WO2019023920A1 - 一种口蹄疫病毒样颗粒疫苗及其制备方法 - Google Patents

一种口蹄疫病毒样颗粒疫苗及其制备方法 Download PDF

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WO2019023920A1
WO2019023920A1 PCT/CN2017/095376 CN2017095376W WO2019023920A1 WO 2019023920 A1 WO2019023920 A1 WO 2019023920A1 CN 2017095376 W CN2017095376 W CN 2017095376W WO 2019023920 A1 WO2019023920 A1 WO 2019023920A1
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foot
mouth disease
disease virus
cells
gene
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French (fr)
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安海谦
冉波
赵荣茂
方华明
任玉珍
王鹏
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金普诺安生物科技（苏州）有限公司
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Priority to CN201780004617.2A priority Critical patent/CN109601007B/zh
Priority to KR1020187037779A priority patent/KR102132730B1/ko
Priority to PCT/CN2017/095376 priority patent/WO2019023920A1/zh
Publication of WO2019023920A1 publication Critical patent/WO2019023920A1/zh

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    • C—CHEMISTRY; METALLURGY
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/125—Picornaviridae, e.g. calicivirus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/125—Picornaviridae, e.g. calicivirus
    • A61K39/135—Foot- and mouth-disease virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
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    • A61K2039/5258—Virus-like particles
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    • C12N2770/00011—Details
    • C12N2770/32011—Picornaviridae
    • C12N2770/32111—Aphthovirus, e.g. footandmouth disease virus
    • C12N2770/32123—Virus like particles [VLP]
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    • C12N2770/00011—Details
    • C12N2770/32011—Picornaviridae
    • C12N2770/32111—Aphthovirus, e.g. footandmouth disease virus
    • C12N2770/32134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
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    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/32011—Picornaviridae
    • C12N2770/32111—Aphthovirus, e.g. footandmouth disease virus
    • C12N2770/32151—Methods of production or purification of viral material

Definitions

  • the invention relates to the field of genetic engineering technology, in particular to a foot-and-mouth disease virus-like particle and a preparation method thereof, and a foot-and-mouth disease vaccine prepared thereby.
  • Foot-and-mouth disease is an acute, highly contagious animal disease. Mainly infested with cloven-hoofed animals, occasionally in humans and other animals. Susceptible animals mainly include more than 70 hoofed domestic and wild mammals in 20 families including cattle, buffalo, sheep, goats, camels and pigs. Due to the high degree of infectivity of foot-and-mouth disease and its significant impact on the agricultural economy, OIE lists it as the first infectious disease of Class A animals.
  • the attenuated vaccine adopts the classic weakening virus method, and the mother poison is weakened by culturing the guinea pig, the chicken embryo or the cell seed through multiple generations, so that the virus gradually undergoes gene mutation to produce an attenuated strain. After the attenuated strain is extensively expanded in the tissue culture, an adjuvant is added to prepare a vaccine. But because the virus is toxic, the virus is gone. Insecure factors such as incomplete living, live virus escape vaccine factories, and the outbreak of foot-and-mouth disease in some parts of the world seem to be related to the live virus remaining in the inactivated vaccine, prompting people to seek a safer and more effective foot-and-mouth disease vaccine.
  • the foot-and-mouth disease attenuated vaccine can produce a certain level of protection during the process of immune prevention, and has a certain control effect on the prevalence of foot-and-mouth disease.
  • the attenuated vaccine is also a live virus, the state of the animal carrying the virus during the vaccination process, in the process of long-term survival in susceptible animals, the attenuated foot-and-mouth disease strain is likely to restore virulence.
  • the proliferation of attenuated virus in susceptible animals will also have a deterrent effect on the protection of immunity, and will not achieve the best immune effect.
  • attenuated vaccines have been phased out, but inactivated vaccines are still widely used in many countries and regions.
  • VLP vaccine is a molecular or biological technique that expresses one or more structural proteins of a virus. These structural proteins have a natural self-assembly ability to form a spatial configuration and an epitope similar to a natural viral particle but no viral nucleic acid. It is highly immunogenic and non-infectious.
  • the conformational epitope is preserved and can be presented in the same way as the whole virus infects the body.
  • Immune cells effectively induce humoral and cellular immunity in the body's immune system, shortening the window period.
  • the VLP vaccine also has the advantage of avoiding the destruction of some important antigenic determinants during inactivation.
  • the amino acid sequence of the antigen can be arbitrarily modified according to its needs, so that it can better stimulate the body to produce a protective immune response.
  • Another object of the present invention is to provide a foot-and-mouth disease vaccine.
  • a further object of the present invention is to provide a recombinant expression vector containing both the foot-and-mouth disease virus P12A gene and the 3C gene.
  • the present invention utilizes insect cell preference codons to codon-optimize the nucleic acid sequence.
  • GenBank sequence LOCUS AET43040 2332 aa linear VRL 15-FEB-2012 DEFINITION polyprotein[Foot-and-mouth disease virus-type O]ACCESSION AET43040 the main strain (serum O type) and its gene sequence leading to the epidemic of foot-and-mouth disease in China were identified.
  • the main strain serum O type
  • chemically synthesized gene fragments required for foot-and-mouth disease VLP The packaging of foot-and-mouth disease VLP requires P12A and 3C proteins, so the chemical synthesis sequences correspond to the P12A gene and the 3C gene, respectively.
  • the specific sequence is as follows:
  • the P12A gene has the nucleotide sequence shown in SEQ ID NO. 1, and the sequence composition includes the RsrII-Kozak sequence-start codon-HBS signal peptide-VP4-VP2-VP3-VP1-2A-stop codon-SV40PolyA Signal - EcoRI.
  • the 3C gene has the nucleotide sequence shown in SEQ ID NO. 2, and the sequence composition is as follows, and is indicated by different identifiers: SphI-Polyhedrin promoter-vector sequence-start codon-HBS signal peptide-3C-termination code sub-HindIII
  • the present invention firstly provides a recombinant expression vector comprising a foot-and-mouth disease virus P12A gene and a 3C gene; the nucleotide sequence of the foot-and-mouth disease virus P12A gene is shown in SEQ ID No. 1 in the sequence listing; or by SEQ ID No.
  • the nucleotide sequence shown in Figure 1 is substituted or deleted by one or several nucleotides to obtain a nucleotide sequence encoding the P12A protein;
  • the nucleotide sequence of the foot-and-mouth disease virus 3C gene is as shown in SEQ ID No. 2 or by SEQ
  • the nucleotide sequence shown by ID No. 2 is substituted or deleted by one or several nucleotides to obtain a nucleotide sequence encoding the 3C protein.
  • the recombinant expression vector of the present invention is prepared by double-digesting the 3C gene fragment and pFastBac HT-B with Sph I and Hind III, and the 3C gene fragment is ligated with pFastBac HT-B to obtain pFastBac.
  • HT-B-3C; the P12A gene fragment and pFastBac HT-B-3C were digested with Rsr II and EcoR I, digested and ligated, and the recombinant expression plasmid pFastBac HT-B-P12A-3C was constructed.
  • Host cells containing the above recombinant expression vectors of the present invention are within the scope of the present invention.
  • the present invention provides a foot-and-mouth disease virus-like particle obtained by the following method: (1) transfecting the above recombinant expression vector into an insect cell to obtain an insect cell capable of expressing a foot-and-mouth disease virus-like granule protein; (2) culturing the cell After 5-7 days, the cells and supernatant were harvested, and the supernatant was subjected to ultrafiltration concentration, centrifuged, and the supernatant was collected. The supernatant was purified by ion exchange chromatography, hydrophobic chromatography or molecular sieve chromatography to obtain foot-and-mouth disease virus-like particles. .
  • the insect cell is an sf9 cell.
  • a hollow fiber column having a cut-off pore diameter of 200 to 500 nm is used for microfiltration clarification, and then concentrated by ultrafiltration using a hollow fiber column having a cut-off pore diameter of preferably 300 kDa.
  • the concentrated sample was subjected to sucrose density gradient centrifugation, centrifuged at 25,000 rpm for 16 h or 35000 rpm for 3 h; the centrate was subjected to hollow fiber column chromatography and ion exchange chromatography, eluted, and the purified foot-and-mouth disease virus-like particles were collected through the peak.
  • Bulk preparation VLPs were purified by molecular sieve chromatography and ion exchange chromatography in two steps.
  • the invention provides the above application of the recombinant expression vector containing the foot-and-mouth disease virus P12A gene and the 3C gene in preparing a foot-and-mouth disease virus vaccine.
  • the present invention provides the use of a host cell comprising the above recombinant expression vector for the preparation of a foot-and-mouth disease virus vaccine.
  • the invention provides the application of the foot-and-mouth disease virus-like particles prepared by the above method in preparing a foot-and-mouth disease virus vaccine.
  • the present invention provides the above recombinant expression vector or host cell containing the same Or the use of the above-mentioned foot-and-mouth disease virus-like particles in preventing animal-infected foot-and-mouth disease virus.
  • the present invention provides a foot-and-mouth disease vaccine comprising the foot-and-mouth disease virus-like particle of the present invention.
  • the invention provides a method for preparing a foot-and-mouth disease virus-like particle vaccine, which is prepared by mixing the foot-and-mouth disease virus-like particles of the present invention with different adjuvants such as 206, 201 and aluminum adjuvant to prepare a vaccine containing 100 ⁇ g/ml of virus-like particles.
  • the present invention adopts an insect baculovirus system which is non-toxic and harmless to humans and animals, and a serum-free, protein-free medium cultured cell without any animal component source, and does not have any pathogenic factor for infecting animals, and does not require height. Closed production site. This not only eliminates the safety hazards in the production of foot-and-mouth disease inactivated vaccines, but also reduces the cost of vaccine production.
  • the foot-and-mouth disease virus-like particle vaccine prepared by the invention does not contain viral genetic material, so it is not infectious, and there is no risk of factory poisoning and incomplete poisoning, and the safety of the vaccine can be completely ensured;
  • the foot-and-mouth disease virus-like particle vaccine has the same immunological characteristics as the intact virus. It has a similar structure under an electron microscope and can maintain the natural antigen epitope without inactivation. Therefore, not only can the body fluid be induced but also the cellular immunity can be induced. Induces a strong and long-lasting immune response with complete protective immunity and a longer immune protection period;
  • the foot-and-mouth disease virus-like particle vaccine does not contain the non-structural proteins 3A, 3B, and 3C of the virus, and can effectively perform differential diagnosis of infected animals and vaccinated animals according to 3A, 3B, and 3C antibodies without any interference;
  • VLP foot-and-mouth disease vaccination animals can produce rapid immune protection, short window period, and immune immunity produced by immunized animals, can obtain complete anti-infection protection, can effectively prevent immunized animals from becoming long-term virus carrying And dangerous sources of infection, which are used for immunization Ways to control and eliminate foot and mouth disease are absolutely necessary;
  • VLP protein expression level obtained by the present invention was measured at 120 mg/liter of culture supernatant. Optimized cell culture conditions can reach higher levels; the expression level of BHK cells is tens to hundreds of times higher than inactivated vaccines.
  • the purified samples were observed by transmission electron microscopy to show virus-like particles with a particle diameter of about 30 nm. The particles were intact and regular.
  • the invention has low production cost, can carry out high-density cell culture, does not require carbon dioxide, has a temperature of 28 ° C and the output of foreign protein is much higher than that of suspension culture BHK cells, and is suitable for industrial large-scale production.
  • the present invention provides a foot-and-mouth disease virus-like particle without viral nucleic acid, no potential carcinogenic risk and risk of sporadic toxicity, good safety, immunological properties and biological activity, and can be prepared and purified on a large scale, and can be used for preparing foot-and-mouth disease.
  • VLP vaccine has good economic value and application prospects.
  • Figure 1 shows the restriction endonuclease Sph I and Hind III on the agarose gel electrophoresis after double-digestion of the plasmid containing the 3C gene and the vector pFastBac HT-B
  • 1 is a 1 kb DNA Ladder Marker
  • 2 is a 3C gene
  • Figure 2 shows the restriction enzymes Rsr II and EcoR I for the identification of the correct plasmid HT-B-3C and the plasmid containing the P12A gene, and the digestion product was subjected to agarose gel electrophoresis, wherein 1: HT -B-3C ED RE, 2-3: P12A fragment ED RE, 4: 1 kb DNA Ladder Marker.
  • Figure 3 shows the recombinant expression vector pFastBac HT-B-P12A-3C E. coli transformation clone detection by PCR.
  • 1 is 1 kb DNA Ladder Marker
  • 2 is recombinant expression vector pFastBac HT-B-P12A-3C
  • 5 is pFastBac HT-B-P12A.
  • Figure 4 shows the supernatant and cells of Sf9 cells infected with recombinant expression vector pFastBac HT-B-P12A-3C.
  • Western blot analysis showed that 81kD, 47kD, 35kD and 24kD target lines appeared in Sf9 cells infected with recombinant virus.
  • Band corresponding to foot-and-mouth disease virus P1 protein, VP31 protein, VP0 protein and VP3/VP2/VP1 protein, and expected protein The quantity is the same.
  • Fig. 5 is a diagram showing the purification of a foot-and-mouth disease virus-like particle peak by using a molecular sieve.
  • the first peak is a VLP
  • the second and third peaks are proteins having a smaller molecular weight.
  • Figure 6 is a peak diagram of an anion exchange chromatography column on foot-and-mouth disease virus-like particles.
  • Fig. 7 is a SDS-PAGE identification diagram of the purified foot-and-mouth disease virus-like particles. Among them, 1 is Marker and 2 is purified FMD VLP.
  • Figure 8 is a photograph showing the results of Western blot analysis of purified foot-and-mouth disease virus-like particles.
  • Lane 1 is Protein Marker (170, 130, 100, 70, 55, 40, 35, 25, 15, 10 kDa) and
  • Lane 2 is the purified FMDV VLP.
  • the strip at 30 kDa is VP0
  • the strip at 25 kDa is VP1.
  • Figure 9 is a transmission electron micrograph of purified foot-and-mouth disease virus-like particles (magnification: 200000 times).
  • the baculovirus expression system (including the vector pFastBacHT-B, the suspension cultured Sf9 cells, and the competent E. coli DH10Bac) used in the examples of the present invention was purchased from Invitrogen, USA.
  • Transfection reagent II Reagent (Cat no.10362-100) is a product of Invitrogen; GPM-115 insect cell serum-free medium is the company's product; SF900III SFM is life technology company product; restriction endonuclease Rsr II, EcoRI, SphI, Hind III and DNA ligase (T4DNA ligase) are products of New England Biolabs; Goldview dye, Taq DNA polymerase, pfu DNA polymerase and dNTPs are products of Cypress®; kanamycin, gentamicin, tetracycline, blu- Gal and IPTG are products of Bayer; guinea pig anti-A FMDV polyclonal antibody is the product of Lanzhou Veterinary Research Institute of Chinese Academy of Agricultural Sciences; Dlight 800 goat anti-guinea pig IgG antibody (catalog number GtxGP-003-D800NHSX) is the product of Immuno Reagents; The gel recovery kit (catalog number
  • the PCR instrument (Model: Mastercycler gradient) is a product of Eppendorf, Germany; the constant temperature incubator (Model: DHP-9082) is a product of Shanghai Yiheng Technology Co., Ltd.; the microcentrifuge (Model: Lx-300) is limited by Kylin-Bell experimental equipment. Company product; water bath (model: DK-8K) is Shanghai Jinghong Experimental Equipment Co., Ltd.; electrophoresis instrument (model: DYY-6C) is Beijing Liuyi Instrument Factory; gel phase system (Model: 2020D) It is a product of BINTA; Odyssey (model: LI-COR) is a product of Li-Cor; and an inverted microscope (model: 7S100) is a product of Nikon Corporation of Japan.
  • GenBank serial number AET43040 (2332 aa linear VRL 15-FEB-2012; DEFINITION polyprotein [Foot-and-mouth disease virus-type O] ACCESSION AET43040, the main strain causing the recent epidemic of foot-and-mouth disease in China (serum O type) was identified. And its gene sequence. On this basis, according to the preference of codon in the process of insect cell translation, the coding gene fragment required for foot-and-mouth disease VLP was designed. The packaging of foot-and-mouth disease VLP requires P12A and 3C proteins, so the chemical synthesis sequences correspond to P12A gene and 3C gene.
  • the P12A gene has the nucleotide sequence shown in SEQ ID NO. 1, and the sequence structure is: RsrII-Kozak sequence-start codon-HBS signal peptide-VP4-VP2-VP3-VP1-2A-stop codon- SV40PolyA signal - EcoRI.
  • the 3C gene has a nucleotide sequence as shown in SEQ ID NO. 2, and the sequence composition is: SphI-Polyhedrin promoter-vector sequence-start codon-HBS signal peptide-3C-stop codon-HindIII.
  • Foot-and-mouth disease virus P12A gene and 3C gene are inserted in tandem into the transfer vector pFastBac HT-B
  • the 3C gene fragment was ligated with pFastBac HT-B to obtain pFastBac HT-B-3C.
  • the P12A gene fragment and pFastBac HT-B-3C were digested with Rsr II and EcoR I, and then ligated and transformed. .
  • the results of the digestion are shown in Figures 1 and 2.
  • the reaction mixture was mixed and centrifuged, and then ligated overnight at 16 °C. Remove the frozen E. coli DH5 ⁇ competent cells at -80 °C until they melted, add the ligation product, mix the light bulb wall, ice bath for 30 min, heat shock at 42 °C for 90 sec, immediately ice bath for 1 min, add 800 ⁇ L
  • the LB medium without antibiotics was shaken at 150 rpm for 50 min at 37 ° C, and 100 ⁇ L of the bacterial solution was applied to Amp/LB plates, and cultured at 37 ° C for 12-16 h to observe the colony growth.
  • the cryopreserved E. coli DH10Bac competent cells were removed from the ice at -80 °C until they were thawed. 100 ⁇ L of the recombinant Eppendor F tube containing 2 ⁇ L of the recombinant plasmid was added, and the tube wall was lightly shaken for 30 min, 42 ° C. The hot bath was hot for 45 s, and immediately ice bathed for 5 min. 800 ⁇ L of antibiotic-free LB medium was added, and the cells were shaken at 200 rpm for 5 h at 37 ° C. 10 ⁇ L of the bacterial solution was applied to the LB selective plate, and cultured at 37 ° C for 48 hours to observe the colony growth.
  • Solution II (0.2mol / L NaOH, 1% SDS) gently mix, set at room temperature for 5min, add 200 ⁇ L 3mol / L potassium acetate (pH 5.5), gently invert and mix, ice bath 10min after room temperature 12,000 Centrifuge at rpm for 10 min, the supernatant will be Transfer to another new Eppendorf tube, add an equal volume of isopropanol, mix by inversion, stand at room temperature for 10 min and centrifuge at 12,000 rpm for 15 min.
  • the precipitate was washed with 70% ethanol, centrifuged at 12,000 rpm for 5 min at room temperature, and the precipitate was dried, and then dissolved by adding 40 ⁇ L of TE buffer (10 mmol/L Tris-HCl, 1 mmol/L EDTA, pH 8.0).
  • the inserted gene was PCR-identified using the M13 primer at both ends of the recombinant Bacmid mini-attTn 7.
  • the M13 primer sequence (synthesized by Shanghai Shenggong Bioengineering Co., Ltd.) is as follows:
  • the PCR amplification conditions were pre-denaturation at 94 ° C for 5 min; denaturation at 94 ° C for 50 s, annealing at 55 ° C for 50 s, extension at 72 ° C for 8 min; 30 cycles and extension at 72 ° C for 10 min.
  • Sf9 suspension cells were cultured with SF900III SFM, and cultured at 27 ° C, shaking at 110 rpm. Sf9 cells were passaged to a 6-well plate 1 d before transfection, and the density per well was 8 ⁇ 10 5 .
  • Solution A 10 ⁇ L of recombinant Bacmid DNA and 90 ⁇ L of serum-free, antibiotic-free SF900III medium were mixed.
  • Solution B 6 ⁇ L of liposome Cellfectin II transfection reagent (fully mixed 5 to 10 times before use) was mixed with 94 ⁇ L of serum-free, antibiotic-free SF900III medium.
  • the cells After 5 days of transfection, if the cells showed typical CPE (the cells appeared round and swollen), the normal control Sf9 cells were uniform in size, the cells were dense, and no cell lesions appeared, indicating that the recombinant baculovirus packaging was successful. The supernatant was harvested, centrifuged at 500 g for 5 min at room temperature, and dispensed into a sterile cryotube, which is a primary virus containing recombinant baculovirus. If the cells do not show typical CPE, harvest the supernatant and re-infect the new cells.
  • the recombinant baculovirus was inoculated into the cells to amplify the poisonous species. After culture for 5-7 days, the culture supernatant was harvested, centrifuged at 500 g for 5 min at room temperature, and dispensed into sterile cryopreservation. The tubes were stored at -80 ° C, and the cells were washed with PBS (pH 7.4), and then pelleted by centrifugation. The supernatant and cell pellet were used for subsequent Western blot analysis.
  • Sample preparation the cells expressing the recombinant baculovirus were lysed with cell lysate (0.1 M sodium bicarbonate solution), and the supernatant was centrifuged, and respectively, 6 ⁇ SDS protein electrophoresis loading buffer was added, and boiled at 100 ° C for 5 min; Each well was loaded with 10-20 ul, two-color pre-stained Marker 2 ⁇ L, protein electrophoresis concentrated gel 80v, and separation gel 120v.
  • cell lysate 0.1 M sodium bicarbonate solution
  • Transfer film 60V constant pressure for 1h; blocking: 5% skim milk powder, overnight at 4 °C; primary antibody: guinea pig anti-FMDV polyclonal antibody (5% skim milk diluted 1:500) at room temperature for 3 h; wash membrane: Wash the membrane three times with PBST (Tween 1 ⁇ ) for 5 min each time; secondary antibody: goat anti-guinea pig Dlight800 antibody (5% skim milk diluted 1:2000), incubate for 40 min at room temperature in the dark; wash membrane: use PBST (Tween is 1 ⁇ ) Wash the membrane three times for 10 min each time; Detection: Scanning and identification using Odyssey's 700 and 800 fluorescence channels.
  • the SF9 cells were infected with the recombinant baculovirus. After 5-7 days of infection, the infected cell culture supernatant was collected and centrifuged at 6000 rpm for 10 min at 4 ° C to remove the supernatant and cells. Identification and purification. Western blot analysis showed in Figure 4. The results showed that 81kD, 47kD, 35kD and 24kD target bands appeared in Sf9 cells infected with recombinant virus, corresponding to foot-and-mouth disease virus P1 protein, VP31 protein, VP0 protein and VP3/VP2/VP1 protein. , consistent with the expected protein molecular weight. The results suggest that P1 protein is expressed in insect cells, and part of P1 is successfully cleaved into VP31, VP0, VP3/VP2/VP1 under the cleavage of 3C protein.
  • Example 3 infected with SF9 cells was collected, filtered at 0.8 um and placed at 4 ° C, and the cells were stored at -20 ° C or immediately used for purification.
  • Cell disruption The collected cells were weighed, and 20 volumes of 0.1 M NaHCO3 (pH 8.3) were added and lysed at room temperature for 30 min. Centrifuge at 12000 rpm for 15 min @ 4 ° C (3 times), collect the supernatant, and discard the cells. Filtration was carried out with 0.8 um, 0.45 um and 0.2 um filters in that order.
  • sucrose solutions were prepared and diluted in 1X PBS solution.
  • 60%, 50%, 40%, 30%, and 20% sucrose solutions (1 ml each) were sequentially added to an ultracentrifuge tube (Beckman) and then added to the sample to be removed. Make a mark. Centrifuge at 25,000 rpm for 16 hr @ 4 ° C. The centrifuge was a Beckman Optima X-100. The centrifuged components were sequentially aspirated, and identified by SDS-PAGE and western blotting, and the corresponding components were dialyzed to remove sucrose. Purified sample.
  • the supernatant of FMDV insect cell culture was concentrated 30 times by 300KDa hollow fiber column, using PBS buffer, and the chromatographic medium was Sepharose 6FF column (XK16 ⁇ 70 column, chromatographic medium height 63.5 cm), flow rate 1 ml/min, layer
  • the VLP and the heteroprotein were separated.
  • the results are shown in Figure 5.
  • the second column chromatography was performed on a CaptoQ ImpRes ion exchange chromatography column, and the VLP peak of the first column was dialyzed against 10 mM Tris-HCl pH 8.0.
  • the eluent was 10 mM Tris-HCl pH 8.0, 1 M NaCl.
  • the peaks were collected as purified VLPs and the results are shown in Figure 6.
  • Figure 7 is a SDS-PAGE map of the purified VLP having corresponding bands at the VP1 and VP0 positions constituting the VLP
  • Figure 8 further demonstrates that the proteins constituting the VLP are specific by immunoblot hybridization.
  • Lowry method to detect the final concentration of the purified protein obtained in the present invention is 1800 ⁇ g / ml, in the vaccine
  • sample concentration is 0.1 mg / ml, negatively stained by floating method, that is, take 15 ⁇ l of sample beads, cover the liquid bead with a copper mesh with a support film for 2 minutes, remove the copper mesh Use a filter paper to remove excess sample from the edge, and then cover the copper mesh on 15 ⁇ l of 0.5% uranium acetate dye solution beads for 2 minutes, remove the copper mesh, and use filter paper to remove excess dye solution from the edge. Can be observed by electron microscope. The results are shown in Fig. 9, which shows virus-like particles with a particle diameter of about 30 nm, and the particles are intact and regular.
  • VLP samples were emulsified with 206 adjuvants and immunized with FMD-negative pigs, and were randomly divided into 3 groups in the control group and 7 in the test group. Serum was taken on days 7, 14, 21, and 28 after immunization. Elisa identified the titer of foot-and-mouth disease antibody and counted the incidence.
  • Example 5 uses a tandem expression FMDV shell protein vector constructed in a different manner from Example 2.
  • a recombinant expression plasmid was constructed by cloning a single ORF containing the P12A3C gene under the promoter SV40 into pFastBac HT-B.
  • the FMDV-related protein gene was placed in tandem under the SV40 promoter.
  • the P12A gene was PCR-amplified by the primers FMDF1 and FMDR1, and the primers FMDF2 and the primer FMDR2 were used for PCR amplification of the 3B3 and 3C genes.
  • the amplified PCR products were purified and then ligated together using a seamless ligation kit.
  • a tandem expression box that makes up a single promoter. The resulting sequence is shown in SEQ ID NO.
  • Preparation method of mixed emulsion of FMDV VLP antigen prepared in Example 4 and Montanide ISA 201VG adjuvant 1. Take equal mass of FMDV VLP aqueous phase antigen and adjuvant.
  • the emulsion preparation is completed. After standing at 20 degrees for one night, do quality control test. The foot-and-mouth disease virus-like particles are adsorbed with different adjuvants to prepare a vaccine containing 50-5000 ⁇ g/ml of virus-like particles.
  • Dosage form water-in-water-in-water type. Take a clean pipette and draw a small amount of vaccine to the surface of the clean cold water. It should spread in a cloud.
  • the invention provides a foot-and-mouth disease virus-like particle vaccine and a preparation method thereof.
  • the pFastBac HT-B-P12A-3C recombinant expression plasmid was constructed by inserting the codon-optimized foot-and-mouth disease virus P12A gene and 3C gene into the transfer vector pFastBac HT-B, and transformed into DH10Bac competent cells to obtain recombinant Bacmid-DNA. The cells were stained with sf9 to obtain suspended insect cells capable of expressing foot-and-mouth disease virus-like granule proteins.
  • the cells were cultured in a serum-free, protein-free medium, and the supernatant was collected, concentrated by ultrafiltration, centrifuged, and purified by molecular sieve and ion exchange column chromatography to prepare a foot-and-mouth disease virus-like particle vaccine.
  • the foot-and-mouth disease virus-like particle vaccine provided by the invention has good immunogenicity, safety, immunological characteristics and biological activity, and can be prepared and purified on a large scale, and can be used for preparing a vaccine for preventing foot-and-mouth disease infection, and the gene mutation causes the foot-and-mouth disease virus strain.
  • the new strain which is mutated and popular, has a procedure of rapid seedling formation, no need for virus attenuation, adaptation to host cells, etc., and can block the virus epidemic as soon as possible.
  • the low cost of not contacting the virus itself and the production of model organisms has good economic value and application prospects.

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Abstract

本发明提供了一种口蹄疫病毒样颗粒疫苗及其制备方法。将密码子优化的FMDV的P12A和3C基因两个ORF串联插入转移载体构建重组表达质粒；或通过启动子SV40下的包含P12A3C基因的单一ORF克隆入载体构建重组表达质粒，转化感受态细胞得到重组Bacmid-DNA，转染sf9细胞，得到能够表达口蹄疫病毒样颗粒蛋白的昆虫细胞。将该细胞培养后收集上清，超滤浓缩、离心、层析纯化VLP，制备口蹄疫病毒样颗粒疫苗。

Description

一种口蹄疫病毒样颗粒疫苗及其制备方法 技术领域
本发明涉及基因工程技术领域，具体地，涉及一种口蹄疫病毒样颗粒及其制备方法，以及以其制备的口蹄疫疫苗。
背景技术
口蹄疫(Foot-and-mouth disease，FMD)是一种急性、高度传染性的动物疾病。主要侵害偶蹄目动物，偶见于人和其它动物。易感动物主要包括牛、水牛、绵羊、山羊、骆驼和猪等20个科的70多种偶蹄类家养和野生哺乳动物。由于口蹄疫的高度传染性和对农业经济的重大影响，国际兽疫局(OIE)将其列为A类动物传染病的首位。
2005年亚洲1型口蹄疫的传入，2009年A型口蹄疫的传入，特别是近年来O型中和逃避变异株的广泛流行，已对我国的养猪业和养牛业造成不可估量的损失。对于大多数国家而言，以免疫预防为主的策略是控制和消灭口蹄疫的唯一有效途径，而免疫预防的难题来自口蹄疫病毒的高度变异性和多型性、动物机体特别是猪体对口蹄疫病毒疫苗免疫的顿感性、以及发展中国家较低水平的口蹄疫疫苗制备工艺和产品质量。根据充分的调查和研究，我国现有的口蹄疫全病毒灭活疫苗不但免疫效力差，而且副反应重，流行的O型变异株可基本上可逃逸现有疫苗的保护作用，这导致国家免费发放的疫苗少被使用、口蹄疫周而复始地流行。每次流行的间歇期是自然感染免疫而不是疫苗接种的结果；由于不同血清型病毒和变异株的同时存在和流行，口蹄疫感染常重复发生，所以研发和生产新型O型口蹄疫疫苗迫在眉睫。
传统疫苗包括灭活疫苗和弱毒疫苗两种类型。其中弱毒疫苗采用经典的弱化病毒的方式，将母毒通过在豚鼠，鸡胚或者细胞种经过多代的培养弱化，使病毒逐渐发生基因突变，产生弱毒毒株。弱毒毒株在组织培养物中经过大量扩增后，加入佐剂等制成疫苗。但由于病毒毒力返强、病毒灭 活不彻底、活病毒逃逸疫苗加工厂等不安全因素，世界上一些地区口蹄疫的暴发似乎与灭活疫苗中残存的活病毒有关，促使人们寻求一种更加安全有效的口蹄疫疫苗。口蹄疫弱毒疫苗在进行免疫预防的过程中，能产生一定水平的保护，对口蹄疫的流行有一定的控制作用。但是由于弱毒疫苗也是活病毒，在预防接种的过程中造成动物携带病毒的状态，在易感动物体内长期存活的过程中，弱毒的口蹄疫毒株很有可能恢复毒力。同时，弱毒病毒在易感动物体内的增殖也会对产生的保护免疫有抑止的作用，不能达到最佳的免疫效果。由于上述的原因，弱毒疫苗已经逐渐淘汰，但灭活疫苗还在许多国家和地区广泛应用。
由于口蹄疫病毒的不断变异和新病毒的出现，传统灭活和裂解疫苗对人类和牲畜的保护率在下降，灭活不彻底导致的散毒时有发生，研发新型亚单位疫苗尤为迫切。病毒样颗粒(Virus Like Particle，VLP)疫苗的出现为研发新型安全有效的疫苗提供了一个新的契机。VLP疫苗是通过分子生物学技术，表达病毒的一个或多个结构蛋白，这些结构蛋白具有天然的自组装配能力，可形成与天然病毒颗粒相似的空间构型和抗原表位但无病毒核酸，免疫原性强，又没有感染性，不存在灭活不完全或毒力回复的风险，并且表面有高密度的病毒抗原，保存了构象表位，可通过和全病毒感染机体一样的途径呈递给免疫细胞，有效诱导机体免疫系统产生体液免疫和细胞免疫，缩短空窗期。VLP疫苗的优点还在于避免了灭活过程中可能破坏一些重要的抗原决定簇。此外，还有一个优点是可根据其需要任意改造抗原的氨基酸序列，使之能更好地刺激机体产生保护性免疫反应。
口蹄疫VLP疫苗商业化的关键是能够大量高效的制备口蹄疫VLP产品。目前重组疫苗常用的表达系统原核表达系统和酵母表达系统无法同时表达多个衣壳蛋白并自组装成为病毒样颗粒，昆虫细胞和杆状病毒系统能够很好解决这一问题。对于需要表达诸如口蹄疫等的由多个蛋白组成的病毒衣壳，这个系统具有巨大成本优势和产品的更佳免疫原性。相比原核表达系统和酵母系统表达的单个蛋白，经过纯化后再人工结合成为VLP, 昆虫细胞和杆状病毒系统生产VLP疫苗过程大大简化，产率可以提高10-100倍，可以实现大规模生产。
发明内容
本发明的目的在于提供一种从昆虫杆状病毒表达系统获得的口蹄疫病毒样颗粒。
本发明的另一目的在于提供一种口蹄疫疫苗。
本发明的再一目的在于提供一种同时含有口蹄疫病毒P12A基因和3C基因的重组表达载体。
为了提高蛋白的表达量，本发明利用昆虫细胞偏爱密码子对所述核酸序列进行了密码子优化。根据GenBank序列LOCUS AET43040 2332 aa linear VRL 15-FEB-2012 DEFINITION polyprotein[Foot-and-mouth disease virus-type O]ACCESSION AET43040，确定了导致我国口蹄疫流行的主要毒株(血清O型)及其基因序列。在此基础之上，按照昆虫细胞转译过程对密码子的偏爱性，化学合成口蹄疫VLP所需的编码基因片段。口蹄疫VLP的包装需要P12A和3C蛋白，所以化学合成序列分别对应P12A基因和3C基因。具体序列如下：
P12A基因，其核苷酸序列如SEQ ID NO.1所示，序列组成结构包括RsrII-Kozak sequence-起始密码子-HBS信号肽-VP4-VP2-VP3-VP1-2A-终止密码子-SV40PolyA信号-EcoRI。
3C基因，其核苷酸序列如SEQ ID NO.2所示，序列组成结构如下，分别用不同标识表示：SphI-Polyhedrin启动子-载体序列-起始密码子-HBS信号肽-3C-终止密码子-HindIII
本发明首先提供了一种重组表达载体，其含有口蹄疫病毒P12A基因和3C基因；所述口蹄疫病毒P12A基因其核苷酸序列如序列表中SEQ ID No.1所示；或由SEQ ID No.1所示核苷酸序列经取代或缺失一个或几个核苷酸，得到编码P12A蛋白的核苷酸序列；
所述口蹄疫病毒3C基因其核苷酸序列是如SEQ ID No.2所示或由SEQ  ID No.2所示核苷酸序列经取代或缺失一个或几个核苷酸，得到编码3C蛋白的核苷酸序列。
本发明的重组表达载体，通过以下方法制备得到：用Sph I和Hind III对3C基因片段和pFastBac HT-B进行双酶切，酶切后3C基因片段与pFastBac HT-B进行连接转化，得到pFastBac HT-B-3C；用Rsr II和EcoR I对P12A基因片段和pFastBac HT-B-3C进行双酶切，酶切后进行连接转化，构建得到pFastBac HT-B-P12A-3C重组表达质粒。
含有本发明上述重组表达载体的宿主细胞属于本发明的保护范围。
进一步地，本发明提供一种口蹄疫病毒样颗粒，通过以下方法制备得到：(1)将上述重组表达载体转染昆虫细胞，得到能够表达口蹄疫病毒样颗粒蛋白的昆虫细胞；(2)培养该细胞5～7d后，收获细胞和上清，对上清进行超滤浓缩，离心，收集上清液，上清液经离子交换层析、疏水层析或分子筛层析，纯化后获得口蹄疫病毒样颗粒。
所述昆虫细胞为sf9细胞。
步骤(2)中，采用截留孔径为200-500nm的中空纤维柱进行微滤澄清，再用截留孔径优选300kDa的中空纤维柱超滤浓缩。
浓缩后样品进行蔗糖密度梯度离心，离心条件为25000rpm离心16h或35000rpm离心3h；离心液经中空纤维柱层析和离子交换层析，洗脱，收集穿过峰得到纯化的口蹄疫病毒样颗粒。批量制备VLP采用分子筛层析和离子交换层析两步法纯化VLP。
本发明提供了上述含有口蹄疫病毒P12A基因和3C基因的重组表达载体在制备口蹄疫病毒疫苗中的应用。
本发明提供了含有上述重组表达载体的宿主细胞在制备口蹄疫病毒疫苗中的应用。
本发明提供了上述方法制得的口蹄疫病毒样颗粒在制备口蹄疫病毒疫苗中的应用。
本发明提供了上述重组表达载体或含有该重组表达载体的宿主细胞 或上述口蹄疫病毒样颗粒在预防动物感染口蹄疫病毒中的应用。
更进一步地，本发明提供了一种口蹄疫疫苗，含有本发明的口蹄疫病毒样颗粒。
本发明提供一种制备口蹄疫病毒样颗粒疫苗的方法，将本发明的口蹄疫病毒样颗粒用不同佐剂如206、201、铝佐剂配苗后制备成含100μg/ml病毒样颗粒的疫苗。
本发明的有益效果在于：
(1)利用最新发现的流行口蹄疫O型毒株有别于现有疫苗毒株的遗传信息(NCBI Genbank ACCESSION AET43040)，人工制备只包含病毒全部衣壳蛋白的病毒样颗粒疫苗。有别于现生产毒株的抗原表位的氨基酸序列。
(2)本发明采用对人类和动物无毒无害的昆虫杆状病毒系统和没有任何动物成份来源的无血清、无蛋白培养基培养细胞，不存在任何感染动物的致病因子，不需要高度密闭性生产场所。这不但消除了口蹄疫灭活疫苗生产中的安全隐患，也因此降低了疫苗的生产成本。本发明制得的口蹄疫病毒样颗粒疫苗不含有病毒遗传物质,因此不具有感染性，不存在工厂化散毒和灭活不彻底散毒的风险，可完全保证疫苗的安全性；
(3)口蹄疫病毒样颗粒疫苗具有与完整病毒相同的免疫学特性，电子显微镜下结构类似，不经灭活可以保持天然抗原表位，所以不但可以诱导体液而且可以诱导细胞免疫，接种动物后可诱导坚强而持久的免疫反应，具有完全的保护性免疫和较长的免疫保护期；
(4)口蹄疫病毒样颗粒疫苗不含有病毒的非结构蛋白3A、3B、3C，可依据3A、3B、3C抗体有效地进行感染动物和免疫接种动物的鉴别诊断，不存在任何干扰；
(5)VLP口蹄疫疫苗接种动物后，能产生快速的免疫保护、窗口期短，而且免疫动物产生的免疫力坚强、可获得完全的抗感染保护，可有效地防止免疫动物变成长期的病毒携带者和危险的传染源，这对于用免疫接 种方法控制和清除口蹄疫的策略是完全必要的；
(6)Lowry和Bradford法检测本发明的获得的VLP蛋白表达水平在120毫克/升培养上清。优化细胞培养条件可以达到更高水平；相比灭活疫苗在BHK细胞的表达水平高几十至上百倍。经透射电镜观察纯化样品呈现病毒样颗粒，颗粒直径在30nm左右，颗粒完整、规则；
(7)本发明生产成本低，能进行高密度细胞培养，不需要二氧化碳，温度在28℃且外源蛋白产量大大高于悬浮培养的BHK细胞，适合于工业化大生产。
总之，本发明提供口蹄疫病毒样颗粒不带有病毒核酸，无潜在致癌危险和散毒风险，具有良好的安全性，免疫特性和生物学活性，并可以大规模制备和纯化，能够用于制备口蹄疫VLP疫苗，具有较好的经济价值和应用前景。
附图说明
图1为限制性内切酶Sph I和Hind III对含3C基因的质粒和载体pFastBac HT-B进行双酶切后琼脂糖凝胶电泳图，1为1kb DNA Ladder Marker，2为3C基因，3为pFastBac HT-B。
图2为用限制性内切酶Rsr II和EcoR I对鉴定正确的质粒HT-B-3C和含P12A基因的质粒进行双酶切，酶切产物进行琼脂糖凝胶电泳图，其中1:HT-B-3C ED RE，2-3:P12A片段ED RE，4：1kb DNA Ladder Marker。
图3为重组表达质粒PCR鉴定重组表达载体pFastBac HT-B-P12A-3C大肠杆菌转化克隆检测结果：其中，1为1kb DNA Ladder Marker，2为重组表达载体pFastBac HT-B-P12A-3C，3为pFastBac HT-B，4为pFastBac HT-B-3C，5为pFastBac HT-B-P12A。
图4为重组表达载体pFastBac HT-B-P12A-3C感染Sf9细胞后的上清和细胞进行了Western blot检测，结果显示在感染重组病毒的Sf9细胞细胞中出现81kD、47kD、35kD和24kD的目的条带，分别对应口蹄疫病毒P1蛋白、VP31蛋白、VP0蛋白和VP3/VP2/VP1蛋白，与预期蛋白分 子量一致。
图5为利用分子筛纯化口蹄疫病毒样颗粒峰图，第一个峰为VLP，第二、第三个峰为分子量较小的蛋白。
图6为口蹄疫病毒样颗粒上阴离子交换层析柱的峰图。
图7为对纯化后的口蹄疫病毒样颗粒进行SDS-PAGE鉴定图。其中，1为Marker，2为纯化后的FMD VLP。
图8为纯化后的口蹄疫病毒样颗粒Western blot分析的结果照片。泳道1为Protein Marker(170、130、100、70、55、40、35、25、15、10kDa)，泳道2为纯化后的FMDV VLP。其中，30kDa处的条带为VP0，25kDa处的条带为VP1。
图9为纯化的口蹄疫病毒样颗粒透射电镜照片(放大倍数：200000倍)。
具体实施方式
以下实施例进一步说明本发明的内容，但不应理解为对本发明的限制。在不背离本发明精神和实质的情况下，对本发明方法、步骤或条件所作的修改或替换，均属于本发明的范围。
下列实施例中未注明具体条件的实验方法，通常按照常规实验条件进行。
本发明实施例采用的杆状病毒表达系统(包括载体pFastBacHT-B、悬浮培养的Sf9细胞和感受态E.coli DH10Bac)购自美国Invitrogen公司产品。转染试剂
Figure PCTCN2017095376-appb-000001
II Reagent(Cat no.10362-100)为Invitrogen公司产品；GPM-115昆虫细胞无血清培养基为本公司产品；SF900III SFM为life technologies公司产品；限制性核酸内切酶Rsr II、EcoRI、SphI、Hind III和DNA连接酶(T4DNA ligase)为New England Biolabs公司产品；Goldview染料、TaqDNA聚合酶、pfu DNA聚合酶和dNTPs为赛百盛公司产品；卡那霉素、庆大霉素、四环素、blu-gal和IPTG为拜尔迪公司产品；豚鼠抗A型FMDV多克隆抗体为中国农业科学院兰州兽医研究所产品；Dlight 800山羊 抗豚鼠IgG抗体(目录号GtxGP-003-D800NHSX)为Immuno Reagents公司产品；凝胶回收试剂盒(目录号DP103-02)为Tiangen公司产品；质粒提取试剂盒(目录号MK014-2)为Tiangen公司产品。
PCR仪(型号：Mastercycler gradient)为德国Eppendorf公司产品；恒温孵箱(型号：DHP-9082)为上海一恒科技有限公司产品；微型离心机(型号：Lx-300)为Kylin-Bell实验设备有限公司产品；水浴锅(型号：DK-8K)为上海精宏实验设备有限公司产品；电泳仪(型号：DYY-6C)为北京市六一仪器厂产品；凝胶成相系统(型号：2020D)为BINTA公司产品；Odyssey(型号：LI-COR)为Li-Cor公司产品；倒置显微镜(型号：7S100)为日本尼康公司产品。
实施例1编码口蹄疫的P12A及3C蛋白的基因序列优化
根据GenBank序列号AET43040，(2332 aa linear VRL 15-FEB-2012；DEFINITION polyprotein[Foot-and-mouth disease virus-type O]ACCESSION AET43040，确定了最近导致我国口蹄疫流行的主要毒株(血清O型)及其基因序列。在此基础之上，按照昆虫细胞转译过程对密码子的偏爱性，设计了口蹄疫VLP所需的编码基因片段。口蹄疫VLP的包装需要P12A和3C蛋白，所以化学合成序列分别对应P12A基因和3C基因。
P12A基因，其核苷酸序列如SEQ ID NO.1所示，序列组成结构为：RsrII-Kozak sequence-起始密码子-HBS信号肽-VP4-VP2-VP3-VP1-2A-终止密码子-SV40PolyA信号-EcoRI。
3C基因，其核苷酸序列如SEQ ID NO.2所示，序列组成结构为：SphI-Polyhedrin启动子-载体序列-起始密码子-HBS信号肽-3C-终止密码子-HindIII。
实施例2重组表达载体pFastBac HT-B-P12A-3C的构建
1、口蹄疫病毒P12A基因和3C基因串联插入转移载体pFastBac HT-B
用Sph I和Hind III对3C基因片段和pFastBac HT-B进行双酶切，酶 切后3C基因片段与pFastBac HT-B进行连接转化，得到pFastBac HT-B-3C；用Rsr II和EcoR I对P12A基因片段和pFastBac HT-B-3C进行双酶切，酶切后进行连接转化。酶切结果见图1和图2。
将上述反应混合液混匀离心后于16℃连接过夜。取出-80℃冻存的E.coli DH5α感受态细胞置冰上至其融化，加入连接产物，轻弹管壁混匀，冰浴30min，42℃水浴热休克90sec，立即冰浴1min，加入800μL无抗生素的LB培养基，在37℃条件下以150rpm摇菌50min，吸取100μL菌液涂布于Amp/LB平板上，37℃培养12～16h，观察菌落生长情况。从平板上挑取3个单菌落，分别接种于4mL的Amp/LB液体培养基中，37℃条件下200rpm摇菌培养过夜。提取质粒后，利用Rsr II/EcoR I和Sph I/Hind III分别进行双酶切鉴定。于37℃酶切反应3h，用1％琼脂糖凝胶电泳分析，挑取酶切鉴定正确的克隆测序确证。
从-80℃取出冻存的E.coli DH10Bac感受态细胞置冰上至其融化，吸出100μL加入含重组质粒2μL的1.5mL的EppendorF管中，轻弹管壁混匀，冰浴30min，42℃水浴热击45s，立即冰浴5min，加入800μL无抗生素的LB培养基，37℃下200rpm摇菌5h，吸取10μL菌液涂布于LB选择性平板上，37℃培养48h，观察菌落生长情况。
从平板上挑取白色单菌落3～4个，分别划线于LB选择性培养平板，于37℃培养24h，观察菌落表型变化。从不同菌落来源的划线平板上随机挑取3个仍为白斑的菌落，加入3mL的KTG/LB(含卡那霉素50μg/mL，庆大霉素7μg/mL，四环素10μg/mL)培养基中，于37℃、200rpm振摇培养16h。取1.5mL的培养物，室温条件下13,000rpm离心1min，收集沉淀。向沉淀中加入200μL溶液I(50mmol/L葡萄糖，25mmol/L Tris-HCl,10mmol/L EDTA,100μg/mL RNase A，pH 8.0)用涡旋器混匀重悬细菌沉淀后，加入200μL新鲜配制的溶液II(0.2mol/L NaOH,1％SDS)轻轻混匀，置室温放置5min，加入200μL 3mol/L的乙酸钾(pH 5.5)，轻柔颠倒混匀后，冰浴10min后室温下12,000rpm离心10min，将上清 移至另一新的Eppendorf管中，加入等倍体积的异丙醇，颠倒混匀，室温放置10min后12,000rpm离心15min。将沉淀用70％的乙醇洗涤后，室温下12,000rpm离心5min，沉淀干燥后加入40μL TE缓冲液(10mmol/L Tris-HCl,1mmol/L EDTA,pH 8.0)溶解。
2、带有目的基因的重组Bacmid DNA的PCR鉴定
利用重组Bacmid mini-attTn 7两端的M13引物，可对插入基因进行PCR鉴定。
M13引物序列(由上海生工生物工程有限公司合成)如下：
M13F:5’GTTTTCCCAGTCACGAC 3’
M13R:5’CAGGAAACAGTCATGAC 3’
PCR扩增条件为94℃预变性5min；94℃变性50s，55℃退火50s，72℃延伸8min；30个循环，72℃延伸10min。
取5μL PCR扩增产物，用0.5％琼脂糖凝胶电泳分析。根据产物的大小判断是否转座成功。PCR鉴定结果见图3。说明正确构建得到表达载体pFastBac HT-B-P12A-3C。
实施例3重组杆状病毒目的蛋白的表达鉴定
1、rBacmid DNA转染Sf9细胞
1)Sf9细胞的培养 用SF900III SFM培养Sf9悬浮细胞，于27℃、110rpm振荡培养。转染前1d将Sf9细胞传代至6孔板，每孔密度为8×105。
2)转染 取两个Eppendorf管，分别加入A液和B液，体系如下：
A液：将重组Bacmid DNA 10μL和90μL的无血清、无抗生素的SF900III培养基混合。
B液：取6μL脂质体Cellfectin II转染试剂(使用前充分颠倒混匀5～10次)与94μL无血清、无抗生素的SF900III培养基混合。
A液和B液在室温下静置5min后，将A液和B液混合，轻弹管壁混匀，室温下放置30min。用2mL无血清、无抗生素的SF900III培养基洗2次细胞，每孔加入800μL无血清、无抗生素的SF900III培养基， 再将上述A液和B液混合物逐滴加至每孔中，并轻摇混匀，置27℃孵育5h。将转染液移去，更换为新鲜的SF900III SFM培养基，置27℃培养，每日观察细胞变化。
2、重组杆状病毒的鉴定
在转染5d后，若细胞出现典型CPE(细胞出现变圆、肿胀)，而正常对照Sf9细胞大小均匀、细胞核致密，无细胞病变出现，表明重组杆状病毒包装成功。收获上清，室温条件下500g离心5min，并分装至无菌冻存管中，此即为含重组杆状病毒的原代毒种。若细胞未出现典型CPE，收获上清，重新感染新的细胞。
将细胞传代至0.5-1×106/ml时，将重组杆状病毒接种细胞扩增毒种，培养5～7d后收获培养上清，室温条件下500g离心5min，分装至无菌冻存管中，置-80℃备存，同时将细胞用PBS(pH 7.4)洗涤后离心沉淀，上清和细胞沉淀用于后续的Western blot鉴定。
重组杆状病毒表达目的蛋白的Western blot鉴定
样品制备：将收取的表达重组杆状病毒的细胞用细胞裂解液(0.1M碳酸氢钠溶液)裂解后，离心取上清，分别加入6×SDS蛋白电泳加样缓冲液，100℃煮沸5min；每孔上样10-20ul，双色预染Marker 2μL，进行蛋白电泳浓缩胶80v，分离胶120v。
蛋白印迹：将凝胶放在PVDF膜(PVDF膜预先置于甲醇溶液中30s激活)上，上下各放3张滤纸，将上述物品放至转膜电泳缓冲液中浸泡15min，以驱除留于滤膜上的气泡。按顺序安装电转移装置，在阴性电极板上依次放置3张滤纸、凝胶、PVDF膜、3张滤纸，确保各层精确对齐(从下到上)，并驱除各层间气泡，标记方位，合上阳极板。
转膜：60V恒压1h；封闭：用5％脱脂奶粉、4℃过夜；一抗：用豚鼠抗FMDV多克隆抗体(5％脱脂奶以1:500稀释)室温下孵育膜3h；洗膜：用PBST(吐温为1‰)洗膜三次，每次5min；二抗：山羊抗豚鼠Dlight800抗体(5％脱脂奶以1:2000稀释)，室温下避光孵育40min；洗膜：用PBST (吐温为1‰)洗膜三次，每次10min；检测：利用Odyssey的700和800两个荧光通道进行扫描鉴定。
将细胞传代至0.5-1×106/ml时，用重组杆状病毒感染SF9细胞，感染5-7d后收集感染细胞培养上清，于4℃6000rpm离心10min，留取上清和细胞，以备鉴定与纯化。Western blot鉴定见图4，结果显示在感染重组病毒的Sf9细胞细胞中出现81kD、47kD、35kD和24kD的目的条带，分别对应口蹄疫病毒P1蛋白、VP31蛋白、VP0蛋白和VP3/VP2/VP1蛋白，与预期蛋白分子量一致。结果提示，P1蛋白在昆虫细胞中实现了表达，且部分P1在3C蛋白的切割下成功切割为VP31、VP0、VP3/VP2/VP1。
实施例4重组杆状病毒表达口蹄疫VLP的纯化与鉴定
1、中空纤维柱超滤浓缩口蹄疫VLP
收集实施例3感染SF9细胞的培养上清，经0.8um过滤后置于4℃，细胞可储存于-20℃或立即用于纯化。细胞破碎：将收集的细胞称重，加入20倍体积的0.1M NaHCO3(pH8.3)，置于室温裂解30min。12000rpm离心15min@4℃(3次)，收集上清，弃细胞。依次用0.8um,0.45um和0.2um滤膜进行过滤。浓缩：中空纤维柱清洗与平衡：用大量纯水冲洗NaOH浸泡的中空纤维柱(300kDa截流)，然后用PBS(pH7.4)平衡。连接好中空纤维柱、泵和待浓缩液体，形成液体回路循环。接通泵(10ml/min)开始纯化，将原液浓缩所需的体积。用大量PBS(pH7.4)进行清洗(10倍体积)，去除杂蛋白。回收剩余的液体，用0.2um滤器进行过滤，获得目的VLP颗粒。
2、超速离心法纯化口蹄疫VLP
分别配制20％、30％、40％、50％和60％的蔗糖溶液，用1XPBS溶液稀释。依次加入60％、50％、40％、30％和20％蔗糖溶液(各1ml)于超速离心管(Beckman)，然后加入待超离样本。做好标记。25000rpm离心16hr@4℃,离心机为Beckman Optima X-100。依次吸取各离心组分，经SDS-PAGE和western blotting鉴定后，将相应的组分透析除去蔗糖，获得 纯化的样品。
3、柱层析法纯化口蹄疫VLP
FMDV昆虫细胞培养上清经300KDa中空纤维柱30倍浓缩，利用PBS缓冲液，层析介质为Sepharose 6FF柱(XK16×70层析柱，层析介质高度63.5厘米)，流速1毫升/分钟，层析分离VLP和杂蛋白。结果见图5。第二次柱层析采用CaptoQ ImpRes离子交换层析柱，经10mM Tris-HCl pH8.0透析第一柱的VLP峰上样，洗脱液为10mM Tris-HCl pH8.0，1M NaCl。收集穿过峰作为纯化的VLP，结果见图6。
4、纯化后FMD VLP的SDS-PAGE和Western blotting
结果见图7、8。图7为VLP纯化后SDS-PAGE图谱，在组成VLP的VP1和VP0位置具有对应条带，图8进一步用免疫印迹杂交证明组成VLP的蛋白是特异的。
Lowry法检测本发明的获得纯化蛋白终浓度为1800μg/ml，在疫苗配
置上，很容易操作达到注射需要的抗原浓度。
5、电镜观察VLP
120KV透射电子显微镜的样品处理：样品浓度为0.1毫克/毫升，用漂浮法负染色，即取15微升样品液珠，用带支持膜的铜网盖在液珠上2分钟，取下铜网，用滤纸从边缘吸去多余的样品，再把铜网盖在15微升0.5％的醋酸铀染液液珠上2分钟，取下铜网，用滤纸从边缘吸去多余的染液，即可电镜观察。结果见图9，呈现病毒样颗粒，颗粒直径在30nm左右，颗粒完整、规则。
6、攻毒实验
免疫动物：将VLP样品用206佐剂乳化后免疫口蹄疫阴性猪，随机分为对照组3头和试验组7头。免疫后第7、14、21、28天抽取血清，Elisa鉴定口蹄疫抗体的效价并统计发病情况。
(1)免疫后O型LB-ELISA检测抗体情况，见表1。
表1VLP免疫后O型抗体检测
Figure PCTCN2017095376-appb-000002
(2)攻毒情况，结果见表2。
表2对照组动物攻毒实验结果
Figure PCTCN2017095376-appb-000003
在攻毒试验中，7头动物中6头获得完全保护，没有口蹄疫病变发生。只有1头动物即第436号动物四蹄发病，但是对照抗体的检测发现，这头动物抗体水平在7头动物中最高，注射14天后达到720，其它在11-180。这说明检测抗体所用的试剂盒来自于全病毒多克隆抗体，对VLP疫苗特异性不好。但是VLP疫苗抗原能够很好保护猪免于口蹄疫病毒感染，是一个很有效的抗原制备技术。
实施例5采用与实施例2不同方法构建的串联表达FMDV壳蛋白载体
通过启动子SV40下的包含P12A3C基因的单一ORF克隆入pFastBac HT-B构建重组表达质粒。按照VP0(VP2+VP4)—VP3—VP1—2A—3B3—3C的串联方式，将FMDV相关蛋白基因串联置于SV40启动子下表达。通过引物FMDF1和FMDR1进行PCR扩增P12A基因，引物FMDF2和引物FMDR2进行PCR扩增3B3和3C基因。纯化扩增得到的PCR产物，然后用无缝连接试剂盒将它们连接在一起。组成单个启动子的串联表达框。最终得到的序列如SEQ ID NO.9所示。
引物FMDF1：
Figure PCTCN2017095376-appb-000004
引物FMDR1：
Figure PCTCN2017095376-appb-000005
引物FMDF2:
Figure PCTCN2017095376-appb-000006
引物FMDR2:
Figure PCTCN2017095376-appb-000007
参照实施例3、4的方法进行表达鉴定、VLP的纯化与鉴定，最终该串联方法得到的VLP得率约为33毫克/升培养液。
实施例6口蹄疫病毒样颗粒疫苗的制备
实施例4制得的FMDV VLP抗原与Montanide ISA 201VG佐剂的混合乳液的配制方法：1、取等质量的FMDV VLP水相抗原与佐剂。
2、水浴加热至31℃。
3、将装有佐剂的烧杯置于水浴中，搅拌器距离杯底1cm。搅拌速度 350rpm。
4、向佐剂中快速加入水相抗原。搅拌5min。
5、停止搅拌。降温至20度，静置1h。
6、乳液制备完成。在20度下静置一夜后，做质控检测。将口蹄疫病毒样颗粒用不同佐剂吸附后制备成含50-5000μg/ml病毒样颗粒的疫苗
各项指标检测结果：
性状：外观为略带粘滞性乳剂。
剂型：水包油包水型。取一清洁吸管，吸取少量疫苗滴于清洁冷水表面，应呈云雾状扩散。
稳定性：吸取疫苗10mL，加入离心管中，以3000r/min离心15min，离心管底部析出的水相应不超过0.5mL。
工业实用性
本发明提供了一种口蹄疫病毒样颗粒疫苗及其制备方法。通过将密码子优化后的口蹄疫病毒的P12A基因和3C基因串联插入转移载体pFastBac HT-B构建pFastBac HT-B-P12A-3C重组表达质粒，将其转化DH10Bac感受态细胞得到重组Bacmid-DNA，转染sf9悬浮细胞，得到能够表达口蹄疫病毒样颗粒蛋白的悬浮昆虫细胞。将该细胞在无血清、无蛋白培养基培养后收集上清，超滤浓缩、离心，经过分子筛和离子交换柱层析纯化VLP，制备口蹄疫病毒样颗粒疫苗。本发明提供的口蹄疫病毒样颗粒疫苗具有良好的免疫原性、安全性、免疫特性和生物学活性，并可以大规模制备和纯化，可用于制备预防口蹄疫感染的疫苗，对于基因突变导致口蹄疫毒株变异进而流行的新毒株，具有成苗快、不需要病毒减毒、适应宿主细胞等的程序，可以最快阻断病毒流行。不接触病毒本身和模式生物生产的低成本使其具有较好的经济价值和应用前景。
Figure PCTCN2017095376-appb-000008
Figure PCTCN2017095376-appb-000009
Figure PCTCN2017095376-appb-000010
Figure PCTCN2017095376-appb-000011
Figure PCTCN2017095376-appb-000012
Figure PCTCN2017095376-appb-000013
Figure PCTCN2017095376-appb-000014

Claims (10)

  1. 一种重组表达载体，其特征在于，含有口蹄疫病毒P12A基因和3C基因；所述口蹄疫病毒P12A基因如序列表中SEQ ID No.1所示；或由SEQ ID No.1所示核苷酸序列经取代或缺失或插入重复一个或几个核苷酸，得到编码P12A蛋白的氨基酸序列或近似序列；
    所述口蹄疫病毒3C基因其核苷酸序列如SEQ ID No.2所示或由SEQ ID No.2所示核苷酸序列经取代或缺失或插入重复一个或几个核苷酸，得到编码3C蛋白的核苷酸序列或近似序列。
  2. 如权利要求1所述的重组表达载体，其特征在于，通过以下方法制备得到：用Sph I和Hind III对3C基因片段和pFastBac HT-B进行双酶切，酶切后3C基因片段与pFastBac HT-B进行连接转化，得到pFastBac HT-B-3C；用Rsr II和EcoR I对P12A基因片段和pFastBac HT-B-3C进行双酶切，酶切后进行连接转化，构建得到pFastBac HT-B-P12A-3C重组表达质粒。
  3. 含有权利要求1～2任一所述的重组表达载体的宿主细胞，包括但不限于真菌、昆虫、脊椎动物或植物细胞。
  4. 一种口蹄疫病毒样颗粒，其特征在于，通过以下方法制备得到：(1)将权利要求1～2任一所述的重组表达载体转染昆虫细胞，得到能够表达口蹄疫病毒样颗粒蛋白的昆虫细胞；(2)培养该细胞后，收获细胞和上清，对上清进行超滤浓缩，离心，收集上清液，上清液经离子交换层析、疏水层析或分子筛层析，纯化后获得口蹄疫病毒样颗粒。
  5. 如权利要求4所述的口蹄疫病毒样颗粒，其特征在于，所述昆虫细胞为sf9、sf21和High5细胞。
  6. 如权利要求4所述的口蹄疫病毒样颗粒，其特征在于，步骤(2)中，采用截留孔径为200-500nm的中空纤维柱进行培养液澄清，截留孔径选300kDa的超滤浓缩；回收剩余液体进行层析分离VLP。
  7. 权利要求1～2任一所述的重组表达载体或权利要求3所述的宿主 细胞或权利要求4～6任一所述的口蹄疫病毒样颗粒在制备口蹄疫病毒疫苗中的应用。
  8. 权利要求1～2任一所述的重组表达载体或权利要求3所述的宿主细胞或权利要求4～6任一所述的口蹄疫病毒样颗粒在预防动物感染口蹄疫病毒中的应用。
  9. 一种口蹄疫疫苗，其特征在于，含有权利要求4～6任一所述的口蹄疫病毒样颗粒。
  10. 一种制备权利要求9所述疫苗的方法，其特征在于，将口蹄疫病毒样颗粒用不同佐剂吸附后制备成含50-5000μg/ml病毒样颗粒的疫苗。
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