Microcarrier with surface modified with herpes virus receptor and application of microcarrier

Abstract

The invention discloses a microcarrier with the surface modified with a herpes virus receptor and application of the microcarrier, and relates to the technical field of animal virology technologies and veterinary biological products. The microcarrier is composed of a microcarrier matrix and herpes virus specific receptor protein fixed to the surface of the microcarrier matrix through chemical coupling; the microcarrier matrix is made of a biocompatible material, and the particle size of the microcarrier matrix is 1-10 [mu] m; the herpes virus specific receptor protein is an extracellular region fragment of a nectin-1 receptor. The method has the advantages that the virus infection efficiency and the virus yield are remarkably improved by accurately regulating and controlling the particle size of the microcarrier and adopting a receptor fixing technology, and the performance is particularly excellent under the condition of low infection complex number; meanwhile, the process is greatly simplified, and the pollution risk is reduced; and the method can be widely applied to herpes virus production, vaccine preparation and difficult-to-culture virus amplification by replacing receptors, and has a wide application prospect.

Description

Technical Field

[0001] This invention relates to the field of animal virology and veterinary biological products, specifically to a virus production tool and its application. More specifically, it relates to a microcarrier with a surface modified with herpesvirus receptors and its application. Background Technology

[0002] Herpesviruses, such as pseudorabies virus (PRV) and feline herpesvirus (FHV), are important pathogens that endanger the livestock industry and pet health. In basic virology research, vaccine preparation and diagnostic reagent development, the efficient and stable amplification of high-titer viral fluid is a core step.

[0003] Currently, traditional virus amplification methods are widely used in laboratories and industry. The standard procedure involves removing the original cell culture medium after the host cells have grown to a suitable density, adding an inoculum containing the virus, allowing for a period of static adsorption, and finally adding maintenance medium to continue culturing. However, this "withdrawal-adsorption-addition" procedure has several inherent drawbacks: First, its infection efficiency is low and difficult to control. The entire process relies entirely on the passive random diffusion of virus particles, resulting in an effective adsorption ratio far lower than the theoretical value, directly affecting the titer and uniformity of the final product. Second, the multiple liquid changes during amplification increase the risk of microbial contamination, easily leading to batch production failure. Furthermore, the virus adsorption efficiency is affected by multiple variables, including cell state, liquid volume, time, and temperature. Any slight fluctuation can cause significant differences in yield between batches, severely restricting the stability and large-scale application of the process. Current technologies lack effective means to actively guide virus targeting and infection, thereby improving infection efficiency.

[0004] To address the aforementioned issues, there is an urgent need in this field for a new technology that can proactively guide viral targeting and infection, thereby fundamentally improving infection efficiency and process stability. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a microcarrier with a surface modified with herpesvirus receptors and its application, thus solving the problems mentioned in the background section.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: A microcarrier with a surface modified with a herpesvirus receptor, the microcarrier comprising a microcarrier matrix and a herpesvirus-specific receptor protein immobilized on its surface by chemical coupling; Biocompatible materials refer to materials that do not produce significant toxicity or adverse effects on cells in the application environment. In addition to agarose, they also include cellulose, chitosan, polylactic acid-glycolic acid copolymer (PLGA), etc. Chemical coupling refers to the method of stably attaching receptor proteins to the surface of microcarriers through covalent bonds. Its mechanism includes, but is not limited to, the reaction of epoxy groups and amino groups, the condensation of carbodiimide (EDC / NHS)-mediated carboxyl groups and amino groups, and the cross-linking of glutaraldehyde. Its core is to create a high density of active sites for the directional immobilization of biomolecules on the surface of microcarriers. The microcarrier matrix is ​​made of biocompatible material with a particle size of 1-10 μm; The herpesvirus-specific receptor protein is an extracellular fragment of the nectin-1 receptor.

[0007] Furthermore, the material of the microcarrier matrix is ​​selected from agarose, dextran, gelatin, polystyrene or silica, preferably with a particle size of 3-5 μm; A size of 1-10 μm ensures that the barrier carrier can be fully suspended in the fluid environment of conventional cell culture through Brownian motion and gentle stirring, thereby achieving uniform contact with the virus and cells. 3-5 μm is preferred because this size is much smaller than animal cells, which can avoid excessive occupation of cell growth space or physical damage. At the same time, its specific surface area is large enough to fix a sufficient amount of receptors and form an ideal "virus enrichment zone" around the cell, thereby maximizing infection efficiency.

[0008] Furthermore, the nectin-1 receptor is porcine or feline nectin-1, and its extracellular region contains a V domain and a C2 structure that specifically bind to the viral gD protein. The nectin-1 receptor is a key host cell receptor for herpesvirus invasion. Its extracellular fragment was obtained through recombinant expression and purification using genetic engineering techniques, fully retaining its native conformation and biological activity. The V domain has been shown to be the core region that directly binds to the viral gD protein with high affinity and is responsible for initiating viral infection, while the C2 domain stabilizes the receptor fragment structure by maintaining its spatial conformation. The strategy of using this extracellular fragment to replace the full-length receptor effectively avoids interference between the transmembrane and intracellular regions, significantly improving its immobilization efficiency and functional display on the microcarrier surface.

[0009] A method for the above-mentioned microcarrier includes the following steps: S1. Microcarrier activation: The microcarrier matrix is ​​suspended in an alkaline buffer solution, and epichlorohydrin is added to react and introduce epoxy-active functional groups on its surface. In this step, the alkaline buffer is preferably 0.1M sodium carbonate-sodium bicarbonate buffer, pH 9.0-10.0. The volume ratio of epichlorohydrin added is usually 5%-15% of the reaction system. The reaction is usually carried out at 30-40℃ with shaking at 100-200 rpm for 6-12 hours to ensure sufficient introduction of epoxy groups. After the reaction is completed, it is necessary to wash with a large amount of deionized water or PBS (phosphate buffer) until neutral to remove unreacted reagents. S2. Receptor immobilization: The nectin-1 receptor protein solution and the activated microcarrier obtained in step S1 are mixed in an alkaline buffer solution with pH 8.5-9.5 and a coupling reaction is carried out at 2-8℃. After the reaction is completed, a blocking agent is added to quench the unreacted active sites. After washing, the microcarrier is obtained. In this step, 2-8℃ and pH 8.5-9.5 are intended to maintain the biological activity of the receptor protein while promoting the ring-opening reaction between its primary amino group (-NH2) and the epoxy group on the surface of the microcarrier to form a stable carbon-nitrogen covalent bond. The concentration of the protein solution is usually between 0.1-0.5 mg / mL to ensure that the fixed receptor density achieves the best effect.

[0010] Furthermore, the coupling reaction described in step S2 is carried out in a carbonate buffer solution at pH 9.0 with shaking at 4°C for 12-16 hours, and the blocking agent is a 1M ethanolamine solution; The blocking step involves quenching all unreacted epoxy groups with small molecule compounds such as ethanolamine to prevent non-specific adsorption in subsequent applications, thus ensuring the functional purity of the microcarrier and the reliability of the results. After blocking, the microcarrier is thoroughly washed with PBS containing surfactants (such as 0.05%-0.1% Tween-20) to remove physically adsorbed proteins. Finally, the microcarrier is resuspended in PBS and stored at 4°C. The protein immobilization amount of the final product can be quantified by the BCA method, or qualitatively / semi-quantitatively characterized by fluorescent labeling, infrared spectroscopy, or other means.

[0011] The application of the aforementioned microcarriers in improving the production efficiency of herpesviruses is as follows: In an adherent host cell culture system, without discarding the original culture medium, virus solution and the aforementioned microcarriers are added simultaneously. The final concentration of the microcarriers used is 10. 5 -10 6 pcs / cm 2 After adsorption is complete, add maintenance solution directly and continue culturing; Not discarding the original culture medium can effectively simplify the process, reducing the traditional three steps of "liquid withdrawal-adsorption-liquid replenishment" to a single step of "direct addition", which greatly improves operational efficiency; reduces the risk of contamination, avoids multiple openings and liquid transfers, and significantly reduces the chance of microbial contamination; and can maintain the stability of the cell microenvironment, so that cells do not have to experience temperature, pH and nutrient fluctuations caused by culture medium replacement, resulting in better cell condition and more conducive to subsequent virus replication.

[0012] Furthermore, the herpesvirus is pseudorabies virus (PRV) or feline herpesvirus (FHV), and the host cell is PK-15 cell or CRFK cell; PK-15 (porcine kidney epithelial cells) is a classic cell line for proliferating PRV, while CRFK (cat kidney cells) is a commonly used cell line for proliferating FHV. The corresponding species nectin-1 receptor immobilized on the surface of the microcarrier can efficiently and specifically capture PRV or FHV viral particles, forming a "virus-microcarrier" complex. This complex moves freely in the culture system, greatly increasing the probability and efficiency of viral contact with cell surface receptors, and forming a local high viral concentration at the cell-microcarrier interface, thereby strongly initiating the viral infection process.

[0013] The above-mentioned microcarriers are used in the preparation of herpesvirus biological products, which include herpesvirus vaccines or herpesvirus diagnostic antigens; The microcarrier system can rapidly, efficiently, and stably produce high-titer herpesvirus fluid. This viral fluid can be used directly to prepare attenuated live vaccines, or it can be inactivated (e.g., treated with formaldehyde or β-propiolactone), purified, and then mixed with suitable adjuvants to prepare inactivated vaccines. In the preparation of diagnostic antigens, the high-purity, high-immunogenicity virus particles obtained by this invention can be directly used as coating antigens to develop enzyme-linked immunosorbent assay (ELISA) kits, immunofluorescence test strips, or colloidal gold test strips, etc., for detecting specific antibodies in animal serum, serving disease diagnosis and immune monitoring.

[0014] The application of the above-mentioned microcarriers in amplifying difficult-to-culture herpesviruses, wherein the difficult-to-culture herpesviruses include human herpesvirus type 6 or 7; Human herpesviruses such as HHV-6 and HHV-7 often exhibit low replication efficiency in traditional cell culture systems, making it difficult to obtain high-titer viral reserves and hindering related research. By replacing the receptors immobilized on the surface of microcarriers with specific receptors corresponding to particular difficult-to-culture viruses (e.g., the main receptor for HHV-6 is the human CD46 molecule), this "active capture and enrichment" mechanism can be applied to the target virus. This can fundamentally solve the amplification bottleneck caused by low initial infection efficiency, providing a powerful tool for the efficient production of these difficult-to-culture viruses at the cellular level and has broad application prospects.

[0015] Beneficial effects: By precisely controlling the microcarrier particle size and receptor immobilization technology, the viral infection efficiency and viral yield are significantly improved, with particularly outstanding performance at low infection multiples; at the same time, the process is greatly simplified and the risk of contamination is reduced; and by changing the receptor, it can be widely used in herpes virus production, vaccine preparation and amplification of difficult-to-culture viruses, with broad application prospects. Attached Figure Description

[0016] Figure 1 The fluorescence contrast results are for a microcarrier with a surface modified with herpesvirus receptors proposed in this invention. Figure 2 The peak analysis results are for a microcarrier with a surface modified with herpesvirus receptors proposed in this invention. Detailed Implementation

[0017] The present invention will be further described in detail below through specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0018] Example 1: A method for preparing a microcarrier with a surface modified with herpesvirus receptors. S1. Microcarrier activation: Weigh 1.0 g of agarose microspheres, wash three times with 100 mL of deionized water for 5 minutes each time, centrifuge and discard the supernatant to obtain microspheres, suspend the washed microspheres in 20 mL of 0.1 M carbonate buffer (pH 9.0), add 2.0 mL of epichlorohydrin, and react at 30 °C and 150 rpm for 8 hours. After the reaction, wash thoroughly three times with 0.1 M PBS (pH 7.4) to obtain activated microspheres with epoxy groups on the surface, and store at 4 °C for later use. S2. Receptor immobilization: Take 200 mg of activated microspheres, add 10 mL of 0.1 M carbonate buffer (pH 9.0) to suspend them, add 2.0 mL of nectin-1 protein solution, and slowly shake at 4 °C for 16 hours. After the reaction is complete, add 5 mL of 1 M ethanolamine solution and block at room temperature for 2 hours. Then, wash 3 times with PBST and 2 times with PBS to completely remove unfixed proteins and reagents. Resuspend the obtained functionalized microcarriers in 10 mL of PBS and store at 4 °C to obtain microcarriers with herpesvirus receptors modified on the surface.

[0019] Characterization results Fourier transform infrared (FTIR) spectroscopy analysis: Activated microspheres at 910 cm⁻¹ -1 The presence of a characteristic absorption peak of the epoxy group nearby, and the significant weakening or disappearance of this peak after the acceptor is immobilized and blocked, indicates that the epoxy group has successfully reacted with the protein.

[0020] BCA protein quantification analysis: By measuring the protein concentration difference in the solution before and after immobilization, it was calculated that approximately 18 μg of nectin-1 protein was immobilized per milligram of microcarrier, with an immobilization efficiency of over 85%.

[0021] Fluorescence microscopy observation: Fixation with FITC-labeled nectin-1 resulted in uniform green fluorescence observed on the microsphere surface under a fluorescence microscope, indicating successful and uniform fixation of the receptor protein. Fluorescence contrast results are referenced. Figure 1 .

[0022] Table 1 Peak Analysis Results

[0023] Table 2 Results of BCA protein quantification analysis

[0024] Data analysis: FTIR analysis directly demonstrated the chemical activation of the microsphere surface and the subsequent successful covalent immobilization of the receptor protein. BCA quantitative analysis accurately showed that the immobilization method of this invention can immobilize approximately 18 μg of nectin-1 protein per milligram of microcarrier, with an immobilization efficiency exceeding 85%, demonstrating the reliability and efficiency of this preparation process in efficiently loading bioactive molecules. Example 2: In-depth analysis of its application and mechanism in improving the production efficiency of pseudorabies virus (PRV) 1. Experimental Design Using PK-15 cells, four groups were set up for comparison: Experimental group 1 (traditional method): Discard the old culture medium, add 1 mL of PRV virus solution (MOI=0.1), and add maintenance solution after 2 hours of adsorption.

[0025] Experimental group 2 (virus + unmodified microspheres): 100 μL of PRV virus solution (MOI=0.1) and 500 μL of unmodified agarose microsphere suspension were added.

[0026] Experimental group 3 (virus + blank microspheres): 100 μL of PRV virus solution (MOI=0.1) and 500 μL of activated but unfixed receptor blank microsphere suspension were added.

[0027] Experimental group 4 (this invention): 100 μL of PRV virus solution (MOI=0.1) and 500 μL of functionalized microcarrier suspension (concentration 5×10⁻⁶) were added. 5 pcs / cm 2 ).

[0028] After all groups were adsorbed at 37℃ and 5% CO2 for 2 hours, 2 mL of maintenance solution was added, and the culture was continued for another 48 hours.

[0029] 2. Results and Analysis Infection efficiency and virus adsorption rate: By indirect immunofluorescence (IFA) detection, the positive cell rate of the present invention group (experimental group 4) was as high as about 85% 24 hours after infection, which was significantly higher than that of the traditional method group (about 25%) and other control groups (all less than 30%). By qPCR quantitative detection of the unadsorbed viral genome in the supernatant after adsorption, the virus adsorption rate of the present invention group exceeded 92%, which was much higher than that of the traditional method group (about 35%).

[0030] Infection synchronicity: Flow cytometry analysis of early viral protein (gB) expression at different time points (6, 12, 18 hours) after viral infection showed that more than 80% of cells in the invention group expressed gB 12 hours after infection, while only about 30% of cells in the conventional method group were positive at this time, indicating that the invention significantly improved infection synchronicity.

[0031] Virus yield: via TCID 50 After 48 hours of incubation, the viral titer of the group in this invention reached 1.0 × 10⁻⁶. 8 TCID 50 / mL, which is the traditional method group (2.5×10 7 TCID 50 It is 4 times that of ( / mL).

[0032] Operational advantages and stability: This invention adopts a "no liquid change" operation, which simplifies the steps, ensures no contamination throughout the process, and has a batch-to-batch coefficient of variation (CV) of less than 5%, demonstrating excellent stability.

[0033] Table 3. Results of PRV infection efficiency and viral yield

[0034] Example 3: Application and performance verification in vaccine and diagnostic antigen preparation Vaccine preparation and immunogenicity evaluation: The high-titer PRV virus solution obtained by amplification using the microcarrier system of this invention was inactivated by 0.1% formaldehyde solution at 4°C for 72 hours, and then purified by ultracentrifugation. The purified viral antigen was emulsified with ISA 206 oil adjuvant at a volume ratio of 1:1 to successfully prepare a pseudorabies inactivated vaccine. After immunizing BALB / c mice, the PRV-specific neutralizing antibody titer in the serum of the vaccine group of this invention (1:2560) was significantly higher than that of the vaccine group prepared using traditional virus solution (1:640).

[0035] Diagnostic kit development and performance: After purifying the amplified feline herpesvirus (FHV), it was used as the coating antigen. The conditions were optimized by the square matrix titration method to develop an indirect ELISA kit for detecting FHV antibodies. The test results of 100 clinical feline serum samples showed that the sensitivity was 98%, the specificity was 99%, and the concordance rate was 98.5%, which is better than the performance of the commercially available kits.

[0036] Example 4: Preliminary Practice in Amplifying the Difficult-to-Culture Virus HHV-6 Based on the core principle of this invention, the receptor fixed on the surface of the microcarrier is replaced with an extracellular fragment of the human CD46 protein, the main cellular receptor of human herpesvirus type 6 (HHV-6).

[0037] Preparation of functionalized microcarriers: Following the method in Example 1, activated agarose microspheres were covalently immobilized with recombinant human CD46 extracellular protein.

[0038] Virus amplification experiment: HHV-6 was cultured using human umbilical cord blood lymphocytes (HSB-2 cells). The experimental group was inoculated with the virus (MOI=0.01) and CD46-functionalized microcarriers were added simultaneously, while the control group was infected using the conventional method.

[0039] Results: After 5 days of culture, the viral genome copy number in the experimental group (determined by qPCR) was 8-10 times that of the control group, and the cytopathic effect (CPE) appeared about 48 hours earlier, indicating that the microcarrier system of the present invention can be effectively applied to the efficient amplification of difficult-to-culture viruses such as HHV-6.

[0040] Example 5: Stability and process repeatability evaluation of microcarrier systems Storage stability: The prepared functionalized microcarriers (for PRV) were stored in PBS at 4°C. Samples were taken at 0, 1, 3 and 6 months, and their activity was evaluated by virus adsorption experiments. The results showed that their activity in promoting viral infection efficiency did not decrease significantly within 6 months.

[0041] Batch-to-batch reproducibility: Three batches of functionalized microcarriers were independently prepared for PRV infection experiments. The viral endpoint titers (TCID) obtained from the three batches were analyzed. 50 The coefficient of variation (CV) of the sample was less than 4%, which proves that the preparation process has good reproducibility.

[0042] Example 6: Application Exploration in Suspension Cell Culture System This invention is not only applicable to adherent cells, but can also be extended to suspension cell culture systems. Taking the production of pseudorabies virus from suspension-cultured BHK-21 cells as an example: During cell suspension culture, PRV virus solution (MOI=0.05) and functionalized microcarriers were added simultaneously. Due to the presence of the microcarriers, the virus was specifically captured and enriched on the surface of the microcarriers, which greatly increased the chance of the virus colliding and contacting with the suspended cells. Ultimately, the virus yield was about 5 times higher than that of the traditional suspension infection method, and the problem of low infection efficiency caused by virus dilution in the traditional method was avoided.

[0043] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the claimed invention.

Claims

1. A microcarrier with a surface modified with herpesvirus receptors, characterized in that, The microcarrier consists of a microcarrier matrix and a herpesvirus-specific receptor protein fixed to its surface by chemical coupling. The microcarrier matrix is ​​made of biocompatible material with a particle size of 1-10 μm; The herpesvirus-specific receptor protein is an extracellular fragment of the nectin-1 receptor.

2. The microcarrier with a surface modified with herpesvirus receptors according to claim 1, characterized in that, The microcarrier matrix is ​​selected from agarose, dextran, gelatin, polystyrene or silica, with a preferred particle size of 3-5 μm.

3. The microcarrier with a surface modified with herpesvirus receptors according to claim 1, characterized in that, The nectin-1 receptor is porcine or feline nectin-1, and its extracellular region contains a V domain and a C2 domain that specifically bind to the viral gD protein.

4. A method for preparing microcarriers as described in any one of 1-3, characterized in that, Includes the following steps: S1. Microcarrier activation: The microcarrier matrix is ​​suspended in an alkaline buffer solution, and epichlorohydrin is added to react and introduce epoxy-active functional groups on its surface. S2. Receptor immobilization: The nectin-1 receptor protein solution and the activated microcarrier obtained in step S1 are mixed in an alkaline buffer solution with a pH of 8.5-9.5 and a coupling reaction is carried out at 2-8°C. After the reaction is completed, a blocking agent is added to quench the unreacted active sites. After washing, the microcarrier is obtained.

5. The method for preparing microcarriers according to claim 4, characterized in that, The coupling reaction described in step S2 was carried out in a carbonate buffer solution at pH 9.0 with shaking at 4°C for 12-16 hours, and the blocking agent was a 1M ethanolamine solution.

6. The application of the microcarrier as described in any one of claims 1-3 in improving the production efficiency of herpesvirus, characterized in that, The application method is as follows: In an adherent host cell culture system, without discarding the original culture medium, add the virus solution and the microcarrier simultaneously. The final concentration of the microcarrier is 10. 5 -10 6 pcs / cm 2 After adsorption is complete, maintenance medium is added directly to continue culturing.

7. The application according to claim 6, characterized in that, The herpesvirus is pseudorabies virus (PRV) or feline herpesvirus (FHV), and the host cell is PK-15 cell or CRFK cell.

8. The application of the microcarrier as described in any one of claims 1-3 in the preparation of herpesvirus biological products, characterized in that, The biological products include herpesvirus vaccines or herpesvirus diagnostic antigens.

9. The application of the microcarrier as described in any one of claims 1-3 in the amplification of difficult-to-culture herpesvirus, characterized in that, The difficult-to-culture herpesviruses include human herpesvirus 6 or 7.

Images