Quantitative detection method of vesicular stomatitis virus and application thereof

Abstract

The invention provides a quantitative detection method of vesicular stomatitis virus and application thereof, and the method comprises the following steps: separating a sample solution containing vesicular stomatitis virus by using a size exclusion high performance liquid chromatograph, and detecting and analyzing by using a multi-angle laser light scatterometer to obtain the particle concentration of vesicular stomatitis virus; a mobile phase of the size exclusion high performance liquid chromatography comprises a buffer solution, inorganic salt and a stabilizer. According to the method, the size exclusion high performance liquid chromatography and the multi-angle laser light scattering technology (SEC-MALLS) are combined, the particle concentration of the vesicular stomatitis virus (VSV) can be accurately and efficiently quantified, the measured virus particle number and the dilution multiple have better correlation, the virus particle number and the virus titer have high correlation, and the accuracy and the accuracy of the detection result are improved. The method can be used for accurately reflecting the infection activity titer of the virus, has higher accuracy, and can be widely applied to basic research of the virus and quality control of vaccines.

Description

Technical Field

[0001] The invention belongs to the technical field of virus particle detection, and particularly relates to a quantitative detection method for vesicular stomatitis virus and application thereof. Background Art

[0002] Vesicular Stomatitis Virus (VSV) is a single-stranded, negative-strand RNA virus belonging to the Rhabdoviridae family with a broad host range and robust replication capacity. Its genome is simple and encodes five major proteins: nucleoprotein (N), phosphoprotein (P), matrix protein (M), envelope glycoprotein (G), and RNA polymerase protein (L). VSV genome replication and transcription occur in the cytoplasm and do not integrate into the host genome, resulting in a low integration risk and high safety profile. Furthermore, the VSV genome is easily modifiable and can efficiently express exogenous proteins, making it of great application value in biomedical research.

[0003] Due to its unique biological properties and easily modifiable genome, VSV has been widely used in various fields. In vaccine development, VSV has been successfully used to construct a variety of recombinant vaccines, such as those for Ebola and COVID-19. These vaccines have demonstrated good safety and immunogenicity in clinical trials, effectively inducing neutralizing antibodies and cellular immune responses. Furthermore, VSV has been used to study the entry mechanisms of highly pathogenic viruses and screen for antiviral drugs, providing an important tool for biomedical research. In oncolytic virus research, VSV has also been developed as a promising oncolytic viral vector for cancer therapy. VSV has also been widely used in the construction of pseudovirus systems. By inserting exogenous genes (such as the viral spike protein gene) into the VSV genome, pseudoviruses carrying the exogenous protein can be constructed. These pseudoviruses retain the replication and packaging capabilities of VSV but lack the pathogenicity of the wild-type virus. Therefore, they can be used to study viral entry mechanisms, screen for neutralizing antibodies, and evaluate vaccines. For example, recombinant VSV pseudoviruses for SARS-CoV-2 have been used to evaluate the neutralizing ability of existing vaccines and antibodies against the novel coronavirus, providing an important tool for vaccine development and antiviral drug research.

[0004] In order to further improve the safety and immunogenicity of VSV vectors, researchers have developed a variety of modification strategies. For example, by replacing or truncating the envelope glycoprotein (G) gene of VSV, the attenuation of the viral vector can be achieved and the vector-specific pre-existing immunity can be eliminated. In addition, by changing the matrix protein (M) gene sequence, the toxicity of the VSV vector can be reduced and the safety can be improved. Gene rearrangement technology has also been used to adjust the transcription order of the VSV gene to enhance the expression of the target antigen and achieve viral attenuation. These modification strategies not only improve the safety and immunogenicity of the VSV vector, but also expand its application range in vaccine development and biomedical research.

[0005] The concentration, activity, and physical properties of viral particles are crucial for virus research and vaccine development. Commonly used methods for measuring vesicular stomatitis virus titers include the plaque formation assay and the TCID50 assay, which quantify the virus by measuring the pathological effects or cell death observed after virus infection. qPCR can also be used to quantify vesicular stomatitis virus genome copy number, indirectly reflecting the number of viral particles and indirectly assessing viral genome content and infectivity. However, it cannot directly provide physical characteristics of viral particles, such as particle size, distribution, and aggregation.

[0006] Virus particle concentration is crucial for vaccine dosage control and safety assessment. Accurate quantification of virus particle concentration can further optimize the purification process, ensure the consistency and reproducibility of vaccine production, and help evaluate the stability of viral preparations. Therefore, there is an urgent need to develop an accurate and efficient virus quantification detection method. Summary of the Invention

[0007] In response to the deficiencies in the prior art, the present invention aims to provide a method for quantitatively detecting vesicular stomatitis virus and its application. The method of the present invention can achieve accurate quantification of vesicular stomatitis virus, has a high linear correlation between the number of virus particles and the dilution factor, and also shows a high correlation between the number of virus particles and the virus titer. The method can be widely used in the quality control of virus preparations and the optimization of the preparation process.

[0008] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0009] In a first aspect, the present invention provides a method for quantitatively detecting vesicular stomatitis virus, the method comprising: separating a sample solution containing vesicular stomatitis virus using a size exclusion high performance liquid chromatograph, detecting and analyzing the sample solution using a multi-angle laser light scattering instrument to obtain a particle concentration of the vesicular stomatitis virus;

[0010] The mobile phase of the size exclusion high performance liquid chromatography comprises a buffer, an inorganic salt and a stabilizer.

[0011] The present invention combines size-exclusion high-performance liquid chromatography with multi-angle laser light scattering (SEC-MALLS) technology to accurately and efficiently quantify the particle concentration of vesicular stomatitis virus (VSV). The measured number of viral particles has a better correlation with the dilution factor, with high accuracy, and can be widely used in basic virus research and vaccine quality control.

[0012] Preferably, the vesicular stomatitis virus comprises any one of a wild-type vesicular stomatitis virus, a mutant vesicular stomatitis virus, a recombinant vesicular stomatitis virus or a vesicular stomatitis virus vector, or a combination of at least two thereof.

[0013] Preferably, the concentration of the buffer in the mobile phase is 5-200mM, for example, it can be 5mM, 10mM, 20mM, 50mM, 80mM, 100mM, 120mM, 150mM, 180mM or 200mM; the concentration of the inorganic salt is 5-500mM, for example, it can be 5mM, 10mM, 50mM, 100mM, 150mM, 200mM, 250mM, 300mM, 350mM, 400mM, 450mM or 500mM; the mass percentage of the stabilizer is 10-30%, for example, it can be 10%, 12%, 14%, 16%, 18%, 20%, 22%, 24%, 26%, 28% or 30%, etc.

[0014] Preferably, the pH value of the mobile phase is 6.0-8.0, for example, it can be 6.0, 6.2, 6.4, 6.6, 6.8, 7.0, 7.2, 7.4, 7.6, 7.8 or 8.0.

[0015] Other specific point values within the above numerical ranges can be selected and will not be described in detail here.

[0016] Preferably, the buffer comprises any one of HEPES buffer, Tris-HCl buffer or phosphate buffer, or a combination of at least two thereof.

[0017] Preferably, the inorganic salt includes any one or a combination of at least two of sodium sulfate, magnesium sulfate, potassium sulfate, ammonium sulfate, sodium nitrate, magnesium nitrate, potassium nitrate, ammonium nitrate, sodium chloride, magnesium chloride, potassium chloride or ammonium chloride.

[0018] Preferably, the stabilizer comprises any one or a combination of at least two of arginine, glycine, glycerol, trehalose, ethylenediaminetetraacetic acid, polyethylene glycol, sucrose, ethanol or dimethyl sulfoxide.

[0019] The present invention adds a stabilizer to the mobile phase to improve protein stability and separation effect, which is beneficial to improving the relevance and accuracy of virus particle concentration detection.

[0020] Preferably, the stabilizer comprises arginine, glycine and glycerol.

[0021] Arginine, glycine and glycerol in the present invention have a synergistic effect and are added to the mobile phase as stabilizers to improve the integrity and stability of virus particles, better separate and purify samples, and improve the relevance and accuracy of virus particle concentration detection.

[0022] Preferably, the mass ratio of arginine, glycine and glycerol is (1-10):(1-10):(1-10).

[0023] The specific point values in the first one (1-10) can be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, etc.

[0024] The specific point values in the second one (1-10) can be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, etc.

[0025] The specific point value in the third one (1-10) can be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, etc.

[0026] Other specific point values within the above numerical ranges can be selected and will not be described in detail here.

[0027] Preferably, the injection volume of the size exclusion HPLC is 1-100 μL, for example, it can be 1 μL, 5 μL, 10 μL, 15 μL, 20 μL, 30 μL, 40 μL, 50 μL, 60 μL, 70 μL, 80 μL, 90 μL or 100 μL; the flow rate is 0.1-1.0 mL / min, for example, it can be 0.1 mL / min, 0.2 mL / min, 0.3 mL / min, 0.4 mL / min, 0.5 mL / min, 0.6 mL / min, 0.7 mL / min, 0.8 mL / min, 0.9 mL / min or 1.0 mL / min; the elution time is 30-60 min, for example, it can be 30 min, 32 min, 35 min, 38 min, 40 min, 42 min, 45 min, 48 min, 50 min, 52 min, 55 min, 58 min or 60 min.

[0028] Other specific point values within the above numerical ranges can be selected and will not be described in detail here.

[0029] Preferably, the size exclusion high performance liquid chromatography column comprises any one of Agilent Bio SEC-5, TSKgel G3000SWXL, TSKgel G4000SWXL, SRT SEC 2000, SRT SEC 1000 or TSKgel G5000PWXL.

[0030] The selection of chromatographic column is crucial for the separation effect of vesicular stomatitis virus. The above chromatographic column has a better separation effect for vesicular stomatitis virus.

[0031] Agilent Bio SEC-5, TSKgel G3000SWXL or TSKgel G4000SWXL chromatographic columns are further preferred, as they have better selectivity for vesicular stomatitis virus.

[0032] Preferably, the column temperature of the chromatographic column is 4-40°C, for example, 4°C, 5°C, 10°C, 15°C, 20°C, 25°C, 30°C, 35°C or 40°C.

[0033] Preferably, the detection wavelength of the size exclusion HPLC is 500-800 nm, for example, it can be 500 nm, 550 nm, 600 nm, 610 nm, 620 nm, 630 nm, 640 nm, 650 nm, 660 nm, 670 nm, 680 nm, 690 nm, 700 nm, 750 nm or 800 nm.

[0034] Other specific point values within the above numerical ranges can be selected and will not be described in detail here.

[0035] Preferably, the number of detection angles of the multi-angle laser light scattering instrument is 3-18, for example, it can be 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18, etc.

[0036] Other specific point values within the above numerical ranges can be selected and will not be described in detail here.

[0037] Preferably, the analysis model includes a Number intensity analysis model.

[0038] Preferably, the detector for the detection further includes any one of an ultraviolet detector, a differential detector, a fluorescence detector, a viscosity detector or a dynamic laser scatterer, or a combination of at least two thereof.

[0039] The present invention combines size exclusion high performance liquid chromatography with multi-angle laser light scattering (SEC-MALLS) technology to quantitatively detect the concentration of vesicular stomatitis virus (VSV) particles. At the same time, an ultraviolet detector, a differential detector, a fluorescence detector, a viscosity detector, and a dynamic laser light scatterer can be added to assist in the detection of multiple physical properties.

[0040] Preferably, the sample solution containing vesicular stomatitis virus comprises a cell culture fluid infected with vesicular stomatitis virus.

[0041] Preferably, the cell culture fluid infected with vesicular stomatitis virus undergoes a pretreatment step before separation using size exclusion high performance liquid chromatography.

[0042] Preferably, the pretreatment step specifically includes: (1) centrifuging or filtering the cell culture fluid infected with vesicular stomatitis virus to remove impurities and collect the liquid; (2) adding nuclease reaction, then adding polyethylene glycol or ammonium sulfate, and letting it stand; (3) centrifuging, collecting the precipitate and resuspending it with buffer.

[0043] The present invention sets a specific sample pretreatment step, which can further purify the sample before size exclusion high performance liquid chromatography separation, remove interfering impurities and concentrate virus particles, thereby improving the relevance and accuracy of virus particle concentration detection.

[0044] Preferably, in step (1), the centrifugal speed is 2000-4000g, for example, it can be 2000g, 2200g, 2400g, 2600g, 2800g, 3000g, 3200g, 3400g, 3600g, 3800g or 4000g; the time is 5-15min, for example, it can be 5min, 6min, 7min, 8min, 9min, 10min, 11min, 12min, 13min, 14min or 15min, etc.

[0045] Preferably, the filtration pore size is 0.1-2 μm, for example, it can be 0.1 μm, 0.2 μm, 0.3 μm, 0.4 μm, 0.5 μm, 0.6 μm, 0.7 μm, 0.8 μm, 0.9 μm, 1 μm, 1.2 μm, 1.5 μm, 1.8 μm or 2 μm, etc.

[0046] Other specific point values within the above numerical ranges can be selected and will not be described in detail here.

[0047] Preferably, in step (2), the dosage of the nuclease is 10-100 U / mL, for example, 10 U / mL, 20 U / mL, 30 U / mL, 40 U / mL, 50 U / mL, 60 U / mL, 70 U / mL, 80 U / mL, 90 U / mL or 100 U / mL, etc.

[0048] Preferably, the reaction temperature is 25-37°C, for example, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C or 37°C; the reaction time is 10-60 min, for example, 10 min, 15 min, 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, 50 min, 55 min or 60 min.

[0049] Preferably, the molecular weight of the polyethylene glycol is 4000-12000, for example, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 11000 or 12000.

[0050] Preferably, the mass percentage of the polyethylene glycol in the pretreatment system is 5-20%, for example, it can be 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20%, etc.

[0051] Preferably, the concentration of ammonium sulfate in the pretreatment system is 0.5-2.0 M, for example, it can be 0.5 M, 0.6 M, 0.7 M, 0.8 M, 0.9 M, 1.0 M, 1.1 M, 1.2 M, 1.3 M, 1.4 M, 1.5 M, 1.6 M, 1.7 M, 1.8 M, 1.9 M or 2.0 M, etc.

[0052] Preferably, the standing time is 0.5-3 h, for example, 0.5 h, 0.6 h, 0.8 h, 1.0 h, 1.2 h, 1.5 h, 1.8 h, 2.0 h, 2.2 h, 2.5 h, 2.8 h or 3 h.

[0053] Other specific point values within the above numerical ranges can be selected and will not be described in detail here.

[0054] Preferably, in step (3), the centrifugal speed is 8000-15000g, for example, it can be 8000g, 9000g, 10000g, 11000g, 12000g, 13000g, 14000g or 15000g, etc.; the time is 15-30min, for example, it can be 15min, 16min, 17min, 18min, 19min, 20min, 21min, 22min, 23min, 24min, 25min, 26min, 27min, 28min, 29min or 30min, etc.

[0055] Other specific point values within the above numerical ranges can be selected and will not be described in detail here.

[0056] Preferably, the buffer comprises any one of HEPES buffer, Tris-HCl buffer or phosphate buffer.

[0057] In a second aspect, the present invention provides application of the method described in the first aspect in vaccine quality control.

[0058] Compared with the prior art, the present invention has the following beneficial effects:

[0059] The present invention combines size-exclusion high-performance liquid chromatography with multi-angle laser light scattering (SEC-MALLS) technology to accurately quantify the particle concentration of vesicular stomatitis virus. Compared with conventional titer detection, this method has a better correlation between the number of virus particles measured and the dilution factor, has high accuracy, and is widely used in basic virus research and vaccine quality control. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 This is a linear fitting graph of the concentration and dilution ratio of vesicular stomatitis virus VSV-H1N1 particles detected in Example 1;

[0061] Figure 2 This is the linear fitting graph of the TCID50 method for the detection of vesicular stomatitis virus VSV-H1N1 particle concentration and dilution multiple;

[0062] Figure 3 The linear fitting diagram of the detection method of Example 1 and the TCID50 method;

[0063] Figure 4 It is a fitting diagram of the ultraviolet absorption spectrum (dashed line) of the virus particles after size exclusion high performance liquid chromatography separation in Example 1 and the concentration spectrum (solid line) of the virus particles after detection and analysis by multi-angle laser light scattering instrument. DETAILED DESCRIPTION

[0064] In order to further illustrate the technical means and effects adopted by the present invention, the technical solutions of the present invention are further described below in conjunction with the preferred embodiments of the present invention, but the present invention is not limited to the scope of the embodiments.

[0065] If no specific techniques or conditions are specified in the examples, the experiments were carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0066] Multi-angle laser light scattering instrument (Wyatt Technology Corporation, HELEOS); Agilent BioSEC-5 (Agilent Technologies, column length 300 mm, inner diameter 7.8 mm, particle size 5 μm, pore size ); SRT SEC1000 (Saifen Technology, column length 300 mm, inner diameter 7.8 mm, particle size 5 μm, pore size ); TSKgel G5000PWXL (Tosoh (Shanghai) Biotechnology Co., Ltd., column length 300 mm, inner diameter 7.8 mm, particle size 10 μm, pore size ); Vero cells (ATCC CCL-81); nuclease (MilliporeSigma TM ); All water used in this experiment meets the requirements of first-class water in GB / T6682.

[0067] Viral vector preparation: Based on the complete genome sequence of the Indiana strain of VSV (GenBank: NC 001560.1), reverse genetic elements such as a hammerhead ribozyme, a T7 promoter, an eGFP tag sequence, and multiple restriction sites were added. The modified VSV genome sequence was cloned into the pCDNA3.1(+) vector to construct the backbone plasmid P-VSV-eGFP (viral VSV-eGFP). Subsequent modifications were based on this viral vector. The H1N1 (influenza virus H1N1) HA gene was amplified by PCR and inserted into the P-VSV-eGFP plasmid to construct the recombinant plasmid P-VSV-H1N1. The N, P, L, and G genes of VSV were cloned into the pCDNA3.1(+) vector using the NheI and NotI restriction sites, respectively, to construct the helper plasmids P-VSV-N, P-VSV-P, P-VSV-L, and P-VSV-G. The recombinant plasmid P-VSV-H1N1 was co-transfected with a helper plasmid into BSR cells, where VSV genomic RNA was efficiently transcribed using the T7 RNA polymerase. After transfection, the cells developed syncytia and green fluorescence. Cytopathic effects and green fluorescence were observed after passage into Vero E6 cells, indicating successful rescue of the recombinant virus VSV-H1N1. To construct the full-length VSV pseudovirus plasmid for the VSV-Spike recombinant vector, the S gene of the SARS-CoV-2 mink variant was amplified by PCR and inserted into the P-VSV-eGFP plasmid, replacing the G gene. This recombinant plasmid, P-VSV-Spike, was constructed using the same procedures as above.

[0068] Virus infection of Vero cells: Vero cells in logarithmic growth phase were cultured in DMEM medium containing 10% FBS (1% double antibody added) to a volume of 2×10 5 / mL cell suspension, inoculate 100 μL / well into a 96-well cell culture plate and incubate at 37°C, 5% CO2 for 24 hours to obtain a monolayer of adherent Vero cells for later use. Rapidly thaw the virus at 37°C and serially dilute it into serum-free DMEM medium to obtain a gradient of virus concentrations. Add the serially diluted virus solution to a 96-well cell culture plate, setting up 8 replicates for each gradient. Incubate at 37°C, 5% CO2 for 48 hours, and collect the virus-infected Vero cell culture fluid.

[0069] Example 1

[0070] This embodiment provides a method for quantitative detection of vesicular stomatitis virus, the method comprising:

[0071] 1. Sample solution pretreatment:

[0072] (1) 2 mL of cell culture medium infected with vesicular stomatitis virus (VSV-H1N1) was centrifuged at 3000 g for 10 min to remove impurities and collect the liquid;

[0073] (2) Add 50 U / mL nuclease and react at 37°C for 30 min, then add PEG 6000 (the mass percentage in the pretreatment system is 10%) and let it stand for 1 h;

[0074] (3) Centrifuge at 12000g for 20 min, collect the precipitate and resuspend in 200 μL HEPES buffer.

[0075] 2. Separation, detection and analysis:

[0076] (1) Separate the pretreated sample solution using size exclusion high performance liquid chromatography:

[0077] Mobile phase: 50 mM HEPES buffer, 100 mM sodium sulfate, 5% arginine, 6% glycine, and 4% glycerol, pH 7.4;

[0078] Chromatographic column: Agilent Bio SEC-5, column temperature 25°C;

[0079] Parameter settings: injection volume 100 μL, flow rate 1.0 mL / min, elution time 30 min, detection wavelength 658 nm;

[0080] (2) The scattered light signal was imported into the number intensity analysis model by using a multi-angle laser light scattering instrument (8 detection angles), the particle refractive index (RI) was set to 1.55, and the sphere model was selected. After analysis and processing, the particle concentration of vesicular stomatitis virus was obtained.

[0081] Example 2

[0082] This embodiment provides a method for quantitative detection of vesicular stomatitis virus, the method comprising:

[0083] 1. Sample solution pretreatment:

[0084] (1) 2 mL of cell culture medium infected with vesicular stomatitis virus (VSV-Spike) was centrifuged at 2000 g for 15 min to remove impurities and collect the liquid;

[0085] (2) Add 100 U / mL nuclease, react at 25°C for 60 min, then add PEG 4000 (the mass percentage in the pretreatment system is 15%), and let it stand for 0.5 h;

[0086] (3) Centrifuge at 8000 g for 30 min, collect the precipitate and resuspend in 200 μL Tris-HCl buffer.

[0087] 2. Separation, detection and analysis:

[0088] (1) Separate the pretreated sample solution using size exclusion high performance liquid chromatography:

[0089] Mobile phase: 100 mM Tris-HCl buffer, 300 mM magnesium nitrate, 10% arginine, 10% glycine, and 10% glycerol, pH 6.0;

[0090] Chromatographic column: TSKgel G3000SWXL, column temperature 4°C;

[0091] Parameter settings: injection volume 80 μL, flow rate 0.8 mL / min, elution time 40 min, detection wavelength 658 nm;

[0092] (2) The scattered light signal was imported into the number intensity analysis model by using a multi-angle laser light scattering instrument (18 detection angles), the particle refractive index (RI) was set to 1.55, and the sphere model was selected. After analysis and processing, the particle concentration of vesicular stomatitis virus was obtained.

[0093] Example 3

[0094] This embodiment provides a method for quantitative detection of vesicular stomatitis virus, the method comprising:

[0095] 1. Sample solution pretreatment:

[0096] (1) 2 mL of cell culture fluid infected with vesicular stomatitis virus (VSV)-eGFP was filtered through a 0.22 μm pore size sterile filter membrane to remove impurities and collect the liquid;

[0097] (2) Add 10 U / mL nuclease, react at 30°C for 10 min, then add ammonium sulfate (the concentration in the pretreatment system is 1.0 M) and let it stand for 3 h;

[0098] (3) Centrifuge at 15000g for 15 min, collect the precipitate and resuspend in 200 μL phosphate buffer.

[0099] 2. Separation, detection and analysis:

[0100] (1) Separate the pretreated sample solution using size exclusion high performance liquid chromatography:

[0101] Mobile phase: 200 mM phosphate buffer, 100 mM potassium chloride, 4% arginine, 3% glycine, and 3% glycerol, pH 8.0;

[0102] Chromatographic column: TSKgel G4000SWXL, column temperature 40°C;

[0103] Parameter settings: injection volume 70 μL, flow rate 0.5 mL / min, elution time 60 min, detection wavelength 658 nm;

[0104] (2) The scattered light signal was imported into the number intensity analysis model by using a multi-angle laser light scattering instrument (3 detection angles), the particle refractive index (RI) was set to 1.55, and the sphere model was selected. After analysis and processing, the particle concentration of vesicular stomatitis virus was obtained.

[0105] Example 4

[0106] This embodiment provides a method for quantitative detection of vesicular stomatitis virus, the method comprising:

[0107] 1. Sample solution pretreatment:

[0108] (1) 2 mL of cell culture medium infected with vesicular stomatitis virus (VSV-H1N1) was centrifuged at 3000 g for 10 min to remove impurities and collect the liquid;

[0109] (2) The collected liquid was centrifuged at 12000 g for 20 min, and the precipitate was collected and resuspended in 200 μL HEPES buffer.

[0110] 2. Separation, detection and analysis:

[0111] (1) Separate the pretreated sample solution using size exclusion high performance liquid chromatography:

[0112] Mobile phase: 50 mM HEPES buffer, 100 mM sodium sulfate, 5% arginine, 6% glycine, and 4% glycerol, pH 7.4;

[0113] Chromatographic column: Agilent Bio SEC-5, column temperature 25°C;

[0114] Parameter settings: injection volume 100 μL, flow rate 1.0 mL / min, elution time 30 min, detection wavelength 658 nm;

[0115] (2) The scattered light signal was imported into the number intensity analysis model by using a multi-angle laser light scattering instrument (8 detection angles), the particle refractive index (RI) was set to 1.55, and the sphere model was selected. After analysis and processing, the particle concentration of vesicular stomatitis virus was obtained.

[0116] Example 5

[0117] This embodiment provides a method for quantitative detection of vesicular stomatitis virus, which differs from Example 1 only in that the chromatographic column is replaced from "Agilent Bio SEC-5" to "SRT SEC 1000", and the other steps remain unchanged.

[0118] Example 6

[0119] This embodiment provides a method for quantitative detection of vesicular stomatitis virus, which differs from Example 1 only in that the chromatographic column is replaced from "Agilent Bio SEC-5" to "TSKgel G5000PWXL", and the other steps remain unchanged.

[0120] Example 7

[0121] This example provides a quantitative detection method for vesicular stomatitis virus, which differs from Example 1 only in that arginine is not added to the mobile phase (pH 7.4), and its reduced amount is proportionally distributed to glycine and glycerol, while other steps remain unchanged.

[0122] Example 8

[0123] This example provides a quantitative detection method for vesicular stomatitis virus, which differs from Example 1 only in that glycine is not added to the mobile phase (pH 7.4), and its reduced amount is proportionally distributed to arginine and glycerol, while other steps remain unchanged.

[0124] Example 9

[0125] This example provides a quantitative detection method for vesicular stomatitis virus, which differs from Example 1 only in that glycerol is not added to the mobile phase (pH 7.4), and its reduced amount is proportionally distributed to arginine and glycine, while other steps remain unchanged.

[0126] Example 10

[0127] This example provides a method for quantitative detection of vesicular stomatitis virus, which differs from Example 1 only in that the mobile phase (pH 7.4) is 50 mM HEPES buffer, 100 mM sodium sulfate, 5% arginine, 6% glycine, 4% glycerol, and 10% ethanol, and the other steps remain unchanged.

[0128] Comparative Example 1

[0129] This comparative example provides a quantitative detection method for vesicular stomatitis virus, which differs from Example 1 only in that arginine, glycine and glycerol are not added to the mobile phase, and other steps remain unchanged.

[0130] Test Example 1

[0131] After 48 hours of infection of Vero cells with graded dilutions of vesicular stomatitis virus VSV-H1N1, the cell morphology and fluorescence changes were observed, the number of cytopathic effect (CPE) wells was determined, and the TCID50 of the virus was calculated using the Reed-Muench method. No pathogen solution was added as a blank control.

[0132] Log10(TCID50)=L+d(s-0.5)+log10(1 / v)

[0133] L = Log10 highest dilution (e.g., the highest dilution is 10-fold dilution, L = 1)

[0134] V = initial volume of cell culture medium per well (mL / well)

[0135] d = Log10 dilution (e.g. 10-fold dilution, d = 1)

[0136] s = sum of GFP ratios of each gradient;

[0137] Example 1 Detection of the linear fit of vesicular stomatitis virus VSV-H1N1 particle concentration and dilution multiple Figure 1 As shown in Figure 2, the detection method of the present invention can achieve accurate quantification of vesicular stomatitis virus, and there is a higher correlation between the number of virus particles and the dilution factor (R 2 =0.99); the linear fitting of TCID50 method for detecting vesicular stomatitis virus VSV-H1N1 particle concentration and dilution multiple is as follows Figure 2 As shown in Figure 2, the correlation between the number of virus particles and the dilution factor (R 2 =0.95) is lower than the present invention; the linear fit of the detection method of Example 1 and the TCID50 method is as follows Figure 3 As shown, the detection trend of the detection method of the present invention is consistent with that of the traditional TCID50 method; Figure 4 It is a fitting diagram of the ultraviolet absorption spectrum (dashed line) of the virus particles after size exclusion high performance liquid chromatography separation in Example 1 and the concentration spectrum (solid line) of the virus particles after detection and analysis by multi-angle laser light scattering instrument.

[0138] Linear fitting correlation R between viral particle concentration and dilution factor detected in Examples 1-10 and Comparative Example 1 2The values are shown in Table 1: In the present invention, nuclease is added during sample pretreatment, and precipitation is performed with polyethylene glycol or ammonium sulfate, which can remove interfering impurities and concentrate virus particles, thereby improving the relevance and accuracy of virus particle concentration detection; AgilentBio SEC-5, TSKgel G3000SWXL, and TSKgel G4000SWXL chromatographic columns are further preferred, which have better selectivity for vesicular stomatitis virus; arginine, glycine, and glycerol have a synergistic effect, which can improve the integrity and stability of virus particles in the mobile phase, further improving the accuracy of detection; the mobile phase composition setting of the present invention is conducive to the separation of vesicular stomatitis virus particles, and the addition of organic solvents in the mobile phase will affect the separation effect.

[0139] Table 1

[0140]

[0141]

[0142] The present invention uses the above-described embodiments to illustrate the quantitative detection method for vesicular stomatitis virus and its applications. However, the present invention is not limited to the above-described embodiments, and implementation of the present invention is not necessarily dependent on the above-described embodiments. Those skilled in the art will appreciate that any improvements to the present invention, equivalent substitutions for raw materials in the products of the present invention, addition of auxiliary ingredients, and selection of specific methods, etc., fall within the scope of protection and disclosure of the present invention.

[0143] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the scope of protection of the present invention.

[0144] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any appropriate manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. A quantitative detection method for vesicular stomatitis virus, characterized in that: The method comprises: separating a sample solution containing vesicular stomatitis virus using a size exclusion high performance liquid chromatograph, detecting and analyzing the sample solution using a multi-angle laser light scattering instrument to obtain the particle concentration of the vesicular stomatitis virus; The mobile phase of the size exclusion high performance liquid chromatography comprises a buffer, an inorganic salt and a stabilizer.

2. The method according to claim 1, characterized in that The concentration of the buffer in the mobile phase is 5-200 mM, the concentration of the inorganic salt is 5-500 mM, and the mass percentage of the stabilizer is 10-30%; Preferably, the pH value of the mobile phase is 6.0-8.

0.

3. The method according to claim 1 or 2, characterized in that The buffer comprises any one of HEPES buffer, Tris-HCl buffer or phosphate buffer or a combination of at least two thereof; Preferably, the inorganic salt comprises any one or a combination of at least two of sodium sulfate, magnesium sulfate, potassium sulfate, ammonium sulfate, sodium nitrate, magnesium nitrate, potassium nitrate, ammonium nitrate, sodium chloride, magnesium chloride, potassium chloride or ammonium chloride; Preferably, the stabilizer comprises any one or a combination of at least two of arginine, glycine, glycerol, trehalose, ethylenediaminetetraacetic acid, polyethylene glycol, sucrose, ethanol or dimethyl sulfoxide; Preferably, the stabilizer comprises arginine, glycine and glycerol.

4. The method according to any one of claims 1 to 3, characterized in that The injection volume of the size exclusion HPLC is 1-100 μL, the flow rate is 0.1-1.0 mL / min, and the elution time is 30-60 min; Preferably, the size exclusion high performance liquid chromatography column comprises any one of Agilent Bio SEC-5, TSKgelG3000SWXL, TSKgel G4000SWXL, SRT SEC 2000, SRT SEC 1000 or TSKgel G5000PWXL; Preferably, the column temperature of the chromatographic column is 4-40°C; Preferably, the detection wavelength of the size exclusion high performance liquid chromatography is 500-800 nm.

5. The method according to any one of claims 1 to 4, characterized in that The multi-angle laser light scattering instrument has a detection angle number of 3-18; Preferably, the analysis model includes a Number intensity analysis model; Preferably, the detector for the detection further includes any one of an ultraviolet detector, a differential detector, a fluorescence detector, a viscosity detector or a dynamic laser scatterer, or a combination of at least two thereof.

6. The method according to any one of claims 1 to 5, characterized in that The sample solution containing vesicular stomatitis virus includes a cell culture fluid infected with vesicular stomatitis virus; Preferably, the cell culture fluid infected with vesicular stomatitis virus undergoes a pretreatment step before separation using size exclusion high performance liquid chromatography; Preferably, the pretreatment step specifically comprises: (1) centrifuging or filtering the cell culture fluid infected with vesicular stomatitis virus to remove impurities and collect the liquid; (2) Add nuclease reaction, then add polyethylene glycol or ammonium sulfate, and let it stand; (3) Centrifuge, collect the precipitate and resuspend it in buffer.

7. The method according to claim 6, characterized in that In step (1), the centrifugation speed is 2000-4000g and the time is 5-15min; Preferably, the pore size of the filter is 0.1-2 μm.

8. The method according to claim 6, characterized in that In step (2), the dosage of the nuclease is 10-100 U / mL; Preferably, the reaction temperature is 25-37°C and the reaction time is 10-60 min; Preferably, the molecular weight of the polyethylene glycol is 4000-12000; Preferably, the mass percentage of the polyethylene glycol in the pretreatment system is 5-20%; Preferably, the concentration of ammonium sulfate in the pretreatment system is 0.5-2.0 M; Preferably, the standing time is 0.5-3h.

9. The method according to claim 6, characterized in that In step (3), the centrifugal speed is 8000-15000g and the time is 15-30min; Preferably, the buffer comprises any one of HEPES buffer, Tris-HCl buffer or phosphate buffer.

10. Use of the method according to any one of claims 1 to 9 in vaccine quality control.

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