A virus-like particle assembly solution and its preparation method and application

Abstract

The present invention provides a virus-like particle assembly solution and a preparation method and application thereof, belonging to the technical field of biological products. The virus-like particle assembly solution includes the following substances: Tris, sodium chloride, an amphoteric surfactant, sucrose, glycine and water for injection; each liter of assembly solution contains 2-5g of Tris, 15-30g of sodium chloride, 1-4g of an amphoteric surfactant, 10-80g of sucrose and 10-30g of glycine, and the remainder is water for injection. The present invention also includes a preparation method and application of the virus-like particle assembly solution. The virus-like particle assembly solution of the present invention can solve the problems of low assembly efficiency of virus-like particles and severe protein aggregation, and has broad application prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of biological products, and specifically relates to a virus-like particle assembly solution and a preparation method and application thereof. Background Art

[0002] Foot-and-mouth disease (FMD) is an acute, febrile and highly contagious disease caused by the foot-and-mouth disease virus (FMDV), which poses a serious threat to the health of even-toed ungulates (such as pigs, cattle, sheep, camels, deer, etc.) around the world. Currently, inactivated vaccines are often used for immunization and prevention of foot-and-mouth disease, but the preparation process of the vaccine still has potential biosafety risks. For example, incomplete inactivation may cause the immunized animals to become asymptomatic carriers of the virus and then spread the virus. In addition, inactivated vaccines cannot effectively distinguish between immunized animals and naturally infected animals, which brings additional challenges to the prevention and control of the disease.

[0003] Virus-like particles (VLPs) are a type of hollow particles that are highly similar in morphology to natural viruses but do not contain viral nucleic acids. They have attracted much attention because of their immunogenicity comparable to that of intact viruses. They can simulate the infection process of natural viruses in the body and induce strong cellular immune responses. The capsid of FMDV is composed of three proteins: VP0, VP3, and VP1, which have the ability to self-assemble into VLPs in vitro. Compared with inactivated vaccines, the production of VLPs vaccines does not involve live viruses and does not contain any non-structural proteins of the virus. Therefore, naturally infected and immune animals can be easily identified through non-structural protein-based detection methods, which is of great significance for the purification and eradication of the disease.

[0004] Although the foot-and-mouth disease virus VLPs produced by the prokaryotic expression system have the advantages of low cost, simple and rapid production process, and many VLPs vaccines have been successfully used in the field of human vaccines, there are still a series of severe challenges in the research and development and production of foot-and-mouth disease VLPs vaccines. Among them, the content of VLPs is a key factor in determining the quality and immune efficacy of vaccines, but the in vitro self-assembly efficiency of VLPs is generally low, and protein aggregation often occurs during the assembly process, resulting in the spatial conformational epitopes of VLPs being inferior to those of natural virus particles, thereby affecting the immune effect of the vaccine. More seriously, when the A-type foot-and-mouth disease virus-like particles are mass-produced, not only is the assembly efficiency low, but the aggregation problem of VLPs is particularly prominent. Although some studies have reported the use of nucleic acid fragments and negatively charged polymers as scaffolds to improve the in vitro assembly efficiency of VLPs, these methods still cannot fundamentally solve the assembly problem of A-type foot-and-mouth disease virus-like particles in large-scale production.

[0005] Therefore, the existing VLPs assembly solution technology urgently needs to be improved to overcome key problems such as low assembly efficiency and severe protein aggregation, and to provide more advanced and efficient technical and product support for the prevention and control of foot-and-mouth disease. Summary of the invention

[0006] The purpose of the present invention is to provide a virus-like particle assembly solution and a preparation method and application thereof, wherein the virus-like particle assembly solution can solve the problems of low virus-like particle assembly efficiency and severe protein aggregation.

[0007] The technical solution adopted to achieve the above purpose is a virus-like particle assembly solution, including the following substances: Tris, sodium chloride, amphoteric surfactant, sucrose, glycine and water for injection; each liter of assembly solution contains 2-5g of Tris, 15-30g of sodium chloride, 1-4g of amphoteric surfactant, 10-80g of sucrose and 10-30g of glycine, and the balance is water for injection.

[0008] The beneficial effects of the above technical scheme adopted in the present invention are as follows: Tris, sodium chloride and amphoteric surfactants in the virus-like particle assembly solution of the present invention play an important role in improving the assembly efficiency of virus-like particles: Tris as a buffer can maintain the pH value of the solution stable, and provide a suitable acid-base environment for the assembly of virus-like particles; while sodium chloride affects the distribution of surface charges of virus-like particles by adjusting the ionic strength of the solution, thereby promoting the interaction and assembly between particles; at the same time, the amphoteric surfactant can reduce the hydrophobicity of the protein surface, reduce the aggregation of proteins during the assembly process, and improve the assembly efficiency; sucrose and glycine play a role in optimizing the morphology of virus-like particles in the assembly solution, and sucrose can adjust and optimize the internal structure and stability of virus-like particles; glycine can be used as a protective agent to reduce the loss of virus-like particles during the assembly and purification process, and also helps to maintain the integrity and morphology of the particles. The combined effect of these components makes the morphology of the virus-like particles finally obtained more regular and uniform, which is beneficial to improving the immunogenicity and protective effect of the vaccine.

[0009] Preferably, each liter of the assembly solution contains 2.42-4.85 g of Tris, 17.53-29.22 g of sodium chloride, 1.23-3.07 g of an amphoteric surfactant, 15-40 g of sucrose and 10-25 g of glycine, with the remainder being water for injection.

[0010] More preferably, each liter of the assembly solution contains 2.43 g of Tris, 23.38 g of sodium chloride, 3.07 g of an amphoteric surfactant, 20 g of sucrose and 25 g of glycine, with the remainder being water for injection.

[0011] More preferably, the amphoteric surfactant is CHAPS.

[0012] The present invention also provides a method for preparing a virus-like particle assembly solution, comprising the following steps: (1) Dissolve Tris, sodium chloride and amphoteric surfactant in water for injection and stir until clear to obtain a mixed solution; (2) Sucrose and glycine are added to the mixed solution in sequence and stirred until clear, the pH is adjusted to 8.0-8.5 and the volume is fixed with water for injection to obtain a virus-like particle assembly solution.

[0013] Preferably, the pH is adjusted in step (2) using HCl or NaOH.

[0014] The invention also provides application of the virus-like particle assembly solution in the assembly and production of type A foot-and-mouth disease virus-like particles.

[0015] Preferably, the use of the virus-like particle assembly solution in the assembly and production of type A foot-and-mouth disease virus-like particles comprises the following steps: The structural protein of type A foot-and-mouth disease virus is placed in a virus-like particle assembly solution and assembled at 3-5°C for 10-20 hours to complete the assembly and production of type A foot-and-mouth disease virus-like particles.

[0016] More preferably, the assembly temperature is 4°C and the time is 16 hours.

[0017] The present invention has the following beneficial effects: The virus-like particle assembly solution of the present invention can significantly improve the self-assembly efficiency of type A foot-and-mouth disease virus-like particles in an in vitro environment, while effectively inhibiting unnecessary aggregation of virus-like particles, so that the spatial conformational epitopes of the virus-like particles are close to those of natural virus particles, further improving the immune effect of the vaccine; at the same time, the preparation method of the present invention is simple, does not require complicated processing procedures, and the materials are relatively cheap and easy to obtain, which is suitable for large-scale industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 is a diagram showing the results of SDS-PAGE analysis of virus-like particles assembled by the assembly solution of the present invention; Figure 2 is a transmission electron microscopy analysis result diagram of virus-like particles assembled by the assembly solution of the present invention; Figure 3 is a particle size result diagram of the dynamic light scattering detection of virus-like particles assembled by the assembly solution of the present invention; Figure 4 It is a high performance liquid chromatography analysis spectrum of virus-like particles assembled by the assembly solution of the present invention. DETAILED DESCRIPTION

[0019] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with embodiment.It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention, that is, the embodiments described are only part of the embodiments of the present invention, rather than all embodiments.If the specific conditions are not indicated in the embodiments, they are carried out according to the conditions recommended by the normal conditions or the manufacturers.If the manufacturer is not indicated in the reagents or instruments used, they are all conventional products that can be obtained by commercial purchase.

[0020] The instruments and equipment used in the present invention and their sources: high-speed centrifuge TGL-16, from Shuke Company; ultraspeed refrigerated centrifuge Aptima XE-90, from Beckman Company; high-performance liquid chromatograph LC-40, from Shimadzu Company; fully automatic density gradient preparation instrument, from BiAcAmp Company; nanoparticle size analyzer NanA-ZS, from Malvern Company; electrophoresis instrument from BiA-Rad Company. Reagents: Tris, from VWR Life Science Company; sodium chloride, CHAPS, sucrose and glycine, all from Sinopharm Chemical Reagent Co., Ltd. Type A foot-and-mouth disease virus structural protein: Type A foot-and-mouth disease virus structural protein is prepared and preserved by the R&D Center of Huapai Bioengineering Group.

[0021] Therefore, the following detailed description of the embodiments of the present invention is not intended to limit the scope of the invention claimed for protection, but merely represents selected embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without making creative work are within the scope of protection of the present invention.

[0022] The features and performance of the present invention are further described in detail below in conjunction with embodiments.

[0023] Example 1 A virus-like particle assembly solution comprises the following substances: Tris, sodium chloride, CHAPS, sucrose, glycine and water for injection; each liter of the assembly solution contains 4.85g of Tris, 17.53g of sodium chloride, 1.23g of CHAPS, 30g of sucrose and 10g of glycine, and the remainder is water for injection.

[0024] This embodiment also provides a method for preparing the above-mentioned virus-like particle assembly solution, comprising the following steps: (1) Weigh Tris, sodium chloride and CHAPS and dissolve them in 900 mL of water for injection. Stir until the solution is clear to obtain a mixed solution. (2) Add sucrose to the mixed solution and stir until the solution is clear, then add glycine and stir until the solution is clear, adjust the pH to 8.0, and then use water for injection to make the volume to 1 L, and store it at 4°C for future use, thereby obtaining a virus-like particle assembly solution.

[0025] Example 2 A virus-like particle assembly solution comprises the following substances: Tris, sodium chloride, CHAPS, sucrose, glycine and water for injection; each liter of the assembly solution contains 2.43g of Tris, 29.22g of sodium chloride, 2.46g of CHAPS, 40g of sucrose and 20g of glycine, and the remainder is water for injection.

[0026] This embodiment also provides a method for preparing the above-mentioned virus-like particle assembly solution, comprising the following steps: (1) Weigh Tris, sodium chloride and CHAPS and dissolve them in 900 mL of water for injection. Stir until the solution is clear to obtain a mixed solution. (2) Add sucrose to the mixed solution and stir until the solution is clear, then add glycine and stir until the solution is clear, adjust the pH to 8.0, and then use water for injection to make the volume to 1 L, and store it at 4°C for future use, thereby obtaining a virus-like particle assembly solution.

[0027] Example 3 A virus-like particle assembly solution comprises the following substances: Tris, sodium chloride, CHAPS, sucrose, glycine and water for injection; each liter of the assembly solution contains 2.43g of Tris, 23.38g of sodium chloride, 3.07g of CHAPS, 20g of sucrose and 25g of glycine, and the remainder is water for injection.

[0028] This embodiment also provides a method for preparing the above-mentioned virus-like particle assembly solution, comprising the following steps: (1) Weigh Tris, sodium chloride and CHAPS and dissolve them in 900 mL of water for injection. Stir until the solution is clear to obtain a mixed solution. (2) Add sucrose to the mixed solution and stir until the solution is clear, then add glycine and stir until the solution is clear, adjust the pH to 8.0, and then use water for injection to make the volume to 1 L, and store it at 4°C for future use, thereby obtaining a virus-like particle assembly solution.

[0029] Example 4 A virus-like particle assembly solution comprises the following substances: Tris, sodium chloride, CHAPS, sucrose, glycine and water for injection; each liter of the assembly solution contains 4.85g of Tris, 23.38g of sodium chloride, 3.07g of CHAPS, 15g of sucrose and 20g of glycine, and the remainder is water for injection.

[0030] This embodiment also provides a method for preparing the above-mentioned virus-like particle assembly solution, comprising the following steps: (1) Weigh Tris, sodium chloride and CHAPS and dissolve them in 900 mL of water for injection. Stir until the solution is clear to obtain a mixed solution. (2) Add sucrose to the mixed solution and stir until the solution is clear, then add glycine and stir until the solution is clear, adjust the pH to 8.0, and then use water for injection to make the volume to 1 L, and store it at 4°C for future use, thereby obtaining a virus-like particle assembly solution.

[0031] Experimental Example: Assembly and testing of type A foot-and-mouth disease virus-like particles The purified enzyme-digested 25 kDa structural protein of type A foot-and-mouth disease virus was placed in a dialysis bag in the assembly solution of Example 3 and assembled at 4° C. for 16 h.

[0032] The assembled virus-like particles (VLPs) were subjected to SDS-PAGE denaturing gel electrophoresis test. Figure 1 As shown, from Figure 1 As can be seen, clear FMD VP0, VP1 and VP3 bands can be seen in the assembly product.

[0033] The assembled VLPs were observed by transmission electron microscopy. Figure 2 shown; from Figure 2 Complete type A VLPs can be seen in the figure, indicating that the virus-like particle assembly solution of the present invention can achieve complete assembly of type A VLPs.

[0034] The particle size of the VLPs sample was detected by a nanoparticle size analyzer, and the results showed that the size of the assembled VLPs was about 30 nm ( Figure 3 ).

[0035] The VLPs of the assembled products were subjected to sucrose gradient centrifugation and high performance liquid chromatography to detect the VLPs content. The specific method is as follows: The assembled VLPs were taken out and centrifuged at 12000rpm for 5min at 4℃. 15~18% of the supernatant of the sucrose density gradient system was added to the prepared 20~45% sucrose density gradient upper layer. The centrifuge tube with the assembled VLPs was placed in an ultra-high speed centrifuge and centrifuged at 35000rpm for 3h at 2~8℃. Using a high performance liquid chromatography system, the needle was inserted into the bottom of the centrifuge tube and fixed. The data acquisition wavelength was 280nm. The VLPs target chromatographic peak was integrated and the VLPs content of the sample was calculated according to the standard curve. The results are as follows: Figure 4 shown.

[0036] from Figure 4It can be seen that the VLPs content obtained by substituting the integrated area into the standard curve formula is 426 μg / mL, indicating that the virus-like particle assembly solution of the present invention can improve the efficiency of virus-like particle assembly, reduce the severity of protein aggregation, and achieve high-yield and efficient assembly of type A foot-and-mouth disease virus-like particles.

[0037] The present invention has been described according to the above embodiments. It should be understood that the above embodiments do not limit the present invention in any form. Any technical solutions obtained by equivalent replacement or equivalent transformation shall fall within the scope of the present invention.

Claims

1. A virus-like particle assembly solution, characterized in that: The invention comprises the following substances: Tris, sodium chloride, amphoteric surfactant, sucrose, glycine and water for injection; each liter of the assembly solution contains 2-5g of Tris, 15-30g of sodium chloride, 1-4g of amphoteric surfactant, 10-80g of sucrose and 10-30g of glycine, and the balance is water for injection.

2. The virus-like particle assembly solution according to claim 1, characterized in that Each liter of the assembly solution contains 2.42-4.85 g of Tris, 17.53-29.22 g of sodium chloride, 1.23-3.07 g of an amphoteric surfactant, 15-40 g of sucrose and 10-25 g of glycine, and the remainder is water for injection.

3. The virus-like particle assembly solution according to claim 2, characterized in that Each liter of the assembly solution contains 2.43 g of Tris, 23.38 g of sodium chloride, 3.07 g of an amphoteric surfactant, 20 g of sucrose and 25 g of glycine, and the remainder is water for injection.

4. The virus-like particle assembly solution according to claim 1 or 3, characterized in that The amphoteric surfactant is CHAPS.

5. The method for preparing the virus-like particle assembly solution according to any one of claims 1 to 4, characterized in that: The following steps are involved: (1) Dissolve Tris, sodium chloride and amphoteric surfactant in water for injection and stir until clear to obtain a mixed solution; (2) Sucrose and glycine are added to the mixed solution in sequence and stirred until clear, the pH is adjusted to 8.0-8.5 and the volume is fixed with water for injection to obtain a virus-like particle assembly solution.

6. The method for preparing a virus-like particle assembly solution according to claim 5, characterized in that: In the step (2), the pH is adjusted using HCl or NaOH.

7. Use of the virus-like particle assembly solution according to any one of claims 1 to 4 in the assembly and production of type A foot-and-mouth disease virus-like particles.

8. The use according to claim 7, characterized in that The following steps are involved: The structural protein of type A foot-and-mouth disease virus is placed in a virus-like particle assembly solution and assembled at 3-5°C for 10-20 hours to complete the assembly and production of type A foot-and-mouth disease virus-like particles.

9. The use according to claim 8, characterized in that The assembly temperature is 4°C and the assembly time is 16 hours.

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