A recombinant virus rPIV5-H3 and its preparation method, application and vaccine

Abstract

The present invention belongs to the field of biotechnology and discloses a recombinant virus rPIV5-H3, whose full gene sequence is shown in SEQ NO.1. The recombinant virus rPIV5-H3 comprises a parainfluenza virus type 5 genome, wherein the genome comprises the gene sequence of the influenza virus H3HA protein; the gene sequence is inserted into the nucleotide sequence of the small hydrophobic protein gene of the parainfluenza virus type 5 genome. At the same time, the present invention also provides a preparation method, application and vaccine of the recombinant virus.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and specifically to a recombinant virus rPIV5-H3 and its preparation method, application and vaccine. Background Art

[0002] Canine influenza virus (CIV), belonging to the genus Influenza A virus in the family Orthomyxoviridae, is a type of influenza virus that can cause contact respiratory infections in dogs. Currently, multiple subtypes of influenza viruses have been found to infect dogs, with H3N8 and H3N2 subtypes being widely prevalent in canine populations.

[0003] Dogs are important companion animals for humans, and influenza viruses in dogs may increase the possibility of infecting humans through close contact between humans and dogs.

[0004] Currently, the most effective strategy for preventing CIV infection is vaccination. While vaccines targeting the disease are commercially available in countries with high incidences of canine influenza, such as the United States and South Korea, there is no effective vaccine in China. Therefore, developing a new, highly effective canine influenza vaccine is of great public health significance. CIV is a single-stranded, negative-sense RNA virus consisting of eight RNA segments. The virus's two proteins, hemagglutinin (HA) and neuraminidase (NA), are the most important antigens for inducing protective immunity in the host. HA mediates the binding of the virus to sialic acid receptors on the host cell membrane, promoting entry of the viral genome into the host. HA glycosylation plays a key role in the immune response.

[0005] Viral vector vaccines have many advantages. Because they are live viral vectors, they can replicate in the body of immunized animals and induce a strong immune response. They can be immunized through natural infection, such as nasal drops and sprays. They can produce humoral immunity and cellular immunity. In addition to stimulating systemic immune responses, they can also stimulate local immune responses, such as the production of sIgA antibodies in the respiratory tract, exerting a strong immune protection effect. Currently, in the development of new crown vaccines, there are many viral vector vaccines in the development process, and they have good immune protection effects in animal models, such as yellow fever virus, poxvirus, Newcastle disease virus, vesicular stomatitis virus, etc. Parainfluenza virus type 5 (PIV5) has been used in dogs as a vaccine to prevent kennel cough for decades, and it has not been proven to cause disease in any other animals, including humans. Therefore, the development of an effective canine influenza virus vector vaccine is the main direction of research on new subunit vaccines for canine influenza virus. Summary of the Invention

[0006] The object of the present invention is to provide a recombinant virus rPIV5-H3 expressing influenza virus H3 HA protein, wherein the recombinant virus rPIV5-H3 comprises a parainfluenza virus type 5 genome, wherein the genome comprises the gene sequence of influenza virus H3 HA protein; the gene sequence is inserted into the nucleotide sequence of the small hydrophobic protein gene of the parainfluenza virus type 5 genome.

[0007] At the same time, the present invention also provides a preparation method, application and vaccine of the recombinant virus.

[0008] To achieve the above-mentioned purpose, the present invention provides the following technical solution: a recombinant virus rPIV5-H3, whose complete gene sequence is shown in SEQ NO.1.

[0009] At the same time, the present invention also discloses a method for preparing the recombinant virus rPIV5-H3 as described above, comprising the following steps:

[0010] Step 1: inserting the full genome cDNA of parainfluenza virus type 5 into a cloning vector to obtain a recombinant plasmid pPIV5; the sequence of the full genome cDNA of parainfluenza virus type 5 is shown in SEQ NO. 2;

[0011] Step 2: Insert the influenza virus HA protein encoding gene sequence into the small hydrophobic protein gene nucleotide sequence of the recombinant plasmid pPIV5. The recombinant plasmid is named pPIV5-H3. The influenza virus HA protein encoding gene sequence is shown in SEQ NO. 3.

[0012] Step 3: co-transfect the host cells with the recombinant plasmid pPIV5-H3, the auxiliary recombinant plasmid NP expressing the auxiliary protein, the auxiliary recombinant plasmid P, and the auxiliary recombinant plasmid L;

[0013] The auxiliary recombinant plasmid NP is a vector containing the NP gene with the nucleotide sequence shown in SEQ NO.4; the auxiliary recombinant plasmid P is a vector containing the P gene with the nucleotide sequence shown in SEQ NO.5; and the auxiliary recombinant plasmid L is a vector containing the L gene with the nucleotide sequence shown in SEQ NO.6.

[0014] In the above-mentioned method for preparing the recombinant virus rPIV5-H3, the cloning vector is a pBlueScript SK II (+) vector;

[0015] The vector in step 3 is pcDNA3.1(+).

[0016] In the above-mentioned method for preparing the recombinant virus rPIV5-H3, the host cell is a BSR-T7 cell, an MDBK cell, a Vero cell or a BHK cell.

[0017] At the same time, the present invention also discloses a use of the recombinant virus rPIV5-H3 as described above for preparing a vaccine.

[0018] Finally, the present invention discloses a vaccine containing the recombinant virus rPIV5-H3 as described above.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The recombinant parainfluenza virus rPIV5-H3 of the present invention comprises a parainfluenza virus type 5 genome, wherein the genome comprises the gene sequence of influenza virus H3 HA protein; the gene sequence is inserted into the nucleotide sequence of the small hydrophobic protein gene of the parainfluenza virus type 5 genome. (1) At the gene level, its genome is relatively stable and is not prone to mutations such as deletions; the PIV5 intergenic region facilitates the insertion and regulation of exogenous genes and is conducive to maintaining the stability of exogenous genes. Its genome can carry 4 to 5 kb of exogenous genes. (2) At the cellular level, its host cell range is wide, does not cause cytopathic effects, does not cause death and damage to host cells, and can obtain titer viruses in a variety of cells. (3) At the organism level, PIV5 can infect most mammals, including humans, but does not cause typical clinical symptoms. It has good safety and is usually administered intranasally, which is simple and safe and can produce efficient humoral immunity and cellular immunity. (4) In clinical practice, canine parainfluenza virus is a type of canine quadruple vaccine preventive virus. It has been routinely used for many years and has good safety.

[0021] On the 1st and 14th days of the experiment, rPIV5-H3 was administered intranasally and subcutaneously to BALB / c mice and beagle dogs, respectively. Four weeks after the first vaccination, influenza virus was administered intranasally and intravenously to BALB / c mice and beagle dogs, respectively. Throat swabs on days 1, 3, 5, 7, 9, 11, and 13 after infection and serum-specific antibody levels on days 0, 3, 7, 9, and 13 after infection were collected and tested, and the weight, temperature changes, and clinical symptoms of BALB / c mice and beagle dogs after infection were monitored. At the same time, tissues such as the lungs and trachea were dissected and removed on day 3 after infection, and the virus load of each group of organs was tested. There was no significant change in the weight, temperature, and clinical symptoms of the animals after immunization, and no virus was detected in the nasopharyngeal swabs of the dogs. rPIV5-H3 has an excellent immune protection effect. The present invention has important application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the enzyme digestion verification diagram of the PIV5 full-length cDNA cloning vector.

[0023] Figure 2 Diagram for the expression of NP, P and L proteins to verify the enzyme digestion of the auxiliary plasmid.

[0024] Figure 3Figure 2 shows the rescue identification of rPIV5 using immunofluorescence (A) and western blotting (B).

[0025] Figure 4 Validation diagram of molecular genetic markers in rPIV5.

[0026] Figure 5 This is a diagram showing the expression identification of the exogenous HA gene of rPIV5-H3 using immunofluorescence;

[0027] Figure 6 This is a bar graph showing the amount of specific antibodies against HA protein produced in mice immunized with recombinant viruses rPIV5-H3, rPIV5-H3-147, and rPIV5-H3-1109;

[0028] Figure 7 This is the plasmid map of the recombinant plasmid pPIV5. DETAILED DESCRIPTION

[0029] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0030] Reagents and instruments

[0031] Table 1 Reagents or consumables

[0032]

[0033]

[0034] Table 2 Instruments and equipment

[0035]

[0036]

[0037] Example 1 Preparation of recombinant parainfluenza virus type 5

[0038] 1.1 Construction of PIV5 reverse genetics operating system plasmid

[0039] 1. Synthetic vector virus

[0040] PIV5 is the canine parainfluenza (PIV5) vaccine strain stored in our laboratory. RNA was extracted from this strain using the Roche RNA extraction kit and analyzed using PrimeScript TMII 1st Strand cDNA Synthesis Kit was used for reverse transcription to obtain the cDNA of the strain.

[0041] The full-length 15246nt of PIV5 (SEQ ID NO.2) was divided into three fragments of approximately 5-5.5kb in size, AC. Fragment A is the 1st bp-5030bp of the full-length (5'-3') of PIV5, fragment B is the 5016bp-9906bp of the full-length (5'-3') of PIV5, and fragment C is the 9892-15246bp of the full-length (5'-3') of PIV5.

[0042] Figure 1 This is the enzyme digestion verification diagram of the PIV5 full-length cDNA cloning vector; pPIV5 was digested with StuI enzyme to verify that it can be cut into two DNA fragments with sizes of 2751bp and 15531bp respectively. The fragment sizes and positions are correct, and the pPIV5 plasmid was successfully constructed.

[0043] PCR amplification was performed using primers (as shown in Table 3 below: AF / R for fragment A, BF / R for fragment B, and CF / R for fragment C). The recovered DNA fragments A, B, and C were inserted into the pEasy-Blunt Zero vector (Beijing Quanshijin Biotechnology Co., Ltd., CB501-01). After sequencing to confirm the correct sequences, the three plasmids were retained and named pZero-A, pZero-B, and pZero-C, respectively.

[0044] Table 3 Primer list

[0045]

[0046] 2. The three plasmids obtained in step 1 (pZero-A, pZero-B, and pZero-C) were amplified by fusion PCR using primers A-TY-F / C-TY-R (see Table 4) to obtain the fragment PIV5-ABC;

[0047] The fragment PIV5-ABC was ligated with the pBlueScript SK II (+) (Ubao Bio, VT1328) vector containing a T7 RNA polymerase promoter sequence at the 5' end, a hepatitis B nuclease sequence at the 3' end, and a T7 RNA polymerase transcription terminator sequence (recipe reference Table 7) to obtain the recombinant plasmid pPIV5 (i.e., the sequence was inserted into the multiple cloning site of pBlueScript SK II (+)). The plasmid map is referenced to Figure 7 , sequencing verified that it was correct and retained.

[0048] Table 4 Primer sequence list

[0049]

[0050] The PIV5-ABC gene fragment was amplified by fusion PCR. The reaction system was as follows:

[0051] Table 5 PIV5-ABC fragment amplification reaction system formula

[0052]

[0053] Add the above ingredients in sequence, mix well and separate instantly;

[0054] The PCR amplification reaction program is as follows:

[0055] Table 6 Reaction procedure of PIV5-ABC fragment amplification reaction system

[0056]

[0057] pBlueScript SK II (+) was double-digested with HindIII and BamHI. The enzyme digestion system is as follows:

[0058] Table 7 Recipe for enzyme digestion and ligation of recombinant plasmid pPIV5

[0059]

[0060] At 37°C, enzyme digestion was performed overnight according to the conditions in Table 7. The PIV5-ABC fragment was digested to obtain the target gene described in Table 8, and the pBlueScript SK II (+) was digested to obtain the vector described in Table 8.

[0061] The target gene and vector are connected, usually at a ratio of 1:3-1:8. The system is as follows:

[0062] Table 8 Recipe for connecting target gene and vector

[0063]

[0064] Mix well and centrifuge immediately, incubate at 50°C for 15 min.

[0065] 3. Using pPIV5 as the vector, amplify the SphI fragment with SphI-F and SphI-R primers, and introduce a PmeI restriction site at the 3' end of the fragment; amplify the BbvCI fragment with BbvCI-F and BbvCI-R primers, and introduce a PmeI restriction site at the 5' end of the primers. Recover the two fragments (fragment SphI and fragment BbvCI) for later use.

[0066] Table 9 Primer sequence list

[0067]

[0068] 4. Use SphI-F and BbvCI-R primers to amplify fragments SphI and BbvCI to obtain fragment ΔSH (nucleotide sequence shown in SEQ ID NO: 7) with the SH small hydrophobic gene deleted and a PmeI restriction site, which is retained for future use.

[0069] 5. The pPIV5 vector was double-digested with SphI and BbvCI enzymes, recovered, and homologously recombined with the fragment △SH to obtain the vector vPIV5-PmeI carrying the PmeI restriction site.

[0070] Use homologous recombinase to connect the target gene and vector after enzyme digestion. The connection ratio is usually 1:3 to 1:8. The system is as follows:

[0071] Table 10 Formula for the synthesis of vector vPIV5-PmeI

[0072]

[0073] 6. The recombinant plasmid pPIV5 obtained in step 2 was amplified to obtain the NP gene sequence from position 152 to position 1681 of the full-length genome (shown in SEQ ID NO: 4), the P gene sequence from position 1850 to position 3026 of the full-length genome (shown in SEQ ID NO: 5), and the L gene sequence from position 8414 to position 15181 of the full-length genome (shown in SEQ ID NO: 6). Figure 2 To express NP, P and L proteins, the enzyme digestion verification diagram of the auxiliary plasmid; Figure 2 In the experiment, HindIII enzyme was used to digest NP, P and L for identification. pcDNA-NP and pcDNA-P could be digested to produce vector fragments and insert fragments (two fragments each). Since the L coding sequence of pcDNA-L plasmid contains three HindIII digestion sites, L could be digested into four DNA fragments. All recombinant plasmids were constructed correctly.

[0074] Table 11 Primer sequence list

[0075]

[0076] The NP gene sequence was inserted into the HindIII site of pcDNA3.1(+) (Cat. No. V790-20) purchased from Invitrogen to obtain the recombinant plasmid pcDNA-NP that can express the viral protein NP in eukaryotic cells; the P gene sequence was inserted into the HindIII site of pcDNA3.1(+) to obtain the recombinant plasmid pcDNA-P that can express the viral protein P in eukaryotic cells; the L gene sequence was inserted into the HindIII site of pcDNA3.1(+) to obtain the recombinant plasmid pcDNA-L that can express the viral protein L in eukaryotic cells.

[0077] PCR amplified the NP\P\L gene fragments, and the reaction system was as follows:

[0078] Table 12 NP / P / L fragment amplification reaction system formula

[0079]

[0080] Add the above ingredients in sequence, mix well and separate instantly;

[0081] The PCR amplification reaction program is as follows:

[0082] Table 13 NP / P / L fragment amplification reaction system reaction program

[0083]

[0084] pcDNA3.1(+) was digested with HindIII. The digestion system was as follows:

[0085] Table 14 pcDNA3.1 (+) enzyme digestion formula

[0086]

[0087] At 37°C, perform overnight enzyme digestion according to the conditions in Table 14 and homologously recombine with the NP / P / L fragments. The system is as follows:

[0088] Table 15 Synthesis formula

[0089]

[0090] 1.2 Obtaining infectious clones of recombinant rPIV5 virus

[0091] 1. Co-transfect the purified pPIV5, pcDNA-NP, pcDNA-P, and pcDNA-L obtained in step 1.1 into BSR T7 / 5 cells stably expressing T7 RNA polymerase.

[0092] 2. 72 hours after completing step 1, freeze-thaw the transfected BSR T7 / 5 cells and supernatant at -80°C, inoculate MDBK cells, and replace the culture medium with DMEM containing 2% FBS 4 hours later.

[0093] 3. 72 hours after completing step 2, aspirate the cell supernatant and inoculate new MDBK cells. After 4 hours, replace the culture medium with DMEM containing 2% FBS.

[0094] 4. 72 hours after completing step 3, repeat step 3 once and inoculate new MDBK cells with the cell supernatant. After 72 hours, aspirate the MDBK cell supernatant for hemagglutination testing. The supernatant that can produce hemagglutination is the supernatant containing the successfully rescued recombinant virus rPIV5, and subsequent testing and identification work is carried out;

[0095] Figure 3 Figure 1 shows the rescue and identification of rPIV5 using immunofluorescence (A) and Western blotting (B). MDBK cells were infected with hemagglutinating recombinant virus liquid rPIV5 and parental virus PIV5 WT, respectively, using the parental virus as a positive control. Uninfected cells served as a negative control. After 24 hours of culture, Western blotting and immunofluorescence were performed using antibodies against the P protein to verify the expression of viral proteins. The results showed that in cells infected with the recombinant virus, P protein expression was detected by immunofluorescence (Figure A); Western blotting detected P protein-specific protein bands (Figure B), indicating the presence of PIV5 virus in samples harvested during blind virus rescue.

[0096] Figure 4 This is a diagram verifying the molecular genetic marker in rPIV5. Sequencing verified the presence of the KpnI restriction site, a molecular genetic marker in rPIV5 (Figure A). The 460bp fragment obtained by RT-PCR of the rPIV5 recombinant virus was digested with KpnI. After KpnI digestion, the fragment was cleaved into two DNA fragments of 225bp and 235bp, respectively. However, this fragment of the parental virus PIV5WT could not be digested by KpnI (Figure B).

[0097] Example 2 Preparation of Recombinant Parainfluenza Virus Type 5 Expressing Influenza Virus H3 HA Protein

[0098] 2.1 Construction of PIV5 reverse genetics system plasmid expressing H3 HA protein

[0099] 1. The HA gene of influenza virus A / canine / South Carolina / 132956 / 2017 was amplified using Primestar HS high-fidelity enzyme. The HA region sequence was amplified using primer pairs H3 HA-F, H3 HA-R1, H3 HA-F2, and H3 HA-R, respectively. The two amplified DNA fragments were named HA1 and HA2, and the two fragments were recovered after electrophoresis identification.

[0100] The nucleotide sequence of HA1 is shown in SEQ ID NO: 8, and the nucleotide sequence of HA2 is shown in SEQ ID NO: 9.

[0101] Table 16 Primer sequence table

[0102]

[0103] 2. In the same PCR reaction system, primers H3 HA-F and H3 HA-R were used to amplify fragments HA1 and HA2 as templates to obtain the H3 HA sequence, a fusion product of the HA1 and HA2 fragments. After the two fusion fragments were confirmed by electrophoresis, the DNA product was recovered for later use. The H3 HA sequence is the HA sequence in the sequence listing (SEQ ID NO: 3).

[0104] PCR amplified the HA gene fragment, and the reaction system was as follows:

[0105] Table 17H3 HA fragment amplification formula

[0106]

[0107] The cDNA in Table 12 is a mixture of fragment HA1 and fragment HA2 at a ratio of 1:1;

[0108] Add the above ingredients in sequence, mix well and separate instantly

[0109] The PCR amplification reaction program is as follows:

[0110] Table 18H3 Parameters for HA fragment amplification program

[0111]

[0112] 3. Use PmeI enzyme to perform enzyme digestion and linearization treatment on the vPIV5-PmeI vector in 1.1 carrying the PmeI restriction site, cut the SH gene fragment region, obtain the vector fragment pPIV5-PmeI, and recover it for future use.

[0113] The H3 HA PCR product and the vector fragment pPIV5-PmeI were digested with PmeI. The enzyme digestion system is as follows:

[0114] Table 19 PmeI enzyme digestion formula

[0115]

[0116] Enzyme digestion time: 2h, reaction conditions: 55℃.

[0117] 4. Use the vector fragment pPIV5-PmeI after enzyme digestion in step 3 as the vector, and connect it with the H3HA fragment after enzyme digestion in step 2 by T4 DNA ligase to obtain the recombinant plasmid rPIV5-H3. After sequencing, keep it for future use.

[0118] Use T4 DNA ligase produced by NEB to ligate the target gene and vector after enzyme digestion. The ligation ratio is usually 1:3-1:8. The system is as shown in Table 15:

[0119] Table 20 T4 DNA ligase ligation formula

[0120]

[0121]

[0122] The recombinant plasmid rPIV5-H3 is obtained by inserting the DNA molecule shown in SEQ ID NO: 3 into the PmeI recognition site of the vPIV5-PmeI vector.

[0123] 2.2 Rescue of recombinant PIV5 expressing H3 HA protein

[0124] 1. The recombinant plasmids rPIV5-H3, pcDNA-NP, pcDNA-P and pcDNA-L obtained by purification and extraction in 2.1 were co-transfected into BSR T7 / 5 cells expressing T7 RNA polymerase.

[0125] 2. 72 hours after completing step 1, freeze-thaw the BSR T7 / 5 cells and supernatant at -80°C, then inoculate MDBK cells. After 4 hours, replace the culture medium with DMEM containing 2% FBS.

[0126] 3. 72 hours after completing step 2, aspirate the cell supernatant and inoculate new MDBK cells. After 4 hours, replace the culture medium with DMEM containing 2% FBS.

[0127] 4. 72 hours after completing step 3, repeat step 3 once and inoculate new MDBK cells. After 72 hours, aspirate the MDBK cell supernatant for hemagglutination assay. The supernatant that produces hemagglutination is the supernatant containing the successfully rescued recombinant virus rPIV5-H3 and is then used for subsequent testing and identification.

[0128] Example 3 Identification of recombinant virus rPIV5-H3

[0129] 3.1 Gene identification

[0130] The recombinant virus rPIV5-H3 genomic RNA was extracted and reverse transcribed to obtain cDNA. The viral genomic region containing the inserted H3 HA gene was PCR amplified using primers SH-JF and SH-JR (sequences see Table 16 below) to obtain a PCR amplification product.

[0131] Table 21 Primer sequence list

[0132]

[0133] The size of the PCR amplification product fragment was consistent with expectations, and Sanger sequencing confirmed that the H3 HA gene sequence had been completely inserted into the PIV5 genome and the sequence was accurate.

[0134] 3.2 Indirect immunofluorescence identification

[0135] 1. Take a 12-well cell culture plate, add 1 mL of DMEM culture medium containing 10% FBS containing 2×10 5 MDBK cells, and culture overnight at 37°C, 5% CO 2 until the MDBK cells grow to 90% density.

[0136] 2. After completing step 1, wash the cells twice with PBS buffer (pH 7.2, 0.01 M), discard the liquid, replace with 1 mL of DMEM medium containing 2% FBS, and inoculate 103 TCID50 of recombinant virus rPIV5-H3, and continue culturing for 48 hours.

[0137] 3. After completing step 2, discard the supernatant from the cell wells and wash twice with pH 7.2, 0.01M PBS buffer. Then, add 1 mL of pre-cooled fixative (ethanol and acetone mixed in a 3:2 volume ratio) to each well and let it stand for 20 minutes to fix and permeabilize the cells.

[0138] 4. After completing step 3, discard the liquid in the wells and wash each well with 1 mL of PBS buffer three times, 5 minutes each time; then add 500 μL of mouse anti-PIV5 diluent to each well and incubate overnight at 4°C for 8 hours. Replace mouse anti-PIV5 with rabbit anti-influenza virus HA protein antibody diluent to conduct experiments to detect the expression of influenza virus HA protein. Both antibody diluents are composed of 1 volume of antibody and 199 volumes of pH 7.2, 0.01M PBS buffer (mouse anti-PIV5 diluent was purchased from Wuhan Huamei Biological, catalog number: CSB-MA000160, rabbit anti-influenza virus HA protein).

[0139] 5. After completing step 4, discard the liquid and wash three times with 1 mL of PBS buffer, each time for 5 minutes; then add TRITC-labeled goat anti-mouse secondary antibody dilution (the dilution method is to dilute the goat anti-mouse secondary antibody (BioVision, product number: 6921-100) with PBS buffer at a volume ratio of 1:400) for antigen-antibody binding staining, and incubate at 37°C in the dark for 1 hour.

[0140] 6. After completing step 5, discard the liquid in the wells and wash three times with 1 mL of PBS buffer, each time for 5 minutes; then add FITC-labeled goat anti-rabbit secondary antibody dilution (the dilution method is to dilute the goat anti-rabbit secondary antibody (Biyuntian Biotechnology Co., Ltd., product number: A0562) with PBS buffer at a volume ratio of 1:200) for antigen-antibody binding staining, and incubate at 37°C in the dark for 1 hour.

[0141] 7. After completing step 6, discard the liquid in the wells and wash three times with 1 mL of PBST buffer, each time for 5 minutes. Leave the PBST buffer in the wells for the last wash and observe under an inverted fluorescence microscope.

[0142] The recombinant virus rPIV5-H3 was replaced with the recombinant wild virus rPIV5 as a control.

[0143] Figure 5 Figure 2: Immunofluorescence analysis of the expression of the exogenous HA gene in rPIV5-H3. To verify HA gene expression in rPIV5-H3-infected cells, MDBK cells were infected with the parental virus, PIV5 WT, at an MOI of 0.1. Immunofluorescence analysis was performed 48 hours after infection. Anti-influenza virus HA antibodies were used for detection, and HA gene expression was only detected in rPIV5-H3-infected cells. Therefore, rPIV5-H3 can express the HA gene after infection.

[0144] The results showed that 48 hours after the recombinant virus rPIV5-H3 infected MDBK cells, the expressed PIV5P protein (red) and influenza virus HA protein (green) could be detected in the cells; while after the recombinant wild virus rPIV5 infected the cells, only the PIV5 P protein (red) could be detected, and the expression of the influenza virus HA protein could not be detected.

[0145] 3.3 Western blot identification

[0146] 1. Take a 6-well cell culture plate and add 2 mL of cell culture medium containing MDBK cells (about 4×10 5 MDBK cells / well) were cultured at 37°C under 5% CO2 overnight until the MDBK cells grew into a monolayer with a density of about 90%.

[0147] 2. After completing step 1, wash the cells three times with 2 mL PBS and inoculate the recombinant virus rPIV5-H3 or rPIV5 (inoculation dose is 10 4 TCID 50 ) and replaced with DMEM culture medium containing 2% FBS, and cultured at 37°C, 5% CO2 for 48h.

[0148] 3. Take the cells that have completed step 2, wash them three times with PBS, digest them with trypsin, and centrifuge to collect the cells.

[0149] 4. After completing step 3, take the cells and lyse them using IP lysis buffer to obtain cell lysate. Centrifuge at 10,000 g for 10 minutes, carefully aspirate the supernatant, add Western Protein Loading Buffer, denature at 100°C for 10 minutes, and then cool to room temperature.

[0150] 5. After completing step 4, the cell lysate was subjected to SDS-PAGE and Western blot. Rabbit anti-influenza virus HA protein antibody was used as the primary antibody and horseradish peroxidase (HRP)-labeled goat anti-rabbit antibody was used as the secondary antibody to detect HA protein; PIV5 P protein was used as an internal reference, mouse anti-P protein V5 tag monoclonal antibody (Wuhan Huamei Biotechnology Co., Ltd., Catalog No.: CSB-MA000160) was used as the primary antibody, and HRP-labeled goat anti-mouse antibody (Abcam, Catalog No.: ab6789) was used as the secondary antibody.

[0151] HA protein-specific bands were detected in MDBK cells infected with the recombinant virus rPIV5-H3, while no such bands were detected in MDBK cells infected with rPIV5, indicating that the recombinant virus rPIV5-H3 can express influenza virus H3 HA protein after infecting cells.

[0152] Comparative Example 1

[0153] The HA gene of influenza virus A / canine / Quanzhou / 1224-1109 / 2018 (H3N2) was amplified using Primestar HS high-fidelity enzyme. The HA region sequence was amplified using primer pairs H3 HA1-F, H3 HA1-R, H3 HA2-F, and H3 HA2-R, respectively. The two amplified DNA fragments were named HA1 and HA2, respectively. After electrophoresis identification, the two fragments were recovered.

[0154] The classification name of A / canine / Quanzhou / 1224-1109 / 2018 (H3N2) is H3N2 subtype canine influenza virus; the deposit number is CGMCC NO.18180, and the depositor is: General Microbiology Center of China Culture Collection Administration; the deposit address is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; the deposit date is August 7, 2019.

[0155] The method was the same as that of Example 2 for preparing recombinant parainfluenza virus type 5 expressing influenza virus H3 HA protein. The other steps were generally the same as those of Example 2. After rescue, the recombinant virus rPIV5-H3-1109 was obtained.

[0156] Comparative Example 2

[0157] The HA gene of influenza virus A / canine / Beijing / 0528-147 / 2017 147 (H3N2) was amplified using Primestar HS high-fidelity enzyme. The HA region sequence was amplified using primers H3 HA1-F, H3 HA1-R, H3 HA2-F, and H3 HA2-R. The two amplified DNA fragments were named HA1 and HA2, respectively. After electrophoresis identification, the two fragments were recovered. The method was the same as that of Example 2 for the preparation of recombinant parainfluenza virus type 5 expressing influenza virus H3 HA protein. The other steps were roughly the same as those of Example 2. After rescue, the recombinant virus rPIV5-H3-147 was obtained.

[0158] The classification name of A / canine / Beijing / 0528-147 / 2017 147(H3N2) is H3N2 subtype canine influenza virus; the deposit number is CGMCC NO.17589, the depositor is: General Microbiology Center of China Culture Collection Administration; the deposit address is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; the deposit date is April 29, 2019.

[0159] Example 4 Evaluation of the immune protection effect of recombinant virus rPIV5-H3 against H3N2 subtype canine influenza virus

[0160] 4.1 Mouse vaccine efficacy test

[0161] Forty 6-week-old BALB / c mice were randomly divided into four groups, with 10 mice in each group. The experimental group was inoculated with 50 μL of recombinant virus rPIV5-H3, rPIV5-H3-1109, and rPIV5-H3-147 through the nasal cavity, while the control group was inoculated with 50 μL of recombinant virus rPIV5 through the nasal cavity. The inoculation dose was 10 6 TCID 50 / mouse. Repeat the above procedure once 15 days after the first vaccination. After immunization, continue to observe each group of mice for clinical symptoms, including loss of appetite, ruffled fur, difficulty breathing, and decreased activity.

[0162] Observations revealed that the rPIV5 control group and the rPIV5-H3, rPIV5-H3-147, and rPIV5-H3-1109 immunization groups showed no obvious clinical symptoms, ate normally, and maintained a relatively stable weight. This suggests that the recombinant rPIV5 and recombinant virus expressing the influenza virus H3 HA protein constructed in this study had no side effects or adverse reactions in mice and were considered safe.

[0163] Blood was collected from mice every 7 days after the first immunization using the retroorbital venous plexus method. The blood was incubated at 37°C for 50 minutes and centrifuged at 8000 rpm for 15 minutes. Serum was collected and stored at -80°C after aliquoting. Antibody levels against HA protein in mice were measured using an indirect ELISA.

[0164] 1) Coat the plate. Dilute the HA antigen protein to 1 μg / mL with ELISA coating buffer, add 50 μL to each well, and incubate at 4°C overnight.

[0165] 2) Remove the antigen solution and wash three times with PBS.

[0166] 3) Blocking: add 100 μL of blocking solution (containing 5% skim milk PBS buffer) to each well and incubate at 37° C. for at least 1 hour.

[0167] 4) Remove the blocking solution and wash the plate three times with PBS;

[0168] 5) Incubate with serum. Dilute the serum to the correct ratio with antibody diluent (i.e., blocking solution). Dilution of mouse serum starts at 1:25. Add 100 μL of serum diluent to the wells according to the plate layout requirements and incubate at 37°C for 2 hours. The negative control is the serum of a healthy mouse. The positive control must be detected.

[0169] 6) Remove the serum diluent and wash the plate 5 times with PBST.

[0170] 7) Incubate with secondary antibody (HRP-conjugated goat anti-mouse IgG, Abcam, cat. no. ab6789). Dilute the secondary antibody in antibody dilution buffer at a dilution ratio of 1:5000 for anti-mouse IgG. Add 100 μL of the secondary antibody dilution buffer to each well according to the plate layout requirements and incubate at 37°C for 1.5 hours.

[0171] 8) Remove the secondary antibody buffer and wash the plate 5 times with PBST.

[0172] 9) Add 100 μL of TMB colorimetric solution (Beyotime, P0209) to each well and gently tap the plate to mix evenly. Incubate at room temperature for a period of time. When the negative wells tend to turn blue, add 100 μL of stop solution to each well to terminate the reaction.

[0173] 10) Record the absorbance at 450 nm using a microplate reader.

[0174] The results showed that the mice immunized with the recombinant viruses rPIV5-H3, rPIV5-H3-147, and rPIV5-H3-1109 all produced specific antibodies against the HA protein, while no specific antibodies against the HA protein were detected in the control group mice. At the same time, the test results showed that the mice immunized with the rPIV5-H3 recombinant virus produced more specific antibodies against the HA protein in their bodies than the mice immunized with the rPIV5-H3-147 and rPIV5-H3-1109 recombinant viruses ( Figure 6 ). Therefore, immunizing mice with rPIV5-H3 recombinant virus can stimulate mice to produce more specific antibodies against HA protein.

[0175] Six weeks after the first vaccination, each mouse was infected with influenza virus by intranasal drip at a dose of 10 4 TCID50, and then the clinical symptoms of the mice were observed every day, and the body weight was weighed. On the third day after the infection, 5 mice in each group were dissected, the mouse lungs were collected, the tissues were ground, and the supernatant was used to extract RNA. The level of influenza virus RNA in the mouse organs was detected by real-time fluorescence quantitative PCR. The results showed that only the mice in the immune recombinant virus rPIV5-H3 group were completely unable to replicate in the mouse lungs after being infected with the influenza virus. Therefore, the rPIV5-H3 group had better immune protection than other immune groups.

[0176] 4.2 Canine Vaccine Efficacy Test

[0177] Twenty-one 8-week-old beagle dogs were randomly divided into 7 groups, including 3 control groups, 3 immunization groups, and 1 blank control group. All of them were negative for H3 subtype influenza virus antibody in HI test. The blank control group was monitored for natural infection with H3 subtype influenza virus throughout the experimental period. The 3 control groups were subcutaneously injected with 0.5 mL of recombinant virus rPIV5, and the 3 immunization groups were subcutaneously injected with 0.5 mL of rPIV5-H3, rPIV5-H3-147, rPIV5-H3-1109 (10 8.5 EID 50 / mL) were immunized twice, with an interval of 14 days between each immunization.

[0178] On the 15th day after vaccination, dogs in control group 1 and rPIV5-H3 vaccination group were injected intravenously with 2 mL of 10 6 EID50 A / canine / South Carolina / 132956 / 2017;

[0179] Dogs in control group 2 and rPIV5-H3-147 immunization group were injected intravenously with 2 mL 10 6 EID50 of A / canine / Beijing / 0528-147 / 2017 147;

[0180] The dogs in the control group 3 and rPIV5-H3-1109 immunization group were injected intravenously with 2 mL 10 6 EID 50 A / canine / Quanzhou / 1224-1109 / 2018. Blood was collected on days 0, 3, 7, 9, and 13 after challenge, and serum was prepared. The serum was treated with RDE, and the antibody levels were tested by HI.

[0181] Serum:RDE=1:3 (volume ratio) was placed in a 37°C water bath for 18-20 hours, and then placed in a 56°C water bath for 30 minutes to inactivate RDE.

[0182] Hemagglutination inhibition test:

[0183] 1. Add 25 μL of normal saline to wells 1-11 of the hemagglutination plate, add 50 μL of normal saline to well 12, and mark the serum number to be tested on the hemagglutination plate;

[0184] 2. Add 25 μL of the serum to be tested to the first well of each row. Pipet the liquid in the first well repeatedly 6-8 times, then aspirate 25 μL to the second well. Be careful not to create bubbles. Pipet it 6-8 times, then aspirate 25 μL to the third well. Dilute the solution to the tenth well in the same manner. Pipet it in the tenth well, then aspirate 25 μL and discard it. Set the 11th well as the antigen control and the 12th well as the red blood cell control.

[0185] 3. Starting from well 11, add 25 μL of the prepared 4 units of antigen from right to left, shake gently after addition, and let it stand at room temperature for 30 minutes;

[0186] 4. Shake the 1% chicken red blood cell suspension and pour it into the sample reservoir. Starting from the 12th well, add 25 μL of 1% chicken red blood cell suspension in the air. Mix it by tapping gently. Let it stand at room temperature for 25 minutes before reading.

[0187] 5. Tilt the plate 45° and read the plate. If the red blood cells slide down in the 12th well and the 11th well is completely agglutinated, the test is considered successful. The highest dilution factor in the well that completely slides down is the antibody titer of the serum.

[0188] From 1d to 14d after the challenge, the clinical symptoms of the dogs in each group were observed daily, and the body temperature changes of the dogs in each group were monitored in the morning and evening. From 1d to 14d after the challenge, nasopharyngeal swabs were collected from the dogs in each group on the 1st, 3rd, 5th, 7th, 9th, 11th, and 13th days, and the excretion of each dog was detected by the chicken embryo quantitative method. The results showed that no virus was detected in the nasopharyngeal swabs of the dogs in the blank control group, indicating that no natural infection with the H3 subtype influenza virus occurred during the experiment. At the same time, no virus was detected in the rPIV5-H3 in the immunized group after the challenge, but the virus was detected in the nasopharyngeal swabs of the dogs in the control group, and trace amounts of virus replication were also detected in the rPIV5-H3-147 and rPIV5-H3-1109 immunized groups. Therefore, the above results indicate that rPIV5-H3 in the immunized group has a better immune protection effect.

[0189] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the invention can be embodied in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as illustrative and non-restrictive, and the scope of the invention is defined by the appended claims, not the foregoing description, and all variations within the meaning and range of equivalents of the claims are intended to be included therein. Any reference sign in a claim should not be construed as limiting the claim to which it relates.

Claims

1. A recombinant virus rPIV5-H3, characterized in that The full gene sequence is shown in SEQ NO.

1. The preparation method of the recombinant virus rPIV5-H3 comprises the following steps: Step 1: inserting the full genome cDNA of parainfluenza virus type 5 into a cloning vector to obtain a recombinant plasmid pPIV5; the sequence of the full genome cDNA of parainfluenza virus type 5 is shown in SEQ NO. 2; Step 2: Insert the influenza virus HA protein encoding gene sequence into the small hydrophobin gene nucleotide sequence of the recombinant plasmid pPIV5. The recombinant plasmid is named pPIV5-H3. The influenza virus HA protein encoding gene sequence is shown in SEQ NO.

3. Step 3: co-transfect the host cells with the recombinant plasmid pPIV5-H3, the auxiliary recombinant plasmid NP expressing the auxiliary protein, the auxiliary recombinant plasmid P, and the auxiliary recombinant plasmid L; The auxiliary recombinant plasmid NP is a vector containing the NP gene with the nucleotide sequence shown in SEQ NO.4; the auxiliary recombinant plasmid P is a vector containing the P gene with the nucleotide sequence shown in SEQ NO.5; and the auxiliary recombinant plasmid L is a vector containing the L gene with the nucleotide sequence shown in SEQ NO.

6.

2. The recombinant virus rPIV5-H3 according to claim 1, characterized in that The cloning vector is pBlueScript SK II (+) vector; The vector in step 3 is pcDNA3.1(+).

3. The recombinant virus rPIV5-H3 according to claim 1, characterized in that The host cell is a BSR-T7 cell, an MDBK cell, a Vero cell or a BHK cell.

4. A use of the recombinant virus rPIV5-H3 as described in claim 1 to prepare a vaccine.

5. A vaccine, characterized in that Contains the recombinant virus rPIV5-H3 as described in claim 1.

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