A dual subunit vaccine of canine distemper virus and canine parvovirus and its preparation method and application

Abstract

The present invention belongs to the technical field of veterinary biological products, and specifically relates to a bivalent subunit vaccine of canine distemper virus and canine parvovirus, and a preparation method and application thereof. The present invention innovatively realizes a method for simultaneously producing two virus subunit vaccines in a single expression and purification process. The purification process is simple, and the enzymes used in production can be recycled and reused, thereby reducing production costs. The molar ratio of the canine distemper virus H protein in the dimer form and the canine parvovirus VP2 protein in the VLPs form in the bivalent subunit vaccine of the present invention is 1:1, which improves the purity of the vaccine antigen, makes the immunogenicity stronger, and reduces the cost.

Description

Technical Field

[0001] The invention belongs to the technical field of veterinary biological products, and specifically relates to a bivalent subunit vaccine of canine distemper virus and canine parvovirus, and a preparation method and application thereof. Background Art

[0002] Canine distemper is a highly contagious disease caused by canine distemper virus (CDV) that infects canines, mustelids and some raccoons. It can cause biphasic fever, acute catarrhal respiratory tract inflammation, gastroenteritis and encephalitis. CDV belongs to the family Paramyxoviridae and the genus Morbillivirus. It is a single-stranded negative-strand RNA virus with no segmentation. Its genome encodes 6 structural proteins (N, P, M, F, H, L) and 2 non-structural proteins (C and V). Among them, the H gene is 1947 nt in length and contains only one open reading frame. The H protein it encodes is a type II transmembrane glycoprotein. The H protein of mature virus particles is a homodimer connected by disulfide bonds, embedded in the surface of the virus envelope, and can interact with receptors on the surface of target cells (such as SLAM and nectin-4) to mediate the virus invasion of the host. There are many neutralizing antigenic epitopes on the H protein, which is the main antigen that induces the body to produce neutralizing antibodies.

[0003] Canine parvovirus disease is a highly lethal acute infectious disease caused by canine parvovirus (CPV) of the genus Parvovirus in the family Parvoviridae. The disease can occur all year round and has the characteristics of rapid onset, short course, high mortality, and strong infectivity. It is one of the most serious infectious diseases that endangers my country's dog breeding industry. VP2 protein is the main component of canine parvovirus capsid protein, which can self-assemble in vitro to form virus-like particles. This protein contains the neutralizing epitope of the virus and has strong immunogenicity. It is the main target antigen for vaccine development.

[0004] There are also related studies on canine distemper and canine parvovirus bivalent subunit vaccines in the prior art, such as CN114317459A, which provides a canine distemper virus strain and a bivalent vaccine based on canine distemper virus and canine parvovirus and its application, involving two production processes of canine distemper virus culture and baculovirus expression of canine parvovirus VP2, and the production process is complicated and costly. CN201810463652.7 relates to a canine distemper and parvovirus bivalent subunit vaccine, which adopts a method of expressing canine distemper virus and canine parvovirus quantitatively and then mixing them, and the antigens are not purified, and the cost of two expressions is higher than this technology. In addition, the existing canine distemper and canine parvovirus bivalent subunit vaccines also use viruses of two proteins to infect insect cells at the same time, resulting in different infection efficiencies of the viruses expressing each protein, and uncontrollable protein production. When cells are infected with multiple baculoviruses, high-frequency recombination occurs, resulting in the production of defective viruses and the inability to express the expected multi-protein products. Summary of the invention

[0005] The purpose of the present invention is to provide a canine distemper virus and canine parvovirus bivalent subunit vaccine and a preparation method and application thereof, which innovatively realizes a method for simultaneously producing two virus subunit vaccines in a single expression and purification process, and the purification filler and enzyme for enzyme cleavage can be reused, the purification process is simple, and the production cost is low. The molar ratio of canine distemper virus to canine parvovirus in the bivalent subunit vaccine of the present invention is 1:1, the vaccine antigen has high purity, stronger immunogenicity, and lower cost.

[0006] The invention provides a bivalent subunit vaccine of canine distemper virus and canine parvovirus, wherein the bivalent subunit vaccine comprises CHO-H-VP2; a gene encoding the CHO-H-VP2 is a recombinant CHO-H-VP2 gene, and the recombinant CHO-H-VP2 gene comprises a nucleotide sequence encoding a Mouse IgG kappa signal peptide, a CDV H gene, a canine IgG FC, a TEV restriction endonuclease motif and a CPV VP2 gene.

[0007] Preferably, the amino acid sequence of the CHO-H-VP2 is shown in SEQ ID NO.7.

[0008] Preferably, the nucleotide sequence of the recombinant CHO-H-VP2 gene is as shown in SEQ ID NO.6.

[0009] Preferably, the nucleotide sequence encoding Mouse IgG kappa signal peptide is shown as SEQ ID NO.1, the nucleotide sequence of the CDV H gene is shown as SEQ ID NO.2, the nucleotide sequence of the canine IgG FC is shown as SEQ ID NO.3, the nucleotide sequence of the TEV cleavage motif is shown as SEQ ID NO.4, and the nucleotide sequence of the CPV VP2 gene is shown as SEQ ID NO.5.

[0010] The present invention also provides a method for preparing a bivalent subunit vaccine of canine distemper virus and canine parvovirus, comprising the following steps: transfecting cells with a recombinant expression vector comprising the recombinant CHO-H-VP2 gene described in the above technical solution to obtain a cell expression supernatant;

[0011] Purifying the cell expression supernatant to obtain a purified target protein;

[0012] Performing enzyme digestion on the purified target protein to obtain digested protein;

[0013] The enzyme-cleaved protein is subjected to virus-like particle self-assembly and then mixed with an adjuvant to obtain the bivalent subunit vaccine.

[0014] Preferably, the protease used for enzymatic cleavage of the purified target protein is TEV protease.

[0015] Preferably, the vector used to prepare the recombinant expression vector includes a plasmid, a viral vector, a cosmid or a phage.

[0016] Preferably, the transfected cells include CHO cells.

[0017] Preferably, the CHO cells include CHO-K1 cells.

[0018] Beneficial effects:

[0019] The present invention provides a bivalent subunit vaccine of canine distemper virus and canine parvovirus, wherein the bivalent subunit vaccine comprises CHO-H-VP2, wherein the CHO-H-VP2 comprises a truncated canine distemper virus H protein, wherein amino acids 1 to 147 at the N-terminus that are irrelevant to dimer formation are deleted, and the intracellular region, the transmembrane region and the DUF4217 domain are included to form a homodimeric protein; at the same time, an FC tag is added to the bivalent subunit vaccine to promote the truncated canine distemper virus H protein to form a dimer, prolong the half-life of the protein, promote antigen presentation, and facilitate downstream purification;

[0020] At the same time, by adding the TEV protease cleavage motif to the CHO-H-VP2, the canine parvovirus VP2 protein produced by TEV protease cleavage is closest to the natural virus sequence, with only one more glycine at the N-terminus, which does not affect the formation of VLPs.

[0021] Furthermore, the bivalent subunit vaccine of the present invention includes canine distemper virus H protein in dimer form and canine parvovirus VP2 protein in VLPs form, and a subunit vaccine including two viruses is obtained through a single expression and purification process, thereby ensuring that the molar ratio of canine distemper virus to canine parvovirus in the bivalent subunit vaccine is 1:1, with stronger immunogenicity and lower cost.

[0022] The present invention also provides a method for preparing a two-unit vaccine of canine distemper virus and canine parvovirus, wherein commercial protein A filler is used to purify the protein, and both the purification filler and the enzyme for enzymatic cleavage can be reused, the purification process is simple, and complex processes such as protein renaturation and ion exchange chromatography are not required. At the same time, the protease used in the preparation process can be recovered and reused for the next production, saving costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0024] Figure 1 It is the 3D structure prediction diagram of the canine distemper virus H protein in the present invention;

[0025] Figure 2 This is the pEE12.4 vector map. DETAILED DESCRIPTION

[0026] The invention provides a bivalent subunit vaccine of canine distemper virus and canine parvovirus, wherein the bivalent subunit vaccine comprises CHO-H-VP2; a gene encoding the CHO-H-VP2 is a recombinant CHO-H-VP2 gene, and the recombinant CHO-H-VP2 gene comprises a nucleotide sequence encoding a Mouse IgG kappa signal peptide, a CDV H gene, a canine IgG FC, a TEV restriction endonuclease motif and a CPV VP2 gene.

[0027] Figure 1

[0028] The present invention also provides a method for preparing a bivalent subunit vaccine of canine distemper virus and canine parvovirus, comprising the following steps: transfecting cells with a recombinant expression vector comprising the recombinant CHO-H-VP2 gene described in the above technical scheme to obtain a cell expression supernatant; purifying the cell expression supernatant to obtain a purified target protein; and subjecting the purified target protein to virus-like particle self-assembly and then mixing with an adjuvant to obtain the bivalent subunit vaccine.

[0029] Before transfecting cells with the recombinant expression vector comprising the recombinant CHO-H-VP2 gene, the present invention preferably further comprises preparing the recombinant expression vector.

[0030] When the recombinant expression vector of the recombinant CHO-H-VP2 gene of the present invention is prepared, the vector used to prepare the recombinant expression vector includes a plasmid, a viral vector, a cosmid or a bacteriophage, preferably a plasmid, and more preferably a pEE12.4 vector. The present invention does not specifically limit the specific process of preparing the recombinant vector, and conventional preparation methods and steps in the art can be used.

[0031] After obtaining the recombinant expression vector, the present invention transfects cells with the recombinant expression vector including the recombinant CHO-H-VP2 gene to obtain cell expression supernatant.

[0032] In the present invention, when the recombinant expression vector comprising the recombinant CHO-H-VP2 gene is transfected, the transfected cells preferably include CHO cells, more preferably CHO-K1 cells. The present invention has no particular limitation on the steps of transfecting cells, and conventional steps in the art can be used.

[0033] After obtaining the cell expression supernatant, the present invention purifies the cell expression supernatant to obtain a purified target protein. The present invention preferably uses affinity chromatography to purify the cell expression supernatant, and the filler used is preferably Protein A filler. The present invention does not specifically limit the source of the filler, and conventional commercial fillers in the art can be used. The present invention does not specifically limit the purification process, and conventional purification steps in the art can be used.

[0034] After obtaining the purified target protein, the present invention performs self-assembly of the purified target protein into virus-like particles (VLPs). The protease used in the self-assembly of the purified target protein into virus-like particles is preferably TEV protease.

[0035] After the self-assembly of the virus-like particles (VLPs) is completed, the present invention preferably removes and recovers TEV protease to obtain an antigen solution containing canine distemper virus H protein dimer and canine parvovirus VLPs. The recovered TEV protease can be directly used in the enzyme cleavage process of the next production.

[0036] After obtaining the antigen liquid containing canine distemper virus H protein dimer and canine parvovirus VLPs, the present invention mixes the antigen liquid containing canine distemper virus H protein dimer and canine parvovirus VLPs with an adjuvant to obtain the bivalent subunit vaccine. The adjuvant of the present invention preferably includes ISA201 VG. The concentration of the antigen liquid containing canine distemper virus H protein dimer and canine parvovirus VLPs of the present invention is preferably 60 μg / mL. The volume ratio of the antigen liquid containing canine distemper virus H protein dimer and canine parvovirus VLPs to the adjuvant of the present invention is preferably 46:54.

[0037] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0038] Example 1

[0039] sequence synthesis

[0040] With reference to the canine distemper virus H gene sequence (DQ778941) and the canine parvovirus VP2 gene sequence (MN840830.1), the CHO-H-VP2 sequence was synthesized after codon optimization. The CHO-H-VP2 sequence is as follows: Mouse IgG kappa signal peptide, 63 bases in size, and its nucleotide sequence is shown in SEQ ID NO.1; CDV H gene sequence, 1380 bases in size, and its nucleotide sequence is shown in SEQ ID NO.2; canine IgG FC sequence, 702 nucleotides in size, and its nucleotide sequence is shown in SEQ ID NO.3; TEV restriction motif, 21 bases in size, and its nucleotide sequence is shown in SEQ ID NO.4; CPV VP2 gene sequence, 1752 bases in size, and its nucleotide sequence is shown in SEQ ID NO.5; the synthesized CHO-H-VP2 sequence contains HindIII and EcoRI restriction sites at both ends, and the total length is 3936 bases, as shown in SEQ ID NO.6. The amino acid sequence encoded by the CHO-H-VP2 sequence is shown in SEQ ID NO.7.

[0041] Example 2

[0042] Construction of CHO-H-VP2-pEE12.4 recombinant plasmid

[0043] The CHO-H-VP2 sequence synthesized in Example 1 and the pEE12.4 vector (the map of the pEE12.4 vector is shown in FIG. 1 ) were digested with HindIII and EcoRI. Figure 2 As shown), the CHO-H-VP2 sequence and pEE12.4 sequence fragments after enzyme digestion were recovered, and ligated, transformed and cloned, and sequenced to construct the CHO-H-VP2-pEE12.4 recombinant plasmid.

[0044] Example 3

[0045] CHO-K1 cell transfection and screening of high-expressing cell lines

[0046] 1. Prepare cells:

[0047] Take a dish (10 cm cell culture dish) of CHO-K1 cells with a confluence of 80%-90% within 24 hours of subculture, discard the culture medium, add 10 mL PBS to wash the cells once, discard the PBS, add 15 mL serum-free and antibiotic-free DMEM / F12 culture medium, and place at 37°C 5% CO 2 Culture in a cell culture incubator.

[0048] 2. Transfection plasmid

[0049] according to 2000 instruction manual, dilute 24 μg of E2-FC-Cap-pEE12.4 recombinant plasmid and 60 ul of 2000, mix well and place at room temperature for 5 minutes. Mix the two tubes of liquid and place at room temperature for 20 minutes. Take the prepared CHO-K1 cells, add the above mixed reagent drop by drop, and then place in 37℃ 5% CO 2 After 4-6 hours of culture, discard the culture medium, add 10 mL of DMEM / F12 culture medium (10% serum, 1% double antibody, without glutamine), and place at 37°C 5% CO 2 Culture in a cell culture incubator.

[0050] 3. Pressurized screening

[0051] 24 h after transfection, take one dish each of transfected CHO-K1 cells and untransfected CHO-K1 cells (negative control), discard the supernatant culture medium, add 10 mL DMEM / F12 culture medium (10% serum + 25 uM MSX, 1% double antibody, without glutamine), and screen under pressure for 7 days. Observe the cells in the middle. When there are many dead cells, replace the medium with the same culture medium.

[0052] 4. Screening of stable cell lines

[0053] 25uM MSX pressure screening until the negative control cells are basically dead, about 7 days, start cell line screening. Take the transfected CHO-K1 cells, discard the culture medium, wash once with PBS, add 500uL 0.25% trypsin, digest at room temperature for 2-5 minutes until the cells are single cells, add 10mL DMEM / F12 culture medium (10% serum + 25uM MSX, 1% double antibody, without glutamine) to terminate the digestion reaction, blow the cells with a pipette, and count the cells. Dilute the cells to 10 with DMEM / F12 culture medium (10% serum + 25uM MSX, 1% double antibody, without glutamine) 4 cells / mL, transferred to 96-well plate, 200ul per well, and placed in 37℃ 5% CO 2 Culture in a cell culture incubator. When the 96-well plate is full, take the supernatant and perform ELISA test. Continue to expand the culture and freeze the positive cell lines with high expression.

[0054] Example 4

[0055] Serum-free suspension culture of high-expressing cell lines

[0056] Resuscitate high-expressing cell lines in DMEM / F12 medium (10% serum, 1% double antibody, without glutamine), and continue to use this medium for 2 to 3 passages until cell growth is stable. When the cell density reaches 2×10 6 cells / mL, and then subcultured. The cell density of subcultured cells was controlled at 0.3-0.5×10 5 cells / mL, the culture medium was DMEM / F12 medium with 10% serum and The basal culture medium (purchased from Jianshun Biotechnology) was mixed in a ratio of 75:25, and the cells were placed at 37°C, 5% CO 2 , and cultured in a constant temperature shaker at 110 rpm. When the cell density reached 2×10 6 cells / mL and the cell viability was >90%, and the DMEM / F12 medium with 10% serum was used for subculturing. The basal medium was mixed in a 50:50 ratio, and the cell density was controlled at 0.3-0.5×10 6 If the cells grow slowly, the supernatant can be centrifuged, 20% of the original culture medium can be retained, and 80% of the DMEM / F12 culture medium containing 10% serum can be added. The ratio of basal medium to culture medium is 75:25, and the culture is continued; repeat the above steps and gradually increase The proportion of basal medium is up to 100% Basal culture medium;

[0057] Example 5

[0058] Shake flask fermentation

[0059] Remove the shake flask cells from the constant temperature shaker and dilute the cells to 0.3-0.5×10 5 cells / mL inoculated with 30mL Transfer basal medium to a 125 mL shake flask and place at 37°C with 5% CO 2 , cultured in a constant temperature shaker at 110 rpm. Glucose concentration was tested every day. When the glucose concentration was less than 4 g / L, glucose solution was added to the culture medium to a glucose concentration of 4 g / L. 3% of Feed medium. On day 12, harvest cell culture supernatant.

[0060] Example 6

[0061] Protein purification

[0062] The cell culture supernatant in Example 5 was collected, centrifuged at 8000g for 30 minutes at 4°C, and the supernatant was taken and filtered through a 0.8 μm filter. The Protein A column material was equilibrated with 5-10 column volumes of PBS, and the treated cell supernatant was repeatedly loaded onto the column 3 times, the column material was washed with 10 times PBST, the column material was washed with 2 times PBS, and the protein was eluted with 2 times the column volume of 0.1M glycine (pH3.0), the eluate was collected, and 1M Tris (pH9.0) was added to neutralize to pH7.5, which was the purified protein.

[0063] Example 7

[0064] Protein cleavage and refolding

[0065] According to the instructions of the BCA protein quantitative detection kit (purchased from Shanghai Biotech), the concentration of purified protein was determined, the total mass of protein was calculated, 1U TEV protease (His-tag, purchased from Biotech) was added for every 8μg protein, and 10× enzyme digestion buffer (500mM NaH 2 PO 4 , 150mM NaCl, 10mM EDTA, 10mM DTT, 1% Tween-20, pH8.0), and digested at 4°C for 12-16 hours. After digestion, the digested protein was placed in a dialysis bag (3500D) and the dialysis fluid was 50mM NaH 2 PO 4 , 500mM NaCl, pH8.0, dialyze for 12-16 hours, changing the solution 2-3 times during the period. After the first dialysis, stand at 4°C for 8-12 hours to complete protein refolding. Replace the dialysis solution with PBS buffer, dialyze for 12-16 hours, changing the solution 2-3 times during the period.

[0066] Example 8

[0067] Removal and recovery of TEV protease

[0068] Take the nickel column filled with filler, balance the nickel column with PBS 2 to 5 times the volume of column material, repeat the sample of the liquid after dialysis in Example 7 for 3 times, and the flow-through liquid is the antigen liquid containing canine distemper virus H protein dimer and canine parvovirus VLPs. Elute the nickel column with PBS buffer containing 200mM imidazole, collect the eluate, which is the recovered TEV protease, and can be directly used in the enzymatic cleavage process of the next production after enzyme activity determination.

[0069] Example 9

[0070] Vaccine preparation

[0071] The antigen solution of Example 8 was diluted with PBS buffer to a final concentration of 60 μg / mL, and mixed and emulsified with ISA 201 VG adjuvant at a volume ratio of 46:54 to obtain a canine distemper and canine parvovirus bivalent subunit vaccine.

[0072] Example 10

[0073] Application of canine distemper and canine parvovirus two-unit vaccine

[0074] 1. Immunization regimen

[0075] Twenty weaned beagle dogs aged 4 to 12 weeks (purchased from Qingdao Bolong Experimental Animal Co., Ltd.) were used. Ten dogs in the immunization group were injected subcutaneously in the neck with 1 mL of the bivalent subunit vaccine per dog. Ten dogs in the blank control group were injected subcutaneously in the neck with 1 mL of PBS per dog. A booster immunization was performed 14 days after immunization.

[0076] 2. Challenge protection test

[0077] 2.1 Immune protection against canine distemper virus

[0078] Fourteen days after booster immunization, five dogs from each group were challenged with canine distemper virus CVCC AV299 strain (purchased from China Veterinary Drug Administration) by subcutaneous injection of 2 mL (containing 10 6.5 TCID50 / mL virus). The survival of the dogs was observed within 14 days after the challenge. The results showed that all dogs in the blank control group died after the challenge, while all dogs in the two-unit vaccine immunization group survived.

[0079] 2.2 Immune protection against canine parvovirus

[0080] Fourteen days after booster immunization, five dogs from each group were challenged with canine parvovirus CVCC AV298 strain (purchased from the China Veterinary Drug Administration) by oral administration of 10 mL (containing 10 6.0TCID50 / mL virus). The survival of the dogs was observed within 14 days after the challenge. The results showed that all dogs in the blank control group died after the challenge, while all dogs in the two-unit vaccine immunization group survived.

[0081] It can be concluded from the above examples that the bivalent subunit vaccine of canine distemper virus and canine parvovirus of the present invention has good immunogenicity and protects dogs from canine distemper virus and canine parvovirus.

[0082] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A two-unit vaccine of canine distemper virus and canine parvovirus, characterized in that: The bivalent subunit vaccine includes CHO-H-VP2; the gene encoding the CHO-H-VP2 is a recombinant CHO-H-VP2 gene, and the recombinant CHO-H-VP2 gene includes a nucleotide sequence encoding a Mouse IgG kappa signal peptide, a CDVH gene, a canine IgGFC, a TEV restriction motif, and a CPVVP2 gene; The bivalent subunit vaccine is obtained by the following preparation method: Transfecting cells with the recombinant expression vector of the recombinant CHO-H-VP2 gene to obtain cell expression supernatant; Purifying the cell expression supernatant to obtain a purified target protein; Performing enzyme digestion on the purified target protein to obtain digested protein; The enzyme-cleaved protein is subjected to virus-like particle self-assembly and then mixed with an adjuvant to obtain the bivalent subunit vaccine; The molar ratio of canine distemper virus to canine parvovirus in the bivalent subunit vaccine is 1:1; The amino acid sequence of the CHO-H-VP2 is shown in SEQ ID NO.

7.

2. The bivalent subunit vaccine according to claim 1, characterized in that: The nucleotide sequence of the recombinant CHO-H-VP2 gene is shown in SEQ ID NO.

6.

3. The bivalent subunit vaccine according to claim 1, characterized in that: The nucleotide sequence encoding the Mouse IgG kappa signal peptide is shown in SEQ ID NO.1, the nucleotide sequence of the CDVH gene is shown in SEQ ID NO.2, the nucleotide sequence of the canine IgGFC is shown in SEQ ID NO.3, the nucleotide sequence of the TEV cleavage motif is shown in SEQ ID NO.4, and the nucleotide sequence of the CPVVP2 gene is shown in SEQ ID NO.

5.

4. The bivalent subunit vaccine according to claim 1, characterized in that: The protease used in the enzymatic cleavage of the purified target protein is TEV protease.

5. The bivalent subunit vaccine according to claim 1, characterized in that: The recombinant expression vector used to prepare the recombinant expression vector includes plasmid, viral vector, cosmid or phage.

6. The bivalent subunit vaccine according to claim 1, characterized in that: The transfected cells include CHO cells.

7. The bivalent subunit vaccine according to claim 6, characterized in that: The CHO cells include CHO-K1 cells.

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