Canine distemper vaccine as well as preparation method and application thereof

Abstract

The invention provides a canine distemper vaccine as well as a preparation method and application thereof, and the method comprises the following steps: designing mRNA vaccine template plasmids aiming at canine distemper virus H protein by taking pUC57 as a carrier; carrying out single enzyme digestion on the mRNA vaccine template plasmid, and recovering a single enzyme digestion product; carrying out RNA in-vitro transcription on the single enzyme digestion product to obtain a transcribed RNA stock solution; the RNA stock solution is purified, the purified RNA is dissolved, and HmRNA is obtained; the method comprises the following steps: adding H mRNA into a citric acid buffer solution to obtain an mRNA-citric acid buffer solution, preparing an LNP premixed solution, and mixing the mRNA-citric acid buffer solution and the LNP premixed solution to obtain the H mRNA-LNP. The canine distemper vaccine prepared by the invention has the advantages of high flexibility, high preparation speed and capability of simultaneously causing the organism to generate humoral immunity and cellular immunity.

Description

Technical Field

[0001] The present invention relates to the technical field of canine distemper vaccine preparation, and particularly relates to a canine distemper vaccine, a preparation method thereof, and an application thereof. Background Art

[0002] Canine distemper (CDV) is a highly contagious disease caused by the canine distemper virus of the genus Morbillivirus in the family Paramyxoviridae. The virus infects canids through the mouth and nose, and then causes severe immunosuppression. The infected animals mainly show symptoms such as biphasic fever, diarrhea, and anorexia. CDV is also a single-stranded negative-strand RNA virus, and the genome size is about 15 kbp.

[0003] Among the current methods for dealing with canine distemper, the most effective and economical method is vaccination. Most of the currently marketed canine distemper vaccines for dogs are attenuated vaccines, which have good protective effects. However, due to the phenomenon of virus mutation in each region, the commercial vaccines are not ideal for protecting dogs. In addition, during the vaccine production process, the canine distemper virus needs to be cultured in cells expressing the canine SLAM receptor to obtain a high virus titer, which greatly increases the cost of vaccine production. Summary of the Invention

[0004] The purpose of the present invention is to provide a canine distemper vaccine, a preparation method thereof, and an application thereof, aiming to develop a canine distemper mRNA vaccine based on the mRNA vaccine platform, provide an effective vaccine for the prevention and treatment of the virus, and have the advantages of high flexibility, fast preparation speed, and the ability to simultaneously induce humoral immunity and cellular immunity in the body.

[0005] In the first aspect, the present invention provides a preparation method of a canine distemper vaccine, and the method includes:

[0006] Using pUC57 as a vector, designing an mRNA vaccine template plasmid for the H protein of the canine distemper virus;

[0007] Performing single enzyme digestion on the mRNA vaccine template plasmid, and recovering the single enzyme digestion product;

[0008] Performing in vitro transcription on the single enzyme digestion product to obtain a transcribed RNA stock solution;

[0009] Purifying the RNA stock solution, and dissolving the purified RNA with RNase-Free ddH2O to obtain HmRNA;

[0010] Adding H mRNA into a citrate buffer solution to obtain an mRNA-citrate buffer solution, preparing an LNP premix solution, and mixing the mRNA-citrate buffer solution and the LNP premix solution to obtain H mRNA-LNP.

[0011] Further, the steps of designing an mRNA vaccine template plasmid targeting the H protein of canine distemper virus with pUC57 as the vector include:

[0012] The vector includes a T7 promoter, a UTR sequence, and a codon-optimized H protein sequence;

[0013] The DNA sequence of the mRNA vaccine template plasmid is shown in SEQ ID NO.1.

[0014] Further, the steps of single enzyme digestion of the mRNA vaccine template plasmid include:

[0015] Construct an enzyme digestion system, which includes: mRNA vaccine template plasmid, enzyme digestion Buffer, restriction endonuclease Sal I, and ultrapure water;

[0016] Set the enzyme digestion reaction conditions as: incubate at 35 - 40 °C for 8 - 12 h.

[0017] Further, the steps of performing in vitro transcription on the single enzyme digestion product to obtain the transcribed RNA stock solution include:

[0018] The reaction system for in vitro transcription includes: 10×Transcription Buffer, ATP Solution, GTP Solution, CTP Solution, N1-Me-Pseudo UTP, T7 RNA Polymerase Mix, CAG Trimer, template DNA, RNase-Free ddH2O;

[0019] The reaction conditions for in vitro transcription are: incubate at 35 - 40 °C for 4 - 6 h.

[0020] Further, the steps of purifying the RNA stock solution and dissolving the purified RNA with RNase-Free ddH2O to obtain H mRNA include:

[0021] (1) Add the pre-prepared lithium chloride precipitation solution and RNase-Free ddH2O to the RNA stock solution and mix thoroughly to obtain a mixed solution;

[0022] (2) Place the mixed solution in a -20 °C refrigerator for 30 min;

[0023] (3) Centrifuge the mixed solution;

[0024] (4) Discard the supernatant, add 70% ethanol solution to wash the RNA precipitate, and repeat 3 times;

[0025] (5) Discard the supernatant, air-dry the precipitated RNA, add RNase-Free ddH2O to dissolve the RNA, and obtain H mRNA.

[0026] Further, the steps of preparing the LNP premix include:

[0027] Dissolve the four lipids of ionizable lipid, distearoyl phosphatidylcholine, cholesterol, and polyethylene glycol in absolute ethanol respectively, and prepare lipid solutions with a concentration of 8 - 12 mg / mL each;

[0028] Mix the four lipid solutions of ionizable lipid, distearoyl phosphatidylcholine, cholesterol, and polyethylene glycol in a ratio of 48 - 52%: 8 - 12%: 38 - 39%: 1 - 2% to prepare a 10 mg / mL LNP premix.

[0029] Further, the mixing volume ratio of the mRNA-citrate buffer and the LNP premix is 2.5 - 3.5:1.

[0030] Further, the steps of preparing the mRNA-citrate buffer include:

[0031] Add H mRNA to a 50 mM, pH = 4 citrate buffer to make the concentration of H mRNA in the solution 100 - 120 ng / μL, and prepare the mRNA-citrate buffer.

[0032] In a second aspect, the present invention provides a canine distemper vaccine, which is prepared according to the above-mentioned preparation method of the canine distemper vaccine.

[0033] In a third aspect, the present invention provides an application of the above-mentioned canine distemper vaccine in the preparation of a drug for treating canine distemper.

[0034] Compared with the prior art, the present invention has the following advantages:

[0035] 1. The canine distemper vaccine prepared by the present invention can detect the presence of CDV-specific neutralizing antibodies in the mouse serum at 28 days, 42 days, and 56 days after the first immunization, and the level of the neutralizing antibody depends on the immunization dose of the vaccine. The average neutralizing antibody level of the 10 μg H mRNA-LNP immunization group is significantly higher than that of the 5 μg H mRNA-LNP immunization group. After the third immunization, the neutralizing antibody level of the mice in the H mRNA-LNP immunization group increased significantly, and compared with the neutralizing antibody level at 42 days after the first immunization, the neutralizing antibody level at 56 days after the first immunization did not show an obvious downward trend, indicating that H mRNA-LNP can maintain a relatively high neutralizing antibody level for a certain period of time after booster immunization. At 42 days after the first immunization, the highest neutralizing antibody titer of individual mice can reach 1:708. Description of the Drawings

[0036] Figure 1 This is the Western blot result diagram after H mRNA transfected BHK-21 cells in an embodiment of the present invention;

[0037] Figure 2 This is a schematic diagram of the serum neutralizing antibody level after immunizing mice with H mRNA-LNP for 28 days in an embodiment of the present invention;

[0038] Figure 3 This is a schematic diagram of the serum neutralizing antibody level after immunizing mice with H mRNA-LNP for 42 days in an embodiment of the present invention;

[0039] Figure 4 This is a schematic diagram of the serum neutralizing antibody level after immunizing mice with H mRNA-LNP for 56 days in an embodiment of the present invention;

[0040] Figure 5 This is a schematic diagram of the serum neutralizing antibody level after immunizing mice with H mRNA-LNP in an embodiment of the present invention.

[0041] The following specific embodiments will further illustrate the present invention in conjunction with the above-mentioned drawings. Specific Embodiments

[0042] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention. Unless otherwise defined, the technical terms or scientific terms used herein shall have the ordinary meanings understood by those of ordinary skill in the art to which the present invention pertains. The words such as "including" used herein mean that the elements or items appearing before this word cover the elements or items listed after this word and their equivalents, without excluding other elements or items.

[0043] Example 1

[0044] 1. Construction of the mRNA template plasmid of canine distemper virus H protein

[0045] Using pUC57 as a vector, an mRNA vaccine template plasmid was designed against the H protein of canine distemper virus. The vector includes a T7 promoter, a UTR sequence, and a codon-optimized H protein sequence (the C-terminus of the H protein has a flag tag). The mRNA vaccine template plasmid sequence was fully synthesized by Genewiz Biotechnology Company, and the codons were optimized according to the mammalian expression system. The plasmid DNA sequence is shown in SEQ ID NO.1 as follows:

[0046]

[0047] 2. Preparation of Canine Distemper Virus H Protein mRNA

[0048] 2.1 Single Enzyme Digestion: Perform single enzyme digestion on the plasmid of Canine Distemper Virus H protein mRNA template. The enzyme digestion system includes: 10 μg of plasmid DNA, 5 μL of enzyme digestion Buffer, 5 μL of restriction endonuclease Sal I, and 30 μL of ultrapure water. The enzyme digestion reaction conditions are: incubate at 37°C for 10 h.

[0049] Recover the single enzyme digestion product: Subsequently, use the micro DNA recovery kit of Guangzhou Feiyang Biotechnology Co., Ltd. to recover the digested DNA according to the steps in the kit instruction manual.

[0050] In Vitro Transcription: After recovery, use the Novoprotein one-step capping mRNA synthesis kit to perform in vitro transcription of mRNA. The reaction system includes: 2 μL of 10×Transcription Buffer, 1 μL of ATP Solution (100 mM), 1 μL of GTP Solution (100 mM), 1 μL of CTP Solution (100 mM), 1 μL of N1-Me-Pseudo UTP (100 mM), 2 μL of T7 RNA Polymerase Mix, 1 μL of CAG Trimer, 4 μL of template DNA, and 7 μL of RNase-Free ddH2O. The reaction conditions are: incubate at 37°C for 5 h.

[0051] Purification: Purify the mRNA using the lithium chloride purification method. The purification steps are: (1) Add 30 μL of lithium chloride precipitation solution (7.5 M lithium chloride, 50 mM EDTA) and 30 μL of RNase-Free ddH2O to 20 μL of the RNA stock solution and mix thoroughly; (2) Place the mixed solution in a -20°C refrigerator for 30 min; (3) Centrifuge the mixed solution at a centrifugation speed of 12000 rpm for 15 min; (4) Discard the supernatant, add 500 μL of 70% ethanol solution to wash the RNA precipitate, and repeat 3 times; (5) Discard the supernatant, air-dry the precipitated RNA, add 50 μL of RNase-Free ddH2O to dissolve the RNA to obtain H mRNA.

[0052] 3. Detection of mRNA Expression in vitro Cells

[0053] BHK-21 cells were seeded in a 12-well cell culture plate. When the cell density reached 80%-90%, H mRNA was transfected into BHK-21 cells using Novoprotein transfection reagent ExFect Transfection Reagent. The transfection system included: 3 μL of ExFect Transfection Reagent, 4 μg of H mRNA, and 200 μL of opti-MEM medium. After the transfection system was completely mixed, it was left standing at room temperature for 20 min. Subsequently, the mixed solution was transferred entirely into the 12-well cell culture plate, and the cell plate was placed in an incubator at 37°C for 36 h. Then, the cell supernatant was discarded, and the cell samples were collected for Western blot analysis to detect the expression of H protein. The results of the Western blot experiment were as Figure 1 shown. The size of the H protein expressed by H mRNA in BHK-21 cells was approximately 76 kD, indicating that the results were consistent with the expectations and that H mRNA was successfully expressed in vitro cells.

[0054] 4. Preparation of mRNA-LNP

[0055] Since naked mRNA itself is extremely unstable, easily hydrolyzed by nucleases and rapidly degraded, and it is also difficult to enter cells. Therefore, mRNA was encapsulated in self-made lipid nanoparticles (LNP) to protect mRNA from enzymatic degradation during delivery and effectively deliver it into cells. The LNP used in this invention is a nanoparticle composed of four lipids: ionizable lipid (DLin-MC3-DMA), distearoylphosphatidylcholine (DSPC), cholesterol, and polyethylene glycol (DMG-PEG2000). The four lipids, DLin-MC3-DMA, DSPC, cholesterol, and DMG-PEG2000, were dissolved in absolute ethanol, and solutions with a concentration of 10 mg / mL were prepared for each. Subsequently, the four lipid solutions were mixed according to the molar percentage of DLin-MC3-DMA:DSPC:cholesterol:DMG-PEG2000 = 50%:10%:38.5%:1.5% to prepare a 10 mg / mL LNP premix. HmRNA was added to a 50 mM, pH = 4 citric acid buffer to make the concentration of H mRNA in the solution 110 ng / μL, resulting in an mRNA-citric acid buffer. The mRNA-citric acid buffer and the LNP premix were mixed at a volume ratio of 3:1 and vortexed for 30 s using a vortex oscillator to obtain H mRNA-LNP.

[0056] Test Example 1

[0057] Evaluation of the immune effect of mRNA-LNP in mice

[0058] Fifteen Balb / c mice were evenly divided into three groups:

[0059] The first group was immunized with H mRNA-LNP. The immunization dose of mRNA-LNP for each mouse was 10 μg, and the immunization volume was 100 μL. (The H mRNA-LNP used was prepared in Example 1)

[0060] The second group of mice was immunized with H mRNA-LNP. The immunization dose of mRNA-LNP for each mouse was 5 μg, and the immunization volume was 100 μL. (The H mRNA-LNP used was prepared in Example 1)

[0061] The third group was injected with firefly luciferase Luc mRNA-LNP respectively, with an immunization dose of 10 μg and a volume of 100 μL.

[0062] The immunization route for the mice was intramuscular injection in the hind legs. The three groups of mice were given booster immunizations with the same dose 14 days and 28 days after the first immunization, and blood was collected from the orbital cavities 28 days, 42 days, and 56 days after the first immunization. Subsequently, neutralization tests were performed on the mouse sera to detect the levels of canine distemper virus-specific antibodies in the sera. The results of the neutralization experiments are as Figures 2 to 5 shown. CDV-specific neutralizing antibodies were detected in the mouse sera at 28 days, 42 days, and 56 days after the first immunization, and the level of neutralizing antibodies depended on the immunization dose of the vaccine. The average neutralizing antibody level in the 10 μg H mRNA-LNP immunization group was significantly higher than that in the 5 μg H mRNA-LNP immunization group. After booster immunization, the neutralizing antibody level in the H mRNA-LNP immunization group of mice increased significantly, and there was no obvious downward trend in the neutralizing antibody level 56 days after the first immunization compared with that 42 days after the first immunization, indicating that H mRNA-LNP could maintain a relatively high neutralizing antibody level for a certain period of time after booster immunization. For the first group, 28 days after the first immunization, the highest neutralizing antibody titer of individual mice could reach 1:32; 42 days after the first immunization, the highest neutralizing antibody titer of individual mice could reach 1:708; 56 days after the first immunization, the highest neutralizing antibody titer of individual mice could reach 1:629;

[0063] For the second group, 28 days after the first immunization, the highest neutralizing antibody titer of individual mice could reach 1:13; 42 days after the first immunization, the highest neutralizing antibody titer of individual mice could reach 1:355; 56 days after the first immunization, the highest neutralizing antibody titer of individual mice could reach 1:200.

[0064] For the third group, 28 days after the first immunization, the neutralizing antibody titers of individual mice were all less than 1:2; 42 days after the first immunization, the neutralizing antibody titers of individual mice were all less than 1:2; 56 days after the first immunization, the neutralizing antibody titers of individual mice were all less than 1:2.

[0065] The method of the neutralization test is described as follows: First, dilute the mouse serum with DMEM in a 2-fold serial dilution. Dilute the canine distemper virus (CDV) solution with a known virus titer to 200 TCID 50 / 100 μL. Vortex-mix the diluted CDV virus solution at 200 TCID 50 / 100 μL with the serum solution of each dilution of the mouse serum in a volume ratio of 1:1, and incubate it in an incubator at 37 °C for 1 h. Pre-inoculate vero (slam-vero) cells expressing canine slam protein into a 96-well plate, add the incubated virus-mouse serum mixture to the slam-vero cells, and additionally set up virus positive wells, virus negative wells, and a regression curve group as controls. Subsequently, culture the slam-vero cells in a cell culture incubator under conditions of 37% and 5% CO2 for 3 days. Then discard the cell supernatant, add 80% acetone solution, and place it in a -20 °C refrigerator for 1 h to fix the cells. After cell fixation, wash the cells 3 times with PBS solution, add 30 μL of FITC-CDV monoclonal antibody (purchased from vmrd company) to each well, and incubate it in a 4 °C refrigerator for 6 h. Discard the FITC-CDV monoclonal antibody, wash the cells 3 times with PBS solution, then observe the cell lesion conditions under a fluorescence microscope, and calculate the CDV specific neutralizing antibody titer of the mouse serum using the Reed-Muench method.

[0066] Although the embodiments of the present invention have been described in detail above, it is obvious to those skilled in the art that various modifications and changes can be made to these embodiments. However, it should be understood that such modifications and changes are all within the scope and spirit of the present invention described in the claims. Moreover, the present invention described herein can have other embodiments and can be implemented or realized in various ways.

Claims

1. A method for preparing a canine distemper vaccine, characterized in that: The method comprises: Using pUC57 as a vector, an mRNA vaccine template plasmid was designed for the H protein of canine distemper virus; Perform single enzyme digestion on the mRNA vaccine template plasmid and recover the single enzyme digestion product; Performing RNA in vitro transcription on the single enzyme digestion product to obtain the transcribed RNA stock solution; Purifying the RNA stock solution, and dissolving the purified RNA using RNase-Free ddH2O to obtain H mRNA; The H mRNA is added to a citric acid buffer to obtain an mRNA-citric acid buffer, an LNP premix is ​​prepared, and the mRNA-citric acid buffer and the LNP premix are mixed to obtain the H mRNA-LNP.

2. The method for preparing a canine distemper vaccine according to claim 1, characterized in that: The steps of using pUC57 as a vector to design an mRNA vaccine template plasmid for canine distemper virus H protein include: The vector includes a T7 promoter, a UTR sequence and a codon-optimized H protein sequence; The DNA sequence of the mRNA vaccine template plasmid is shown in SEQ ID NO.

1.

3. The method for preparing a canine distemper vaccine according to claim 1, characterized in that: The step of performing single enzyme digestion on the mRNA vaccine template plasmid comprises: Constructing an enzyme digestion system, the enzyme digestion system comprising: mRNA vaccine template plasmid, enzyme digestion buffer, restriction endonuclease SalI, and ultrapure water; Set the enzyme digestion reaction conditions as follows: incubate at 35-40°C for 8-12 hours.

4. The method for preparing a canine distemper vaccine according to claim 1, characterized in that: The step of performing RNA in vitro transcription on the single enzyme digestion product to obtain the transcribed RNA stock solution comprises: The reaction system for in vitro transcription includes: 10×Transcription Buffer, ATP Solution, GTP Solution, CTP Solution, N1-Me-Pseudo UTP, T7 RNA Polymerase Mix, CAG Trimer, template DNA, RNase-Free ddH2O; The reaction conditions for in vitro transcription are: incubation at 35-40°C for 4-6 hours.

5. The method for preparing a canine distemper vaccine according to claim 1, characterized in that: The step of purifying the RNA stock solution and dissolving the purified RNA using RNase-Free ddH2O to obtain HmRNA comprises: (1) Add pre-prepared lithium chloride precipitation solution and RNase-Free ddH2O to the RNA stock solution and mix thoroughly to obtain a mixed solution; (2) Place the mixture in a -20°C refrigerator for 30 min; (3) centrifuging the mixed solution; (4) Discard the supernatant and add 70% ethanol solution to wash the RNA precipitate, repeating three times; (5) Discard the supernatant, air-dry the precipitated RNA, add RNase-Free ddH2O to dissolve the RNA, and obtain H mRNA.

6. The method for preparing a canine distemper vaccine according to claim 1, characterized in that: The steps of preparing the LNP premix include: Dissolve four lipids, namely, ionizable lipid, distearoylphosphatidylcholine, cholesterol and polyethylene glycol, in anhydrous ethanol to prepare lipid solutions with a concentration of 8-12 mg / mL; The four lipid solutions of ionizable lipid, distearoylphosphatidylcholine, cholesterol and polyethylene glycol were mixed in the ratio of 48-52%: 8-12%: 38-39%: 1-2% to prepare a 10 mg / mL LNP premix.

7. The method for preparing a canine distemper vaccine according to claim 1, characterized in that: The mixing volume ratio of mRNA-citrate buffer and LNP premix is ​​2.5-3.5:

1.

8. The method for preparing a canine distemper vaccine according to claim 1, characterized in that: The steps for preparing mRNA-citrate buffer include: H mRNA was added to 50 mM, pH = 4 citrate buffer solution to make the concentration of H mRNA in the solution 100-120 ng / μL, thereby preparing mRNA-citrate buffer solution.

9. A canine distemper vaccine, characterized in that: The canine distemper vaccine is prepared according to the method for preparing the canine distemper vaccine according to any one of claims 1 to 8.

10. Use of the canine distemper vaccine according to claim 9 in the preparation of a drug for treating canine distemper.

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