WO2022077592A1 - Virus preservation reagent - Google Patents

Virus preservation reagent Download PDF

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WO2022077592A1
WO2022077592A1 PCT/CN2020/125326 CN2020125326W WO2022077592A1 WO 2022077592 A1 WO2022077592 A1 WO 2022077592A1 CN 2020125326 W CN2020125326 W CN 2020125326W WO 2022077592 A1 WO2022077592 A1 WO 2022077592A1
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virus
vaccine
reagent
preservation
virus preservation
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PCT/CN2020/125326
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French (fr)
Chinese (zh)
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黄文林
周晓鸿
田烁
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广州达博生物制品有限公司
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
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    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
    • C12N2710/10343—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
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    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention belongs to the technical field of biomedicine, and particularly relates to a virus preservation reagent.
  • Gene therapy is a therapeutic method in which exogenous genes are introduced into target cells to express the corresponding proteins. Gene therapy can cure diseases by correcting or compensating for gene defects or abnormal gene expression. In recent years, a number of gene therapy projects have been approved for marketing or entered into clinical trials in many countries around the world.
  • gene therapy mainly integrates foreign genes into different types of viral vectors, and then uses the recombinant viruses to introduce them into target cells. Due to their high efficiency and modifiability, viral vectors have also become powerful tools for vaccine development.
  • viruses are relatively harsh, and generally need to be stored in a relatively stable -80°C environment.
  • meeting such transportation conditions or storage conditions requires higher equipment and higher costs, and is prone to temperature instability, resulting in virus inactivation. Therefore, a virus preservation reagent that can keep the virus active at higher temperatures plays a crucial role in reducing logistics costs, maintaining virus activity, and ensuring the effectiveness of viral vaccines and gene therapy products.
  • Existing virus preservation reagents have complex components or contain antibiotics, and are only suitable for in vitro diagnostic tests. For vaccines or therapeutic recombinant viruses that need to be injected into the human body, most of the existing virus preservation reagents do not have sufficient safety and effectiveness.
  • Cida 201711464688.9 discloses a virus quality control product of a stably preserved hepatitis C virus gene detection kit, the quality control product includes a hepatitis C virus (HCV) virus preservative, and the preservative contains auric acid tricarboxylic acid Ammonium salt, preservative, bestatin hydrochloride and further control the concentration of ammonium triformate, preservative, bestatin hydrochloride in the quality control product were 100-500 ⁇ M, 80-180mM, 20-60nM, effectively inhibits the degradation of the quality control virus particles, making it more stable, easier to store, and prolonging the validity period.
  • HCV hepatitis C virus
  • Chinese patent 201910611068.6 discloses a virus preservation solution that can effectively preserve viruses and other samples for a long time, and is composed of the following components: calcium chloride 0.1-3.5g, potassium chloride 0.07-1.49g, sodium chloride 0.76-2.76g, Magnesium chloride 0.01-0.91g, magnesium sulfate 0.01-1.93g, sugar 0.1-0.361g, potassium dihydrogen phosphate 0.01-0.72g, disodium hydrogen phosphate 0.05-0.12g, sodium bicarbonate 0.007-0.67g, bovine serum albumin 0.1 -1g, antibiotic 0.6-5.3g, 4-hydroxyethylpiperazine ethanesulfonic acid 0.236-0.96g, phenol red 0.01-1g, L-cysteine hydrochloride 0.01-3.52g, L-glutamic acid 0.28 -2.95g, dilute to 100mL with water, and adjust pH to 7.2-7.8.
  • the preservation reagent has many outstanding effects, but
  • Chinese Patent No. 202010571607.0 discloses a virus preservation solution that is highly compatible with a magnetic bead method virus nucleic acid extraction kit, which contains the following components: guanidine hydrochloride, tris(hydroxymethyl)aminomethane hydrochloride, ethylenediaminetetraacetic acid, isopropylamine Propanol or ethanol.
  • the virus preservation solution has simple components, readily available raw materials, and low cost. It can store samples at room temperature for a long time without high temperature inactivation. It is very safe for operators and the environment. At the same time, it reduces the possibility of RNA degradation and can obtain more nucleic acid, reducing the missed detection rate.
  • the preservation solution can be applied to vaccines or therapeutic recombinant viruses injected into the human body needs further research.
  • the present invention provides a virus preservation reagent.
  • the preservation reagent provided by the present invention has simple components, low toxicity to cells, and can stabilize the virus for a long time and maintain the virus activity under different environmental conditions.
  • the present invention provides a virus preservation reagent.
  • the virus preservation reagent includes the following components: NaCl, Tris-base, MgCl 2 ⁇ 6H 2 O, glycerol, and water.
  • NaCl content is 5.85 ⁇ 0.585g
  • Tris-base is 1.21 ⁇ 0.121g
  • MgCl 2 ⁇ 6H 2 O is 0.2 ⁇ 0.02g
  • glycerol is 100 ⁇ 10mL
  • water for injection is supplemented to 1000mL .
  • each component is: in each 1L virus preservation reagent: NaCl content is 5.85g, Tris-base is 1.21g, MgCl 2 ⁇ 6H 2 O is 0.2g, glycerol is 100mL, and water for injection is supplemented to 1000mL .
  • the pH of the virus preservation reagent is 7.4-8.4; preferably 7.6-8.2; the pH value is adjusted by HCl.
  • viruses include but are not limited to adenoviruses, lentiviruses, and retroviruses.
  • the virus is a recombinant adenovirus.
  • the present invention provides the application of the aforementioned virus preservation reagent in the preparation of vaccines.
  • the vaccine includes a viral vector.
  • viruses targeted by the vaccine include but are not limited to coronaviruses and influenza viruses; preferably SARS-CoV-2 coronaviruses.
  • each 1 mL of virus preservation reagent contains 0.5 ⁇ 10 11 to 1 ⁇ 10 12 vp virus vector, preferably 1 ⁇ 10 11 vp.
  • the viral vaccine is an adenovirus vector vaccine.
  • the vaccine is a SARS-CoV-2 coronavirus vaccine; the SARS-CoV-2 coronavirus vaccine includes a type 5 adenovirus of subclass C with complete deletion, partial deletion or no deletion of E1 and E3 regions.
  • the present invention provides a vaccine.
  • the vaccine includes the aforementioned virus vaccine preservation reagent.
  • the vaccine includes a viral vector.
  • viruses targeted by the vaccine include but are not limited to coronaviruses and influenza viruses; preferably SARS-CoV-2 coronaviruses.
  • each 1 mL of virus preservation reagent contains 0.5 ⁇ 10 11 to 1 ⁇ 10 12 vp virus vector, preferably 1 ⁇ 10 11 vp.
  • the viral vaccine is an adenovirus vector vaccine.
  • the vaccine is a SARS-CoV-2 coronavirus vaccine; the SARS-CoV-2 coronavirus vaccine includes a type 5 adenovirus of subclass C with complete deletion, partial deletion or no deletion of E1 and E3 regions.
  • the present invention provides a method for preparing the aforementioned virus preservation reagent.
  • the described preparation method comprises the following steps:
  • Necessary items in the step (4) include but are not limited to: cytotoxicity, bacterial content, stability, and reconstitution stability.
  • the present invention provides a method for preparing the aforementioned vaccine.
  • the preparation method includes: replacing the solvent of the crude recombinant coronavirus vaccine product with the aforementioned virus preservation solution by using a hollow fiber column for ultrafiltration replacement.
  • Described ultrafiltration replacement step is as follows:
  • Fig. 1 is the cytotoxicity of virus preservation reagent
  • Figure 2 shows the stability test results of recombinant coronavirus vaccines in different pH virus storage reagents under the condition of 37°C;
  • Figure 3 shows the stability test results of recombinant coronavirus vaccines in different virus storage reagents at 4°C;
  • Figure 4 shows the stability test results of recombinant coronavirus vaccines in different virus storage reagents at 25°C;
  • Figure 5 shows the accelerated stability test results of recombinant coronavirus vaccines in different virus preservation reagents at 37°C;
  • Figure 6 shows the results of repeated freeze-thaw stability testing of recombinant coronavirus vaccines in different virus storage reagents.
  • Embodiment 1 A kind of virus vaccine preservation reagent
  • the virus vaccine preservation reagent of this embodiment includes the following components: NaCl, Tris-base, MgCl 2 ⁇ 6H 2 O, glycerol, and water.
  • Example 1 The cytotoxicity of the virus vaccine preservation reagent provided in Example 1 was detected, and a comparative example was set.
  • the components of the virus preservation reagent of the comparative example are as follows:
  • Comparative Example 1 PBS preservation solution: Na 2 HPO 4 8 mM, KH 2 PO 4 42 mM, NaCl 136 mM and KCl 2.6 mM.
  • Comparative Example 2 (Hank's preservation solution, conventional formula): NaCl, KCl, KH 2 PO 4 , Na 2 HPO 4 , NaHCO 3 , CaCl 2 , MgCl 2 , MgSO 4 .
  • 293 cells were digested and subcultured to about 90% confluence. Cells were routinely digested, and cell suspensions were collected and counted. Prepare 4 mL of 1 ⁇ 10 5 /mL 293 cell suspension with 10% FBS DMEM (FBS was purchased from YOSHI, the product number is A1025; DMEM was purchased from Gibco, the product number is C11995500BT). , 100 ⁇ L/well, that is, 1 ⁇ 10 4 cells per well. Only 100 ⁇ L of medium was added to each well of the remaining four sides, and cultured at 37° C. and 5% CO 2 for 24 h.
  • FBS FBS was purchased from YOSHI, the product number is A1025
  • DMEM was purchased from Gibco, the product number is C11995500BT
  • Group B (10%FBS DMEM 95 ⁇ L+PBS preservation solution 5 ⁇ L) ⁇ 8
  • Hank's Balanced Salt Solution and PBS are phosphate buffers commonly used in cell separation or culture. They combine the buffering capacity of the buffer with the isotonicity of normal saline, and can maintain osmotic pressure and maintain pH stability. Hank's Balanced Salt Solution is basically harmless to cells. After the virus preservation solution is added to cells, the inhibition rate of cell growth is 12%, which is less than the inhibition rate of Hank's solution of 13.2%. In general, the virus preservation solution is less toxic to cells, lower than Hank's balanced salt solution, and slightly higher than PBS.
  • Example 3 A recombinant coronavirus vaccine
  • Recombinant coronavirus vaccine was prepared by adopting the virus vaccine preservation reagent provided in Example 1, mainly by means of ultrafiltration replacement.
  • the recombinant coronavirus vaccine adopted in this example is the SARS-CoV-2 coronavirus vaccine, and the preparation steps of the vaccine before ultrafiltration replacement are as follows:
  • S gene of the SARS-CoV-2 coronavirus is optimized, and the sequence of the optimized S gene is SEQ ID NO: 3.
  • the amplification primers are V1 and V2, their respective sequences are shown in SEQ ID NOs: 1-2, VI and V2 are a pair, and the C-terminal fragment S1C of the S1 gene is amplified.
  • the ligation product was transformed into Escherichia coli DH5a, positive clones were screened by ampicillin resistance, and clones were selected for colony PCR identification. Plasmids were extracted from positive colonies after culture and sent for sequencing to confirm the sequence.
  • the collection of viruses adopts the method of picking plaques: low melting point agarose is added to the culture medium, and small plaques can be seen under the microscope on the 10th to 21st day after transfection. After plaque formation, the plaques were picked up with agarose and placed in 1 mL of fresh medium overnight. Usually, 3-6 plaques are picked, and then the titers are compared, and the plaque with the highest titer is used for subsequent experiments.
  • the virus in the medium was added to the fresh 293 cell culture medium for a small amount of virus amplification.
  • plaques appeared in the cells again the cells and the supernatant were collected, and the virus was collected by repeated freezing and thawing three times, and this virus was used as the P1 generation virus.
  • the product obtained after purification is the purified product of the SARS-CoV-2 vaccine.
  • the SARS-CoV-2 vaccine contains the S1C gene of the SARS-CoV-2 virus and a defective adenovirus.
  • the defective adenovirus is a type 5 adenovirus of subclass C with complete deletion of the E1 and E3 regions, and cannot replicate in ordinary human cells.
  • the ultrafiltration replacement steps are as follows:
  • Example 4 Stability detection of recombinant coronavirus vaccine in different pH virus preservation reagents under the condition of 37°C
  • step (6) the virus preservation reagents without pH adjustment are divided into 5 groups, and different amounts of HCl are added respectively, and are monitored with a pH meter to obtain pH values of 7.63, 7.79, Virus preservation reagents of 8.03, 8.21, 8.43.
  • Example 3 it is distinguished from the ultrafiltration replacement step (3) in the solution replacement step, and the sample after the ultrafiltration concentration is carried out solution replacement with the virus preservation solution of different pH respectively, to obtain the different recombinant coronavirus vaccines of solution pH, and The series of recombinant coronavirus vaccines were tested for stability.
  • coronavirus vaccines were grouped, and each group was placed at 37°C for 24 hours, and the corresponding virus infection titers were detected.
  • the detection method is as follows:
  • the virus preservation solution When the pH of the virus preservation solution is between 7.63-8.21, the virus can maintain a high activity.
  • Example 5 Stability detection of recombinant coronavirus vaccines in different virus preservation reagents at 4°C
  • Stability detection was carried out on the recombinant coronavirus vaccine provided in Example 3, and a comparative example was set.
  • the components of the virus preservation reagent of the comparative example are the same as those of Comparative Example 1 and Comparative Example 2 in Example 2.
  • the recombinant coronavirus vaccines provided in Example 3 were grouped, and each group was placed at 4°C for 3 days, 7 days, and 14 days, and the virus infection titers under the corresponding days were detected.
  • the detection method refers to Example 4.
  • the results are shown in the following table and Figure 3.
  • the following table shows the results of the virus infection titer test.
  • the virus preservation solution of this application PBS preservation solution Hank's Preservative Solution Day 3 100.2% 100% 100% Day 7 98% 75.8% 82.3% Day 14 95% 39.5% 39.7%
  • the recombinant coronavirus vaccine was stored in the virus preservation solution of the present application for 14 days, and the virus infection titer only decreased by 5%. Under the same conditions, when the recombinant coronavirus vaccine was stored in PBS preservation solution for 14 days, the virus infection titer decreased by 60.5%; when stored in Hank's preservation solution for 14 days, the virus infection titer decreased by 60.3%.
  • the recombinant coronavirus vaccine can still maintain a high infectious activity within 14 days in the virus preservation solution provided in this application.
  • Example 6 Stability detection of recombinant coronavirus vaccine in different virus preservation reagents under the condition of 25°C
  • Stability detection was carried out on the recombinant coronavirus vaccine provided in Example 3, and a comparative example was set.
  • the components of the virus preservation reagent of the vaccine of the comparative example are the same as those of Comparative Example 1 and Comparative Example 2 in Example 2.
  • the recombinant coronavirus vaccines provided in Example 3 were grouped, and each group was placed at 25°C for 2h, 4h, 8h, and 24h, and the virus infection titers at the corresponding time points were detected.
  • the detection method refers to Example 4.
  • the results are shown in the following table and Figure 4.
  • the following table shows the results of the virus infection titer test.
  • the virus preservation solution of this application PBS preservation solution Hank's Preservative Solution 2h 99.2% 100% 100% 4h 99.2% 100% 80% 8h 99.2% 75.1% 64.3% 24h 93% 62.5% 32%
  • the recombinant coronavirus vaccine was stored in this virus preservation solution for 8 hours, and the virus infection titer remained basically unchanged; after 24 hours of storage, the virus infection titer only decreased by 7%.
  • the viral infection titer was lower than 80%, and the infection titer decreased at a higher rate.
  • the recombinant coronavirus vaccine can still maintain a high infectious activity within 24 hours in this virus preservation solution.
  • Stability detection was carried out on the recombinant coronavirus vaccine provided in Example 3, and a comparative example was set.
  • the components of the virus preservation reagent in the vaccine of the comparative example are the same as those of Comparative Example 1 and Comparative Example 2 in Example 2.
  • the recombinant coronavirus vaccines provided in Example 3 were grouped, and each group was placed at 37°C for 1 day, 3 days, 5 days, and 7 days, and the virus infection titers at the corresponding time points were detected.
  • the detection method refers to Example 4.
  • the results are shown in the following table and Figure 5.
  • the following table shows the results of the virus infection titer test.
  • the virus preservation solution of this application PBS preservation solution Hank's Preservative Solution 1 day 98% 83.5% 62.8% 3 days 91.2% 69.7% 62.8% 5 days 91.2% 69.7% 50.3%
  • the recombinant coronavirus vaccine is stored in the virus preservation solution provided by the present invention for 5 days, and the virus infection titer is basically maintained, which can be maintained above 90%. Under the same conditions, after the recombinant coronavirus vaccine was stored in PBS or Hank's preservation solution for 5 days, the viral infection titer dropped sharply.
  • the recombinant coronavirus vaccine can still maintain a high infectious activity within 5 days in the virus preservation solution of the present application; while in other types of preservation solutions, the recombinant coronavirus vaccine After the vaccine was stored for 1 day, the viral infection titer activity decreased significantly.
  • Example 8 Repeated freeze-thaw stability detection of recombinant coronavirus vaccine in different virus preservation reagents
  • Example 3 Repeated freeze-thaw stability testing was performed on the recombinant coronavirus vaccine provided in Example 3, and a comparative example was set.
  • the components of the virus preservation reagent in the vaccine of the comparative example are the same as those of Comparative Example 1 and Comparative Example 2 in Example 2.
  • the recombinant coronavirus vaccines provided in Example 3 were grouped, and after each group was freeze-thawed 3 times, 5 times, and 10 times, the corresponding virus infection titers were detected.
  • the detection method refers to Example 4.
  • the results are shown in the following table and Figure 6.
  • the following table shows the results of the virus infection titer test.
  • This virus preservation solution PBS preservation solution Hank's Preservative Solution 3 times 99.2% 85.9% 85.9% 5 times 101.2% 89.8% 89.8% 10 times 98.6% 89.8% 79%
  • the viral infection titer decreased to varying degrees.
  • the recombinant coronavirus vaccine can maintain high viral activity after repeated freezing and thawing 3-10 times in this virus preservation solution.

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Abstract

Provide is a virus preservation reagent. Every 1 L of the virus preservation reagent comprises 5.85±0.585 g of NaCl, 1.21±0.121 g of Tris-base, 0.2±0.02 g of MgCl2·6H2O, 100±10 mL of glycerin, and 1000 mL of injection water complemented. The preservation reagent is simple in component and low in cytotoxicity, has the effects of stabilizing viruses and maintaining virus activity for a long time under different environmental conditions, and can be applied to preparation of vaccines taking viruses as vectors.

Description

一种病毒保存试剂A virus preservation reagent 技术领域technical field
本发明属于生物医药技术领域，具体涉及一种病毒保存试剂。The invention belongs to the technical field of biomedicine, and particularly relates to a virus preservation reagent.
背景技术Background technique
基因治疗(Gene therapy)是一种将外源基因导入靶细胞，以表达相应蛋白的治疗手段。基因治疗能通过纠正或补偿基因缺陷或基因的表达异常，从而达到治疗疾病的目的。近几年来，多项基因治疗项目相继在世界上许多国家获得批准上市或进入临床实验。Gene therapy is a therapeutic method in which exogenous genes are introduced into target cells to express the corresponding proteins. Gene therapy can cure diseases by correcting or compensating for gene defects or abnormal gene expression. In recent years, a number of gene therapy projects have been approved for marketing or entered into clinical trials in many countries around the world.
目前，基因治疗主要通过将外源基因整合入不同类型的病毒载体中，再利用该重组病毒将其导入靶细胞中。由于其高效性及可改造性，病毒载体目前也成为了疫苗开发的的有力工具。At present, gene therapy mainly integrates foreign genes into different types of viral vectors, and then uses the recombinant viruses to introduce them into target cells. Due to their high efficiency and modifiability, viral vectors have also become powerful tools for vaccine development.
病毒的保存条件相对苛刻，一般需要存储于相对稳定的-80℃环境中。但实际情况中，达到这样的运输条件或储存条件对设备的要求较高，成本较大，且容易发生温度不稳定，造成病毒失活的情况。因此，一款能让病毒在较高温度依然维持活性的病毒保存试剂，对于减少物流成本，维持病毒活性，保证病毒类疫苗和基因治疗产品的效果有着至关重要的作用。The storage conditions of viruses are relatively harsh, and generally need to be stored in a relatively stable -80°C environment. However, in actual situations, meeting such transportation conditions or storage conditions requires higher equipment and higher costs, and is prone to temperature instability, resulting in virus inactivation. Therefore, a virus preservation reagent that can keep the virus active at higher temperatures plays a crucial role in reducing logistics costs, maintaining virus activity, and ensuring the effectiveness of viral vaccines and gene therapy products.
现有的病毒保存试剂成分复杂或含有抗生素，只适用于体外诊断检测。而对于需要注射入人体的疫苗或治疗类的重组病毒，现有的病毒保存试剂多不具备足够的安全性和有效性。Existing virus preservation reagents have complex components or contain antibiotics, and are only suitable for in vitro diagnostic tests. For vaccines or therapeutic recombinant viruses that need to be injected into the human body, most of the existing virus preservation reagents do not have sufficient safety and effectiveness.
中国专利201711464688.9中公开了一种可稳定保存的丙型肝炎病毒基因检测试剂盒的病毒质控品，该质控品中包括丙型肝炎病毒(HCV)病毒保存剂，保存剂包含金精三甲酸铵盐、防腐剂、苯丁抑制素盐酸盐并进一步控制金精三甲酸铵盐、防腐剂、苯丁抑制素盐酸加入后在质控品中的浓度分别为100-500μM、80-180mM、20-60nM，有效抑制了质控品病毒颗粒的降解，使其更稳定，更易保存，延长了有效期。但其针对性较强，不适于广泛地应用，且安全性不确定。Chinese Patent No. 201711464688.9 discloses a virus quality control product of a stably preserved hepatitis C virus gene detection kit, the quality control product includes a hepatitis C virus (HCV) virus preservative, and the preservative contains auric acid tricarboxylic acid Ammonium salt, preservative, bestatin hydrochloride and further control the concentration of ammonium triformate, preservative, bestatin hydrochloride in the quality control product were 100-500μM, 80-180mM, 20-60nM, effectively inhibits the degradation of the quality control virus particles, making it more stable, easier to store, and prolonging the validity period. However, it has strong pertinence, is not suitable for wide application, and its safety is uncertain.
中国专利201910611068.6中公开了一种能够长时间有效保存病毒等样本的病毒保存液，由以下成分组成：氯化钙0.1-3.5g、氯化钾0.07-1.49g、氯化钠0.76-2.76g、氯化镁0.01-0.91g、硫酸镁0.01-1.93g、糖0.1-0.361g、磷酸二氢钾0.01-0.72g、磷酸氢二钠0.05-0.12g、碳酸氢钠0.007-0.67g、牛血清白蛋白0.1-1g、抗生素0.6-5.3g、4-羟乙基哌嗪乙磺酸0.236-0.96g、酚红 0.01-1g、L-半胱氨酸盐酸盐0.01-3.52g、L-谷氨酸0.28-2.95g，水定容至100mL，pH值调节至7.2-7.8。该保存试剂具有众多突出的效果，但成分复杂，且难以应用于治疗类病毒。Chinese patent 201910611068.6 discloses a virus preservation solution that can effectively preserve viruses and other samples for a long time, and is composed of the following components: calcium chloride 0.1-3.5g, potassium chloride 0.07-1.49g, sodium chloride 0.76-2.76g, Magnesium chloride 0.01-0.91g, magnesium sulfate 0.01-1.93g, sugar 0.1-0.361g, potassium dihydrogen phosphate 0.01-0.72g, disodium hydrogen phosphate 0.05-0.12g, sodium bicarbonate 0.007-0.67g, bovine serum albumin 0.1 -1g, antibiotic 0.6-5.3g, 4-hydroxyethylpiperazine ethanesulfonic acid 0.236-0.96g, phenol red 0.01-1g, L-cysteine hydrochloride 0.01-3.52g, L-glutamic acid 0.28 -2.95g, dilute to 100mL with water, and adjust pH to 7.2-7.8. The preservation reagent has many outstanding effects, but the composition is complex, and it is difficult to be applied to the treatment of viroids.
中国专利202010571607.0中公开了一种高度兼容磁珠法病毒核酸提取试剂盒的病毒保存液，其包含以下成分：盐酸胍、三(羟甲基)氨基甲烷盐酸盐、乙二胺四乙酸、异丙醇或乙醇。该病毒保存液的成分简单，原料易得，成本较低，可以长时间室温保存样本，无需高温灭活，对操作人员和环境都非常安全，同时降低RNA被降解的可能性，可以获得较多的核酸，降低漏检率。但该保存液能否应用于注射入人体的疫苗或治疗类的重组病毒还需要进一步地研究。Chinese Patent No. 202010571607.0 discloses a virus preservation solution that is highly compatible with a magnetic bead method virus nucleic acid extraction kit, which contains the following components: guanidine hydrochloride, tris(hydroxymethyl)aminomethane hydrochloride, ethylenediaminetetraacetic acid, isopropylamine Propanol or ethanol. The virus preservation solution has simple components, readily available raw materials, and low cost. It can store samples at room temperature for a long time without high temperature inactivation. It is very safe for operators and the environment. At the same time, it reduces the possibility of RNA degradation and can obtain more nucleic acid, reducing the missed detection rate. However, whether the preservation solution can be applied to vaccines or therapeutic recombinant viruses injected into the human body needs further research.
发明内容SUMMARY OF THE INVENTION
为了解决上述问题，本发明提供了一种病毒保存试剂，本发明提供的保存试剂成分简单，对细胞毒性小，且能够在不同的环境条件下起到长期稳定病毒，维持病毒活性的作用。In order to solve the above problems, the present invention provides a virus preservation reagent. The preservation reagent provided by the present invention has simple components, low toxicity to cells, and can stabilize the virus for a long time and maintain the virus activity under different environmental conditions.
一方面，本发明提供了一种病毒保存试剂。In one aspect, the present invention provides a virus preservation reagent.
所述的病毒保存试剂包括以下成分：NaCl、Tris-base、MgCl 2·6H 2O、甘油、水。 The virus preservation reagent includes the following components: NaCl, Tris-base, MgCl 2 ·6H 2 O, glycerol, and water.
所述的病毒保存试剂每1L中：NaCl含量为5.85±0.585g、Tris-base为1.21±0.121g、MgCl 2·6H 2O为0.2±0.02g、甘油为100±10mL、注射用水补足至1000mL。 In each 1L of the virus preservation reagent: NaCl content is 5.85±0.585g, Tris-base is 1.21±0.121g, MgCl 2 ·6H 2 O is 0.2±0.02g, glycerol is 100±10mL, and water for injection is supplemented to 1000mL .
优选地，所述的各成分含量为：每1L病毒保存试剂中：NaCl含量为5.85g、Tris-base为1.21g、MgCl 2·6H 2O为0.2g、甘油为100mL、注射用水补足至1000mL。 Preferably, the content of each component is: in each 1L virus preservation reagent: NaCl content is 5.85g, Tris-base is 1.21g, MgCl 2 ·6H 2 O is 0.2g, glycerol is 100mL, and water for injection is supplemented to 1000mL .
所述的病毒保存试剂pH为7.4-8.4；优选为7.6-8.2；pH值通过HCl调节。The pH of the virus preservation reagent is 7.4-8.4; preferably 7.6-8.2; the pH value is adjusted by HCl.
所述的病毒包括但不限于腺病毒、慢病毒、逆转录病毒。The viruses include but are not limited to adenoviruses, lentiviruses, and retroviruses.
优选地，所述的病毒为重组腺病毒。Preferably, the virus is a recombinant adenovirus.
另一方面，本发明提供了前述病毒保存试剂在制备疫苗中的应用。In another aspect, the present invention provides the application of the aforementioned virus preservation reagent in the preparation of vaccines.
所述的疫苗中包括病毒载体。The vaccine includes a viral vector.
所述的疫苗针对的病毒包括但不限于冠状病毒、流感病毒；优选为SARS-CoV-2冠状病毒。The viruses targeted by the vaccine include but are not limited to coronaviruses and influenza viruses; preferably SARS-CoV-2 coronaviruses.
所述的疫苗中，每1mL病毒保存试剂中含有0.5×10 11-1×10 12vp病毒载体，优选为1×10 11vp。 In the vaccine, each 1 mL of virus preservation reagent contains 0.5×10 11 to 1×10 12 vp virus vector, preferably 1×10 11 vp.
优选地，所述的病毒疫苗为腺病毒载体疫苗。Preferably, the viral vaccine is an adenovirus vector vaccine.
所述的疫苗为SARS-CoV-2冠状病毒疫苗；所述的SARS-CoV-2冠状病毒疫苗中包括E1、E3区完全缺失、部分缺失或不缺失的C亚类的5型腺病毒。The vaccine is a SARS-CoV-2 coronavirus vaccine; the SARS-CoV-2 coronavirus vaccine includes a type 5 adenovirus of subclass C with complete deletion, partial deletion or no deletion of E1 and E3 regions.
再一方面，本发明提供了一种疫苗。In yet another aspect, the present invention provides a vaccine.
所述的疫苗中包括前述的病毒疫苗保存试剂。The vaccine includes the aforementioned virus vaccine preservation reagent.
所述的疫苗中包括病毒载体。The vaccine includes a viral vector.
所述的疫苗针对的病毒包括但不限于冠状病毒、流感病毒；优选为SARS-CoV-2冠状病毒。The viruses targeted by the vaccine include but are not limited to coronaviruses and influenza viruses; preferably SARS-CoV-2 coronaviruses.
所述的疫苗中，每1mL病毒保存试剂中含有0.5×10 11-1×10 12vp病毒载体，优选为1×10 11vp。 In the vaccine, each 1 mL of virus preservation reagent contains 0.5×10 11 to 1×10 12 vp virus vector, preferably 1×10 11 vp.
优选地，所述的病毒疫苗为腺病毒载体疫苗。Preferably, the viral vaccine is an adenovirus vector vaccine.
所述的疫苗为SARS-CoV-2冠状病毒疫苗；所述的SARS-CoV-2冠状病毒疫苗中包括E1、E3区完全缺失、部分缺失或不缺失的C亚类的5型腺病毒。The vaccine is a SARS-CoV-2 coronavirus vaccine; the SARS-CoV-2 coronavirus vaccine includes a type 5 adenovirus of subclass C with complete deletion, partial deletion or no deletion of E1 and E3 regions.
又一方面，本发明提供了前述病毒保存试剂的制备方法。In another aspect, the present invention provides a method for preparing the aforementioned virus preservation reagent.
所述的制备方法包括以下步骤：The described preparation method comprises the following steps:
(1)准备高压灭菌后的配液桶、储液桶、玻璃接头及0.45μm+0.22μm滤器；(1) Prepare the dosing bucket, storage bucket, glass joint and 0.45μm+0.22μm filter after autoclaving;
(2)利用电子天平或精密电子天平称按前述病毒保存试剂的种类及配比称取试剂；加入适量的注射用水，并用磁力搅拌器使其溶解；(2) Use an electronic balance or a precision electronic balance to weigh the reagents according to the types and proportions of the aforementioned virus preservation reagents; add an appropriate amount of water for injection, and dissolve them with a magnetic stirrer;
(3)利用稀盐酸调整溶液pH，并补充注射用水至相应重量，继续使用磁力搅拌器使其充分溶解；(3) utilize dilute hydrochloric acid to adjust the pH of the solution, and supplement the water for injection to the corresponding weight, and continue to use a magnetic stirrer to fully dissolve it;
(4)利用0.45μm+0.22μm滤器将溶液除菌过滤(压力<1bar)至储液桶中，并进行必要项目的检测。(4) Use a 0.45μm+0.22μm filter to sterilize and filter the solution (pressure < 1bar) into the liquid storage tank, and carry out the detection of necessary items.
所述的步骤(4)中必要项目包括但不限于：细胞毒性、含菌量、稳定性，复溶稳定性。Necessary items in the step (4) include but are not limited to: cytotoxicity, bacterial content, stability, and reconstitution stability.
又一方面，本发明提供了前述疫苗的制备方法。In yet another aspect, the present invention provides a method for preparing the aforementioned vaccine.
所述的制备方法中包括：利用中空纤维柱进行超滤置换的方式将重组冠状病毒疫苗粗产品的溶剂替换为前述病毒保存液。The preparation method includes: replacing the solvent of the crude recombinant coronavirus vaccine product with the aforementioned virus preservation solution by using a hollow fiber column for ultrafiltration replacement.
所述的超滤置换步骤如下：Described ultrafiltration replacement step is as follows:
(1)中空纤维柱准备：先后用注射用水、0.5M NaOH、注射用水、病毒保存液清洗中空纤维柱。(1) Preparation of hollow fiber column: Wash the hollow fiber column with water for injection, 0.5M NaOH, water for injection, and virus preservation solution successively.
(2)超滤：在层析冷柜中对SARS-CoV-2疫苗精纯产物进行超滤。(2) Ultrafiltration: Ultrafiltration was performed on the purified product of SARS-CoV-2 vaccine in a chromatography freezer.
(3)溶液置换：在浓缩后样品中，重复3次加入2倍体积的病毒保存液，并重复超滤，直至样品体积缩小至原体积的(50-60)％，并排空中空纤维柱，获得病毒疫苗原液。(3) Solution replacement: In the concentrated sample, add 2 times the volume of virus preservation solution three times, and repeat the ultrafiltration until the sample volume is reduced to (50-60)% of the original volume, and empty the empty fiber column, Obtain viral vaccine stock.
(4)清洗中空纤维柱。(4) Cleaning the hollow fiber column.
附图说明Description of drawings
图1为病毒保存试剂的细胞毒性；Fig. 1 is the cytotoxicity of virus preservation reagent;
图2为37℃条件下，重组冠状病毒疫苗在不同pH病毒保存试剂中的稳定性检测结果；Figure 2 shows the stability test results of recombinant coronavirus vaccines in different pH virus storage reagents under the condition of 37°C;
图3为4℃条件下，重组冠状病毒疫苗在不同病毒保存试剂中的稳定性检测结果；Figure 3 shows the stability test results of recombinant coronavirus vaccines in different virus storage reagents at 4°C;
图4为25℃条件下，重组冠状病毒疫苗在不同病毒保存试剂中的稳定性检测结果；Figure 4 shows the stability test results of recombinant coronavirus vaccines in different virus storage reagents at 25°C;
图5为37℃条件下，重组冠状病毒疫苗在不同病毒保存试剂中的加速稳定性检测结果；Figure 5 shows the accelerated stability test results of recombinant coronavirus vaccines in different virus preservation reagents at 37°C;
图6为重组冠状病毒疫苗在不同病毒保存试剂中反复冻融稳定性检测结果。Figure 6 shows the results of repeated freeze-thaw stability testing of recombinant coronavirus vaccines in different virus storage reagents.
具体实施方式Detailed ways
下面结合具体实施例，对本发明作进一步详细的阐述，下述实施例不用于限制本发明，仅用于说明本发明。以下实施例中所使用的实验方法如无特殊说明，实施例中未注明具体条件的实验方法，通常按照常规条件，下述实施例中所使用的材料、试剂等，如无特殊说明，均可从商业途径得到。The present invention will be described in further detail below with reference to specific embodiments. The following embodiments are not intended to limit the present invention, but are only used to illustrate the present invention. The experimental methods used in the following examples, unless otherwise specified, the experimental methods without specific conditions in the examples are usually in accordance with conventional conditions, and the materials, reagents, etc. used in the following examples, unless otherwise specified, are all Commercially available.
实施例1 一种病毒疫苗保存试剂Embodiment 1 A kind of virus vaccine preservation reagent
本实施例的病毒疫苗保存试剂包括以下成分：NaCl、Tris-base、MgCl 2·6H 2O、甘油、水。 The virus vaccine preservation reagent of this embodiment includes the following components: NaCl, Tris-base, MgCl 2 ·6H 2 O, glycerol, and water.
其配制方法为：Its preparation method is:
(1)准备20L Nalgene配液桶和储液桶。储液桶连接玻璃接头和0.45μm+0.22μm滤器，高压灭菌。(1) Prepare a 20L Nalgene dosing bucket and storage bucket. The storage tank is connected with a glass connector and a 0.45μm+0.22μm filter, and autoclaved.
(2)将磁力搅拌器置于电子秤上，将20L配液桶放在磁力搅拌器上，去皮。(2) Put the magnetic stirrer on the electronic scale, put the 20L liquid dispensing bucket on the magnetic stirrer, and peel it.
(3)按下表中配方，用电子天平称取氯化钠，用精密电子天平称取氨基丁三醇、六水氯化镁，将称好的试剂置于3000mL烧杯中。加注射用水1L，用药匙将烧杯中试剂搅拌起来，倒进配液桶，用2000mL烧杯接注射用水冲洗3000mL烧杯，冲洗水倒入配液桶中。(3) According to the formula in the table, weigh sodium chloride with an electronic balance, tromethamine and magnesium chloride hexahydrate with a precision electronic balance, and place the weighed reagents in a 3000mL beaker. Add 1L of water for injection, stir the reagent in the beaker with a spoon, pour it into the dosing bucket, rinse the 3000 mL beaker with the 2000 mL beaker connected to the water for injection, and pour the flushing water into the dosing bucket.
序号serial number 名称name 20L标准配方20L standard formula
11 氨基丁三醇TrisTromethamine Tris 24.22g±0.024g24.22g±0.024g
22 六水氯化镁MgCl 2·6H 2O Magnesium chloride hexahydrate MgCl 2 ·6H 2 O 4.06g±0.012g4.06g±0.012g
33 甘油glycerin 2000mL±2.0g2000mL±2.0g
44 氯化钠NaClSodium ChlorideNaCl 116.88g±0.1g116.88g±0.1g
55 稀盐酸HClDilute hydrochloric acid HCl 调至pH 8.0Adjust to pH 8.0
66 注射用水Water for Injection 定容至20LFixed volume to 20L
(4)用3000mL烧杯在电子天平称取甘油，倒入配液桶中搅拌，用2000mL烧杯接注射用水冲洗3000mL烧杯三次，冲洗水倒入配液桶中。(4) Use a 3000mL beaker to weigh glycerin on an electronic balance, pour it into the dosing bucket and stir, rinse the 3000mL beaker three times with a 2000mL beaker and water for injection, and pour the rinse water into the dosing bucket.
(5)边搅拌边加注射用水至15kg。(5) Add water for injection to 15kg while stirring.
(6)用pH计在线检测溶液pH，用移液管吸取稀盐酸，缓慢滴加至配液桶，调节pH至8.0±0.1后，加注射用水至最终重量(18.145kg+甘油重量±0.02kg)，搅拌至少5分钟。(6) Use a pH meter to detect the pH of the solution on-line, absorb dilute hydrochloric acid with a pipette, slowly add it dropwise to the dosing bucket, adjust the pH to 8.0±0.1, add water for injection to the final weight (18.145kg+glycerin weight±0.02kg) , stir for at least 5 minutes.
(7)盖好桶盖，在超净台中连接储液桶上的0.45μm+0.22μm滤器，除菌过滤(压力<1bar)至储液桶中，在储液桶上标记溶液名称，批号，配制日期，有效期6个月，室温保存。取3支样品检测无菌、支原体，确保无外源污染。(7) Cover the barrel, connect the 0.45μm+0.22μm filter on the liquid storage tank in the ultra-clean bench, sterilize and filter (pressure <1bar) into the liquid storage tank, and mark the solution name and batch number on the liquid storage tank. Date of preparation, valid for 6 months, stored at room temperature. Take 3 samples to test for sterility and mycoplasma to ensure no exogenous contamination.
实施例2 病毒保存试剂的细胞毒性检测Example 2 Cytotoxicity detection of virus preservation reagent
对实施例1提供的病毒疫苗保存试剂的细胞毒性进行检测，并设置对比例。The cytotoxicity of the virus vaccine preservation reagent provided in Example 1 was detected, and a comparative example was set.
对比例的病毒保存试剂成分如下：The components of the virus preservation reagent of the comparative example are as follows:
对比例1(PBS保存液)：Na 2HPO 4 8mM、KH 2PO 4 42mM、NaCl 136mM和KCl 2.6mM。 Comparative Example 1 (PBS preservation solution): Na 2 HPO 4 8 mM, KH 2 PO 4 42 mM, NaCl 136 mM and KCl 2.6 mM.
对比例2(Hank's保存液，常规配方)：NaCl、KCl、KH 2PO4、Na 2HPO 4、NaHCO 3、CaCl 2、MgCl 2、MgSO 4。 Comparative Example 2 (Hank's preservation solution, conventional formula): NaCl, KCl, KH 2 PO 4 , Na 2 HPO 4 , NaHCO 3 , CaCl 2 , MgCl 2 , MgSO 4 .
细胞毒性检测具体步骤如下：The specific steps of cytotoxicity detection are as follows:
(1)准备细胞：(1) Prepare cells:
取293细胞消化传代培养至约90％汇合度。常规消化细胞，收集细胞悬液计数。用10％FBS DMEM(FBS购于YOSHI，货号为A1025；DMEM购于Gibco，货号为C11995500BT)制备4mL 1×10 5/mL 293细胞悬液，混匀接种于96孔板7-11列B-G行，100μL/孔，即每孔有1×10 4个细胞。余下四边每孔只加培养基100μL，置于37℃、5％CO 2条件下培养24h。 293 cells were digested and subcultured to about 90% confluence. Cells were routinely digested, and cell suspensions were collected and counted. Prepare 4 mL of 1×10 5 /mL 293 cell suspension with 10% FBS DMEM (FBS was purchased from YOSHI, the product number is A1025; DMEM was purchased from Gibco, the product number is C11995500BT). , 100 μL/well, that is, 1×10 4 cells per well. Only 100 μL of medium was added to each well of the remaining four sides, and cultured at 37° C. and 5% CO 2 for 24 h.
(2)准备样品：(2) Prepare the sample:
A组：(10％FBS DMEM 100μL)×8Group A: (10%FBS DMEM 100μL)×8
B组：(10％FBS DMEM 95μL+PBS保存液5μL)×8Group B: (10%FBS DMEM 95μL+PBS preservation solution 5μL)×8
C组：(10％FBS DMEM 95μL+Hank’s保存液5μL)×8Group C: (10%FBS DMEM 95μL+Hank’s preservation solution 5μL)×8
D组：(10％FBS DMEM 95μL+病毒保存液5μL)×8Group D: (10% FBS DMEM 95μL + virus preservation solution 5μL) × 8
分别混匀，备用。Mix well and set aside.
(3)吸弃孔内培养基，每孔按不同组各加入100μL样品，置于37℃、5％CO 2条件下培养直至A组达到80％汇合度左右。 (3) Aspirate and discard the medium in the wells, add 100 μL of samples to each well according to different groups, and culture at 37° C. and 5% CO 2 until group A reaches about 80% confluence.
(4)检测：(4) Detection:
培养48h后对照组细胞汇合度达到80％。每孔加入10μL CCK-8，轻敲板壁混匀，置于37℃、5％CO 2条件下培养。1h后达到橘黄色，放入酶标仪在450nm下读取数值并记录结果。 After 48 hours of culture, the confluence of the control group cells reached 80%. Add 10 μL of CCK-8 to each well, tap the wall of the plate to mix well, and incubate at 37°C and 5% CO 2 . After 1h, it reached orange, put it into the microplate reader and read the value at 450nm and record the result.
结果如下：The result is as follows:
每100μL完全培养基中加入5μL PBS后，对细胞生长平均抑制率为6.0％；每100μL完全培养基中加入5μL Hank's液后，对细胞生长平均抑制率为13.2％；每100μL完全培养基中加入5μL本病毒保存液后，对细胞生长平均抑制率为12.0％；详细数据见下表及附图1。After adding 5 μL of PBS per 100 μL of complete medium, the average inhibition rate of cell growth was 6.0%; after adding 5 μL of Hank’s solution per 100 μL of complete medium, the average inhibition rate of cell growth was 13.2%; After 5 μL of the virus preservation solution, the average inhibition rate of cell growth was 12.0%; the detailed data are shown in the following table and FIG. 1 .
分组grouping 平均吸光值Average absorbance 细胞存活率(％)Cell viability (%) 抑制率(％)Inhibition rate(%)
A(对照组)A (control group) 0.7430.743 100.0100.0 00
B(PBS保存液)B (PBS preservation solution) 0.6960.696 93.793.7 6.06.0
C(Hank's保存液)C(Hank's preservation solution) 0.6440.644 86.686.6 13.213.2
D(病毒保存液)D (virus preservation solution) 0.6520.652 87.887.8 12.012.0
Hank's平衡盐溶液与PBS是细胞分离或培养中常用的磷酸盐缓冲液，均集缓冲液的缓冲能力与生理盐水的等渗性为一体，具有维持渗透压、保持pH稳定的作用。Hank's平衡盐溶液对细胞基本没有伤害作用。本病毒保存液加入细胞后，对细胞生长的抑制率为12％，小于Hank's液13.2％的抑制率。综合而言，本病毒保存液对细胞的毒性较低，低于Hank's平衡盐溶液，略高于PBS。Hank's Balanced Salt Solution and PBS are phosphate buffers commonly used in cell separation or culture. They combine the buffering capacity of the buffer with the isotonicity of normal saline, and can maintain osmotic pressure and maintain pH stability. Hank's Balanced Salt Solution is basically harmless to cells. After the virus preservation solution is added to cells, the inhibition rate of cell growth is 12%, which is less than the inhibition rate of Hank's solution of 13.2%. In general, the virus preservation solution is less toxic to cells, lower than Hank's balanced salt solution, and slightly higher than PBS.
实施例3 一种重组冠状病毒疫苗Example 3 A recombinant coronavirus vaccine
采用实施例1提供的病毒疫苗保存试剂制备重组冠状病毒疫苗，主要通过超滤置换的方式。Recombinant coronavirus vaccine was prepared by adopting the virus vaccine preservation reagent provided in Example 1, mainly by means of ultrafiltration replacement.
本实施例采用的重组冠状病毒疫苗为SARS-CoV-2冠状病毒疫苗，所述的疫苗在超滤置换前的制备步骤如下：The recombinant coronavirus vaccine adopted in this example is the SARS-CoV-2 coronavirus vaccine, and the preparation steps of the vaccine before ultrafiltration replacement are as follows:
(1)对SARS-CoV-2冠状病毒的S基因进行优化，优化后的S基因的序列为SEQ ID NO：3。(1) The S gene of the SARS-CoV-2 coronavirus is optimized, and the sequence of the optimized S gene is SEQ ID NO: 3.
(2)取得SARS-CoV-2S基因的优化序列后，用PCR方法对进行扩增。扩增引物为V1与V2，其各自的序列如SEQ ID NO：1-2所示，VI和V2为一对，扩增S1基因C端片段S1C。(2) After obtaining the optimized sequence of SARS-CoV-2S gene, use PCR method to amplify. The amplification primers are V1 and V2, their respective sequences are shown in SEQ ID NOs: 1-2, VI and V2 are a pair, and the C-terminal fragment S1C of the S1 gene is amplified.
(3)PCR产物经跑胶鉴定与切胶回收后，用SmaI与HindIII在37℃条件下进行酶切。同时用这两种酶对pShuttle载体进行酶切。(3) After the PCR product was identified by running gel and recovered by gel cutting, it was digested with SmaI and HindIII at 37°C. The pShuttle vector was digested with these two enzymes at the same time.
(4)用T4连接酶将酶切后的PCR产物与pShuttle在16℃条件进行过夜连接。(4) Use T4 ligase to ligate the digested PCR product with pShuttle overnight at 16°C.
(5)将连接产物转化大肠杆菌DH5a，利用氨苄抗性筛选阳性克隆，并挑选克隆进行菌落PCR鉴定。阳性菌落经培养后提取质粒，并送测序以确认序列。(5) The ligation product was transformed into Escherichia coli DH5a, positive clones were screened by ampicillin resistance, and clones were selected for colony PCR identification. Plasmids were extracted from positive colonies after culture and sent for sequencing to confirm the sequence.
(6)获得正确的重组SARS-CoV-2S1C基因的pShuttle质粒后，将其与腺病毒骨架质粒pBHGlox(delta)E1,3Cre共同转染至293细胞中以包装重组腺病毒。(6) After obtaining the correct pShuttle plasmid of the recombinant SARS-CoV-2S1C gene, it was co-transfected with the adenovirus backbone plasmid pBHGlox(delta)E1,3Cre into 293 cells to package the recombinant adenovirus.
(7)病毒的收集采用挑空斑的方式：在培养液中加入低溶点琼脂糖，转染后一般在第10-21天可以在显微镜下看到小的空斑。空斑形成后将空斑与琼脂糖一起挑起，放入1mL新鲜培养基中过夜。通常挑取3-6个空斑不等，然后比较滴度，使用滴度最高的一个空斑进行后续实验。(7) The collection of viruses adopts the method of picking plaques: low melting point agarose is added to the culture medium, and small plaques can be seen under the microscope on the 10th to 21st day after transfection. After plaque formation, the plaques were picked up with agarose and placed in 1 mL of fresh medium overnight. Usually, 3-6 plaques are picked, and then the titers are compared, and the plaque with the highest titer is used for subsequent experiments.
(8)将培养基中病毒加入新鲜293细胞培养液中进行病毒少量扩增。至细胞再次出现空斑，收集细胞及上清，反复冻融三次收集病毒，以此病毒为P1代病毒。(8) The virus in the medium was added to the fresh 293 cell culture medium for a small amount of virus amplification. When plaques appeared in the cells again, the cells and the supernatant were collected, and the virus was collected by repeated freezing and thawing three times, and this virus was used as the P1 generation virus.
(9)以P1代病毒感染293细胞，连续进行三代感染，至P4代进行病毒的大量扩增，待空斑形成后收集病毒并对病毒进行柱层析纯化和浓缩。(9) Infect 293 cells with the P1 generation virus, carry out three consecutive generations of infection, and conduct a large number of virus amplification in the P4 generation, collect the virus after the formation of plaques, and carry out column chromatography purification and concentration of the virus.
(10)纯化后获得的产物即为SARS-CoV-2疫苗精纯产物。SARS-CoV-2疫苗包含SARS-CoV-2病毒S1C基因和缺陷型腺病毒。缺陷型腺病毒为E1与E3区完全缺失的C亚类的5型腺病毒，不能在普通的人体细胞内复制。(10) The product obtained after purification is the purified product of the SARS-CoV-2 vaccine. The SARS-CoV-2 vaccine contains the S1C gene of the SARS-CoV-2 virus and a defective adenovirus. The defective adenovirus is a type 5 adenovirus of subclass C with complete deletion of the E1 and E3 regions, and cannot replicate in ordinary human cells.
超滤置换步骤如下：The ultrafiltration replacement steps are as follows:
(1)中空纤维柱准备：先后用注射用水、0.5M NaOH、注射用水、病毒保存液清洗中空纤维柱。(1) Preparation of hollow fiber column: Wash the hollow fiber column with water for injection, 0.5M NaOH, water for injection, and virus preservation solution successively.
(2)超滤：在层析冷柜中对SARS-CoV-2疫苗精纯产物进行超滤。(2) Ultrafiltration: Ultrafiltration was performed on the purified product of SARS-CoV-2 vaccine in a chromatography freezer.
(3)溶液置换：在浓缩后样品中，重复3次加入2倍体积的病毒保存液，并重复超滤，直至样品体积缩小至原体积的(50-60)％，并排空中空纤维柱，获得病毒疫苗原液。(3) Solution replacement: In the concentrated sample, add 2 times the volume of virus preservation solution three times, and repeat the ultrafiltration until the sample volume is reduced to (50-60)% of the original volume, and empty the empty fiber column, Obtain viral vaccine stock.
(4)清洗中空纤维柱。(4) Cleaning the hollow fiber column.
实施例4 在37℃条件下，重组冠状病毒疫苗在不同pH病毒保存试剂中的稳定性检测Example 4 Stability detection of recombinant coronavirus vaccine in different pH virus preservation reagents under the condition of 37°C
含不同pH病毒保存试剂的重组冠状疫苗的制备方法：Preparation method of recombinant coronavirus vaccine containing different pH virus preservation reagents:
参考实施例1，区别为在第(6)步，将未调整pH的病毒保存试剂分为5组，分别加入不同量的HCl，同时用pH计进行监测，以获取pH分别为7.63、7.79、8.03、8.21、8.43的病毒保存试剂。With reference to Example 1, the difference is that in step (6), the virus preservation reagents without pH adjustment are divided into 5 groups, and different amounts of HCl are added respectively, and are monitored with a pH meter to obtain pH values of 7.63, 7.79, Virus preservation reagents of 8.03, 8.21, 8.43.
参考实施例3，区别为超滤置换步骤(3)溶液置换步骤中，分别用不同pH的病毒保存液对超滤浓缩后样品进行溶液置换，以获取溶液pH不同的重组冠状病毒疫苗，并对该系列重组冠状病毒疫苗进行稳定性检测。With reference to Example 3, it is distinguished from the ultrafiltration replacement step (3) in the solution replacement step, and the sample after the ultrafiltration concentration is carried out solution replacement with the virus preservation solution of different pH respectively, to obtain the different recombinant coronavirus vaccines of solution pH, and The series of recombinant coronavirus vaccines were tested for stability.
对上述重组冠状病毒疫苗分组，每组各置于37℃条件下24h，并检测相应的病毒感染滴度。The above recombinant coronavirus vaccines were grouped, and each group was placed at 37°C for 24 hours, and the corresponding virus infection titers were detected.
检测方法如下：The detection method is as follows:
(1)取一瓶293细胞，弃去原瓶中培养液，加入胰酶把细胞消化下来，用完全培养基(含10％血清的DMEM)终止消化；(1) Take a bottle of 293 cells, discard the culture medium in the original bottle, add trypsin to digest the cells, and stop the digestion with complete medium (DMEM containing 10% serum);
(2)细胞悬液计数，稀释至10 5/mL； (2) Count the cell suspension and dilute to 10 5 /mL;
(3)将293细胞接种到12孔板中，每孔1mL，37℃，5％CO 2培养24h-48h； (3) 293 cells were seeded into 12-well plates, 1 mL per well, and cultured at 37°C, 5% CO 2 for 24h-48h;
(4)转染病毒前，取孔板其中一孔吸干培养液，加入胰酶(购自AMRESCO，货号为0458-25G)消化，待显微镜下观察到细胞脱落壁面，加入完全培养基终止消化，取细胞悬液计数，记录孔中细胞数量，此孔保留细胞悬液，当做空白对照孔；(4) Before transfecting the virus, take one of the wells of the well plate to dry the culture medium, add trypsin (purchased from AMRESCO, the product number is 0458-25G) to digest, when the cell wall surface is observed under the microscope, add complete medium to stop the digestion , count the cell suspension, record the number of cells in the well, retain the cell suspension in this well, and use it as a blank control well;
(5)将待测样品稀释，根据估测的样品浓度作1:100稀释(若样品浓度高，可进行1:1000稀释)，按照下图加入不同量的病毒稀释液，72h观察结果；(5) Dilute the sample to be tested, make 1:100 dilution according to the estimated sample concentration (if the sample concentration is high, 1:1000 dilution can be carried out), add different amounts of virus diluents according to the following figure, and observe the results in 72h;
Figure PCTCN2020125326-appb-000001
Figure PCTCN2020125326-appb-000001
(6)计算感染滴度，公式为：滴度＝(细胞数×20÷体积)×10×1000(6) Calculate the infection titer, the formula is: titer=(number of cells×20÷volume)×10×1000
结果如下表及附图2：The results are shown in the following table and attached Figure 2:
pHpH 7.637.63 7.797.79 8.038.03 8.218.21 8.438.43
病毒感染滴度virus infection titer 3.51×10 10 3.51×10 10 3.51×10 10 3.51×10 10 3.51×10 10 3.51×10 10 3.51×10 10 3.51×10 10 2.83×10 10 2.83×10 10
根据以上结果可知：According to the above results, it can be seen that:
病毒保存液pH在7.63-8.21之间时，病毒均能维持较高的活性。When the pH of the virus preservation solution is between 7.63-8.21, the virus can maintain a high activity.
实施例5在4℃条件下，重组冠状病毒疫苗在不同病毒保存试剂中的稳定性检测Example 5 Stability detection of recombinant coronavirus vaccines in different virus preservation reagents at 4°C
对实施例3提供的重组冠状病毒疫苗进行稳定性检测，并设置对比例。Stability detection was carried out on the recombinant coronavirus vaccine provided in Example 3, and a comparative example was set.
对比例的病毒保存试剂成分同实施例2中对比例1和对比例2。The components of the virus preservation reagent of the comparative example are the same as those of Comparative Example 1 and Comparative Example 2 in Example 2.
对实施例3提供的重组冠状病毒疫苗分组，每组各置于4℃条件下3天、7天、14天，并检测相应天数下的病毒感染滴度。The recombinant coronavirus vaccines provided in Example 3 were grouped, and each group was placed at 4°C for 3 days, 7 days, and 14 days, and the virus infection titers under the corresponding days were detected.
检测方法参考实施例4。The detection method refers to Example 4.
结果如下表及附图3。下表为病毒感染滴度检测结果。The results are shown in the following table and Figure 3. The following table shows the results of the virus infection titer test.
   本申请病毒保存液The virus preservation solution of this application PBS保存液PBS preservation solution Hank's保存液Hank's Preservative Solution
第3天Day 3 100.2％100.2% 100％100% 100％100%
第7天Day 7 98％98% 75.8％75.8% 82.3％82.3%
第14天Day 14 95％95% 39.5％39.5% 39.7％39.7%
根据以上结果可知：According to the above results, it can be seen that:
4℃条件下，重组冠状病毒疫苗在本申请病毒保存液中保存14天，病毒感染滴度仅下降5％。而同样条件下，重组冠状病毒疫苗在PBS保存液中保存14天，病毒感染滴度下降了60.5％；在Hank's保存液中保存14天，病毒感染滴度下降了60.3％。Under the condition of 4°C, the recombinant coronavirus vaccine was stored in the virus preservation solution of the present application for 14 days, and the virus infection titer only decreased by 5%. Under the same conditions, when the recombinant coronavirus vaccine was stored in PBS preservation solution for 14 days, the virus infection titer decreased by 60.5%; when stored in Hank's preservation solution for 14 days, the virus infection titer decreased by 60.3%.
总体而言，4℃条件下，相对于其它类型病毒保存液，重组冠状病毒疫苗在本申请提供的病毒保存液中14天内仍能够维持较高的感染活性。In general, at 4°C, compared with other types of virus preservation solutions, the recombinant coronavirus vaccine can still maintain a high infectious activity within 14 days in the virus preservation solution provided in this application.
实施例6 25℃条件下，重组冠状病毒疫苗在不同病毒保存试剂中的稳定性检测Example 6 Stability detection of recombinant coronavirus vaccine in different virus preservation reagents under the condition of 25°C
对实施例3提供的重组冠状病毒疫苗进行稳定性检测，并设置对比例。Stability detection was carried out on the recombinant coronavirus vaccine provided in Example 3, and a comparative example was set.
对比例的疫苗的病毒保存试剂成分同实施例2中对比例1和对比例2。The components of the virus preservation reagent of the vaccine of the comparative example are the same as those of Comparative Example 1 and Comparative Example 2 in Example 2.
对实施例3提供的重组冠状病毒疫苗分组，每组各置于25℃条件下2h、4h、8h、24h，并检测相应时间点的病毒感染滴度。The recombinant coronavirus vaccines provided in Example 3 were grouped, and each group was placed at 25°C for 2h, 4h, 8h, and 24h, and the virus infection titers at the corresponding time points were detected.
检测方法参考实施例4。The detection method refers to Example 4.
结果如下表及附图4。下表为病毒感染滴度检测结果。The results are shown in the following table and Figure 4. The following table shows the results of the virus infection titer test.
   本申请病毒保存液The virus preservation solution of this application PBS保存液PBS preservation solution Hank's保存液Hank's Preservative Solution
2h2h 99.2％99.2% 100％100% 100％100%
4h4h 99.2％99.2% 100％100% 80％80%
8h8h 99.2％99.2% 75.1％75.1% 64.3％64.3%
24h24h 93％93% 62.5％62.5% 32％32%
根据以上结果可知：According to the above results, it can be seen that:
25℃条件下，重组冠状病毒疫苗在本病毒保存液中保存8h，病毒感染滴度基本维持不变；保存24h后，病毒感染滴度也仅下降7％。而同样条件下，重组冠状病毒疫苗在PBS保存液中保存4h后，在Hank's保存液中保存2h后，病毒感染滴度便低于80％，且感染滴度下降速率较高。Under the condition of 25°C, the recombinant coronavirus vaccine was stored in this virus preservation solution for 8 hours, and the virus infection titer remained basically unchanged; after 24 hours of storage, the virus infection titer only decreased by 7%. Under the same conditions, after the recombinant coronavirus vaccine was stored in PBS preservation solution for 4 hours and Hank's preservation solution for 2 hours, the viral infection titer was lower than 80%, and the infection titer decreased at a higher rate.
总体而言，25℃条件下，相对于其它类型病毒保存液，重组冠状病毒疫苗在本病毒保存液中24h内仍能够维持较高的感染活性。In general, at 25°C, compared with other types of virus preservation solutions, the recombinant coronavirus vaccine can still maintain a high infectious activity within 24 hours in this virus preservation solution.
实施例7 37℃条件下，重组冠状病毒疫苗在不同病毒保存试剂中的加速稳定性检测Example 7 Accelerated Stability Detection of Recombinant Coronavirus Vaccines in Different Virus Preservation Reagents at 37°C
对实施例3提供的重组冠状病毒疫苗进行稳定性检测，并设置对比例。Stability detection was carried out on the recombinant coronavirus vaccine provided in Example 3, and a comparative example was set.
对比例的疫苗中病毒保存试剂成分同实施例2中对比例1和对比例2。The components of the virus preservation reagent in the vaccine of the comparative example are the same as those of Comparative Example 1 and Comparative Example 2 in Example 2.
对实施例3提供的重组冠状病毒疫苗分组，每组各置于37℃条件下1天、3天、5天、7天，并检测相应时间点的病毒感染滴度。The recombinant coronavirus vaccines provided in Example 3 were grouped, and each group was placed at 37°C for 1 day, 3 days, 5 days, and 7 days, and the virus infection titers at the corresponding time points were detected.
检测方法参考实施例4。The detection method refers to Example 4.
结果如下表及附图5。下表为病毒感染滴度检测结果。The results are shown in the following table and Figure 5. The following table shows the results of the virus infection titer test.
   本申请病毒保存液The virus preservation solution of this application PBS保存液PBS preservation solution Hank's保存液Hank's Preservative Solution
1天1 day 98％98% 83.5％83.5% 62.8％62.8%
3天3 days 91.2％91.2% 69.7％69.7% 62.8％62.8%
5天5 days 91.2％91.2% 69.7％69.7% 50.3％50.3%
根据以上结果可知：According to the above results, it can be seen that:
37℃条件下，重组冠状病毒疫苗在本发明提供的病毒保存液中保存5天，病毒感染滴度基本维持不变，均能够维持在90％以上。而同样条件下，重组冠状病毒疫苗在PBS或Hank's保存液中保存5天后，病毒感染滴度急剧下降。Under the condition of 37°C, the recombinant coronavirus vaccine is stored in the virus preservation solution provided by the present invention for 5 days, and the virus infection titer is basically maintained, which can be maintained above 90%. Under the same conditions, after the recombinant coronavirus vaccine was stored in PBS or Hank's preservation solution for 5 days, the viral infection titer dropped sharply.
总体而言，37℃条件下，相对于其它类型病毒保存液，重组冠状病毒疫苗在本申请病毒保存液中5天内仍能够维持较高的感染活性；而在其它类型保存液中，重组冠状病毒疫苗保存1天后，病毒感染滴度活性便大幅度下降。In general, at 37°C, compared with other types of virus preservation solutions, the recombinant coronavirus vaccine can still maintain a high infectious activity within 5 days in the virus preservation solution of the present application; while in other types of preservation solutions, the recombinant coronavirus vaccine After the vaccine was stored for 1 day, the viral infection titer activity decreased significantly.
实施例8 重组冠状病毒疫苗在不同病毒保存试剂中反复冻融稳定性检测Example 8 Repeated freeze-thaw stability detection of recombinant coronavirus vaccine in different virus preservation reagents
对实施例3提供的重组冠状病毒疫苗进行反复冻融稳定性检测，并设置对比例。Repeated freeze-thaw stability testing was performed on the recombinant coronavirus vaccine provided in Example 3, and a comparative example was set.
对比例的疫苗中病毒保存试剂成分同实施例2中对比例1和对比例2。The components of the virus preservation reagent in the vaccine of the comparative example are the same as those of Comparative Example 1 and Comparative Example 2 in Example 2.
对实施例3提供的重组冠状病毒疫苗分组，每组冻融3次、5次、10次后，检测相应的病毒感染滴度。The recombinant coronavirus vaccines provided in Example 3 were grouped, and after each group was freeze-thawed 3 times, 5 times, and 10 times, the corresponding virus infection titers were detected.
检测方法参考实施例4。The detection method refers to Example 4.
结果如下表及附图6。下表为病毒感染滴度检测结果。The results are shown in the following table and Figure 6. The following table shows the results of the virus infection titer test.
   本病毒保存液This virus preservation solution PBS保存液PBS preservation solution Hank's保存液Hank's Preservative Solution
3次3 times 99.2％99.2% 85.9％85.9% 85.9％85.9%
5次5 times 101.2％101.2% 89.8％89.8% 89.8％89.8%
10次10 times 98.6％98.6% 89.8％89.8% 79％79%
根据以上结果可知：According to the above results, it can be seen that:
重组冠状病毒疫苗在本病毒保存液中反复冻融10次后，病毒感染滴度基本维持不变。而重组冠状病毒疫苗在PBS或Hank's保存液中冻融3次、5次、10次后，病毒感染滴度均有不同程度的下降。After the recombinant coronavirus vaccine was repeatedly frozen and thawed in this virus preservation solution for 10 times, the viral infection titer remained basically unchanged. After the recombinant coronavirus vaccine was frozen and thawed in PBS or Hank's preservation solution for 3, 5, and 10 times, the viral infection titer decreased to varying degrees.
总体而言，重组冠状病毒疫苗在本病毒保存液中反复冻融3-10次均能够保持较高的病毒活性。In general, the recombinant coronavirus vaccine can maintain high viral activity after repeated freezing and thawing 3-10 times in this virus preservation solution.

Claims (10)

  1. 一种病毒保存试剂，其特征在于，包括以下成分：NaCl、Tris-base、MgCl 2·6H 2O、甘油和水，并用HCl调整试剂pH。 A virus preservation reagent is characterized in that it comprises the following components: NaCl, Tris-base, MgCl 2 ·6H 2 O, glycerol and water, and the pH of the reagent is adjusted with HCl.
  2. 根据权利要求1所述的病毒保存试剂，其特征在于，每1L病毒保存试剂中：NaCl 5.85±0.585g、Tris-base 1.21±0.121g、MgCl 2·6H 2O 0.2±0.02g、甘油100±10mL、注射用水补足至1000mL。 The virus-preserving reagent according to claim 1, wherein in every 1 L of the virus-preserving reagent: NaCl 5.85±0.585g, Tris-base 1.21±0.121g, MgCl 2 .6H 2 O 0.2±0.02g, glycerol 100±0.02g 10mL and water for injection to make up to 1000mL.
  3. 根据权利要求1所述的病毒保存试剂，其特征在于，所述的试剂pH为7.4-8.4，所述的病毒为腺病毒、慢病毒、逆转录病毒中的至少一种。The virus preservation reagent according to claim 1, wherein the pH of the reagent is 7.4-8.4, and the virus is at least one of adenovirus, lentivirus, and retrovirus.
  4. 根据权利要求2所述的病毒保存试剂，其特征在于，每1L病毒保存试剂中：NaCl 5.85g、Tris-base 1.21g、MgCl 2·6H 2O 0.2g、甘油100mL、注射用水补足至1000mL。 The virus preservation reagent according to claim 2, wherein in each 1 L of the virus preservation reagent: NaCl 5.85g, Tris-base 1.21g, MgCl 2 ·6H 2 O 0.2g, glycerol 100mL, and water for injection to make up to 1000mL.
  5. 根据权利要求3所述的病毒保存试剂，其特征在于，所述的病毒为腺病毒。The virus preservation reagent according to claim 3, wherein the virus is an adenovirus.
  6. 权利要求1-5任一项所述的病毒保存试剂在制备疫苗中的应用。Application of the virus preservation reagent described in any one of claims 1-5 in the preparation of vaccines.
  7. 根据权利要求6所述的应用，其特征在于，所述的疫苗为重组冠状病毒疫苗。The application according to claim 6, wherein the vaccine is a recombinant coronavirus vaccine.
  8. 根据权利要求7所述的应用，其特征在于，所述的疫苗为SARS-CoV-2冠状病毒疫苗；所述的SARS-CoV-2冠状病毒疫苗中包括E1、E3区完全缺失、部分缺失或不缺失的C亚类的5型腺病毒。The application according to claim 7, wherein the vaccine is a SARS-CoV-2 coronavirus vaccine; the SARS-CoV-2 coronavirus vaccine comprises complete deletion, partial deletion or Adenovirus type 5 of subclass C without deletion.
  9. 一种重组冠状病毒疫苗，其特征在于，所述的疫苗中包括权利要求1-5任一项所述的病毒保存试剂。A recombinant coronavirus vaccine, characterized in that the vaccine comprises the virus preservation reagent described in any one of claims 1-5.
  10. 一种重组冠状病毒疫苗的制备方法，其特征在于，所述的制备方法中包括：利用中空纤维柱进行超滤置换的方式将重组冠状病毒疫苗粗产品的溶剂替换为前述病毒保存液。A preparation method of a recombinant coronavirus vaccine, characterized in that, the preparation method comprises: replacing the solvent of the crude product of the recombinant coronavirus vaccine with the aforementioned virus preservation solution by utilizing a hollow fiber column for ultrafiltration replacement.
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