid bilayer constitutes the basic skeleton of the cell membrane. The hydrophobic end of sodium lauryl sarcosinate can be inserted into the lipid bilayer to destroy its structure.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] 1. The existing technology can only purify virus-like particles from soluble proteins, that is, it can only purify virus-like particles that have been formed. At the same time, there are often a large number of bacterial cell membranes in the inclusion bodies in the precipitation. The purified virus-like particles are often wrapped by the bacterial outer membrane, and the virus-like particles are difficult to release. On the one hand, this patent can destroy the bacterial cell membrane to release the virus-like particles, and on the other hand, it can purify the virus-like particles in the inclusion bodies, which greatly expands the purification method and application scope of virus-like particles. Since large proteins usually exist in an insoluble form after being presented in virus-like particles, this method solves the problem of purifying large proteins after being presented in virus-like particles.

[0019] 2. The method for purifying bacterial membrane-coated nanoparticle vaccines described in this patent can remove the bacterial membrane that wraps the virus-like particles, so that a large number of virus-like particles are released, and the purified virus-like particles have a higher purity. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The present invention will be further described below in conjunction with the accompanying drawings and embodiments:

[0021] Figure 1 Shows the induced expression of the target protein;

[0022] Figure 2 The comparison results of the purification method of the present application and the purification by sucrose density gradient centrifugation are shown; wherein the left figure is the HBcAg-RBM sucrose density gradient centrifugation described in Example 1, and the right figure is the HBcAg sucrose density gradient centrifugation;

[0023] Figure 3 The electron micrographs of virus-like particles obtained by the purification method of the present application and by sucrose density gradient centrifugation purification are compared; wherein the left picture is the HBcAg-RBM virus-like particles obtained in Example 1, and the right picture is the HBcAg virus-like particles;

[0024] Figure 4 is an electron micrograph of the virus-like particles obtained in the comparative example;

[0025] Figure 5 The antibody response of mice after immunization with HBcAg-RBM virus-like particles is shown;

[0026] Figure 6 It shows that the antibodies induced by the nanoparticle vaccine described in Example 1 can recognize the inactivated COVID-19 virus;

[0027] Figure 7 It is shown that the nanoparticle vaccine described in Example 1 has a strong affinity with the antibodies induced therefrom. DETAILED DESCRIPTION

[0028] In order to better understand the present invention, the present invention will be further described in detail below in conjunction with the accompanying drawings so that those skilled in the art can implement the present invention with reference to the description text.

[0029] It should be noted that the experimental methods described in the following experiments, unless otherwise specified, are all conventional methods, and the reagents and materials, unless otherwise specified, can be obtained from commercial channels.

[0030] Example 1

[0031] This embodiment takes the purification of HBcAg-RBM protein as an example to provide a method for purifying a bacterial outer membrane-coated nanoparticle vaccine. RBM is the receptor binding region of the new crown SARS-COV2, and HBcAg is a truncated hepatitis B surface core antigen. HBcAg-RBM is the receptor binding region of the new crown SARS-COV2 inserted into the truncated hepatitis B surface core antigen HBcAg, that is, the truncated hepatitis B surface core antigen HBcAg presents the receptor binding region of the new crown SARS-COV2.

[0032] The specific purification method comprises the following steps:

[0033] (1) Induce the expression of the target protein HBcAg-RBM in Escherichia coli and collect the cells, such as Figure 1 shown.

[0034] (2) Ultrasonic disruption of bacterial cells, centrifugation, and subsequent purification of the precipitate; the ultrasonic conditions are: ultrasonic time: 10 minutes, working time: 10 seconds, interval time: 10 seconds, alarm temperature 4°C, power: 25%.

[0035] (3) The precipitate obtained in step (2) is first washed with a low-concentration sodium lauryl sarcosinate solution having a mass concentration of 0.3%, and then the washed precipitate is dissolved with a high-concentration sodium lauryl sarcosinate solution having a mass concentration of 0.9%, thereby destroying the bacterial cell membrane and releasing virus-like particles, thereby achieving purification of the target protein in the form of inclusion bodies.

[0036] (4) Further subjecting the obtained virus-like particles to sucrose density gradient centrifugation. The specific operation is as follows:

[0037] The dissolved precipitate is reassembled in a buffer solution under low temperature conditions, and purified by sucrose density gradient centrifugation to remove other impurities including damaged cell membranes to obtain purified HBcAg-RBM protein.

[0038] Comparative Example 1

[0039] This comparative example uses traditional sucrose density gradient centrifugation to purify HBcAg carrier, i.e. truncated hepatitis B virus core antigen, and the specific method is as follows:

[0040] (1) Inducing the expression of target protein in bacteria containing HBcAg with IPTG;

[0041] (2) collecting bacterial cells by centrifugation;

[0042] (3) Ultrasonication breaks up bacterial cells, and the target protein HBcAg exists in a soluble form in the ultrasonic supernatant;

[0043] (4) using the ultrasonic supernatant for sucrose density gradient centrifugation according to the traditional method;

[0044] (5) Samples from sucrose density gradient centrifugation were taken from different layers for SDS-PAGE analysis.

[0045] like Figure 2 As shown in Example 1, the target protein finally purified by sucrose density gradient centrifugation was enriched in the 8-10 layer ( Figure 2 Left), while in this comparative example, the distribution of the target protein in sucrose density gradient centrifugation was similar to that of the control vector ( Figure 2 Right) Behavior is similar.

[0046] Figure 2 The target protein HBcAg in the right picture exists in a soluble form. After being treated according to the traditional method, the target protein is enriched in the 7-10 layers of sucrose density gradient centrifugation. And under the electron microscope, it appears as dispersed virus-like particles.

[0047] Figure 2 In the left figure, HBcAg-RBM exists in the form of inclusion bodies after expression and is wrapped by the bacterial cell membrane and cannot be released. After treatment according to the method of the present invention, HBcAg-RBM exists in a soluble form. After sucrose density gradient centrifugation, HBcAg-RBM is enriched in the 7-10 layers of sucrose density gradient centrifugation. And under electron microscopy, it appears as dispersed virus-like particles.

[0048] The comparison of the above results shows that the method of the present invention can resolve virus-like particles in the form of inclusion bodies wrapped by bacterial cell membranes.

[0049] Further electron microscopic observation showed that the virus-like particles purified by the above two methods were Figure 3 shown.

[0050] Figure 3 The picture on the right shows HBcAg in a soluble form. After purification by traditional methods, it exists in the form of dispersed virus-like particles under an electron microscope.

[0051] Figure 3 Left: After purification according to the method described in Example 1 of the present invention, virus-like particles in the form of inclusion bodies wrapped in bacterial cell membranes can be Figure 3 The right picture achieves a similar effect, and the virus-like particles are present in the form of dispersed particles under electron microscopy, which further illustrates the effectiveness and feasibility of the bacterial outer membrane-encapsulated nanoparticle vaccine purification method of the present invention.

[0052] Example 2

[0053] This example purifies the bacterial outer membrane-encapsulated nanoparticle vaccine by referring to the method described in Example 1, with the only difference being that in step (3), the precipitate obtained in step (2) is first washed with a low-concentration sodium lauryl sarcosinate solution having a mass concentration of 0.1%, and then the washed precipitate is dissolved with a high-concentration sodium lauryl sarcosinate solution having a mass concentration of 0.3%.

[0054] Example 3

[0055] This example purifies the bacterial outer membrane-encapsulated nanoparticle vaccine by referring to the method described in Example 1, with the only difference being that in step (3), the precipitate obtained in step (2) is first washed with a low-concentration sodium lauryl sarcosinate solution having a mass concentration of 0.2%, and then the washed precipitate is dissolved with a high-concentration sodium lauryl sarcosinate solution having a mass concentration of 0.6%.

[0056] Example 4

[0057] This example purifies the bacterial outer membrane-encapsulated nanoparticle vaccine by referring to the method described in Example 1, with the only difference being that in step (3), the precipitate obtained in step (2) is first washed with a low-concentration sodium lauryl sarcosinate solution having a mass concentration of 0.3%, and then the washed precipitate is dissolved with a high-concentration sodium lauryl sarcosinate solution having a mass concentration of 0.8%.

[0058] Comparative Example 2

[0059] In order to explore the effect of the concentration of sodium lauryl sarcosyl solution on the purification effect of bacterial outer membrane-encapsulated nanoparticle vaccine, the bacterial outer membrane-encapsulated nanoparticle vaccine was purified by referring to the method described in Example 1, with the only difference being that in step (3), the precipitate obtained in step (2) was first washed with a sodium lauryl sarcosyl solution having a higher concentration of 0.9% by mass, and then the washed precipitate was dissolved with a sodium lauryl sarcosyl solution having a lower concentration of 0.2% by mass.

[0060] The virus-like particles obtained by the comparative method were destroyed by the excessively high concentration of sodium lauryl sarcosine solution, which simultaneously destroyed the structure of the cell membrane and the nanoparticles, and cleaved the cell membrane and the nanoparticles into fragments. The experimental results are shown in FIG. Figure 4 shown.

[0061] Test example: Detection of immunogenicity of purified nanoparticle vaccine

[0062] This test example detected the immunogenicity of the nanoparticle vaccine purified in Example 1. The purified nanoparticle vaccine was subcutaneously immunized in mice three times every two weeks. Serum was collected one week after each immunization and the kinetics of RBM-specific IgG response were analyzed. The specific experimental process was as follows:

[0063] (1) Coating: Coating the ELISA plate with S1 protein;

[0064] (2) Blocking: 2% BSA blocking;

[0065] (3) Primary antibody: collected mouse serum;

[0066] (4) Secondary antibody: HRP-labeled goat anti-mouse IgG antibody.

[0067] (5) Color development: TMB color development.

[0068] like Figure 5 The results shown indicate that a stronger immune response can be induced in mice, indicating that the nanoparticle vaccine purified by the method of Example 1 has stronger immunogenicity.

[0069] Furthermore, the serum of mice immunized with the virus-like particle vaccine purified in Example 1 was tested using the inactivated COVID-19 virus coating, and the serum response of the nanoparticle vaccine was tested by ELISA. The specific experimental process was as follows:

[0070] (1) Coating: Coating the ELISA plate with inactivated COVID-19 virus;

[0071] (2) Blocking: 2% BSA blocking;

[0072] (3) Primary antibody: collected mouse serum;

[0073] (4) Secondary antibody: HRP-labeled goat anti-mouse IgG antibody;

[0074] (5) Color development: TMB color development.

[0075] like Figure 6 The results shown indicate that the antibodies in the serum of mice immunized with the nanoparticle vaccine purified in Example 1 can recognize the inactivated new coronavirus.

[0076] Affinity analysis of the reaction between antiserum induced by nanoparticle vaccine and nanoparticles in ELISA. Nanoparticle vaccine was coated, and different concentrations of NaSCN were added to the serum sample, and the affinity was reflected by calculating the reactivity percentage. The specific experimental process is as follows:

[0077] (1) Coating: Coating the ELISA plate with purified nanoparticle vaccine;

[0078] (2) Blocking: 2% BSA blocking;

[0079] (3) Dilute the collected mouse serum with 2% BSA;

[0080] (4) adding 0M, 1M, 2M, 3M, 4M, and 5M NaSCN to the wells added with serum to destroy the binding of serum antibodies to the nanoparticle vaccine;

[0081] (5) Secondary antibody: HRP-labeled goat anti-mouse IgG antibody;

[0082] (6) Color development: TMB color development.

[0083] Sodium thiocyanate (NaSCN) was used to destroy the binding ability of the nanoparticle vaccine to the antibodies in the serum induced by it by two-way analysis of variance. Figure 7 The results showed that the purified nanoparticle vaccine had a strong affinity with the antibodies it induced.

[0084] Finally, it should be noted that the above is only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred arrangement scheme, a person skilled in the art should understand that the technical solution of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solution of the present invention.

Claims

1. A method for purifying bacterial outer membrane-encapsulated nanoparticle vaccines, characterized in that: The purification method comprises the following steps: (1) Induce the expression of target protein in bacteria and collect bacteria; (2) Ultrasonic disruption of bacterial cells, centrifugation, and subsequent purification of the precipitate; (3) washing the precipitate obtained in step (2) with a low concentration sodium lauryl sarcosyl solution, and then dissolving the washed precipitate with a high concentration sodium lauryl sarcosyl solution to destroy the bacterial cell membrane and release virus-like particles, thereby purifying the target protein in the form of inclusion bodies.

2. The method for purifying bacterial outer membrane-encapsulated nanoparticle vaccines according to claim 1, characterized in that: The method further comprises performing sucrose density gradient centrifugation on the obtained virus-like particles.

3. The method for purifying bacterial outer membrane-encapsulated nanoparticle vaccines according to claim 1 or 2, characterized in that: In step (2), the ultrasonic conditions are: ultrasonic time: 10 minutes, working time: 10 seconds, interval time: 10 seconds, alarm temperature 4°C, power: 25%.

4. The method for purifying bacterial outer membrane-encapsulated nanoparticle vaccines according to claim 1 or 2, characterized in that: In step (3), the mass concentration of the low-concentration sodium lauryl sarcosinate solution is 0.1% to 0.3%, and the mass concentration of the high-concentration sodium lauryl sarcosinate solution is 0.3% to 0.9%.

5. The method for purifying bacterial outer membrane-encapsulated nanoparticle vaccines according to claim 1 or 2, characterized in that: In step (3), the precipitate is first resuspended with a low concentration sodium lauryl sarcosinate solution and then immediately centrifuged at 12000 rpm / min for 10 minutes; the precipitate treated with the low concentration sodium lauryl sarcosinate solution is then resuspended with a high concentration sodium lauryl sarcosinate solution and shaken on a shaker until the precipitate is completely dissolved.

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