Preparation method of influenza D virus-like particles

Abstract

The invention discloses a preparation method of influenza D virus-like particles, and belongs to the field of veterinary biological products. Influenza D virus (IDV) is one of important pathogens of bovine respiratory disease syndrome (BRDC), the disease causes a hundreds of billions of dollars economic loss to the global cattle raising industry every year, however, commercialized vaccines for influenza D do not exist in the market at present. The preparation method comprises the following steps: target gene amplification, construction of recombinant shuttle plasmids, construction of recombinant baculoviruses, rescue of recombinant baculoviruses, and preparation and purification of virus-like particles. The D-type influenza virus-like particles (IDV VLPs) are obtained through the steps. After mice are immunized, the IDV VLPs are found to have high immunogenicity and can be used as a candidate vaccine for preventing the D-type influenza.

Description

Technical Field

[0001] The present invention belongs to the field of veterinary biological products, and more specifically relates to a preparation method and application of influenza D virus-like particles. Background Art

[0002] Influenza D virus (IDV) is the latest member of the Orthomyxoviridae family and is an enveloped single-stranded negative-sense RNA virus. In 2011, the virus was first discovered in pigs in Oklahoma, USA. Relevant research shows that influenza D virus is one of the important pathogens of bovine respiratory disease complex (BRDC), which can cause damage to the respiratory mucosa and increase the risk of secondary bacterial infections, causing billions of dollars in economic losses to the global cattle industry every year. The diameter of IDV is 100-120 nm, and the virus particles have dense spikes on the surface. The main structural proteins encoded by the genome include nucleocapsid protein (NP), matrix protein (M1), hemagglutinin-esterase fusion protein (HEF), and ion channel protein (M2). Among them, the HEF protein is a multifunctional glycoprotein that plays a key role in the adsorption and invasion of the virus to host cells, and is also the core immunogenic protein that induces the host immune response to produce neutralizing antibodies, thereby preventing the invasion and spread of the virus. The M1 matrix protein constitutes the outer shell skeleton of the virus, binds tightly to the outermost envelope of the virus, and plays a role in protecting the virus core and maintaining the spatial structure of the virus.

[0003] Epidemiological studies have shown that since its first discovery in 2011, influenza D virus has spread to more than 20 countries and regions. In addition to pigs and cattle, influenza D virus infections have also been reported in goats, sheep, camels, horses, water buffalo, and deer. Serological investigation studies have shown that specific antibodies against influenza D virus have also been detected in the sera of cattle farm workers and the general population, suggesting that the virus has a potential risk of zoonosis and poses a threat to public health safety.

[0004] Immunization is a key measure for disease control, but there is currently no commercial vaccine against influenza D virus. Virus-like particles (VLPs) vaccines have received extensive attention due to their high immunogenicity and superior safety. VLPs are self-assembled by one or more structural proteins of the virus, with a structure similar to that of the natural virus, which can precisely mimic the natural conformation of the virus, have good immunogenicity, and induce a comprehensive and strong immune response in the body. In addition, virus-like particles do not contain the genetic material of the virus, so there is no risk of virus resurrection and infection of the body at all, greatly reducing the possibility of disease occurrence caused by vaccination. At the same time, in the production process, large-scale production can be achieved with the help of genetic engineering technology, and the production process is convenient for quality control, effectively ensuring the safety and stability of the vaccine.

[0005] Therefore, developing a safe and effective vaccine against influenza D virus is an urgent problem to be solved by those skilled in the art. Summary of the Invention

[0006] The object of the present invention is to provide a method for preparing influenza D virus-like particles based on the insect baculovirus expression system to develop a candidate vaccine for preventing IDV.

[0007] To achieve the above object, the present invention uses the following technical solutions:

[0008] In the first aspect, the present invention provides a virus-like particle vaccine self-assembled by the HEF hemagglutinin-esterase fusion protein and the M1 matrix protein of the influenza D virus D / OK lineage.

[0009] In the second aspect, the present invention provides a method for preparing the virus-like particle vaccine, comprising the following steps:

[0010] 1) Using the cDNA of D / swine / Oklahoma / 1334 / 2011 as a template, amplifying the M1 and HEF gene fragments with specific primers containing vector homologous arms and Kozak sequences;

[0011] 2) Separately ligating the HEF and M1 gene fragments to an expression vector, transforming them into DH5α competent cells, and screening through double digestion identification to obtain recombinant shuttle plasmids pFastBac1-M1 and pFastBac1-HEF containing the IDV M1 and IDV HEF genes respectively;

[0012] 3) The above recombinant shuttle plasmid was transformed into DH10 Bac competent cells for homologous recombination. After blue-white screening and PCR identification using specific primers and M13 universal primers, recombinant bacmids rBacmid-M1 and rBacmid-HEF containing the IDV M1 and IDV HEF genes were obtained respectively.

[0013] 4) The above recombinant bacmids were transfected into the insect baculovirus expression system respectively. After four blind passages, the supernatant was harvested and PCR identification was carried out using specific primers and M13 universal primers to obtain recombinant baculoviruses rBV-M1 and rBV-HEF containing the IDV M1 and IDV HEF genes;

[0014] 5) The recombinant baculovirus rBV-M1 containing the IDV M1 gene and the recombinant baculovirus rBV-HEF containing the IDV HEF gene were co-infected into insect cells. The cell supernatant was harvested and then virus-like particles containing the target protein were obtained by separation and purification; 6) The virus-like particles containing the IDV HEF protein and M1 protein were added with adjuvant to prepare a virus-like particle vaccine;

[0015] Among them, the nucleotide sequence of the IDV M1 gene is shown as Seq ID No.1; the nucleotide sequence of the IDV HEF gene is shown as Seq ID No.2; among them, the primer sequences for amplifying the IDV M1 gene are shown as Seq ID No.3 and Seq ID No.4; the primer sequences for amplifying the IDV HEF gene are shown as Seq ID No.5 and Seq ID No.6, and the universal primer sequences for identification are shown as Seq ID No.7 and Seq ID No.8.

[0016] The insect cells are Sf9 cells.

[0017] The expression vector is pFastBac1 plasmid

[0018] The adjuvant is Freund's adjuvant.

[0019] As an invention concept identical to the above technical solution, the present invention also claims the IDV VLPs prepared by the above method.

[0020] As an invention concept identical to the above technical solution, the present invention also claims the application of the virus-like particles in the preparation of a vaccine for preventing D-type influenza virus infection.

[0021] The beneficial effects of the present invention are:

[0022] The insect baculovirus expression system used in the method of the present invention has the ability for large-scale production, can be used to express large-genome foreign proteins, is suitable for the insertion of various foreign genes, and has high safety. In addition, this system can perform post-translational modification on complex glycoproteins. Compared with the prokaryotic expression system, it is more conducive to expressing various viral structural proteins and ensuring the safety and stability of vaccine production. And virus-like particles (VLPs) have good immunogenicity due to their unique repetitive folded surface structure. Compared with traditional attenuated or inactivated vaccines, VLP vaccines have higher stability, safety, no toxic side effects, and can induce strong immune responses. The D-type influenza virus-like particles (IDV VLPs) prepared by using the nucleotide sequences of the HEF and M1 proteins of the D-type influenza virus with the insect baculovirus expression system in the present invention. The experimental results of immunizing mice show that it can induce a relatively high level of immune response, indicating its potential as a candidate for D-type influenza vaccine. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation to the present invention. In the drawings:

[0024] Figure 1 : Amplification results of the target genes of IDV M1 and IDV HEF. M: DL5000, (A): M1 gene (779 bp); (B): HEF gene (2034 bp);

[0025] Figure 2 : Double digestion identification results of the recombinant shuttle plasmids of IDV M1 and HEF. M: DL5000, (a): Double digestion result of the pFastbac1-M1 plasmid, the size of the pFastbac1 vector fragment is about 4700 bp, and the size of the M1 gene fragment is about 756 bp; (b): Double digestion result of the pFastbac1-HEF plasmid, the size of the pFastbac1 vector fragment is about 4700 bp, and the size of the HEF gene fragment is about 2011 bp;

[0026] Figure 3 : PCR identification results of the recombinant bacmids rBacmid-M1 and rBacmid-HEF. Figure (a) is the identification of rBacmid-M1, M: DL5000, lane 1 is the identification with specific primers, the fragment size is about 779 bp, and lane 2 is the identification with the universal primer M13, the fragment size is about 3079 bp; Figure (b) M: DL5000, lane 1 is the identification with specific primers, the fragment size is about 2034 bp, and lane 2 is the identification with the universal primer M13, the fragment size is about 4334 bp;

[0027] Figure 4: Lesion diagrams of Sf9 cells transfected with recombinant bacmids rBacmid-M1 and rBacmid-HEF. (A) shows normal Sf9 cells. (B) shows cells after 96 hours of infection with p3 generation rBv-M1. (C) shows cells after 96 hours of infection with p3 generation rBv-HEF.

[0028] Figure 5 : PCR identification results of recombinant baculoviruses rBv-M1 and rBv-HEF. In Figure (a), for the identification of rBv-M1, M: DL5000, lane 1 is the identification with specific primers, and the fragment size is approximately 779 bp. Lane 2 is the identification with universal primers, and the fragment size is approximately 3079 bp. In Figure (b), for the identification of rBv-HEF, M: DL5000, lane 1 is the identification with specific primers, and the fragment size is approximately 2034 bp. Lane 2 is the identification with universal primers, and the fragment size is approximately 4334 bp.

[0029] Figure 5 : IFA identification results of the M1 protein and protein HEF of IDV;

[0030] Figure 6 : Western blot identification results of the M1 protein and protein HEF of IDV. Among them, (a) shows the identification result of the M1 protein, and the band size is approximately 27 kDa. (b) shows the identification result of the HEF protein, and the band size is approximately 72 kDa;

[0031] Figure 7 : Electron microscopy identification results of purified IDV VLPs;

[0032] Figure 8 : Western blot identification results of the M1 protein and HEF protein in purified IDV VLPs;

[0033] Figure 9 : Monitoring chart of antibody titers after immunizing mice with virus-like particles IDV VLPs. Specific implementation manners

[0034] 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 described in detail and completely below. For those conditions not specified in the embodiments, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. For reagents or instruments whose manufacturers are not specified, they are all commercially available conventional products.

[0035] Example 1 Construction of recombinant plasmids:

[0036] Specific primers for the M1 and HEF genes containing vector homologous arms were designed respectively according to the nucleic acid sequence of the reference D / swine / Oklahoma / 1334 / 2011 strain and the information of the vector pFastBac1. To improve the expression level, Kozak sequences were added to the upstream primers. The primer sequences are shown in Table 1 in detail. Using the cDNA of D / swine / Oklahoma / 1334 / 2011 as a template, the M1 and HEF gene fragments were amplified with specific primers;

[0037] The results of PCR amplification are as Figure 1 shown. It can be seen from Figure 1 (a) that the amplified fragment of the M1 gene specific primer is about 779 bp; it can be seen from Figure 1 (b) that the amplified fragment of the HEF gene specific primer is about 2034 bp;

[0038] The M1 and HEF PCR products were recovered and ligated to the double-digested pFastBac1 vector by seamless cloning method respectively, and then transformed into DH5α competent cells. The cells were spread on an LB solid plate containing ampicillin resistance and cultured overnight. Positive clone colonies were selected and cultured in an LB liquid medium with ampicillin resistance at 37 °C and 200 rpm for 4 h. After correct double digestion identification and sequencing, the recombinant plasmids were named pFastBac1-M1 and pFastBac1-HEF;

[0039] The results of double digestion identification are as Figure 2 shown. It can be seen from Figure 2 (a) in: the double digestion result of the pFastbac1-M1 plasmid, the size of the pFastbac1 vector fragment is about 4700 bp, and the size of the M1 gene fragment is about 756 bp; it can be seen from Figure 2 (b) in: the double digestion result of the pFastbac1-HEF plasmid, the size of the pFastbac1 vector fragment is about 4700 bp, and the size of the HEF gene fragment is about 2011 bp.

[0040]

[0041]

[0042] Example 2 Construction of recombinant bacmid:

[0043] Take 100 μL of DH10Bac competent cells, add 1 μL of pFastBac1(HEF / M1) plasmid, incubate on ice for 30 min, heat shock at 42 °C for 60 s, and then place back on ice for 2 - 3 min; add 900 μL of SOC culture medium, shake at 37 °C and 200 rpm for 4 h; evenly coat on an LB solid plate containing X-gal, IPTG, kanamycin, gentamicin, and tetracycline; incubate overnight at 37 °C; the next day, pick the white colonies from the blue-white plaque plate into a triple-antibiotic (kanamycin, gentamicin, tetracycline) LB liquid medium, shake at 37 °C and 200 rpm for 4 h, and perform PCR identification using universal primers M13F / R (the primer sequences are shown in detail in Table 2). The bacterial solution with correct results is streaked again. The next day, pick the white colonies, inoculate them into liquid LB, perform PCR identification, and repeat three times. Inoculate the positive bacterial solution after three streaks into 10 mL of triple-antibiotic liquid medium, incubate overnight at 37 °C and 200 rpm. Extract the bacmid according to the plasmid extraction instruction manual and name it rBacmid-M1 and rBacmid-HEF;

[0044] Through PCR identification with specific primers and universal primers, the identification results of rBacmid-M1 are as shown in Figure 3 (a). Lane 1 is the identification with specific primers, and the fragment size is about 779 bp. Lane 2 is the identification with universal primers, and the fragment size is about 3079 bp. The results are consistent with the sizes of the target bands; the identification results of rBacmid-HEF are as shown in Figure 3 (b). Lane 1 is the identification with specific primers, and the fragment size is about 2034 bp. Lane 2 is the identification with universal primers, and the fragment size is about 4334 bp. The results are consistent with the sizes of the target bands.

[0045]

[0046] Example 3 Rescue and identification of recombinant baculovirus:

[0047] Seed well-grown Sf9 cells into a 3.5-cm cell culture dish. When the cell density reaches 70%-80%, perform transfection. Pre-equilibrate serum-free Sf9 insect cell medium, X-treme GENE HP DNA transfection reagent, and recombinant bacmid to room temperature. According to the concentration of the recombinant bacmid, add approximately 200 μL of serum-free insect cell medium, and then add 2 μg of recombinant bacmid and mix well. Add 6 μL of X-treme GENE HP DNA transfection reagent, mix well, and incubate at room temperature for 15 min. Add the incubated DNA complex to the seeded Sf9 cell culture dish and culture for 5-7 days. Collect the cell transfection supernatant into a centrifuge tube to obtain the P1 generation virus solution. Re-inoculate the P1 generation virus solution into Sf9 cells with a density of 70%-80%. Collect the supernatant after about 5-7 days as the P2 generation virus solution. Continuously passage blindly for 4 generations and observe cytopathic effects. Take the P4 generation virus solution and extract the genome using a virus genome extraction kit, and perform PCR identification and IFA expression identification. The correctly identified recombinant baculoviruses are named rBv-M1 and rBv-HEF.

[0048] The cytopathic effect images are as Figure 4 , Figure 4 (a) shows cells without inoculation of recombinant baculovirus, Figure 4 (b), Figure 4 (c) shows cells inoculated with the p4 generation recombinant baculoviruses rBv-M1 and rBv-HEF respectively. It can be seen that the cells as a whole become larger, the cell nuclei rupture, and cytopathic phenomena such as granular degeneration appear; The PCR identification results are as Figure 5 . Through PCR identification with specific primers and universal primers, the identification result of rBv-M1 is as Figure 5 (a) shows. Lane 1 is the identification with specific primers, and the fragment size is approximately 779 bp. Lane 2 is the identification with universal primers, and the fragment size is approximately 3079 bp, which is consistent with the size of the target band; The identification result of rBv-HEF is as Figure 5 (b) shows. Lane 1 is the identification with specific primers, and the fragment size is approximately 2034 bp. Lane 2 is the identification with universal primers, and the fragment size is approximately 4334 bp, which is consistent with the size of the target band; The IFA identification result is as Figure 6 shown. It can be seen that green fluorescence signals appear in each experimental group compared with the uninfected group, indicating that each component protein is correctly expressed.

[0049] Example 4 Assembly, purification, and identification of virus-like particles:

[0050] According to the instructions of the BacPAK baculovirus titer determination kit, determine the virus titers of the recombinant baculoviruses rBv-HEF and rBv-M1. With a total MOI = 5, uniformly add rBv-HEF and rBv-M1 in a ratio of 1:1 to well-grown cells in the logarithmic growth phase with a cell density of about 2×106 Cultured in Sf9 suspension cell solution at a density of

[0051] 1) First, centrifuge the collected supernatant at 3000×g and 4°C for 30 min to remove large cell debris, and take the supernatant.

[0052] 2) Centrifuge the supernatant from the previous step at 8000×g and 4°C to remove small cell debris, and take the supernatant.

[0053] 3) Ultracentrifuge the supernatant from the previous step at 100000×g and 4°C for 1 h, discard the supernatant. Add an appropriate amount of PBS and dissolve overnight at 4°C.

[0054] 4) Use a long needle syringe to add 20% sucrose solution and 60% sucrose solution to the ultracentrifuge tube in sequence, and add the dissolved VLP solution to the top layer of the ultracentrifuge tube. Ultracentrifuge at 100000×g and 4°C for 2 h using a horizontal rotor.

[0055] 5) Take out the white bands in the 20% sucrose solution and 60% sucrose solution. Dilute with an appropriate amount of PBS.

[0056] 6) Ultracentrifuge the diluted solution at 100000×g and 4°C for 1 h.

[0057] 7) Discard the supernatant, add an appropriate amount of PBS to dissolve the precipitate, and filter the fully dissolved solution through a 0.22 μm filter to obtain purified IDV VLPs.

[0058] 8) Observe the purified IDV VLPs by transmission electron microscopy and identify them by Western Blot.

[0059] The transmission electron microscopy results of IDV VLPs are as Figure 7 shown. It can be seen from Figure 7 that the virus-like particles IDV VLPs prepared in the present invention are circular particles similar to real viruses, indicating that the assembly of virus-like particles is successful.

[0060] The immunoblot identification results of each component protein of IDV VLPs are as Figure 8 shown. It can be seen from Figure 8 (a) that the size of the M1 protein band is about 27 kDa; it can be seen from Figure 8 (b) that the HEF protein band is about 72 kDa; the results are consistent with the expected bands.

[0061] Example 5 Preliminary study on the immunogenicity of virus-like particles:

[0062] 1) Immunization protocol formulation

[0063] Twelve 6 - 8 - week - old female BALB / c mice were randomly divided into 2 groups, with 6 mice in each group. The immunogenicity of virus - like particles IDV VLPs was evaluated by intraperitoneal injection. Specifically, the virus - like particles IDV VLPs were added drop - by - drop to Freund's adjuvant at a ratio of 1:1, emulsified by magnetic stirring for 30 min. Then, 200 μl (15 μg) of the fully emulsified virus - like particles IDV VLPs was intraperitoneally injected into the mice, and a booster immunization was carried out 14 days after the first immunization.

[0064] 2) Determination of ELISA antibody titer

[0065] Blood was collected from the tail veins of mice on the 7th, 14th, 21st, 28th, and 35th days after the first immunization, and the serum was separated. The serum was inactivated at 56 °C in a water bath for 30 min. The virus - like particles IDV VLPs were coated overnight on the solid - phase carrier of the ELISA plate at a concentration of 1 μg / well (100 μL). The test serum was diluted 1:1000, and the specific antibody titer was detected by the indirect ELISA method.

[0066] The results are as Figure 9 shown. The antibody titer generated after immunization increased with time, and the antibody titer increased rapidly after the second immunization. The titer rise tended to be stable in the 5th week. Antibodies against IDV VLPs could be rapidly produced in the immunized mice, indicating that IDV VLPs have high immunogenicity and can be used as a new vaccine candidate for the prevention of influenza D.

[0067] The present invention discloses an influenza D virus - like particle, its preparation method and application. Those skilled in the art can draw on the content of this article and appropriately modify the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The products of the present invention have been described through preferred embodiments. Relevant personnel can obviously make changes or appropriate alterations and combinations to the products described herein without departing from the content, spirit, and scope of the present invention to implement and apply the technology of the present invention.

Claims

1. A virus-like particle vaccine, characterized in that, It is a virus-like particle composed of the M1 protein and HEF protein of influenza D virus.

2. A method for preparing D-type influenza virus-like particles, characterized in that, It includes the following steps: Step 1: Construction of recombinant shuttle plasmids Using the cDNA of D / swine / Oklahoma / 1334 / 2011 as a template, specific primers containing vector homologous arms and Kozak sequences were used to amplify the M1 and HEF gene fragments. The M1 and HEF gene fragments were respectively cloned into expression vectors, transformed into competent cells, and screened by double digestion to obtain recombinant shuttle plasmids pFastBac1-M1 and pFastBac1-HEF containing the IDV HEF and IDV M1 genes respectively; Step 2: Construction of recombinant bacmids The recombinant shuttle plasmids constructed in Step 1 were transformed into DH10 Bac competent cells for homologous recombination. After blue-white screening, PCR identification was performed using specific primers and M13 universal primers to obtain recombinant bacmids rBacmid-M1 and rBacmid-HEF containing the IDV M1 and IDV HEF genes respectively; Step 3: Rescue of recombinant baculoviruses By means of liposome-mediated transfection, the recombinant bacmids constructed in Step 2 were respectively transfected into the insect baculovirus expression system. The supernatant was harvested after three blind passages, and PCR identification was performed using specific primers and M13 universal primers to obtain recombinant baculoviruses rBv-M1 and rBv-HEF containing the IDV M1 and IDV HEF genes respectively; Step 4: Preparation and purification of virus-like particles The recombinant baculoviruses rescued in Step 3 were inoculated into the insect baculovirus expression system according to a total MOI = 5, and the cell supernatant was harvested; purification was carried out by sucrose density gradient centrifugation and filtration through a 0.22 μm filter to finally obtain influenza D virus-like particles.

3. The preparation method of a D-type influenza virus virus-like particle according to claim 2, characterized in that, The sequences of the specific primers containing vector homologous arms and Kozak sequences are as follows: M1-F: 5’-CATCGGGCGCGGATCCGCCACCATGGCACAAGAACA-3’ M1-R: 5’-TACCGCATGCCTCGAGTCACTTCCAGTCTCTTTTTAGGGC-3’ HEF-F: 5’-CATCGGGCGCGGATTCCGCCACCATGTTTTTGCTTCT-3’ HEF-R: 5’-TACCGCATGCCTCGAGCTATTTCTTGCAACAGATCCAAAT-3’ In Step 2 and Step 3, the specific primers are the same as those in Step 1, and the sequence of the M13 universal primer is: M13-F: 5’-GTTTTCCCAGTCACGAC-3’ M13-R: 5’-CAGGAAACAGCTATGAC-3’.

4. The preparation method of a D-type influenza virus virus-like particle according to claim 2, characterized in that, In Step 3 and Step 4, the insect baculovirus expression system is insect Sf9 cells, insect High Five cells or insect Sf21 cells.

5. The preparation method of a D-type influenza virus virus-like particle according to claim 2, characterized in that, The recombinant baculoviruses rBv-M1 and rBv-HEF constructed in Step 4 were inoculated according to a virus titer ratio of 1:

1.

6. A D-type influenza virus-like particle prepared by the method for preparing a D-type influenza virus-like particle according to any one of claims 1 to 5.

7. Use of the D-type influenza virus-like particle according to claim 6 in the preparation of a vaccine for preventing D-type influenza.

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