WO2016086576A1 - 一种表达脊髓灰质炎病毒样颗粒蛋白的载体及脊髓灰质炎病毒样颗粒的制备方法 - Google Patents

一种表达脊髓灰质炎病毒样颗粒蛋白的载体及脊髓灰质炎病毒样颗粒的制备方法 Download PDF

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WO2016086576A1
WO2016086576A1 PCT/CN2015/077202 CN2015077202W WO2016086576A1 WO 2016086576 A1 WO2016086576 A1 WO 2016086576A1 CN 2015077202 W CN2015077202 W CN 2015077202W WO 2016086576 A1 WO2016086576 A1 WO 2016086576A1
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poliovirus
vector
virus
type
sequence
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彭涛
许煜华
马书智
王弋
安鸿
尹海滨
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广东华南联合疫苗开发院有限公司
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    • 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
    • A61K39/13—Poliovirus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C12N15/866—Baculoviral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • C12N7/04—Inactivation or attenuation; Producing viral sub-units
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention belongs to the field of biotechnology and biomedicine; relates to a carrier for expressing poliovirus-like granule protein and a preparation method of poliovirus-like particles.
  • Polio is an acute intestinal infectious disease caused by poliomyelitis caused by poliovirus infection. It mainly affects children under the age of five. There is no specific treatment for polio. It is widely spread globally and is extremely harmful. Acute infectious diseases, due to specific pathological changes, and damage to the gray matter cells of the anterior horn of the spinal cord, especially in the gray matter area, it is called polio.
  • the clinical features of muscle paralysis, especially the flaccid paralysis of the body occur mostly in children under 5 years of age, especially infants and young children, so it is also called infantile paralysis, but it is not only harmful to young children, but also among adults. Occurred, mainly hidden infections, only 0.1% to 1% of cases of paralysis. The virus invades the motoneurons of the anterior horn of the spinal cord, causing flaccid muscle paralysis. The condition is different, the light is innocent, and the severe ones involve the life center and die.
  • Poliovirus belongs to the genus Enterovirus of the picornavirus family and contains a single-stranded positive-strand RNA gene.
  • the poliovirus genomic RNA is about 7.5 kb long.
  • the gene component is divided into four parts: a 5' non-coding region, a polyprotein coding region, a 3' non-coding region, and a 3'-end Poly (A) tail.
  • the polyprotein coding region encodes a polyprotein precursor, which is divided into P1, P2 and P3 regions, wherein the P1 region can be hydrolyzed by protease to produce capsid proteins VP1, VP2, VP3 and VP4, and the P2 and P3 regions can be hydrolyzed.
  • the protein 3C produced by hydrolysis of the P3 region performs most of the cleavage function during the processing of the polyprotein precursor, and its precursor 3CD also has 3C activity.
  • Five copies of VP1, VP2, VP3 and VP4 constitute a pentamer, and 12 pentamers constitute an icosahedral nucleocapsid.
  • the main antigenic sites of poliovirus are located on structural proteins VP1, VP2 and VP3.
  • VLPs Virus-like Particles
  • Virus-like Particles (VLPs) vaccine is a new type of subunit vaccine developed in recent years. It is a virus structural protein expressed in vitro that is automatically assembled under specific conditions and is similar in morphology to natural virus. Hollow particles, internal virus-free nucleic acid structure, no infectivity, have the natural spatial conformation of the virus, have strong immunogenicity and biological activity, and therefore have great advantages in the development of new vaccines.
  • VLPs of human hepatitis B virus and human papillomavirus have been successfully developed into vaccines and have been put on the market. As early as 1990, David and Toyohiko in 1989 and Sandra in 1992 successfully obtained polio type I and type III VLPs. Toyohiko et al. also immunized mice with purified type III VLPs to induce neutralizing antibodies in mice. Therefore, research on poliovirus-like particles is the preferred target for the development of poliovirus vaccines.
  • the current strategies for constructing polycistronic vectors mainly include:
  • a plurality of vectors are co-transfected with cells, which are co-expressed with the protein of interest.
  • this method requires simultaneous transfection of multiple vectors into one cell, which has low transfection efficiency, interfering with each other, and unbalanced protein expression.
  • IRES chromosomal entry site
  • the target gene is ligated using a cleavage polypeptide.
  • linker peptides include a 2A sequence, an LP4 sequence, an IRES sequence, an NIa protease and its recognition sequence, and a linker sequence which can be recognized by a host cell protease.
  • the D-x-E-x-N-P-G-P amino acid sequences are collectively referred to as 2A-like sequences.
  • 2A sequence of picornavirus 2A-like sequence of insect virus
  • 2A-like sequence of type C rotavirus 2A-like sequence of type C rotavirus.
  • Porcine teschovirus (PTV, a picornavirus) 2A is a typical self-cleaving polypeptide with a short structure compared to other self-shearing peptides, 22 amino acids in size (GSGATNFSLLKQAGDVEENPGP), shear efficiency High, upstream and downstream gene expression balance is good, making it an ideal tool for constructing polycistronic vectors.
  • the folded 2A causes steric hindrance to the ribosomal peptide group, making the Pro-tRNA amino nucleophilic attack impossible to complete, thus unable to form 2A-tRNA ester bonds, thus the 19th amino acid Shearing occurs between the 20th amino acid and the upstream protein fused to the tail of the 2A polypeptide to form a fusion protein with 2A, while the ribosome can continue to translate the downstream protein to form a complete N-terminal proline.
  • Downstream protein The entire process does not require any protease participation, and the upstream and downstream gene expression is balanced and the shear efficiency is high.
  • Another object of the present invention is to provide a method for preparing poliovirus-like particles.
  • a vector for expressing poliovirus-like granule protein which comprises an expression cassette of the following structure: one of the three genes of poliovirus structural proteins VP0, VP1, VP3 is located downstream of promoter 1; The genes are located downstream of promoter 2 via a 2A sequence; the two promoters initiate expression in the opposite direction;
  • VP0, VP1 and VP3 constitute a complete poliovirus structural protein P1 gene
  • the poliovirus is poliovirus type I virus, poliovirus type II virus or poliovirus type III virus;
  • the 2A sequence is a gene sequence encoding an amino acid sequence comprising D-x-E-x-N-P-G-P.
  • the above-mentioned vector for expressing poliovirus-like granule protein which comprises an expression cassette of the following structure: poliovirus structural protein VP1 gene is located downstream of promoter 1, and constitutes "promoter 1-VP1" Structure; poliovirus structural proteins VP0 and VP3 genes are located downstream of promoter 2 via the 2A sequence, and constitute the structure of "promoter 2-VP3-2A-VP0"; the above two promoters initiate expression in opposite directions;
  • VP0, VP1 and VP3 constitute a complete poliovirus structural protein P1 gene
  • the poliovirus is poliovirus type I virus, poliovirus type II virus or poliovirus type III virus;
  • the 2A sequence is a gene sequence encoding an amino acid sequence comprising D-x-E-x-N-P-G-P.
  • a method for preparing a vector for expressing poliovirus-like granule protein comprises the following steps:
  • the primers are designed to clone any one of VP0, VP1 and VP3 into the skeleton vector, which is located downstream of one promoter of the backbone vector, and then connect the other two genes through the 2A sequence.
  • the ligated fragment is cloned downstream of another promoter of the same backbone vector; the resulting recombinant vector is the vector expressing the poliovirus-like granule protein.
  • the above host cell is Spodoptera frugiperda Spodoptera frugiperda cell Sf9, Saccharomyces cerevisiae or mammalian cell.
  • the above skeleton carrier selects a corresponding skeleton carrier depending on the type of host cell.
  • the above-described backbone vector is pFastBac TM -Dual vector, pESC URA vector, pVIVO2-mcs pBudCE4.1 vector or carrier.
  • the optimized P1 gene sequence is set forth in SEQ ID NO:1;
  • the optimized P1 gene sequence is set forth in SEQ ID NO: 2;
  • the optimized P1 gene sequence is set forth in SEQ ID NO: 3.
  • a method for preparing poliovirus-like particles comprising the steps of:
  • step 2) If the recombinant poliovirus baculovirus is obtained in step 1), the recombinant poliovirus baculovirus is infected into the host cell, and the poliovirus-like particles can be obtained after culture.
  • the method for preparing the above poliovirus-like particle comprises the following steps:
  • the host cell is an insect cell Sf9;
  • the virus-like granule protein is expressed in the host cell, and the expressed virus-like granule protein is assembled into a virus-like particle, secreted into the host cell, and then the cell culture is carried out.
  • the supernatant is centrifuged to remove cell debris, and the supernatant is concentrated by a pellicon membrane package, and then purified by sucrose to obtain poliovirus-like particles, which can be used as an immune sample.
  • the technical scheme provided by the invention adopts a vector to express multiple genes, avoids the low efficiency of virus-like particle VLPs due to the low efficiency of 3C/3CD cleavage of P1, and avoids the influence of the upstream promoter on the expression efficiency of the downstream promoter. Protein expression is unbalanced, which affects the assembly of VLPs.
  • the present invention achieves tandem expression of multiple genes under a single promoter and successfully packages virus-like particles.
  • the preparation of the virus-like particles of the invention can induce high levels of antibody immune response and protective effects, and has the advantages of simple operation, high expression amount, good immunogenicity, and the like, and therefore the invention has broad application prospects in the field of vaccine development.
  • Figure 1 is a schematic diagram showing the construction of various vectors expressing poliovirus-like granule proteins
  • Figure 2 shows the immunofluorescence analysis of type I, II, and III recombinant poliovirus baculovirus; 1 is Bac-I-V3, 2 is Bac-II-V3, 3 is Bac-III-V3, and 4 is a negative control;
  • Figure 3 shows the expression of recombinant poliovirus baculovirus expression samples of type I, II and III by Western blot analysis; 1. Bac-I-V3 supernatant, 2, Bac-I-V3 cells, 3, Bac-II -V3 supernatant, 4, Bac-II-V3 cells, 5, Bac-III-V3 supernatant, 6, Bac-III-V3 cells;
  • Figure 4 shows the sucrose gradient Western blot analysis of type I recombinant virus expression samples; 1-10 is the analysis of 10 components collected from top to bottom after 10-50% sucrose gradient overshoot; The sucrose concentrations were 33.2% and 38%, respectively;
  • Figure 5 is a negative staining electron micrograph of poliovirus-like particle VLPs; A, B, and C are polio type I, type II, and type III virus-like particles, respectively.
  • Figure 6 is a graph showing the analysis of neutralizing antibodies induced by polio type I, type II, and type III virus-like particles;
  • I VLPs-Sabin I curve of sera type I VLPs immune serum neutralizing Sabin I;
  • II VLPs-Sabin II II polio Sabin type VLPs serum and curve II;
  • III VLPs-Sabin III polio type III Sabin VLP s serum and a curve III;
  • IPV-Sabin I IPV in serum and Sabin Curve of I;
  • IPV-Sabin II curve of neutralizing Sabin II in IPV immune serum;
  • IPV-Sabin III curve of neutralizing Sabin III in IPV immune serum.
  • a vector for expressing poliovirus-like granule protein which comprises an expression cassette of the following structure: one of the three genes of poliovirus structural proteins VPO, VP1, VP3 is located downstream of promoter 1; The genes are located downstream of promoter 2 via a 2A sequence; the two promoters initiate expression in opposite directions; wherein three genes of VP0, VP1, and VP3 constitute a complete poliovirus structural protein P1 gene; The inflammatory virus is a poliovirus type I virus, a poliovirus type II virus or a poliovirus type III virus; the 2A sequence is a gene sequence encoding an amino acid sequence comprising DxExNPGP.
  • the above vector for expressing poliovirus-like granule protein comprises an expression cassette of the following structure: poliovirus structural protein VP1 gene is located downstream of promoter 1, and constitutes "promoter 1-VP1" Structure; poliovirus structural proteins VP0 and VP3 genes are located downstream of promoter 2 via a 2A sequence, and constitute the structure of "promoter 2-VP3-2A-VP0"; the above two promoters initiate expression in opposite directions; The three genes of VP0, VP1, and VP3 constitute the complete poliovirus structural protein P1 gene; the poliovirus is polio Type I virus, poliovirus type II virus or poliovirus type III virus; the 2A sequence is a gene sequence encoding an amino acid sequence comprising D-x-E-x-N-P-G-P.
  • a method for preparing a vector for expressing poliovirus-like granule protein comprises the following steps:
  • the primers are designed to clone any one of VP0, VP1 and VP3 into the skeleton vector, which is located downstream of one promoter of the backbone vector, and then connect the other two genes through the 2A sequence.
  • the ligated fragment is cloned downstream of another promoter of the same backbone vector; the resulting recombinant vector is the vector expressing the poliovirus-like granule protein.
  • the above host cell is Spodoptera frugiperda Spodoptera frugiperda cell Sf9, Saccharomyces cerevisiae or mammalian cell.
  • the above skeleton carrier selects a corresponding skeleton carrier depending on the type of host cell.
  • said vector is pFastBac TM -Dual backbone vector, pESC URA vector, pVIVO2-mcs pBudCE4.1 vector or carrier.
  • the optimized P1 gene sequence described above is:
  • the optimized P1 gene sequence is set forth in SEQ ID NO:1;
  • the optimized P1 gene sequence is set forth in SEQ ID NO: 2;
  • the optimized P1 gene sequence is set forth in SEQ ID NO: 3.
  • a method for preparing poliovirus-like particles comprising the steps of:
  • step 2) If the recombinant poliovirus baculovirus is obtained in step 1), the recombinant poliovirus baculovirus is infected into the host cell, and the poliovirus-like particles can be obtained after being cultured;
  • the corresponding vector obtained when yeast or mammalian cells are used as a host, the corresponding vector obtained is transfected into a host cell, and poliovirus-like particles can be directly obtained; when the insect cell is hosted, the corresponding vector obtained is transfected into the host. After the cells, the recombinant poliovirus baculovirus is obtained, and the virus is used to infect the host cells, and the poliovirus-like particles are obtained after the culture.
  • the preparation method of the above poliovirus-like particle comprises the following steps:
  • the host cell is an insect cell Sf9;
  • the virus-like granule protein is expressed in the host cell, and the expressed virus-like granule protein is assembled into a virus-like particle, secreted into the host cell, and then the cell culture is carried out.
  • the supernatant is centrifuged to remove cell debris, and the supernatant is concentrated by a pellicon membrane package, and then purified by sucrose to obtain poliovirus-like particles, which can be used as an immune sample.
  • IPV rabbit anti-antibody (antibody prepared by our company, which is obtained by immunizing rabbits with Pasteur's IPV vaccine);
  • Example 1 A vector for expressing poliovirus-like granule protein
  • the VP1 sequence of the poliovirus type I virus is placed under the P 10 promoter of the pFastBac Dual plasmid, and the 2A sequence gene of the picornavirus family (ie, the encoded amino acid sequence is the base sequence of GSGATNFSLLKQAGDVEENPGP) is ligated to polio.
  • the VP3 gene of type I virus is ligated to the VP0 gene of poliovirus type I at the other end and placed under the P polh promoter of pFastBac Dual plasmid to obtain the shuttle vector pFBD-I VP1-VP3-2A-VP0, which expresses the spinal cord.
  • a vector for poliovirus type I virus-like granule proteins are used to obtain the shuttle vector pFBD-I VP1-VP3-2A-VP0.
  • Example 2 A vector for expressing poliovirus-like granule protein
  • the VP1 sequence of the poliovirus type II virus is placed under the P 10 promoter of the pFastBac Dual plasmid, and the 2A sequence gene of the picornavirus family (ie, the encoded amino acid sequence is the base sequence of GSGATNFSLLKQAGDVEENPGP) is ligated to polio.
  • the VP3 gene of type II virus is ligated to the VP0 gene of poliovirus type II, and placed under the P polh promoter of pFastBac Dual plasmid to obtain the shuttle vector pFBD-II VP1-VP3-2A-VP0, which expresses the spinal cord.
  • a vector for poliovirus type II virus-like granule proteins are used to obtain the shuttle vector pFBD-II VP1-VP3-2A-VP0.
  • Example 3 A vector for expressing poliovirus-like granule protein
  • the VP1 sequence of the poliovirus type III virus is placed under the P 10 promoter of the pFastBac Dual plasmid, and the 2A sequence gene of the picornavirus family (ie, the encoded amino acid sequence is the base sequence of GSGATNFSLLKQAGDVEENPGP) is ligated to polio.
  • the VP3 gene of the type III virus is ligated to the VP0 gene of the poliovirus type III, and placed under the P polh promoter of the pFastBac Dual plasmid to obtain the shuttle vector pFBD-III VP1-VP3-2A-VP0, which expresses the spinal cord.
  • a vector for poliovirus type III virus-like granule proteins are used to obtain the shuttle vector pFBD-III VP1-VP3-2A-VP0.
  • the method for preparing a poliovirus-like granule protein-expressing vector described in Example 1 comprises the following steps:
  • PV type I poliovirus type I virus
  • Sf9 host cell Spodoptera frugiperda Spodoptera frugiperda
  • PUC57 was the vector
  • Xho I, Kpn I was used.
  • the PV type I P1 sequence (SEQ ID NO: 1) was synthesized at the restriction site and cloned into the PUC57 vector to obtain the recombinant vector PUC57-I-P1.
  • the VP1 fragment was amplified with primers I VP1-Xho I F and I VP1-Kpn I R (see Table 1) using PUC57-I-P1 as a template, and ligated into pFastBac TM -Dual skeleton vector.
  • the recombinant vector pFBD-I-VP1 was obtained, wherein IVP1 was located downstream of the promoter Pp10.
  • VP3 and VP0 were ligated through the 2A sequence, ie, I VP3- was amplified using primers I VP3-2A-VP0-XbaI F and I VP3-2A-VP0R (see Table 1) using PUC57-I-P1 as a template.
  • I 2A2-VP0 was amplified with primers I VP3-2A-VP0F1, II VP3-2A-VP0F2, II VP3-2A-VP0F3 and I VP3-2A-VP0-Hind III R (see Table 1), I VP3-2A1 and I 2A2-VP0 were used as templates, and PCR amplification was performed with primers I VP3-2A-VP0-Xba I F and I VP3-2A-VP0-Hind III R (see Table 1) to obtain I VP3-2A- VP0 fragment.
  • the VP3-2A-VP0 fragment was ligated into the recombinant vector pFBD-I-VP1 to obtain a recombinant vector pFBD-I-VP1-VP3-2A-VP0, a vector expressing poliovirus type I virus-like granule protein, wherein VP3-2A-VP0 is located downstream of the promoter Pph.
  • Table 2 The construction of related vectors is shown in Table 2, and the schematic diagram of the construction is shown in Fig. 1.
  • the codonar source was optimized according to the preference of the host cell Spodoptera frugiperda Spodoptera frugiperda (Sf9) cell line, PUC57 was used as vector, and XhoI, KpnI was used as the restriction enzyme.
  • the PV II type P1 sequence (SEQ ID NO: 2) was synthesized at the site and cloned into the PUC57 vector to obtain the recombinant vector PUC57-II-P1.
  • Polio Type II (MEF-1 strains) sequence Pl (SEQ ID NO: 2) in VP0 (SEQ ID NO :7), VP1 (SEQ ID NO: 8), VP3 (SEQ ID NO: 9) sequences, designed to give corresponding primers, as shown in Table 3.
  • the VP1 fragment was amplified with primers II VP1-Xho I F and II VP1-Kpn I R (see Table 3) using PUC57-II-P1 as a template, and ligated into pFastBac TM -Dual skeleton vector.
  • the recombinant vector pFBD-II-VP1 was obtained, wherein VP1 was located downstream of the promoter Pp10.
  • VP3 and VP0 were ligated by 2A sequence, ie, using PUC57-II-P1 as a template, primers II VP3-2A-VP0-XbaI F and II VP3-2A-VP0R (see Table 3) were used to amplify II VP3- 2A1, amplify II 2A2-VP0 with primers II VP3-2A-VP0F1, II VP3-2A-VP0F2, II VP3-2A-VP0F3 and II VP3-2A-VP0-Hind III R (see Table 3), and then II VP3-2A1 and II 2A2-VP0 were used as templates, and PCR amplification was performed with primers II VP3-2A-VP0-Xba I F and II VP3-2A-VP0-Hind III R to obtain II VP3-2A-VP0 fragment.
  • the VP3-2A-VP0 fragment was ligated into the recombinant vector pFBD-II-VP1 to obtain a recombinant vector pFBD-II-VP1-VP3-2A-VP0, which is a vector expressing poliovirus type II virus-like granule protein, wherein II VP3-2A-VP0 is located downstream of the promoter Pph.
  • the construction of related vectors is shown in Table 4, and the schematic diagram of the construction is shown in Fig. 1.
  • the method for preparing a poliovirus-like granule protein-expressing vector described in Example 3 comprises the following steps:
  • the codon source was optimized according to the preference of the host cell Spodoptera frugiperda Spodoptera frugiperda (Sf9) cell line, PUC57 was used as the vector, and XhoI and KpnI were used as the enzyme digestion.
  • the PV III type P1 sequence (SEQ ID NO: 3) was synthesized at the site and cloned into the PUC57 vector to obtain the recombinant vector PUC57-III-P1.
  • the III VP1 fragment was amplified with primers III VP1-Xho I F and III VP1-Kpn I R (see Table 5) using PUC57-I-P1 as a template, and ligated into pFastBac TM -Dual skeleton vector.
  • the recombinant vector pFBD-III-VP1 was obtained, wherein VP1 was located downstream of the promoter Pp10.
  • VP3 and VP0 were ligated by 2A sequence, ie, using PUC57-I-P1 as a template, primers III VP3-2A-VP0-XbaI F and III VP3-2A-VP0R (see Table 5) were used to amplify III VP3- 2A1, amplified with primers III VP3-2A-VP0F1, II VP3-2A-VP0F2, II VP3-2A-VP0F3 and III VP3-2A-VP0-Hind III R (see Table 5) to obtain III 2A2-VP0, and then III VP3-2A1 and III 2A2-VP0 were used as templates, and PCR amplification was performed with primers III VP3-2A-VP0-Xba I F and III VP3-2A-VP0-Hind III R to obtain a III VP3-2A-VP0 fragment.
  • the VP3-2A-VP0 fragment was ligated into the recombinant vector pFBD-III-VP1 to obtain a recombinant vector pFBD-III-VP1-VP3-2A-VP0, which is a vector expressing poliovirus type III virus-like granule protein, wherein III VP3-2A-VP0 is located downstream of the promoter Pph.
  • the construction of the relevant vector is shown in Table 6, and the schematic diagram of the construction is shown in Fig. 1.
  • the recombinant baculovirus vector obtained in the previous step was transfected into Sf9 cells to obtain the corresponding first-generation recombinant baculovirus, namely recombinant poliovirus baculovirus, named Bac-I-VP1-VP3-2A-VP0. -V1 (abbreviated as Bac-I), Bac-II-VP1-VP3-2A-VP0-V1 (abbreviated as Bac-II) and Bac-III-VP1-VP3-2A-VP0-V1 (abbreviated as Bac-III) Each virus was continuously amplified by 0.1 MOI, and the third generation viruses Bac-I-V3, Bac-II-V3 and Bac-III-V3 were obtained.
  • Bac-I-VP1-VP3-2A-VP0. -V1 abbreviated as Bac-I
  • Bac-III-VP1-VP3-2A-VP0-V1 abbreviated as Bac-III
  • Bf-I-V3, Bac-II-V3 and Bac-III-V3 obtained above were infected with Sf9 adherent cells at 1 MOI according to Invitrogen instructions. After 3 days, the supernatant was discarded and fixed with formaldehyde to IPV (inactivated spinal cord). Gray vaccination vaccine) rabbit anti-monoclonal antibody, Donkey Anti-Rabbit Alexa Fluor 488 for immunofluorescence analysis of secondary antibodies, the results shown in Figure 2, Bac-I, Bac-II and Bac-III infected SF9 cells are bright Green fluorescence, no bright green fluorescence was detected in the blank group. It is indicated that they all express the corresponding protein of interest (ie, VP0, VP1 and VP3 proteins) in Sf9 cells.
  • the Bac-I-V3, Bac-II-V3 and Bac-III-V3 recombinant baculoviruses prepared above were prepared according to the Invitrogen instructions.
  • the sf9 suspension cells were infected with 1MOI, and cultured for 6 days with Excell-420 as suspension medium. After the cells were completely lysed, the cells and supernatant were collected, and the cells and supernatant samples were taken for 12% SDS-PAGE electrophoresis, and PVDF was transferred.
  • VP1 (35KDa) was not detected in the cells after 7 days of expression of all recombinant baculovirus, and a large amount of VP1 (35KDa) protein was detected in the supernatant, presumably in the expression system.
  • the released VP1 protein may form mature virus-like particles or secrete extracellularly.
  • a method for preparing poliovirus-like particles comprising the steps of:
  • the suspension was added to the upper layer of 10%-50% sucrose gradient, centrifuged at ultra-high speed for 4 h, and 8 sucrose gradients (1 ml/per gradient) were collected for Western blot analysis and collected according to Western blot results (see Figure 4).
  • the enriched gradient was de-sugared to concentrate the sample.
  • the polio type I, type II, and type III virus-like particles obtained after purification in Example 8 were observed by electron microscopy. Electron microscopy results showed that high-concentration, structurally intact virus-like particles appeared in the field of view with a diameter of 25-30 nm (Fig. 5).
  • Figure 5A and Figure 5C show polio type I and type III virus-like particles, similar to other reported polio type I and type III virus-like particles, and Figure 5B shows polio type II virus-like particles (Fig. 5B). There are no reports of poliovirus type II granules at present). This indicates that the method of the present invention is capable of efficiently producing structurally intact poliovirus-like particles.
  • the polio type I, type II, and type III virus-like particles prepared in Example 8 were diluted and mixed with 1/10 volume of AL(OH) 3 adjuvant (1000 mg/ml) to make a protein concentration of 20 ⁇ g/ In ml, six-week-old BALB/c mothers were immunized three times at 0, 2, and 4 weeks with the above 10 ⁇ g of purified VLP, and each was immunized with 10 ⁇ g each.
  • the IPV inactivated vaccine purchased from Pasteur was set as a positive control and the samely treated uninfected Sf9 cells were used as a negative control. After 5 weeks, blood was collected from the mouse eyeballs, and serum was collected and stored in -80 minutes.
  • Serum samples of each group of mice were inactivated for 56 min, and diluted with a serum-free M199 medium.
  • Type I VLPs immune serum neutralized Sabin I, type II VLPs immune serum neutralized SabinII, type III VLPs neutralized Sabin III, IPV immune serum and negative control serum neutralized SabinI, SabinII and Sabin III, respectively (SabinI, SabinII and SabinIII The polio standard attenuated strain, from the Guangdong Provincial Center for Disease Control, and the Sabin I, Sabin II, and Sabin III attenuated strains were diluted to 100 pfu/100 ⁇ l with M199 medium. 100 ⁇ l of each of the diluted virus solution and the sample was mixed and uniformly placed in a cell incubator at 37 ° C for 2 hours.
  • the Vero cell culture supernatant was aspirated, and 200 ⁇ l of the mixture was added to a 6-well Vero cell, positive and negative controls were added, and incubation was continued at 37 ° C for 2 h. 4 ml of 1% methylcellulose was added to each well, placed in a CO 2 incubator for 4 days, and the medium was removed, and crystal violet staining was performed to count the plaque. The serum dilution which reduced the number of plaques by 50% was determined as serum. The neutralization titer and the plaque reduction rate are calculated as: [(negative control plaque number - sample plaque number) / negative control plaque number] ⁇ 100%.
  • the poliovirus type I virus-like mice prepared by the present invention can induce a neutralization titer of 1:128 or more, and the IPV inactivated vaccine neutralizes the Sabin I effect.
  • the price is about 1:512; poliovirus type II virus-immunized mice can induce a neutralizing titer of 1:256, and the neutralizing titer of IPV inactivated vaccine is slightly higher than 1:512; poliovirus type III virus
  • the granule-immunized mice can induce a neutralizing titer higher than 1:512; the neutralizing titer of the IPV inactivated vaccine is between 1: (1024 and 2048), although the virus-like particles prepared by the present invention are more than Pasteur's
  • the neutralizing potency of the IPV inactivated vaccine is low, but the virus-like particles prepared by the invention have a structure similar to that of the natural virus particles without viral nucleic acid, cannot be autonomously replicated, and have better safety, while the cultivation of the wild strain requires strict Experimental conditions, the possibility of virulence recovery in attenuated strains.
  • the virus-like particles prepared by the present invention are still only preliminary purification, and the purity is different from that of the commercially
  • the above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention. All of them fall within the protection scope of the present invention.
  • the host cells in the above embodiments are changed into yeast cells or mammalian cells, etc., so that the corresponding skeleton vectors are selected according to the host cells, and expression similar to the above experimental examples is performed.
  • the construction of poliovirus-like granule protein vector, as well as the preparation of poliovirus-like particles, are all within the scope of protection claimed in the present invention.

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Abstract

一种表达脊髓灰质炎病毒样颗粒蛋白的载体及脊髓灰质炎病毒样颗粒的制备方法，该载体含有以下结构的表达框：脊髓灰质炎病毒结构蛋白VP0、VP1、VP3三个基因中的任一个基因位于启动子1下游，另外两个基因之间通过2A序列连接至启动子2下游，2个启动子启动表达的方向相反。脊髓灰质炎病毒样颗粒的制备方法是将该载体转染相应的宿主细胞，培养后可获得病毒样颗粒或者重组杆状病毒，如获得的是重组杆状病毒，再将该病毒感染宿主细胞，可获得病毒样颗粒。

Description

一种表达脊髓灰质炎病毒样颗粒蛋白的载体及脊髓灰质炎病毒样颗粒的制备方法 技术领域
本发明属于生物技术和生物医药领域；涉及一种表达脊髓灰质炎病毒样颗粒蛋白的载体及脊髓灰质炎病毒样颗粒的制备方法。
背景技术
脊髓灰质炎是由脊髓灰质炎病毒感染引起的以肢体麻痹为主的急性肠道传染病，主要影响五岁以下的儿童，脊髓灰质炎没有特效治疗方法，是全球性广泛传播的、危害极大的急性传染病，由于具有特异的病理变化，及脊髓前角灰白质细胞的损害，尤其在灰质区，故称脊髓灰质炎。临床特征为肌肉麻痹，特别是机体的弛缓性麻痹，多发生在5岁以下儿童，尤其是婴幼儿，故亦称小儿麻痹症(infantile paralysis)，但其并非仅仅侵害幼儿，成年人中亦多有发生，主要是隐性感染，发生麻痹病例只占0.1％-1％。病毒侵犯脊髓前角运动神经元，造成弛缓性肌肉麻痹，病情轻重不一，轻者无瘫痪出现，严重者累及生命中枢而死亡。
脊髓灰质炎病毒(Poliovirus，PV)属于小RNA病毒科肠道病毒属，内含单股正链RNA基因，脊髓灰质炎病毒基因组RNA长约7.5kb。基因组分为四个部分：5’端非编码区、多聚蛋白编码区、3’端非编码区和3’端Poly(A)尾。其中多聚蛋白编码区编码产生一个多聚蛋白前体，分为P1、P2和P3区，其中P1区可经蛋白酶水解产生衣壳蛋白VP1、VP2、VP3和VP4，P2和P3区则可水解产生蛋白酶、RNA复制酶及用于识别细胞、调节基因的其他蛋白。P3区水解产生的蛋白3C在多聚蛋白前体加工过程中完成绝大多数裂解功能，其前体3CD也有3C的活性。5个拷贝的VP1、VP2、VP3和VP4构成了五聚体，12个五聚体构成二十面体核壳，脊髓灰质炎病毒主要抗原位点位于结构蛋白VP1，VP2和VP3上。脊髓灰质炎病毒有三个血清型，即I型、II型、III型，各型之间无交叉免疫反应。
脊髓灰质炎没有特效治疗方法，只能疫苗预防。目前有上市的有两种脊髓灰质炎疫苗，口服减毒活疫苗(OPV)和灭活疫苗(IPV)。在我国，脊髓灰质炎疫苗属于国家免疫规划，由于成本问题，还主要使用OPV疫苗，但是由于口服炎减毒活疫苗可能有相关麻痹型脊灰(VAPP)和疫苗衍生脊髓灰质炎病毒(VDPV)的风险，VAPP年发生率为每100万发生0.14例，只要继续使用口服脊髓灰质炎疫苗，就不能根除脊灰，世界上很多国家使用巴斯德生产的IPV疫苗，但是IPV生产需要严格的实验条件，成本控制是很大问题，且IPV疫苗中可以检测到猿猴病毒SV40，可以使培养的细胞发生转化，并可导致动物产生肿瘤，因此需要开发新型的安 全的脊髓灰质炎疫苗。
病毒样颗粒(Virus-like Particles，VLPs)疫苗是近年来发展起来的一种新型亚单位疫苗，它是体外表达的病毒结构蛋白在特定的条件下自动装配成的在形态上类似于天然病毒的空心颗粒，内部无病毒核酸结构，没有感染性，具有病毒的天然空间构象，具有很强的免疫原性和生物学活性，因此在新型疫苗开发方面有巨大优势。目前已有人乙型肝炎病毒和人乳头瘤病毒的VLPs已经被成功研制成疫苗且已投放市场。早在1990年David和1989年Toyohiko、1992年Sandra分别成功获得了脊灰I型和III型VLPs，Toyohiko等还用纯化得到的III型VLPs免疫小鼠，能够诱导小鼠产生中和抗体。因此，针对脊髓灰质炎病毒样颗粒研究是发展脊髓灰质炎病毒疫苗的首选目标。
由于形成该类病毒VLP需要多个蛋白参与，需要构建多顺反子载体才能满足该目的。目前构建多顺反子载体的策略主要包括：
(1)多个载体共同转染细胞，是目的蛋白共表达。但该方法需要同时转染多个载体到一个细胞中，其转染效率低，载体之间互相干扰，蛋白表达不平衡。
(2)构建多启动子(multiple promoters)表达载体。通过把多个基因分别连接不同类型的启动子下，通过各个启动子进行控制表达。但是由于不同的启动子的启动效率的差异，也会造成蛋白表达不均衡，蛋白相互作用距离较远。同时各个启动子元件较大，如果使用多个启动子就会加重载体的负担，影响转染和表达效率。
(3)利用IRES系统构建多顺反子表达载体。利用内核糖体进入位点(IRES)连接多个目的基因，构建融合蛋白表达载体，实现在一个载体上多个基因共表达。其原理是在翻译蛋白的过程中不依赖RNA帽子结构，能够独立的募集核糖体，进而翻译下游基因，各个蛋白独立表达。该系统存在以下缺陷：(1)IRES自身结构较大(约为0.5kb)，其应用常受到载体容量的限制，同时会增加载体的负担，导致转染效率降低。(2)IRES介导的上下游基因表达不平衡，通常下游基因的表达量仅是上游的20％到50％。所以在构建多顺反子载体系统中，IRES并不是理想的连接元件。
(4)利用剪切多肽连接目的基因。常用的连接肽有2A序列、LP4序列、IRES序列、NIa蛋白酶及其识别序列及可以被宿主细胞蛋白酶识别的连接序列等。在不同种类的病毒中，具有D-x-E-x-N-P-G-P氨基酸序列统称为2A样序列。例如：小核糖核酸病毒的2A序列、昆虫病毒的2A样序列；C型轮状病毒的2A样序列。猪捷申病毒(Porcine teschovirus，PTV，一种小核糖核酸病毒)2A是自剪切多肽的典型，与其他自剪切肽相比具有结构短，大小为22个氨基酸(GSGATNFSLLKQAGDVEENPGP)，剪切效率高，上下游基因表达平衡性好，使其成为构建多顺反子载体的理想工具之一。
在真核生物体内翻译的过程中，折叠后的2A对核糖体肽基造成空间位阻，使Pro-tRNA氨基亲核进攻无法完成，从而无法形成2A-tRNA酯键，因此在第19个氨基酸和第20个氨基酸之间会发生剪切作用，释放出融合了2A多肽尾巴的上游蛋白，形成与2A的融合蛋白，同时核糖体能继续翻译下游蛋白，形成N端带有一个脯氨酸的完整下游蛋白。整个过程不需要任何蛋白酶参与，上下游基因表达平衡，剪切效率高。
发明内容
本发明的目的在于提供一种表达脊髓灰质炎病毒样颗粒蛋白的载体。
本发明的另一目的在于提供一种及脊髓灰质炎病毒样颗粒的制备方法。
本发明所采取的技术方案是：
一种表达脊髓灰质炎病毒样颗粒蛋白的载体，该载体中含有以下结构的表达框：脊髓灰质炎病毒结构蛋白VP0、VP1、VP3三个基因中任一个基因位于启动子1下游；另外两个基因之间通过2A序列连接位于启动子2下游；2个启动子启动表达的方向相反；
其中，VP0、VP1、VP3的三个基因组成完整的脊髓灰质炎病毒结构蛋白P1基因；
所述脊髓灰质炎病毒为脊髓灰质炎I型病毒、脊髓灰质炎II型病毒或脊髓灰质炎III型病毒；
所述2A序列为编码含有D-x-E-x-N-P-G-P氨基酸序列的基因序列。
进一步的，上述一种表达脊髓灰质炎病毒样颗粒蛋白的载体，该载体中含有以下结构的表达框：脊髓灰质炎病毒结构蛋白VP1基因位于启动子1下游，构成“启动子1-VP1”的结构；脊髓灰质炎病毒结构蛋白VP0和VP3基因通过2A序列连接位于启动子2下游，构成“启动子2-VP3-2A-VP0”的结构；上述2个启动子启动表达的方向相反；
其中，VP0、VP1、VP3的三个基因组成完整的脊髓灰质炎病毒结构蛋白P1基因；
所述脊髓灰质炎病毒为脊髓灰质炎I型病毒、脊髓灰质炎II型病毒或脊髓灰质炎III型病毒；
所述2A序列为编码含有D-x-E-x-N-P-G-P氨基酸序列的基因序列。
一种表达脊髓灰质炎病毒样颗粒蛋白的载体的制备方法，包括以下步骤：
1)根据脊髓灰质炎病毒的P1基因序列，根据宿主细胞的偏好性进行密码子优化，合成优化的P1基因序列；
2)根据优化的P1基因序列，设计引物将VP0、VP1、VP3中任一个基因先克隆至骨架载体中，位于骨架载体的一个启动子的下游，再将另2个基因通过2A序列进行连接，并将连接好的片段克隆至同一骨架载体的另一个启动子的下游；所得重组载体即为表达脊髓灰质炎病毒样颗粒蛋白的载体。
进一步的，上述宿主细胞为Spodoptera frugiperda草地夜蛾细胞Sf9、酿酒酵母或哺乳细胞。
进一步的，上述骨架载体根据宿主细胞的类型选择相应的骨架载体。
进一步的，上述骨架载体为pFastBacTM-Dual载体、pESC URA载体、pVIVO2-mcs载体或pBudCE4.1载体。
进一步的，上述优化的P1基因序列为：
若脊髓灰质炎病毒为I型，优化的P1基因序列如SEQ ID NO：1所示；
若脊髓灰质炎病毒为II型，优化的P1基因序列如SEQ ID NO：2所示；
若脊髓灰质炎病毒为III型，优化的P1基因序列如SEQ ID NO：3所示。
一种脊髓灰质炎病毒样颗粒的制备方法，包括以下步骤：
1)将上述所述的表达脊髓灰质炎病毒样颗粒蛋白的载体转染相应的宿主细胞，培养后可获得脊髓灰质炎病毒样颗粒或者重组脊髓灰质炎杆状病毒；
2)若步骤1)中获得的是重组脊髓灰质炎杆状病毒，再将重组脊髓灰质炎杆状病毒感染宿主细胞，经培养后即可获得脊髓灰质炎病毒样颗粒。
进一步的，上述一种脊髓灰质炎病毒样颗粒的制备方法，包括以下步骤：
1)将表达脊髓灰质炎病毒样颗粒蛋白的载体转化大肠杆菌DH10Bac感受态细胞，培养后提取重组大肠杆菌DH10Bac的质粒，获得重组杆状病毒载体；
2)将上一步获得的重组杆状病毒载体转染宿主细胞，培养后得到包含编码病毒样颗粒蛋白基因的重组杆状病毒；
3)将上一步获得的重组杆状病毒感染宿主细胞，培养后获得病毒样颗粒，即为脊髓灰质炎病毒样颗粒；
其中，所述的宿主细胞为昆虫细胞Sf9；
在上述步骤3)中将重组杆状病毒感染宿主细胞后，在宿主细胞中表达病毒样颗粒蛋白，所表达的病毒样颗粒蛋白组装成病毒样颗粒，并分泌到宿主细胞外，然后将细胞培养上清初步离心去除细胞碎片，再将上清液经pellicon膜包进行浓缩，再经蔗糖纯化，即可获得脊髓灰质炎病毒样颗粒，可作为免疫样品。
本发明的有益效果是：
1、本发明提供的技术方案采用一个载体表达多个基因，避免由于3C/3CD切割P1效率不高导致病毒样颗粒VLPs组装效率不高，避免上游启动子影响下游启动子的表达效率导致的各个蛋白表达不平衡，从而影响VLPs组装。
2、本发明实现了单一启动子下多个基因的串联表达，并成功包装出病毒样颗粒。
3、本发明制备病毒样颗粒能够诱导高水平的抗体免疫反应和保护效果，同时具有操作简单、表达量高、免疫原性好等优点，因此本发明在疫苗开发领域具有广阔的应用前景。
附图说明
图1为各类表达脊髓灰质炎病毒样颗粒蛋白的载体的构建示意图；
图2为I、II、III型重组脊髓灰质炎杆状病毒表达免疫荧光分析；1为Bac-I-V3，2为Bac-II-V3，3为Bac-III-V3，4为阴性对照；
图3为I、II、III型重组脊髓灰质炎杆状病毒表达样品细胞和上清Western blot分析；1、Bac-I-V3上清，2、Bac-I-V3细胞，3、Bac-II-V3上清，4、Bac-II-V3细胞，5、Bac-III-V3上清，6、Bac-III-V3细胞；
图4为I型重组病毒表达样品蔗糖梯度Western blot分析；1-10为在10-50％的蔗糖梯度超离后，从上到下收集的10个组分进行分析；其中6、7组分处的蔗糖浓度分别为33.2％和38％；
图5为脊髓灰质炎病毒样颗粒VLPs负染电镜图；A、B、C分别为脊髓灰质炎I型、II型、III型病毒样颗粒，
图6为脊髓灰质炎I型、II型、III型病毒样颗粒诱导中和抗体的分析图；I VLPs-Sabin I：脊髓灰质炎I型VLPs免疫血清中和Sabin I的曲线；II VLPs-Sabin II：脊髓灰质炎II型VLPs免疫血清中和Sabin II的曲线；III VLPs-Sabin III：脊髓灰质炎III型VLPs免疫血清中和Sabin III的曲线；IPV-Sabin I：IPV免疫血清中和Sabin I的曲线；IPV-Sabin II：IPV免疫血清中和Sabin II的曲线；IPV-Sabin III：IPV免疫血清中和Sabin III的曲线。
具体实施方式
一种表达脊髓灰质炎病毒样颗粒蛋白的载体，该载体中含有以下结构的表达框：脊髓灰质炎病毒结构蛋白VPO、VP1、VP3三个基因中任一个基因位于启动子1下游；另外两个基因之间通过2A序列连接位于启动子2下游；2个启动子启动表达的方向相反；其中，VP0、VP1、VP3的三个基因组成完整的脊髓灰质炎病毒结构蛋白P1基因；所述脊髓灰质炎病毒为脊髓灰质炎I型病毒、脊髓灰质炎II型病毒或脊髓灰质炎III型病毒；所述2A序列为编码含有D-x-E-x-N-P-G-P氨基酸序列的基因序列。
优选的，上述一种表达脊髓灰质炎病毒样颗粒蛋白的载体，该载体中含有以下结构的表达框：脊髓灰质炎病毒结构蛋白VP1基因位于启动子1下游，构成“启动子1-VP1”的结构；脊髓灰质炎病毒结构蛋白VP0和VP3基因通过2A序列连接位于启动子2下游，构成“启动子2-VP3-2A-VP0”的结构；上述2个启动子启动表达的方向相反；其中，VP0、VP1、VP3的三个基因组成完整的脊髓灰质炎病毒结构蛋白P1基因；所述脊髓灰质炎病毒为脊髓灰质炎 I型病毒、脊髓灰质炎II型病毒或脊髓灰质炎III型病毒；所述2A序列为编码含有D-x-E-x-N-P-G-P氨基酸序列的基因序列。
一种表达脊髓灰质炎病毒样颗粒蛋白的载体的制备方法，包括以下步骤：
1)根据脊髓灰质炎病毒的P1基因序列，根据宿主细胞的偏好性进行密码子优化，合成优化的P1基因序列；
2)根据优化的P1基因序列，设计引物将VP0、VP1、VP3中任一个基因先克隆至骨架载体中，位于骨架载体的一个启动子的下游，再将另2个基因通过2A序列进行连接，并将连接好的片段克隆至同一骨架载体的另一个启动子的下游；所得重组载体即为表达脊髓灰质炎病毒样颗粒蛋白的载体。
优选的，上述宿主细胞为Spodoptera frugiperda草地夜蛾细胞Sf9、酿酒酵母或哺乳细胞。
优选的，上述骨架载体根据宿主细胞的类型选择相应的骨架载体。
优选的，上述骨架载体为pFastBacTM-Dual载体、pESC URA载体、pVIVO2-mcs载体或pBudCE4.1载体。
优选的，上述优化的P1基因序列为：
若脊髓灰质炎病毒为I型，优化的P1基因序列如SEQ ID NO：1所示；
若脊髓灰质炎病毒为II型，优化的P1基因序列如SEQ ID NO：2所示；
若脊髓灰质炎病毒为III型，优化的P1基因序列如SEQ ID NO：3所示。
一种脊髓灰质炎病毒样颗粒的制备方法，包括以下步骤：
1)将上述所述的表达脊髓灰质炎病毒样颗粒蛋白的载体转染相应的宿主细胞，培养后可获得脊髓灰质炎病毒样颗粒或者重组脊髓灰质炎杆状病毒；
2)若步骤1)中获得的是重组脊髓灰质炎杆状病毒，再将重组脊髓灰质炎杆状病毒感染宿主细胞，经培养后即可获得脊髓灰质炎病毒样颗粒；
其中，若以酵母或哺乳细胞等为宿主时，获得的相应载体转染到宿主细胞后，可直接获得脊髓灰质炎病毒样颗粒；当以昆虫细胞为宿主时，获得的相应载体转染到宿主细胞后，获得的是重组脊髓灰质炎杆状病毒，需用该病毒再去感染宿主细胞，经培养后才可获得脊髓灰质炎病毒样颗粒。
优选的，上述一种脊髓灰质炎病毒样颗粒的制备方法，包括以下步骤：
1)将表达脊髓灰质炎病毒样颗粒蛋白的载体转化大肠杆菌DH10Bac感受态细胞，培养后提取重组大肠杆菌DH10Bac的质粒，获得重组杆状病毒载体；
2)将上一步获得的重组杆状病毒载体转染宿主细胞，培养后得到包含编码病毒样颗粒蛋白基因的重组杆状病毒；
3)将上一步获得的重组杆状病毒感染宿主细胞，培养后获得病毒样颗粒，即为脊髓灰质炎病毒样颗粒；
其中，所述的宿主细胞为昆虫细胞Sf9；
在上述步骤3)中将重组杆状病毒感染宿主细胞后，在宿主细胞中表达病毒样颗粒蛋白，所表达的病毒样颗粒蛋白组装成病毒样颗粒，并分泌到宿主细胞外，然后将细胞培养上清初步离心去除细胞碎片，再将上清液经pellicon膜包进行浓缩，再经蔗糖纯化，即可获得脊髓灰质炎病毒样颗粒，可作为免疫样品。
下面结合具体实施方式，对本发明的权利要求做进一步的详细说明。
本发明提供的技术方案所使用的材料和试剂：
1主要材料
病毒：Sabin I、Sabin II、Sabin III；
细胞：昆虫细胞Sf9。
1.2主要试剂
PremerStar DNA聚合酶(大连宝生物)；
Bac重组杆状质粒提取试剂盒(Omiga生物)；
卡那霉素、庆大霉素、四环素、X-gal、IPTG(北京鼎国)；
LB培养基(Sigma)；
Grace′s培养基、FBS(Gibco)；
Cellfectin II脂质体(Sigma)；
IPV兔抗(本公司制备，利用巴斯德的IPV疫苗免疫兔子获得的抗体)；
HRP羊抗兔二抗(Invitrogen)；
Donkey Anti-Rabbit Alexa Fluor 488(Invitrogen)。
实施例1一种表达脊髓灰质炎病毒样颗粒蛋白的载体
将脊髓灰质炎I型病毒的VP1序列置于pFastBac Dual质粒的P10启动子下，将小核糖核酸病毒科的2A序列基因(即编码之氨基酸序列为GSGATNFSLLKQAGDVEENPGP的碱基序列)一端连接脊髓灰质炎I型病毒的VP3基因，另一端连接至脊髓灰质炎I型病毒的VP0基因，置于pFastBac Dual质粒的Ppolh启动子下，获得穿梭载体pFBD-I VP1-VP3-2A-VP0，即表达脊髓灰质炎I型病毒样颗粒蛋白的载体。
实施例2一种表达脊髓灰质炎病毒样颗粒蛋白的载体
将脊髓灰质炎II型病毒的VP1序列置于pFastBac Dual质粒的P10启动子下，将小核糖核酸病毒科的2A序列基因(即编码之氨基酸序列为GSGATNFSLLKQAGDVEENPGP的碱基 序列)一端连接脊髓灰质炎II型病毒的VP3基因，另一端连接至脊髓灰质炎II型病毒的VP0基因，置于pFastBac Dual质粒的Ppolh启动子下，获得穿梭载体pFBD-II VP1-VP3-2A-VP0，即表达脊髓灰质炎II型病毒样颗粒蛋白的载体。
实施例3一种表达脊髓灰质炎病毒样颗粒蛋白的载体
将脊髓灰质炎III型病毒的VP1序列置于pFastBac Dual质粒的P10启动子下，将小核糖核酸病毒科的2A序列基因(即编码之氨基酸序列为GSGATNFSLLKQAGDVEENPGP的碱基序列)一端连接脊髓灰质炎III型病毒的VP3基因，另一端连接至脊髓灰质炎III型病毒的VP0基因，置于pFastBac Dual质粒的Ppolh启动子下，获得穿梭载体pFBD-III VP1-VP3-2A-VP0，即表达脊髓灰质炎III型病毒样颗粒蛋白的载体。
实施例4表达脊髓灰质炎病毒样颗粒蛋白的载体的制备方法
实施例1中所述的表达脊髓灰质炎病毒样颗粒蛋白的载体的制备方法，包括以下步骤：
1)基因优化合成
根据脊髓灰质炎I型病毒(PV I型)的P1序列，根据宿主细胞Spodoptera frugiperda草地夜蛾(Sf9)细胞系的偏爱性进行密码子虫源优化，PUC57为载体，以Xho I，Kpn I为酶切位点合成PV I型P1序列(SEQ ID NO：1)，并将其克隆至PUC57载体中，得重组载体PUC57-I-P1。
2)引物设计
以pFastBacTM-Dual为骨架载体，对骨架载体中Pph和P10启动子分析，根据优化后的脊髓灰质炎I型(Mahoney株)P1序列(SEQ ID NO：1)中VP0(SEQ ID NO：4)、VP1(SEQ ID NO：5)、VP3(SEQ ID NO：6)的基因序列，设计引相应引物，如表1所示。
表1引物设计表
Figure PCTCN2015077202-appb-000001
Figure PCTCN2015077202-appb-000002
3)表达脊髓灰质炎I型病毒样颗粒蛋白载体的制备
根据设计方案，以PUC57-I-P1为模板，用引物I VP1-Xho I F和I VP1-Kpn I R(见表1)扩增得VP1片段，并将其连接到pFastBacTM-Dual骨架载体中，得重组载体pFBD-I-VP1，其中IVP1位于启动子Pp10下游。
将VP3和VP0通过2A序列进行连接，即：以PUC57-I-P1为模板，利用引物I VP3-2A-VP0-XbaI F和I VP3-2A-VP0R(见表1)扩增得到I VP3-2A1，用引物I VP3-2A-VP0F1、II VP3-2A-VP0F2、II VP3-2A-VP0F3和I VP3-2A-VP0-Hind III R(见表1)扩增得到I 2A2-VP0，将I VP3-2A1和I 2A2-VP0作为模板，再以引物I VP3-2A-VP0-Xba I F和I VP3-2A-VP0-Hind III R(见表1)进行PCR扩增得I VP3-2A-VP0片段。
将I VP3-2A-VP0片段连接至重组载体pFBD-I-VP1中，得重组载体pFBD-I-VP1-VP3-2A-VP0，即表达脊髓灰质炎I型病毒样颗粒蛋白的载体，其中，VP3-2A-VP0位于启动子Pph的下游。相关载体构建情况如表2所示，构建示意图见图1。
表2重组载体pFBD-I-VP1-VP3-2A-VP0的构建情况
Figure PCTCN2015077202-appb-000003
实施例5表达脊髓灰质炎病毒样颗粒蛋白的载体的制备方法
实施例2中所述的表达脊髓灰质炎病毒样颗粒蛋白的载体的制备方法，包括以下步骤：
1)基因优化合成
根据脊髓灰质炎II型病毒(PV II型)的P1序列，根据宿主细胞Spodoptera frugiperda草地夜蛾(Sf9)细胞系的偏爱性进行密码子虫源优化，PUC57为载体，以XhoI，KpnI为酶切位点合成PV II型P1序列(SEQ ID NO：2)，并将其克隆至PUC57载体中，得重组载体PUC57-II-P1。
2)引物设计
以pFastBacTM-Dual为骨架载体，对骨架载体中Pph和P10启动子分析，根据优化后的脊髓灰质炎II型(MEF-1株)P1序列(SEQ ID NO：2)中VP0(SEQ ID NO：7)、VP1(SEQ ID NO：8)、VP3(SEQ ID NO：9)序列，设计引相应引物，如表3所示。
表3引物设计表
Figure PCTCN2015077202-appb-000004
3)表达脊髓灰质炎II型病毒样颗粒蛋白载体的制备
根据设计方案，以PUC57-II-P1为模板，用引物II VP1-Xho I F和II VP1-Kpn I R(见表3)扩增得VP1片段，并将其连接到pFastBacTM-Dual骨架载体中，得重组载体pFBD-II-VP1，其中VP1位于启动子Pp10下游。
将VP3和VP0通过2A序列进行连接，即：以PUC57-II-P1为模板，利用引物II VP3-2A-VP0-XbaI F和II VP3-2A-VP0R(见表3)扩增得到II VP3-2A1、用引物II VP3-2A-VP0F1、II VP3-2A-VP0F2、II VP3-2A-VP0F3和II VP3-2A-VP0-Hind III R(见表3)扩增得到II 2A2-VP0，再将II VP3-2A1和II 2A2-VP0作为模板，再以引物II VP3-2A-VP0-Xba I F和II VP3-2A-VP0-Hind III R进行PCR扩增得II VP3-2A-VP0片段。
将II VP3-2A-VP0片段连接至重组载体pFBD-II-VP1中，得重组载体pFBD-II-VP1-VP3-2A-VP0，即表达脊髓灰质炎II型病毒样颗粒蛋白的载体，其中，II VP3-2A-VP0位于启动子Pph的下游。相关载体构建情况如表4所示，构建示意图见图1。
表4重组载体pFBD-II-VP1-VP3-2A-VP0的构建情况
Figure PCTCN2015077202-appb-000005
实施例6表达脊髓灰质炎病毒样颗粒蛋白的载体的制备方法
实施例3中所述的表达脊髓灰质炎病毒样颗粒蛋白的载体的制备方法，包括以下步骤：
1)基因优化合成
根据脊髓灰质炎III型病毒(PV III型)的P1序列，根据宿主细胞Spodoptera frugiperda草地夜蛾(Sf9)细胞系的偏爱性进行密码子虫源优化，PUC57为载体，以XhoI，KpnI为酶切位点合成PV III型P1序列(SEQ ID NO：3)，并将其克隆至PUC57载体中，得重组载体PUC57-III-P1。
2)引物设计
以pFastBacTM-Dual为骨架载体，对其Pph和P10启动子分析，根据优化后的脊髓灰质炎III型(Saukett株)P1序列(SEQ ID NO：3)中VP0(SEQ ID NO：10)、VP1(SEQ ID NO： 11)、VP3(SEQ IDNO：12)序列，设计引相应引物，如表5所示。
表5引物设计表
Figure PCTCN2015077202-appb-000006
3)表达脊髓灰质炎III型病毒样颗粒蛋白载体的制备
根据设计方案，以PUC57-I-P1为模板，用引物III VP1-Xho I F和III VP1-Kpn I R(见表5)扩增得III VP1片段，并将其连接到pFastBacTM-Dual骨架载体中，得重组载体pFBD-III-VP1，其中VP1位于启动子Pp10下游。
将VP3和VP0通过2A序列进行连接，即：以PUC57-I-P1为模板，利用引物III VP3-2A-VP0-XbaI F和III VP3-2A-VP0R(见表5)扩增得到III VP3-2A1、用引物III VP3-2A-VP0F1、II VP3-2A-VP0F2、II VP3-2A-VP0F3和III VP3-2A-VP0-Hind III R(见表5)扩增得到III 2A2-VP0，再将III VP3-2A1和III 2A2-VP0作为模板，再以引物III VP3-2A-VP0-Xba I F和III VP3-2A-VP0-Hind III R进行PCR扩增得III VP3-2A-VP0片段。
将III VP3-2A-VP0片段连接至重组载体pFBD-III-VP1中，得重组载体pFBD-III-VP1-VP3-2A-VP0，即表达脊髓灰质炎III型病毒样颗粒蛋白的载体，其中，III VP3-2A-VP0位于启动子Pph的下游。相关载体构建情况如表6所示，构建示意图见图1。
表6重组载体pFBD-III-VP1-VP3-2A-VP0的构建情况
Figure PCTCN2015077202-appb-000007
实施例7重组脊髓灰质炎杆状病毒的制备方法
1)将实施例1～6中所述的表达脊髓灰质炎病毒样颗粒蛋白的载体pFBD-I-VP1-VP3-2A-VP0、pFBD-II-VP1-VP3-2A-VP0和pFBD-III-VP1-VP3-2A-VP0分别通过Invitrogen的Bac-to-Bac系统，转化大肠杆菌DH10Bac，培养后，筛选提取阳性重组大肠杆菌DH10Bac的质粒，获得重组杆状病毒载体；
2)将上一步获得的重组杆状病毒载体转染Sf9细胞获得相应的第一代重组杆状病毒，即重组脊髓灰质炎杆状病毒，分别命名为Bac-I-VP1-VP3-2A-VP0-V1(简称Bac-I)、Bac-II-VP1-VP3-2A-VP0-V1(简称Bac-II)和Bac-III-VP1-VP3-2A-VP0-V1(简称Bac-III)，用0.1MOI对各病毒进行连续扩增，获得的第三代病毒Bac-I-V3、Bac-II-V3和Bac-III-V3。
并对上述制得的重组脊髓灰质炎杆状病毒进行以下相关指标检测。
一、重组杆状病毒蛋白表达免疫荧光分析
按照Invitrogen说明书，将上述获得的Bac-I-V3、Bac-II-V3和Bac-III-V3以1MOI感染Sf9贴壁细胞，3天后，弃上清，用甲醛固定，以IPV(灭活脊髓灰质炎疫苗)兔抗为一抗，Donkey Anti-Rabbit Alexa Fluor 488为二抗进行免疫荧光分析，结果如图2所示，Bac-I、Bac-II和Bac-III感染的SF9细胞都有明亮绿色荧光，空白组未检测到明亮绿色荧光。说明它们都能在Sf9细胞中表达相应的目的蛋白(即VP0、VP1和VP3蛋白)。
二、重组杆状病毒蛋白表达Western blot分析
按照Invitrogen说明书，将上述制得的Bac-I-V3、Bac-II-V3和Bac-III-V3重组杆状病毒 以1MOI感染sf9悬浮细胞，以Excell-420为悬浮培养基，培养6天，待细胞全部裂解后，收集细胞和上清，取细胞和上清样品进行12％的SDS-PAGE电泳，转印PVDF膜，5％的脱脂奶粉封闭过夜，PBST漂洗5次，以1∶4000稀释一抗IPV兔抗，室温孵育2h，PBST稀释5次，HRP标记的二抗羊抗兔室温孵育1.5h，PBST漂洗5次，利用超敏发光液(A液25ml、B液25ml)进行压片显色。检测结果如图3所示，所有重组杆状病毒表达7天后的细胞内基本检测到不到VP1(35KDa)，而上清中都能检测到大量的VP1(35KDa)蛋白，推测在该表达系统下，释放的VP1蛋白可能形成了成熟的病毒样颗粒，或者单独分泌到胞外。
实施例8脊髓灰质炎病毒样颗粒的制备方法
脊髓灰质炎病毒样颗粒的制备方法，包括以下步骤：
1)表达脊髓灰质炎病毒样颗粒蛋白的载体的构建：如实施例4～6所述；
2)重组脊髓灰质炎杆状病毒的制备：如实施例7所述；
3)脊髓灰质炎病毒样颗粒的表达和组装：将实施例7制得的重组脊髓灰质炎杆状病毒Bac-I-V3、Bac-II-V3和Bac-III-V3分别以1MOI感染sf9悬浮细胞，每个样品表达2L，培养6天待细胞完全裂解后收获病毒上清；
4)脊髓灰质炎病毒样颗粒的纯化：将收获的病毒上清装入250ml的离心杯中，配平。使用JA-14转子(Beckman)高速离心收集上清，上清过0.22um的pellicon膜包浓缩10倍得到浓缩液。离心管的底部加入4ml的25％(W/W)蔗糖溶液，上层加入浓缩液。用离心机超高速离心4h。用PBS溶解沉淀得到重悬液。重悬液加到10％-50％蔗糖梯度上层，离心机超高速离心4h，收集8个蔗糖梯度(1ml/每梯度)，进行Western blot检测分析，根据Western blot结果(见图4)，收集富集的梯度，脱糖浓缩样品。
上述Western blot检测结果如图4所示，Western Blot检测Bac-I-V3蔗糖梯度结果表明在组份6、7中有大小为38、36、26和12KDa的条带，其大小分别同脊髓灰质炎I型VP0、VP1、VP2(VP3)和VP4蛋白大小吻合(4中只检测到VP1，可能与上清经过浓缩，其他蛋白得以呈现有关)，组份6、7的蔗糖浓度分别为33.2％和38％；Bac-II-V3和Bac-III-V3型蔗糖梯度样品的Western Blot结果与Bac-I-V3相似，说明病毒样颗粒(VLPs)富集中在这两个组份；即收集蔗糖浓度分别为33.2％和38％处的样品，进行超高速离心脱糖浓缩，PBS溶解沉淀并过0.22um滤膜，分别得到纯化的脊髓灰质炎I型、II型、III型病毒样颗粒。
下面对实施例8中制得的脊髓灰质炎病毒样颗粒进行以下相关检测。
一、脊髓灰质炎病毒样颗粒VLPs的电镜检测
将实施例8中纯化后得到的脊髓灰质炎I型、II型、III型病毒样颗粒分别进行电镜观察。电镜负染结果表明视野中出现高浓度、结构完整的病毒样颗粒，其直径大小为25-30nm(图5)， 图5A和图5C为脊髓灰质炎I型、III型病毒样颗粒，与其他报道的脊髓灰质炎I型、III型病毒样颗粒大小和结构相似，图5B为脊髓灰质炎II型病毒样颗粒(目前未见有关脊髓灰质炎II型病毒样颗粒的报道)。表明本发明方法能够高效产生结构完整的脊髓灰质炎病毒样颗粒。
二、脊髓灰质炎病毒样颗粒疫苗效力分析
1)疫苗的制备和免疫程序
稀释实施例8中制备的脊髓灰质炎I型、II型、III型病毒样颗粒，并分别加入1/10体积的AL(OH)3佐剂(1000mg/ml)混合，使得蛋白浓度为20μg/ml，利用以上10μg纯化的VLP对六周龄的BALB/c母鼠分别在0，2，4周进行免疫三次，每次每只免疫10μg。同时设置从巴斯德购买的IPV灭活疫苗为阳性对照和同样处理的未感染病毒的Sf9细胞作为阴性对照。5周后小鼠眼球采血，收集血清，-80分装保存。
2)免疫小鼠血清中和抗体滴度
取各组小鼠血清样品，56灭活30min，用无血清的M199培养基成倍比稀释。
I型VLPs免疫血清中和Sabin I，II型VLPs免疫血清中和SabinII，III型VLPs中和Sabin III，IPV免疫血清和阴性对照血清都分别中和SabinI、SabinII和Sabin III(SabinI、SabinII和SabinIII，脊髓灰质炎标准减毒株，来自于广东省疾控中心)，将SabinI、SabinII、Sabin III减毒株用M199培养基稀释到100pfu/100μl。稀释后的病毒液、样品各取100μl，混合均匀，在37℃细胞培养箱放置2小时。吸去Vero细胞培养上清，取200μl混合液加入到6孔板Vero细胞中，加入阳性和阴性对照，放入37℃继续培养2h。每孔加入4ml 1％的甲基纤维素，放置于CO2培养箱中4天，去培养基，结晶紫染色，统计出斑情况，能使空斑数减少50％的血清稀释度判定为血清的中和滴度，空斑减少率计算公式为：[(阴性对照空斑数-样品空斑数)/阴性对照空斑数]×100％。
实验结果如图6所示，和阴性对照相比，本发明制备的脊髓灰质炎I型病毒样颗粒免疫小鼠可以诱导1∶128以上的中和效价，IPV灭活疫苗中和Sabin I效价大约为1∶512；脊髓灰质炎II型病毒样颗粒免疫小鼠可以诱导1∶256的中和效价，IPV灭活疫苗中和效价稍高于1∶512；脊髓灰质炎III型病毒样颗粒免疫小鼠可以诱导高于1∶512的中和效价；IPV灭活疫苗中和效价为1∶(1024～2048)之间，虽然本发明制备的病毒样颗粒比巴斯德的IPV灭活疫苗中和效价要低，但是本发明制备的病毒样颗粒具有与天然病毒颗粒相似的结构而没有病毒核酸，不能自主复制，安全性更好，而野毒株的培养需要严格的实验条件，减毒株存在毒力恢复的可能性。且本发明制备的病毒样颗粒目前还只是初步的纯化，与市售的IPV灭活疫苗相比纯度存在差异，可能也会导致中和效价的差异。
上述结果说明，本发明制备的脊髓灰质炎I型、II型和III型VLPs诱导产生的抗体分别对Sabin I型、SabinII型和SabinIII有较强的中和作用，因此本发明制备的脊髓灰质炎病毒样颗粒可作为预防脊髓灰质炎病毒引起的小儿麻痹症的疫苗。
为本领域的专业技术人员容易理解，以上所述仅为本发明专利的较佳实施例，并不用以限制本发明，凡本发明的精神和原则之内所作的任何修改、等同替换和改进等，均落在本发明要求的保护范围之内，如将上述实施例中的宿主细胞改为酵母细胞或哺乳细胞等等，从而根据宿主细胞选择相应的骨架载体，进行同以上实验例类似的表达脊髓灰质炎病毒样颗粒蛋白载体的构建，以及脊髓灰质炎病毒样颗粒的制备等，均落在本发明要求的保护范围之内。

Claims (10)

  1. 一种表达脊髓灰质炎病毒样颗粒蛋白的载体，其特征在于：该载体中含有以下结构的表达框：脊髓灰质炎病毒结构蛋白VP0、VP1、VP3三个基因中任一个基因位于启动子1下游；另外两个基因之间通过2A序列连接位于启动子2下游；2个启动子启动表达的方向相反；
    其中，VP0、VP1、VP3的三个基因组成完整的脊髓灰质炎病毒结构蛋白P1基因；
    所述脊髓灰质炎病毒为脊髓灰质炎I型病毒、脊髓灰质炎II型病毒或脊髓灰质炎III型病毒；
    所述2A序列为编码含有D-x-E-x-N-P-G-P氨基酸序列的基因序列。
  2. 一种表达脊髓灰质炎病毒样颗粒蛋白的载体，其特征在于：该载体中含有以下结构的表达框：脊髓灰质炎病毒结构蛋白VP1基因位于启动子1下游，构成“启动子1-VP1”的结构；脊髓灰质炎病毒结构蛋白VP0和VP3基因通过2A序列连接位于启动子2下游，构成“启动子2-VP3-2A-VP0”的结构；上述2个启动子启动表达的方向相反；
    其中，VP0、VP1、VP3的三个基因组成完整的脊髓灰质炎病毒结构蛋白P1基因；
    所述脊髓灰质炎病毒为脊髓灰质炎I型病毒、脊髓灰质炎II型病毒或脊髓灰质炎III型病毒；
    所述2A序列为编码含有D-x-E-x-N-P-G-P氨基酸序列的基因序列。
  3. 权利要求1～2任一所述的一种表达脊髓灰质炎病毒样颗粒蛋白的载体的制备方法，其特征在于：包括以下步骤：
    1)根据脊髓灰质炎病毒的P1基因序列，根据宿主细胞的偏好性进行密码子优化，合成优化的P1基因序列；
    2)根据优化的P1基因序列，设计引物将VP0、VP1、VP3中任一个基因先克隆至骨架载体中，位于骨架载体的一个启动子的下游，再将另2个基因通过2A序列进行连接，并将连接好的片段克隆至同一骨架载体的另一个启动子的下游；所得重组载体即为表达脊髓灰质炎病毒样颗粒蛋白的载体。
  4. 权利要求3所述的一种表达脊髓灰质炎病毒样颗粒蛋白的载体的制备方法，其特征在于：所述的宿主细胞为Spodopterafrugiperda草地夜蛾细胞Sf9、酿酒酵母或哺乳细胞。
  5. 根据权利要求3所述的一种表达脊髓灰质炎病毒样颗粒蛋白的载体的制备方法，其特征在于：所述骨架载体根据宿主细胞的类型选择相应的骨架载体。
  6. 根据权利要求4或5所述的一种表达脊髓灰质炎病毒样颗粒蛋白的载体的制备方法，其特征在于：所述骨架载体为pFastBacTM-Dual载体、pESC URA载体、pVIVO2-mcs载体或pBudCE4.1载体。
  7. 权利要求3所述的一种表达脊髓灰质炎病毒样颗粒蛋白的载体的制备方法，其特征在于：所述优化的P1基因序列为：
    若脊髓灰质炎病毒为I型，优化的P1基因序列如SEQ ID NO：1所示；
    若脊髓灰质炎病毒为II型，优化的P1基因序列如SEQ ID NO：2所示；
    若脊髓灰质炎病毒为III型，优化的P1基因序列如SEQ ID NO：3所示。
  8. 一种脊髓灰质炎病毒样颗粒的制备方法，其特征在于：包括以下步骤：
    1)将权利要求1～2任一所述的或权利要求3～7任一所述制备方法制备的表达脊髓灰质炎病毒样颗粒蛋白的载体转染相应的宿主细胞，培养后可获得脊髓灰质炎病毒样颗粒或者重组脊髓灰质炎杆状病毒；
    2)若步骤1)中获得的是重组脊髓灰质炎杆状病毒，再将重组脊髓灰质炎杆状病毒感染宿主细胞，经培养后即可获得脊髓灰质炎病毒样颗粒。
  9. 根据权利要求8所述的一种脊髓灰质炎病毒样颗粒的制备方法，其特征在于：包括以下步骤：
    1)将将权利要求1～2任一所述的或权利要求3～7任一所述制备方法制备的表达脊髓灰质炎病毒样颗粒蛋白的载体转化大肠杆菌DH10 Bac感受态细胞，培养后提取重组大肠杆菌DH10 Bac的质粒，获得重组杆状病毒载体；
    2)将上一步获得的重组杆状病毒载体转染宿主细胞，培养后得到包含编码病毒样颗粒蛋白基因的重组杆状病毒；
    3)将上一步获得的重组杆状病毒感染宿主细胞，培养后获得病毒样颗粒，即为脊髓灰质炎病毒样颗粒；
    其中，所述的宿主细胞为昆虫细胞Sf9；
    在上述步骤3)中将重组杆状病毒感染宿主细胞后，在宿主细胞中表达病毒样颗粒蛋白，所表达的病毒样颗粒蛋白组装成病毒样颗粒，并分泌到宿主细胞外，然后将细胞培养上清初步离心去除细胞碎片，再将上清液经pellicon膜包进行浓缩，再经蔗糖纯化，即可获得脊髓灰质炎病毒样颗粒，可作为免疫样品。
  10. 权利要求8～9任一所述的制备方法制备的脊髓灰质炎病毒样颗粒在制备抗脊髓灰质炎I型、II型或III型病毒疫苗药物中的应用。
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