WO2023102862A1 - Method for preparing polyhedra for wrapping foreign proteins - Google Patents

Method for preparing polyhedra for wrapping foreign proteins Download PDF

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WO2023102862A1
WO2023102862A1 PCT/CN2021/136920 CN2021136920W WO2023102862A1 WO 2023102862 A1 WO2023102862 A1 WO 2023102862A1 CN 2021136920 W CN2021136920 W CN 2021136920W WO 2023102862 A1 WO2023102862 A1 WO 2023102862A1
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polh
protein
sequence
polyhedrons
polyhedra
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PCT/CN2021/136920
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French (fr)
Chinese (zh)
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贡成良
薛仁宇
胡小龙
朱敏
曹广力
张梓瑶
冯永杰
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苏州大学
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Priority to PCT/CN2021/136920 priority Critical patent/WO2023102862A1/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • 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
    • C12N15/64—General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host
    • 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
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/91—Cell lines ; Processes using cell lines

Definitions

  • the invention relates to the field of viral genetic engineering, in particular to a method for preparing polyhedrons wrapping foreign proteins.
  • Drug carriers can change the way the drug enters the body, affect the distribution in the body, control the release rate of the drug, and deliver the drug to the target organ.
  • drug carriers There are many types of drug carriers, and the more common ones are microcapsules, microspheres, nanoparticles, etc.
  • microcapsules are drug store-type microcapsules that use polymer materials to wrap solid or liquid drugs; and microspheres refer to drug dispersion. Or a tiny spherical entity formed by being adsorbed in a polymer matrix; nanoparticles generally refer to drug-containing particles of 10-100nm.
  • Microspheres and microcapsules can increase the local effective concentration of drugs in the body, have sustained release and long-term effect, and improve the stability of drugs, etc.; nanoparticles can improve the oral absorption of insoluble drugs, prolong the circulation time of drugs in the body, and enhance the stability of drugs.
  • Drug targeting as a special carrier for biomacromolecules, etc.
  • the carrier materials for making microcapsules, microspheres and nanoparticles usually include gelatin, gum arabic, albumin, starch, chitosan, alginate, cellulose acetate, ethyl cellulose, polylactic acid, polyamino acid, polyhydroxybutyrate Ester, polylactic acid-glycolic acid copolymer, etc.
  • Bioactive proteins (enzymes) or polypeptide drugs are very sensitive to temperature, acid-base, salt, denaturants, enzymes, etc., and are easily degraded.
  • the stability of protein (enzyme) is increased after immobilization, which is convenient for storage, transportation and repeated use. Protein immobilization methods include adsorption, cross-linking, carrier binding and embedding, all of which require human intervention.
  • Cytopolyhedrosis is a protein polyhedron crystal embedded with virus particles formed by insect cytoplasmic polyhedrosis virus (Cypovirus) in infected cells, with a size of 2-3 ⁇ m.
  • Polyhedrons can resist extreme environmental conditions such as acids, salts, and denaturants, and have a protective effect on the virus particles wrapped inside them. Therefore, the plasma polyhedron can be used as a good protein (polypeptide) drug carrier, and the protein (polypeptide) drug coated with the plasma polyhedrin protein microcrystal can not only prevent degradation, but also have a slow and controlled release effect.
  • VP3-tag 75 amino acid residues (VP3-tag) of the N-terminal of the viral tower protein (TP) encoded by the S4 segment of the silkworm plasmopolyhedrosis virus, also known as the viral structural protein 3 (VP3), were fused to the N-terminal of the target protein , can make the fusion protein wrap into polyhedron; the N-terminal 30 amino acid residues of polyhedrin protein (H1-tag) can also guide recombinant protein into polyhedron like VP3-tag. So far, the major capsid protein of norovirus, vascular endothelial growth factor, endostatin, protein kinase C, fibroblast growth factor, etc.
  • Proteins such as leukemia inhibitory factor, bone morphogenetic protein-2, and antigenic protein of carp herpesvirus type II are wrapped into polyhedrin.
  • Existing recombinant proteins mostly exist in the form of inclusion bodies, the purification process of recombinant proteins is complex, separation and purification are difficult, and the biological activity of recombinant proteins is poor, and the safety of products is affected by the presence of a variety of endotoxins in the periplasm .
  • the purpose of the present invention is to provide a method for preparing polyhedrons encapsulating foreign proteins.
  • the polyhedron not only has a protective effect on the encapsulated protein drug or enzyme, but also has a slow and controlled release effect. So far, there is no report on the use of viral structural protein 5 (VP5) encoded by the S7 segment of Bombyx mori plasmopolyhedrosis virus to guide the packaging of target proteins into polyhedrons.
  • VP5 viral structural protein 5
  • the technical solution adopted in the present invention is: a method for preparing a polyhedron wrapping foreign protein, comprising the following steps: (1) cloning the coding sequence of the polyhedrin gene of the silkworm plasmopolyhedrosis virus into the Bac - Downstream of the polyhedron gene promoter of the to-Bac expression system vector, construct a recombinant transfer plasmid; (2) Synthesize multiple cloning sites, protease restriction sites, Bombyx mori plasmopolyhedrosis virus VP5 sequences, and stop codons in series (3) Cloning the MCS-VP5 sequence to the downstream of the P10 promoter of the recombinant transfer plasmid in step (1) to obtain pFast-VP5-Polh; (4) The sequence encoding the target protein that will remove the stop codon Cloning the upstream of the MCS-VP5 sequence in the pFast-VP5-Polh plasmid or the
  • step (6) use the recombinant virus BmNPV-Pr-VP5-Polh obtained in step (6) to inoculate silkworms, collect silkworms after the onset of disease, and purify polyhedrons by differential centrifugation after homogenization , to obtain polyhedrons encapsulating foreign proteins.
  • the invention discloses the exogenous protein-encapsulating polyhedron prepared by the method for preparing the exogenous protein-encapsulating polyhedron, and the application of the exogenous protein-encapsulating polyhedron in preparing or serving as a protein sustained-release system.
  • the invention discloses the use of the silkworm plasmopolyhedrosis virus VP5 sequence in preparing protein carriers or polyhedrons wrapping foreign proteins, or as protein carriers.
  • the target protein is an exogenous protein, such as capsid protein, vascular endothelial growth factor, endostatin, protein kinase C, fibroblast growth factor, leukemia inhibitory factor, bone morphogenetic protein-2, antigenic protein, etc.
  • exogenous protein such as capsid protein, vascular endothelial growth factor, endostatin, protein kinase C, fibroblast growth factor, leukemia inhibitory factor, bone morphogenetic protein-2, antigenic protein, etc.
  • the sequence of VP5 of the silkworm plastopolyhedrosis virus is: GGACTGTCTCCAATAGCTTTGGCCCAGAAAAAACACGAAATGATGCTGCACACACACGAAATCCACAGCATGGACATCGATGGCTCAATT; specifically, as an illustration, the present invention prepares the method steps of the polyhedron wrapping the exogenous protein as follows: (1) the silkworm plastopolyhedrosis virus polyhedrosis protein The coding sequence of the gene (GenBank accession number: GQ924589) was cloned into the downstream of the polyhedron gene promoter of the vector pFastBac TM Dual (product of Invitrogen, USA) to construct the recombinant transfer plasmid pFastBac TM Dual-Polh; in this step, the silkworm plasmotype The coding sequence of the polyhedrosis protein gene of polyhedrosis virus can be obtained by PCR amplification after reverse transcription into c
  • the sequence encoding the target protein can be optimized according to the codon preference of silkworm.
  • the coding sequence of the target protein can be cloned between the P10 promoter and the thrombin cleavage site in pFast-VP5-Polh by restriction enzyme ligation or seamless cloning, and ensure that the reading frame of the target gene is in line with the coagulation
  • the enzyme cleavage site and the reading frame of the VP5-tag are consistent; (5) Transform pFast-Pr-VP5-Polh into DH10/Bac competent cells, and spread it on a medium containing tetracycline, kanamycin, gentamycin, On X-gal and IPTG LB agar plates, cultivate at 37°C, pick white colonies, and extract recombinant Bacmid-Pr-VP5-Polh DNA; in this step, pFast-Pr-VP5-Polh transformed DH10/Bac
  • the contents of tetracycline, kanamycin, gentamycin, IPTG and X-gal on the LB agar culture plate are 10 ⁇ g/ml, 50 ⁇ g/ml, 7 ⁇ g/ml, 40 ⁇ g/ml and 100 ⁇ g/ml, respectively.
  • a small amount of polyhedron can be obtained through step (7); a large amount of polyhedron can be obtained through step (8).
  • the silkworm used for inoculation in step (8) is preferably 4-5 instar larvae or silkworm chrysalis.
  • the polyhedron obtained by the above technical scheme can detect the target fusion protein through SDS-PAGE and Western blotting, and the target protein encapsulated in the polyhedron has biological activity.
  • the present invention has the following advantages compared with the prior art: 1.
  • the present invention uses the VP5-tag to encapsulate foreign proteins into polyhedrons for the first time through the baculovirus expression system, and has a high embedding rate.
  • the present invention can wrap the recombinant protein in the polyhedron; the expression level of the polyhedron is high, and the polyhedron is insoluble in water, has strong resistance to harsh environmental conditions, and can be purified by simple differential centrifugation, and the purification process of the polyhedron It can be carried out under normal temperature conditions.
  • the pure polyhedron is rapidly lysed under alkaline conditions.
  • the pH is adjusted to the isoelectric point of the polyhedrin protein with hydrochloric acid solution, the polyhedrin protein forms a flocculent precipitate, and the polyhedrin protein can be removed by centrifugation, while the target protein remains in the supernatant In this way, the purified recombinant protein can be obtained quickly and conveniently.
  • a specific cleavage site for protease is designed between the target protein and the VP5-tag. This design not only facilitates the detection of recombinant proteins, but also cuts the recombinant proteins with proteases, thereby removing the VP5-tag and enabling The product is closer to nature.
  • recombinant proteins are easily degraded, and usually need to be freeze-dried and stored at low temperature.
  • Polyhedrons can resist extreme environmental conditions such as acids, salts, and denaturants, as well as proteases, and have a protective effect on recombinant proteins wrapped inside them. Therefore, the polyhedrons wrapped with recombinant proteins obtained in the present invention do not need special treatment, and can be used for a long time at room temperature. save.
  • the polyhedron is icosahedral or octahedral protein microcrystals with a size of about 2-3 ⁇ m, which not only prevents the degradation of the wrapped protein, but also has a slow and controlled release effect. Therefore, the polyhedron wrapped with polypeptide drugs can be used as a slow release drug development.
  • Fig. 1 is the sequencing result of the chemically synthesized MCS-TCS-VP5 N331-360 sequence in Example 1.
  • Fig. 2 is the PCR identification of Bacmid-EGFP-VP5-Polh in Example 1.
  • Bacmid-EGFP-VP5-Polh genomic DNA as template, use M13 forward primer (5'-CCCAGTCACGACGTTGTAAAACG-3') (SEQ ID NO: 5) and M13 reverse primer (5'-AGCGGATAACAATTTCACACAGG-3') (SEQ ID NO: 6) PCR amplification was performed.
  • Lane 1 wild-type Bacmid DNA
  • lane M molecular weight standard DNA
  • lane 2 Bacmid-EGFP-VP5-Polh.
  • Fig. 3 is the fluorescence observation of the polyhedrons formed by BmNPV-EGFP-VP5-Polh in the cultured cells in Example 1.
  • Fig. 4 is the Western blot detection of the polyhedron formed by BmNPV-EGFP-VP5-Polh in the silkworm hemolymph in Example 1.
  • Lane NC plastid polyhedron of wild-type silkworm
  • Lane EGFP polyhedron formed by BmNPV-EGFP-VP5-Polh.
  • the primary antibody was mouse anti-GFP monoclonal antibody
  • the secondary antibody was HRP-labeled goat anti-mouse IgG.
  • FIG. 5 is a Western blot detection of the protective effect of polyhedron on embedded green fluorescent protein in Example 1.
  • EGFP-Polyhedra represents the purified polyhedron embedded with green fluorescent protein
  • Tempo °C represents the storage temperature of the polyhedron (25°C or -20°C)
  • the Na 2 CO 3 -NaHCO 3 was lysed, "-” means no lysing, “+” means lysing;
  • polyhedrin means the primary antibody is mouse anti-polyhedrin antibody;
  • GFP means mouse anti-green Monoclonal antibodies to fluorescent proteins.
  • the secondary antibody was HRP-labeled goat anti-mouse IgG.
  • FIG. 7 PCR identification of recombinant Bacmid-bFGF-VP5-Polh in Example 2.
  • Bacmid-bFGF-VP5-Polh genomic DNA as template, use M13 forward primer (5'-CCCAGTCACGACGTTGTAAAACG-3') (SEQ ID NO: 5) and M13 reverse primer (5'-AGCGGATAACAATTTCACACAGG-3') (SEQ ID NO: 6) was amplified by PCR.
  • Lane M molecular weight standard DNA
  • Lane 1 wild-type Bacmid DNA
  • Lane 3 Bacmid-bFGF-VP5-Polh.
  • FIG. 8 is a Western blot detection of polyhedrons formed by BmNPV-bFGF-VP5-Polh in the silkworm hemolymph in Example 2.
  • FIG. Lane NC plastid polyhedron of wild-type silkworm; Lane bFGF, polyhedron formed by BmNPV-bFGFP-VP5-Polh.
  • the primary antibody was rabbit anti-bFGF monoclonal antibody, and the secondary antibody was HRP-labeled goat anti-rabbit IgG.
  • FIG. 9 is a Western blot detection of the proportion of basic fibroblast growth factor expressed in silkworm hemolymph embedded into polyhedrons in Example 2.
  • FIG. BmNPV-bFGF-VP7-Polh infected five-instar silkworm hemolymph 7 days after the cells were crushed, centrifuged at 3000 rpm for 10 minutes, supernatant and precipitate (polyhedron) were obtained, separated by SDS-PAGE, and detected by Western blot Basic fibroblast growth factor in serum and pellet.
  • "bFGF-Serum” represents the supernatant after hemolymph centrifugation
  • bFGF-polyhedra represents the pellet of hemolymph centrifugation.
  • the primary antibody was rabbit anti-bFGF monoclonal antibody, and the secondary antibody was HRP-labeled goat anti-rabbit IgG.
  • Figure 10 shows the biological activity of basic fibroblast growth factor detected by cell culture in Example 2.
  • Cells were stained with Giemsa.
  • Lysate of bFGF-polyhedra represents the culture medium of L-929 cells containing the lysate of bFGF-polyhedra lysed with Na 2 CO 3 -NaHCO 3
  • bFGF-polyhedra represents the culture medium of L-929 cells with bFGF-polyhedra
  • WT BmCPV polyhedra represents the medium used to culture L-929 cells containing wild-type polyhedra
  • Con represents the blank control group L-929 cells.
  • the specific operation methods involved in the present invention are conventional methods, such as the specific operations of cloning and enzyme digestion; the test methods involved are also conventional techniques; except for the sequence of design, the raw material reagents involved are all conventional products, among which pFastBacTM Dual is a product of Invitrogen Corporation of the United States, which belongs to the Bac-to-Bac (Bacteria to Baculovirus) expression system vector.
  • pFastBacTM Dual is a product of Invitrogen Corporation of the United States, which belongs to the Bac-to-Bac (Bacteria to Baculovirus) expression system vector.
  • RNA PCR KitVer Routinely extract the total RNA from the midgut tissue infected with silkworm plasmopolyhedrosis virus, use RNA PCR KitVer.
  • the coding sequence of viral polyhedrin gene (GenBank accession number: GQ924589) designed primer polh-EI (GAATTCATGGCAGACGTAGCAGGAACA, with Eco R site) and polh-XB (TCTAGATCACTGACGGTTACTCAGAGC with Xba Restriction site), by PCR amplification of 5'- and 3'-ends with Eco R and Xba
  • the coding sequence of the plastid polyhedrin gene at the locus was cloned into the T-vector, and after sequencing verification, Eco R and Xba Double-enzyme-digested and cloned into pFastBac TM Dual that was also digested to obtain the recombinant transfer plasmid pFastBac TM
  • the green fluorescence of the polyhedron can be observed under the fluorescence microscope ( Figure 3), indicating that the green fluorescent protein was embedded into the polyhedron under the guidance of the VP5-tag;
  • the intersegmental membrane was punctured to inoculate the 5th instar silkworm, raised at 25°C, and the hemolymph of the infected silkworm was collected after 7 days.
  • the hemolymph of the infected silkworm was subjected to differential centrifugation at 1000 rpm and 3000 rpm (repeated three times). Can be purified to polyhedrons.
  • the protein on the PAGE gel was transferred to PVDF membrane (Roche Company), and Western blot detection was performed with the antibody of green fluorescent protein, and the hybridization signal representing the recombinant green protein could be detected (Fig.
  • the lysed polyhedron was stored at 25°C for 14 days, and the signal bands representing polyhedrin and green fluorescent protein were basically not observed. It shows that the polyhedrin and green fluorescent protein are obviously degraded during the preservation process, and the polyhedrin without cracking can still be observed for 14 days at 25°C or -20°C.
  • the signal bands representing the polyhedrin and green fluorescent protein indicate that Polyhedrons protect the embedded GFP ( Figure 5).
  • the silkworm plastopolyhedron was cracked with 0.1mol/L Na 2 CO 3 -NaHCO 3 at 28°C for 25 minutes, adjusted to the isoelectric point of the polyhedrin with hydrochloric acid, centrifuged at 12,000 rpm for 10 minutes, and After the clear was extracted with phenol, phenol/chloroform (1:1 volume ratio), and chloroform successively, the RNA was precipitated with ethanol, and then reverse-transcribed with RNA PCR KitVer.
  • the primers (polh-EI, polh-XB) in step (1) of Example 1 amplified the coding sequence of the plasmopolyhedrin gene by PCR, cloned into T-vector, and after sequencing verification, used Eco R and Xba Double-enzyme digestion cloned into pFastBac TM Dual that was also digested to obtain the recombinant transfer plasmid pFastBac TM Dual-Polh; (2) Same as step (2) in Example 1.
  • a target band consistent with the theoretical molecular weight (target gene + 2650bp) can be amplified from the recombinant Bacmid DNA ( Figure 7), indicating that the recombinant Bacmid has been correctly constructed as required, named Bacmid-bFGF-VP5-Polh; (6 ) use Bacmid-bFGF-VP5-Polh to replace Bacmid-EGFP-VP5-Polh in step (6) of Example 1, and prepare the P1 generation recombinant virus BmNPV-bFGF-VP5-Polh according to the scheme of step (6) of Example 1; The P1 generation virus infected silkworm cultured cells, and after culturing for 5 days, the supernatant of the virus-infected cultured cells was collected to obtain the P2 generation recombinant virus, and stored at -80°C; (7) Infect the silkworm cultured cells with the P2 generation recombinant virus with a multiplicity of infection of 5, Cultivate at 27°C for 4 days, collect the cultured
  • the polyhedron bFGF-Polyhedra can be purified. After the purified polyhedron was separated by SDS-PAGE, the protein on the PAGE gel was transferred to PVDF membrane (Roche Company), and Western blot detection was performed with the antibody of basic fibroblast growth factor, and the protein representing basic fibroblast growth factor could be detected.
  • the hybridization signal of cell growth factor shows that the recombinant basic fibroblast growth factor is embedded into polyhedron; (9) take the hemolymph in step (8), break the cells, and centrifuge at 3000 rpm for 10 Minutes, get supernatant and precipitate (polyhedron), after SDS-PAGE separation, use rabbit anti-bFGF monoclonal antibody to detect basic fibroblast growth factor in supernatant and precipitate by Western blot ( Figure 9), and analyze by grayscale scanning It was measured that 51% of the recombinant basic fibroblast growth factor expressed in silkworm hemolymph was embedded in polyhedrons; (10) select 4 wells in a 24-well plate, and add 500 ⁇ L of high-glucose medium to each well, Then add the lysate (2.5 ⁇ L) of 5 ⁇ 10 5 bFGF-polyhedra lysed with 0.1 mol/L Na 2 CO 3 -NaHCO 3 , 5 ⁇ 10 5 bFGF-pol
  • L-929 cells were seeded in Transwell chambers, 5 ⁇ 10 3 cells/well, and the cells in the chambers were grown in 100 ⁇ L of high-glucose medium. Put the chamber inoculated with cells into the wells of the above-mentioned 24-well plate containing polyhedron or polyhedron lysate, incubate at 37°C for 48 hours, and then perform Giemsa staining ( Figure 10).
  • Figure 10 The results show that the number of cells in the chamber is determined by bFGF -polyhedra lysate group, bFGF-polyhedra group, and wild-type polyhedron group decreased sequentially, and the number of cells in the wild-type polyhedron group had no significant difference from the blank control. It shows that the recombinant fibroblast growth factor released from bFGF-polyhedra after Na 2 CO 3 -NaHCO 3 cleavage treatment has biological activity and promotes cell growth; Press down for a slow release.

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Abstract

The present invention relates to the field of immobilized proteins, in particular to a method for preparing a polyhedra for wrapping foreign proteins. A bombyx mori cytoplasmic polyhedrin, and a fusion foreign protein having a bombyx mori cytoplasmic polyhedrosis virus structural protein VP5 tag and a protease specific cleavage site are co-expressed in bombyx mori culture cells or bombyx mori by using a baculovirus expression system and by means of a genetic engineering method, so that a polyhedra wrapping the foreign protein is formed. The formed polyhedra can be purified by means of simple differential centrifugation; the polyhedra has protection and slow release effects on the foreign proteins wrapped by the polyhedra. After the purified polyhedra is cleaved under an alkaline condition, the pH value is adjusted to a polyhedrin isoelectric point by using a hydrochloric acid solution, the polyhedrin can be precipitated by means of centrifugation, and the fusion foreign protein is retained in a supernatant, so that the fusion foreign protein can be quickly and conveniently obtained. The fusion foreign protein is subjected to enzyme digestion by means of a corresponding protease to remove the VP5 tag, so that a more natural foreign protein can be conveniently obtained.

Description

一种制备包裹外源蛋白的多角体的方法A method for preparing polyhedrons wrapping foreign proteins 技术领域technical field
本发明涉及病毒基因工程领域，具体涉及一种制备包裹外源蛋白的多角体的方法。The invention relates to the field of viral genetic engineering, in particular to a method for preparing polyhedrons wrapping foreign proteins.
背景技术Background technique
药物载体能改变药物进入体内的方式、影响在体内的分布、控制药物的释放速度、将药物输送到靶向器官。药物载体的种类繁多，较为常见的有微囊与微球、纳米粒等；其中，微囊是用高分子材料将固态或液态药物包裹成为的药库型微型胶囊；而微球是指药物分散或被吸附在高分子聚合物基质中而形成的微小球状实体；纳米粒一般是指10-100nm的含药粒子。Drug carriers can change the way the drug enters the body, affect the distribution in the body, control the release rate of the drug, and deliver the drug to the target organ. There are many types of drug carriers, and the more common ones are microcapsules, microspheres, nanoparticles, etc. Among them, microcapsules are drug store-type microcapsules that use polymer materials to wrap solid or liquid drugs; and microspheres refer to drug dispersion. Or a tiny spherical entity formed by being adsorbed in a polymer matrix; nanoparticles generally refer to drug-containing particles of 10-100nm.
微球和微囊制剂可提高药物在体内的局部有效浓度、具缓释和长效性、提高药物的稳定性等；纳米粒可改善难溶性药物的口服吸收、延长药物的体内循环时间、增强药物的靶向性、用作生物大分子的特殊载体等。制作微囊、微球、纳米粒的载体材料通常有明胶、阿拉伯胶、白蛋白、淀粉、壳聚糖、海藻酸盐、醋酸纤维素、乙基纤维素、聚乳酸、聚氨基酸、聚羟基丁酸酯、聚乳酸-羟基乙酸共聚物等。不管是微球、微囊，还是纳米粒制作工艺较为复杂，影响因素较多。生物活性蛋白（酶）或多肽类药物，对温度、酸碱、盐、变性剂、酶等非常敏感，极易降解。蛋白（酶）固定化后稳定性增加、便于保存运输和重复利用。蛋白固定化方法有吸附法、交联法、载体结合法和包埋法，这些方法均须人为干预。Microspheres and microcapsules can increase the local effective concentration of drugs in the body, have sustained release and long-term effect, and improve the stability of drugs, etc.; nanoparticles can improve the oral absorption of insoluble drugs, prolong the circulation time of drugs in the body, and enhance the stability of drugs. Drug targeting, as a special carrier for biomacromolecules, etc. The carrier materials for making microcapsules, microspheres and nanoparticles usually include gelatin, gum arabic, albumin, starch, chitosan, alginate, cellulose acetate, ethyl cellulose, polylactic acid, polyamino acid, polyhydroxybutyrate Ester, polylactic acid-glycolic acid copolymer, etc. Whether it is microspheres, microcapsules, or nanoparticles, the production process is relatively complicated and there are many influencing factors. Bioactive proteins (enzymes) or polypeptide drugs are very sensitive to temperature, acid-base, salt, denaturants, enzymes, etc., and are easily degraded. The stability of protein (enzyme) is increased after immobilization, which is convenient for storage, transportation and repeated use. Protein immobilization methods include adsorption, cross-linking, carrier binding and embedding, all of which require human intervention.
质型多角体是昆虫质型多角体病毒(Cypovirus)在感染细胞内形成的一种包埋有病毒粒子的蛋白质多面体结晶，大小在2-3µm。多角体能抵御酸、盐、变性剂等极端的环境条件，对包裹在其内部的病毒粒子有保护作用。因此质型多角体可作为一种良好的蛋白（多肽）类药物载体，用质型多角体蛋白微晶包裹蛋白（多肽）类药物不仅可防止降解，而且有缓控释作用。以往研究显示，将家蚕质型多角体病毒S4节段编码的病毒塔蛋白（TP）又称病毒结构蛋白3（VP3）N端的75个氨基酸残基（VP3-标签）融合在目标蛋白的N端，能使融合蛋白包裹进多角体中；多角体蛋白N端30个氨基酸残基（H1-标签）也可像VP3-标签一样将重组蛋白引导至多角体中。至今，通过多角体蛋白与融合H1-标签或VP3-标签的目的蛋白共表达，已将诺如病毒的主要衣壳蛋白、血管内皮生长因子、内皮抑素、蛋白激酶C、成纤维细胞生长因子、白血病抑制因子、骨形态发生蛋白-2、鲤疱疹病毒II型的抗原蛋白等蛋白（酶）包裹进多角体蛋白中。现有重组蛋白多以包涵体的形式存在，重组蛋白的纯化工艺复杂、分离纯化难度大，而且重组蛋白的生物学活性较差、且因细胞周质中含有种类繁多的内毒素而影响制品的安全性。Cytopolyhedrosis is a protein polyhedron crystal embedded with virus particles formed by insect cytoplasmic polyhedrosis virus (Cypovirus) in infected cells, with a size of 2-3 μm. Polyhedrons can resist extreme environmental conditions such as acids, salts, and denaturants, and have a protective effect on the virus particles wrapped inside them. Therefore, the plasma polyhedron can be used as a good protein (polypeptide) drug carrier, and the protein (polypeptide) drug coated with the plasma polyhedrin protein microcrystal can not only prevent degradation, but also have a slow and controlled release effect. Previous studies have shown that the 75 amino acid residues (VP3-tag) of the N-terminal of the viral tower protein (TP) encoded by the S4 segment of the silkworm plasmopolyhedrosis virus, also known as the viral structural protein 3 (VP3), were fused to the N-terminal of the target protein , can make the fusion protein wrap into polyhedron; the N-terminal 30 amino acid residues of polyhedrin protein (H1-tag) can also guide recombinant protein into polyhedron like VP3-tag. So far, the major capsid protein of norovirus, vascular endothelial growth factor, endostatin, protein kinase C, fibroblast growth factor, etc. Proteins (enzymes) such as leukemia inhibitory factor, bone morphogenetic protein-2, and antigenic protein of carp herpesvirus type II are wrapped into polyhedrin. Existing recombinant proteins mostly exist in the form of inclusion bodies, the purification process of recombinant proteins is complex, separation and purification are difficult, and the biological activity of recombinant proteins is poor, and the safety of products is affected by the presence of a variety of endotoxins in the periplasm .
技术问题technical problem
本发明的目的是提供一种制备包裹外源蛋白的多角体的方法。多角体不仅对包裹的蛋白药物或酶有保护作用，而且有缓控释作用。到目前为止，还没有用家蚕质型多角体病毒S7节段编码的病毒结构蛋白5（VP5）引导目标蛋白包裹进多角体的报道。The purpose of the present invention is to provide a method for preparing polyhedrons encapsulating foreign proteins. The polyhedron not only has a protective effect on the encapsulated protein drug or enzyme, but also has a slow and controlled release effect. So far, there is no report on the use of viral structural protein 5 (VP5) encoded by the S7 segment of Bombyx mori plasmopolyhedrosis virus to guide the packaging of target proteins into polyhedrons.
技术解决方案technical solution
为达到上述目的，本发明采用的技术方案是：一种制备包裹外源蛋白的多角体的方法，包括下列步骤：(1) 将家蚕质型多角体病毒多角体蛋白基因的编码序列克隆进Bac-to-Bac表达系统载体的多角体基因启动子下游，构建重组转移质粒；（2）合成由多克隆位点、蛋白酶酶切位点、家蚕质型多角体病毒VP5序列、终止密码子依次串联的MCS-VP5序列；（3）将MCS-VP5序列克隆至步骤（1）的重组转移质粒的P10启动子的下游获得pFast-VP5-Polh；（4）将去除终止密码的编码目的蛋白的序列克隆在pFast-VP5-Polh质粒中的MCS-VP5序列的上游或MCS-VP5序列中的多克隆位点区域，获得pFast-Pr-VP5-Polh；(5)将pFast-Pr-VP5-Polh转化感受态细胞，涂布于LB琼脂平板上，培养后挑取白色菌落，提取重组Bacmid-Pr-VP5-Polh DNA；(6) 将重组Bacmid-Pr-VP5-Polh DNA转染家蚕培养细胞，培养至细胞发病后，收集细胞培养上清，获重组病毒BmNPV-Pr-VP5-Polh；(7) 重组病毒BmNPV-Pr-VP5-Polh接种家蚕培养细胞，培养后，收集细胞，破碎细胞后通过差速离心获包裹外源蛋白的多角体；或(8)用步骤（6）获得的重组病毒BmNPV-Pr-VP5-Polh接种家蚕，发病后，收集家蚕，匀浆后通过差速离心纯化多角体，获包裹外源蛋白的多角体。In order to achieve the above object, the technical solution adopted in the present invention is: a method for preparing a polyhedron wrapping foreign protein, comprising the following steps: (1) cloning the coding sequence of the polyhedrin gene of the silkworm plasmopolyhedrosis virus into the Bac - Downstream of the polyhedron gene promoter of the to-Bac expression system vector, construct a recombinant transfer plasmid; (2) Synthesize multiple cloning sites, protease restriction sites, Bombyx mori plasmopolyhedrosis virus VP5 sequences, and stop codons in series (3) Cloning the MCS-VP5 sequence to the downstream of the P10 promoter of the recombinant transfer plasmid in step (1) to obtain pFast-VP5-Polh; (4) The sequence encoding the target protein that will remove the stop codon Cloning the upstream of the MCS-VP5 sequence in the pFast-VP5-Polh plasmid or the multiple cloning site region in the MCS-VP5 sequence to obtain pFast-Pr-VP5-Polh; (5) pFast-Pr-VP5-Polh transformation Competent cells were spread on LB agar plates, and white colonies were picked after culture to extract recombinant Bacmid-Pr-VP5-Polh DNA; (6) Recombinant Bacmid-Pr-VP5-Polh DNA was transfected into cultured silkworm cells, cultured After the onset of the cells, the cell culture supernatant was collected to obtain the recombinant virus BmNPV-Pr-VP5-Polh; (7) The recombinant virus BmNPV-Pr-VP5-Polh was inoculated into silkworm cultured cells. or (8) use the recombinant virus BmNPV-Pr-VP5-Polh obtained in step (6) to inoculate silkworms, collect silkworms after the onset of disease, and purify polyhedrons by differential centrifugation after homogenization , to obtain polyhedrons encapsulating foreign proteins.
本发明公开了上述制备包裹外源蛋白的多角体的方法制备的包裹外源蛋白的多角体，以及包裹外源蛋白的多角体在制备蛋白缓释体系或者作为蛋白缓释体系中的应用。The invention discloses the exogenous protein-encapsulating polyhedron prepared by the method for preparing the exogenous protein-encapsulating polyhedron, and the application of the exogenous protein-encapsulating polyhedron in preparing or serving as a protein sustained-release system.
本发明公开了家蚕质型多角体病毒VP5序列在制备蛋白载体或者包裹外源蛋白的多角体中的应用，或者在作为蛋白载体中的应用。The invention discloses the use of the silkworm plasmopolyhedrosis virus VP5 sequence in preparing protein carriers or polyhedrons wrapping foreign proteins, or as protein carriers.
本发明中，目的蛋白为外源蛋白，比如衣壳蛋白、血管内皮生长因子、内皮抑素、蛋白激酶C、成纤维细胞生长因子、白血病抑制因子、骨形态发生蛋白-2、抗原蛋白等。In the present invention, the target protein is an exogenous protein, such as capsid protein, vascular endothelial growth factor, endostatin, protein kinase C, fibroblast growth factor, leukemia inhibitory factor, bone morphogenetic protein-2, antigenic protein, etc.
本发明中，家蚕质型多角体病毒VP5序列为：GGACTGTCTCCAATAGCTTTGGCCCAGAAAAAACACGAAATGATGCTGCACACACACGAAATCCACAGCATGGACATCGATGGCTCAATT；具体的，作为示意，本发明制备包裹外源蛋白的多角体的方法步骤如下： (1) 将家蚕质型多角体病毒多角体蛋白基因的编码序列(GenBank登录号：GQ924589)克隆进载体pFastBac TM Dual（美国Invitrogen公司的产品）的多角体基因启动子下游，构建重组转移质粒pFastBac TM Dual-Polh；在该步骤中，家蚕质型多角体病毒多角体蛋白基因的编码序列可以通过以病毒的基因组RNA或病毒感染的中肠组织的总RNA为模板，反转录成cDNA后，通过PCR扩增获得；也可以根据已公开的多角体蛋白基因的编码序列，化学合成获得；(2) 将多克隆位点（MCS）、凝血酶酶切位点（TCS）、家蚕质型多角体病毒S7片段编码的VP5蛋白部分DNA序列依次串联，并在3’端加终止密码子TAA，尔后通过化学合成获MCS-TCS-VP5序列；在该步骤中，VP5-标签上游添加了多克隆位点（MCS）和凝血酶酶切位点（划线部分），便于克隆并考虑到重组蛋白的检测以及从重组蛋白中切除VP5-标签；序列中的凝血酶酶切割位点可以用Xa因子、肠激酶等的切割位点替代；MCS-TCS-VP5的序列如下：MCS- CTCGTGCCTAGAGGTTCAGGACTGTCTCCAATAGCTTTGGCCCAGAAAAAACACGAAATGATGCTGCACACACACGAAATCCACAGCATGGACATCGATGGCTCAATTTAA；（3） MCS-TCS-VP5序列克隆转移质粒pFastBac TM Dual-polh中的P10启动子的下游获pFast-VP5-Polh；在该步骤中，MCS-TCS-VP5序列可以通过酶切连接克隆进pFastBac TM Dual-Polh中的P10启动子下游，也可以通过无缝克隆的方法进行；（4） 将去除终止密码的编码目的蛋白的序列克隆在pFast-VP5-Polh质粒中的MCS-TCS-VP5序列的上游或MCS-VP5序列中的多克隆位点区域，并使目的基因的读码框与凝血酶酶切位点、VP5蛋白部分序列的读码框融合获pFast-Pr-VP5-Polh；在该步骤中，所述的目的蛋白为需要包埋的外源蛋白，例：绿色荧光蛋白、碱性成纤维细胞生长因子等。为了提高表达水平，可以将编码目的蛋白的序列根据家蚕密码子的偏爱性进行优化。目的蛋白的编码序列可以通过酶切连接的方式或无缝克隆的方法克隆在pFast-VP5-Polh中的P10启动子与凝血酶酶切位点之间，并保证目的基因的读码框与凝血酶酶切位点、VP5-标签的读码框一致；(5)将pFast-Pr-VP5-Polh转化DH10/Bac感受态细胞，涂布于含四环素、卡那霉素、庆大霉素、X-gal、IPTG的LB琼脂平板上，于37℃培养，挑取白色菌落，提取重组Bacmid-Pr-VP5-Polh DNA；在该步骤中，pFast-Pr-VP5-Polh转化的DH10/Bac感受态细胞，涂布于含四环素、卡那霉素、庆大霉素、IPTG、X-gal的LB 琼脂培养平板上是为了方便重组Bacmid的筛选。优选的，四环素、卡那霉素、庆大霉素、IPTG 和X-gal在LB 琼脂培养平板上的含量分别为10 µg/ml、50 µg/ml、7 µg/ml、40µg/ml和100 µg/ml；(6) 将重组Bacmid-Pr-VP5-Polh DNA转染家蚕培养细胞，26～27℃培养，细胞发病后，收集细胞培养上清；为了获得高滴度的重组病毒，可以用上述培养上清再次接种家蚕培养细胞，26～27℃培养3～5天后，收集细胞培养上清，获重组病毒BmNPV-Pr-VP5-Polh；(7) 重组病毒BmNPV-Pr-VP5-Polh接种家蚕培养细胞，26～27℃培养3-5天后，收集细胞，破碎细胞后通过差速离心获包裹外源蛋白的多角体；或(8)用步骤（6）获得的重组病毒BmNPV-Pr-VP5-Polh接种家蚕，发病后，收集家蚕，匀浆后通过差速离心纯化多角体，获包裹外源蛋白的多角体。通过步骤（7）可以获取少量的多角体；通过步骤（8）可以获取大量的多角体，考虑到成本因素，步骤（8）中接种所用的家蚕优先使用4－5龄幼虫或蚕蛹。通过上述技术方案获得的多角体，经SDS-PAGE和Western blotting检测，可检测到目的融合蛋白，且包裹在多角体中的目的蛋白具有生物学活性。 In the present invention, the sequence of VP5 of the silkworm plastopolyhedrosis virus is: GGACTGTCTCCAATAGCTTTGGCCCAGAAAAAACACGAAATGATGCTGCACACACACGAAATCCACAGCATGGACATCGATGGCTCAATT; specifically, as an illustration, the present invention prepares the method steps of the polyhedron wrapping the exogenous protein as follows: (1) the silkworm plastopolyhedrosis virus polyhedrosis protein The coding sequence of the gene (GenBank accession number: GQ924589) was cloned into the downstream of the polyhedron gene promoter of the vector pFastBac TM Dual (product of Invitrogen, USA) to construct the recombinant transfer plasmid pFastBac TM Dual-Polh; in this step, the silkworm plasmotype The coding sequence of the polyhedrosis protein gene of polyhedrosis virus can be obtained by PCR amplification after reverse transcription into cDNA by taking the genomic RNA of the virus or the total RNA of the midgut tissue infected by the virus as a template; The coding sequence of the body protein gene was obtained by chemical synthesis; (2) The multiple cloning site (MCS), the thrombin cleavage site (TCS), and the partial DNA sequence of the VP5 protein encoded by the silkworm plasmopolyhedrosis virus S7 fragment were concatenated in sequence , and a stop codon TAA was added at the 3' end, and then the MCS-TCS-VP5 sequence was obtained by chemical synthesis; in this step, a multiple cloning site (MCS) and a thrombin cleavage site ( underlined part), which is convenient for cloning and considers the detection of recombinant protein and the removal of VP5-tag from recombinant protein; the thrombin enzyme cleavage site in the sequence can be replaced by the cleavage site of factor Xa, enterokinase, etc.; MCS-TCS - The sequence of VP5 is as follows: MCS- CTCGTGCCTAGAGGTTCA GGACTGTCTCCAATAGCTTTGGCCCAGAAAAAACACGAAATGATGCTGCACACACACGAAATCCACAGCATGGACATCGATGGCTCAATTTAA; (3) MCS-TCS-VP5 sequence clone transfer plasmid pFastBac TM Dual-polh downstream of the P10 promoter obtained pFast-VP5-P olh; in this step, MCS-TCS - The VP5 sequence can be cloned into the downstream of the P10 promoter in pFastBac TM Dual-Polh by enzyme digestion, or by seamless cloning; (4) The sequence encoding the target protein without the stop codon is cloned into pFast-VP5 -The upstream of the MCS-TCS-VP5 sequence in the Polh plasmid or the multiple cloning site region in the MCS-VP5 sequence, and make the reading frame of the target gene and the reading frame of the thrombin restriction site and the VP5 protein partial sequence pFast-Pr-VP5-Polh is obtained by fusion; in this step, the target protein is a foreign protein that needs to be embedded, for example: green fluorescent protein, basic fibroblast growth factor, etc. In order to increase the expression level, the sequence encoding the target protein can be optimized according to the codon preference of silkworm. The coding sequence of the target protein can be cloned between the P10 promoter and the thrombin cleavage site in pFast-VP5-Polh by restriction enzyme ligation or seamless cloning, and ensure that the reading frame of the target gene is in line with the coagulation The enzyme cleavage site and the reading frame of the VP5-tag are consistent; (5) Transform pFast-Pr-VP5-Polh into DH10/Bac competent cells, and spread it on a medium containing tetracycline, kanamycin, gentamycin, On X-gal and IPTG LB agar plates, cultivate at 37°C, pick white colonies, and extract recombinant Bacmid-Pr-VP5-Polh DNA; in this step, pFast-Pr-VP5-Polh transformed DH10/Bac State cells were spread on LB agar culture plates containing tetracycline, kanamycin, gentamycin, IPTG and X-gal in order to facilitate the screening of recombinant Bacmid. Preferably, the contents of tetracycline, kanamycin, gentamycin, IPTG and X-gal on the LB agar culture plate are 10 µg/ml, 50 µg/ml, 7 µg/ml, 40 µg/ml and 100 µg/ml, respectively. µg/ml; (6) Transfect silkworm cultured cells with recombinant Bacmid-Pr-VP5-Polh DNA, culture at 26-27°C, and collect cell culture supernatant after cell onset; in order to obtain high-titer recombinant virus, you can use The above culture supernatant was inoculated again with silkworm cultured cells, and after culturing at 26-27°C for 3-5 days, the cell culture supernatant was collected to obtain the recombinant virus BmNPV-Pr-VP5-Polh; (7) Inoculation of the recombinant virus BmNPV-Pr-VP5-Polh Bombyx mori cultured cells, cultured at 26-27°C for 3-5 days, collected cells, broken cells and obtained polyhedrons wrapped with foreign proteins by differential centrifugation; or (8) using the recombinant virus BmNPV-Pr- obtained in step (6) VP5-Polh was inoculated with silkworms, and after the onset of disease, the silkworms were collected, homogenized, and polyhedrons were purified by differential centrifugation to obtain polyhedrons encapsulating exogenous proteins. A small amount of polyhedron can be obtained through step (7); a large amount of polyhedron can be obtained through step (8). Considering the cost factor, the silkworm used for inoculation in step (8) is preferably 4-5 instar larvae or silkworm chrysalis. The polyhedron obtained by the above technical scheme can detect the target fusion protein through SDS-PAGE and Western blotting, and the target protein encapsulated in the polyhedron has biological activity.
有益效果Beneficial effect
由于上述技术方案运用，本发明与现有技术相比具有下列优点：1. 本发明首次通过杆状病毒表达系统利用VP5-标签将外源蛋白包裹进多角体，具有高的包埋率。Due to the application of the above-mentioned technical solutions, the present invention has the following advantages compared with the prior art: 1. The present invention uses the VP5-tag to encapsulate foreign proteins into polyhedrons for the first time through the baculovirus expression system, and has a high embedding rate.
2. 常规的大肠杆菌系统表达的重组蛋白多以包涵体的形式存在，重组蛋白的纯化工艺复杂、分离纯化难度大；由于大肠杆菌不具有翻译后的修饰系统，往往导致重组蛋白的生物学活性较差、且因细胞周质中含有种类繁多的内毒素而影响制品的安全性。本发明可以将重组蛋白包裹在多角体内；多角体蛋白表达水平高，且多角体不溶于水，对恶劣环境条件的抵抗性强，可以通过简单的差速离心实现纯化，且多角体的纯化过程可以在常温条件下进行。纯净的多角体在碱性条件下快速裂解，当用盐酸溶液调pH至多角体蛋白等电点时，多角体蛋白形成絮状沉淀，可以通过离心去除多角体蛋白，而目标蛋白保留在上清中，从而可以快速、方便地获得纯化的重组蛋白。在本发明中，在目标蛋白与VP5-标签间设计了蛋白酶的特异性切割位点，该设计不仅能方便重组蛋白的检测，也能通过蛋白酶对重组蛋白进行切割，从而移除VP5-标签使产物更接近天然。2. Most of the recombinant proteins expressed in the conventional Escherichia coli system exist in the form of inclusion bodies, the purification process of recombinant proteins is complicated, and the separation and purification are difficult; since Escherichia coli does not have a post-translational modification system, the biological activity of recombinant proteins often results. It is poor and affects the safety of the product due to the wide variety of endotoxins contained in the periplasm. The present invention can wrap the recombinant protein in the polyhedron; the expression level of the polyhedron is high, and the polyhedron is insoluble in water, has strong resistance to harsh environmental conditions, and can be purified by simple differential centrifugation, and the purification process of the polyhedron It can be carried out under normal temperature conditions. The pure polyhedron is rapidly lysed under alkaline conditions. When the pH is adjusted to the isoelectric point of the polyhedrin protein with hydrochloric acid solution, the polyhedrin protein forms a flocculent precipitate, and the polyhedrin protein can be removed by centrifugation, while the target protein remains in the supernatant In this way, the purified recombinant protein can be obtained quickly and conveniently. In the present invention, a specific cleavage site for protease is designed between the target protein and the VP5-tag. This design not only facilitates the detection of recombinant proteins, but also cuts the recombinant proteins with proteases, thereby removing the VP5-tag and enabling The product is closer to nature.
3. 常规获得的重组蛋白极易发生降解，通常需冻干后在低温下保存。多角体能抵御酸、盐、变性剂等极端的环境条件以及蛋白酶，对包裹在其内部的重组蛋白有保护作用，因此本发明获得的包裹重组蛋白的多角体无需特殊处理，在常温下就可以长期保存。3. Routinely obtained recombinant proteins are easily degraded, and usually need to be freeze-dried and stored at low temperature. Polyhedrons can resist extreme environmental conditions such as acids, salts, and denaturants, as well as proteases, and have a protective effect on recombinant proteins wrapped inside them. Therefore, the polyhedrons wrapped with recombinant proteins obtained in the present invention do not need special treatment, and can be used for a long time at room temperature. save.
4. 多角体为二十面体或八面体、大小为2～3µm左右的蛋白微晶，不仅对包裹的蛋白有防降解作用，而且有缓控释作用，因此包裹多肽药物的多角体可作为缓释药物开发。4. The polyhedron is icosahedral or octahedral protein microcrystals with a size of about 2-3 μm, which not only prevents the degradation of the wrapped protein, but also has a slow and controlled release effect. Therefore, the polyhedron wrapped with polypeptide drugs can be used as a slow release drug development.
附图说明Description of drawings
图1为实施例一中的化学合成的MCS-TCS-VP5 N331-360序列的测序结果。 Fig. 1 is the sequencing result of the chemically synthesized MCS-TCS-VP5 N331-360 sequence in Example 1.
图2为实施例一中的Bacmid-EGFP-VP5-Polh的PCR鉴定。以Bacmid-EGFP-VP5-Polh基因组DNA为模板，用M13正向引物(5'-CCCAGTCACGACGTTGTAAAACG-3') (SEQ ID NO: 5)和M13反向引物 (5'-AGCGGATAACAATTTCACACAGG-3') (SEQ ID NO: 6) 进行PCR扩增。泳道1：野生型Bacmid DNA；泳道M：标准DNA分子量；泳道2：Bacmid-EGFP-VP5-Polh。Fig. 2 is the PCR identification of Bacmid-EGFP-VP5-Polh in Example 1. Using Bacmid-EGFP-VP5-Polh genomic DNA as template, use M13 forward primer (5'-CCCAGTCACGACGTTGTAAAACG-3') (SEQ ID NO: 5) and M13 reverse primer (5'-AGCGGATAACAATTTCACACAGG-3') (SEQ ID NO: 6) PCR amplification was performed. Lane 1: wild-type Bacmid DNA; lane M: molecular weight standard DNA; lane 2: Bacmid-EGFP-VP5-Polh.
图3为实施例一中的BmNPV-EGFP-VP5-Polh在培养细胞中形成的多角体的荧光观察。Fig. 3 is the fluorescence observation of the polyhedrons formed by BmNPV-EGFP-VP5-Polh in the cultured cells in Example 1.
图4为实施例一中BmNPV-EGFP-VP5-Polh在家蚕血淋巴中形成的多角体的Western blot检测。泳道NC，野生型家蚕质型多角体；泳道EGFP，BmNPV-EGFP-VP5-Polh形成的多角体。一抗为鼠抗GFP单克隆抗体，二抗为HRP标记的羊抗鼠IgG。Fig. 4 is the Western blot detection of the polyhedron formed by BmNPV-EGFP-VP5-Polh in the silkworm hemolymph in Example 1. Lane NC, plastid polyhedron of wild-type silkworm; Lane EGFP, polyhedron formed by BmNPV-EGFP-VP5-Polh. The primary antibody was mouse anti-GFP monoclonal antibody, and the secondary antibody was HRP-labeled goat anti-mouse IgG.
图5为实施例一中的Western blot检测多角体对包埋的绿色荧光蛋白的保护作用。“EGFP-Polyhedra”代表纯化的包埋绿色荧光蛋白的多角体；“温度℃”代表多角体保存温度（25℃或-20℃）；“裂解”代表多角体在保存前是否用0.1mol/L的Na 2CO 3-NaHCO 3进行裂解处理，“-”代表没有进行裂解处理，“+”代表进行裂解处理；“polyhedrin”代表一抗为鼠抗多角体蛋白抗体；“GFP”代表鼠抗绿色荧光蛋白的单抗。二抗为HRP标记的羊抗鼠IgG。 FIG. 5 is a Western blot detection of the protective effect of polyhedron on embedded green fluorescent protein in Example 1. FIG. "EGFP-Polyhedra" represents the purified polyhedron embedded with green fluorescent protein; "Temperature ℃" represents the storage temperature of the polyhedron (25°C or -20°C); The Na 2 CO 3 -NaHCO 3 was lysed, "-" means no lysing, "+" means lysing; "polyhedrin" means the primary antibody is mouse anti-polyhedrin antibody; "GFP" means mouse anti-green Monoclonal antibodies to fluorescent proteins. The secondary antibody was HRP-labeled goat anti-mouse IgG.
图6实施例二中的化学合成的碱性成纤维细胞生长因子编码序列的测序鉴定。图中仅显示部分测序结果。Figure 6 Sequencing identification of the coding sequence of the chemically synthesized basic fibroblast growth factor in Example 2. Only partial sequencing results are shown in the figure.
图7实施例二中的重组Bacmid-bFGF-VP5-Polh的PCR鉴定。以Bacmid-bFGF-VP5-Polh基因组DNA为模板，用M13正向引物(5'-CCCAGTCACGACGTTGTAAAACG-3') (SEQ ID NO: 5) 和 M13 反向引物 (5'-AGCGGATAACAATTTCACACAGG-3') (SEQ ID NO: 6) 进行PCR扩增。泳道M：标准DNA分子量；泳道1：野生型Bacmid DNA；泳道3：Bacmid-bFGF-VP5-Polh。Figure 7 PCR identification of recombinant Bacmid-bFGF-VP5-Polh in Example 2. Using Bacmid-bFGF-VP5-Polh genomic DNA as template, use M13 forward primer (5'-CCCAGTCACGACGTTGTAAAACG-3') (SEQ ID NO: 5) and M13 reverse primer (5'-AGCGGATAACAATTTCACACAGG-3') (SEQ ID NO: 6) was amplified by PCR. Lane M: molecular weight standard DNA; Lane 1: wild-type Bacmid DNA; Lane 3: Bacmid-bFGF-VP5-Polh.
图8为实施例二中BmNPV-bFGF-VP5-Polh在家蚕血淋巴中形成多角体的Western blot检测。泳道NC，野生型家蚕质型多角体；泳道bFGF，BmNPV-bFGFP-VP5-Polh形成的多角体。一抗为兔抗bFGF单克隆抗体，二抗为HRP标记的羊抗兔IgG。FIG. 8 is a Western blot detection of polyhedrons formed by BmNPV-bFGF-VP5-Polh in the silkworm hemolymph in Example 2. FIG. Lane NC, plastid polyhedron of wild-type silkworm; Lane bFGF, polyhedron formed by BmNPV-bFGFP-VP5-Polh. The primary antibody was rabbit anti-bFGF monoclonal antibody, and the secondary antibody was HRP-labeled goat anti-rabbit IgG.
图9为实施例二中通过Western blot检测家蚕血淋巴中表达的碱性成纤维细胞生长因子包埋进多角体的比例。BmNPV-bFGF-VP7-Polh感染五龄家蚕7天后的血淋巴经细胞破碎后，3000转/分，离心10分钟，得上清和沉淀（多角体），SDS-PAGE分离后，用Western blot检测上清和沉淀中的碱性成纤维细胞生长因子。“bFGF-Serum”代表血淋巴离心后的上清，“bFGF-polyhedra” 代表血淋巴离心的沉淀。一抗为兔抗bFGF单克隆抗体，二抗为HRP标记的羊抗兔IgG。FIG. 9 is a Western blot detection of the proportion of basic fibroblast growth factor expressed in silkworm hemolymph embedded into polyhedrons in Example 2. FIG. BmNPV-bFGF-VP7-Polh infected five-instar silkworm hemolymph 7 days after the cells were crushed, centrifuged at 3000 rpm for 10 minutes, supernatant and precipitate (polyhedron) were obtained, separated by SDS-PAGE, and detected by Western blot Basic fibroblast growth factor in serum and pellet. "bFGF-Serum" represents the supernatant after hemolymph centrifugation, and "bFGF-polyhedra" represents the pellet of hemolymph centrifugation. The primary antibody was rabbit anti-bFGF monoclonal antibody, and the secondary antibody was HRP-labeled goat anti-rabbit IgG.
图10为实施例二中的细胞培养检测碱性成纤维细胞生长因子的生物学活性。细胞用吉姆萨染色。Lysate of bFGF-polyhedra代表培养L-929细胞的培养液中含有用Na 2CO 3-NaHCO 3裂解bFGF-polyhedra的裂解液，bFGF-polyhedra代表培养L-929细胞的培养液中用含有bFGF-polyhedra，WT BmCPV polyhedra代表培养L-929细胞的培养液中用含有野生型多角体，Con为空白对照组L-929细胞。 Figure 10 shows the biological activity of basic fibroblast growth factor detected by cell culture in Example 2. Cells were stained with Giemsa. Lysate of bFGF-polyhedra represents the culture medium of L-929 cells containing the lysate of bFGF-polyhedra lysed with Na 2 CO 3 -NaHCO 3 , and bFGF-polyhedra represents the culture medium of L-929 cells with bFGF-polyhedra , WT BmCPV polyhedra represents the medium used to culture L-929 cells containing wild-type polyhedra, and Con represents the blank control group L-929 cells.
本发明的实施方式Embodiments of the present invention
本发明涉及的具体操作方法为常规方法，比如克隆、酶切的具体操作；涉及的测试方法也为常规技术；除了特别指出设计的序列外，涉及的原料试剂都为常规产品，其中的pFastBac TMDual是美国Invitrogen公司的产品，属于Bac-to-Bac（Bacteria to Baculovirus）表达系统载体。下面结合附图及实施例对本发明作进一步描述。 The specific operation methods involved in the present invention are conventional methods, such as the specific operations of cloning and enzyme digestion; the test methods involved are also conventional techniques; except for the sequence of design, the raw material reagents involved are all conventional products, among which pFastBacTM Dual is a product of Invitrogen Corporation of the United States, which belongs to the Bac-to-Bac (Bacteria to Baculovirus) expression system vector. The present invention will be further described below in conjunction with the accompanying drawings and embodiments.
实施例一包埋绿色荧光蛋白多角体的构建。Example 1 Construction of green fluorescent protein-embedded polyhedrons.
(1)常规提取家蚕质型多角体病毒感染中肠组织的总RNA，用RNA PCR KitVer.3.0 (Qiagen公司) 按产品录说明书进行反转录，获中肠组织cDNA，根据家蚕质型多角体病毒多角体蛋白基因的编码序列 (GenBank登录号：GQ924589)设计引物polh-EI (GAATTCATGGCAGACGTAGCAGGAACA，带有 EcoR
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酶切位点）和polh-XB (TCTAGATCACTGACGGTTACTCAGAGC，带有 Xba
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酶切位点），通过PCR扩增5’-和3’-端分别带 EcoR
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和 Xba
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位点的质型多角体蛋白基因的编码序列，克隆进T-载体，经过测序验证后，用 EcoR
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和 Xba
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双酶切克隆进同样酶切的pFastBac TMDual，获重组转移质粒pFastBac TM Dual-Polh； (2) 按SEQ ID NO：1化学合成3’端带有 KpnI位点的MCS-TCS-VP5序列，其中MCS序列含有 XhoI、 SphI、 NcoI、 NheI、 PvuII、 NsiII酶切位点；SEQ ID NO: 1：CTCGAGGCATGCCCATGGGCTAGCAGCTGTATGCATCTCGTGCCTAGAGGTTCAGGACTGTCTCCAATAGCTTTGGCCCAGAAAAAACACGAAATGATGCTGCACACACACGAAATCCACAGCATGGACATCGATGGCTCAATTTAA；(3) 步骤（2）合成的序列MCS-TCS-VP5测序验证（图1）后克隆进pFastBac TM Dual-Polh的 XhoI、 KpnI之间，获pFast-VP5-Polh； （4）根据SEQ ID NO:2所示序列人工合成优化绿色荧光蛋白基因序列，并在5’、3’端分别添加 XhoI、 SphI的位点，经过测序验证后，克隆至pFast-VP5-Polh的 XhoI、 SphI的位点获pFast-EGFP-VP5-Polh；SEQ ID NO: 2：ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAG；（5）pFast-EGFP-VP5-Polh转化DH10/Bac感受态细胞，涂布于含四环素（10 µg/ml）、卡那霉素（50 µg/ml）、庆大霉素（7 µg/ml）、IPTG（40µg/ml）、X-gal（100 µg/ml）的LB 琼脂培养平板上，于37℃培养48小时，挑取白色菌落培养，提取重组Bacmid基因组DNA，用M13 正向引物(5'-CCCAGTCACGACGTTGTAAAACG-3')和 M13 反向引物 (5'-AGCGGATAACAATTTCACACAGG-3')对重组Bacmid基因组DNA进行PCR，可从重组Bacmid DNA中扩增出与理论分子量（目的基因+2650bp）一致的目的条带(图2)，说明已按要求正确构建了重组Bacmid DNA，命名为Bacmid-EGFP-VP5-Polh；（6）Bacmid-EGFP-VP5-Polh (4 μg)在脂质体（FuGENE HD transfection reagent，Roche公司）的介导下转染1×10 6家蚕培养细胞，27℃培养5天，收取发病培养细胞上清，获P1代重组病毒BmNPV-EGFP-VP5-Polh。取P1代病毒感染家蚕培养细胞，培养5天后，收取病毒感染的培养细胞上清，获P2代重组病毒，4℃避光保存；（7）用感染复数为5的P2代重组病毒感染家蚕培养细胞，27℃培养4天，收集培养细胞，细胞破碎后，经1000转/分和3000转/分的差速离心（反复三次），获得纯化的多角体EGFP-Polyhedra。该多角体在荧光显微镜可观察到发出的绿色荧光（图3），说明绿色荧光蛋白在VP5-标签的引导下被包埋进多角体；（8）用昆虫针醮取P2代重组病毒，从节间膜处穿刺接种5龄起蚕，25℃饲养，7天后收集发病蚕的血淋巴，感染发病的蚕的血淋巴通过1000转/分和3000转/分的差速离心（反复三次），可纯化到多角体。纯化的多角体经SDS-PAGE分离后，将PAGE胶上的蛋白转移到PVDF膜（Roche公司）上，用绿色荧光蛋白的抗体进行Western blot检测，可检测到代表重组绿色蛋白的杂交信号（图4），说明重组绿色蛋白被包埋进行多角体；（9）取2×10 7个纯化的多角体EGFP-Polyhedra（100 µL） 4份，其中2份用等体积的0.1mol/L的Na 2CO 3-NaHCO 3在28℃裂解25分钟后，分别在25℃和-20℃保存14天；另外2份先分别在25℃和-20℃保存14天后，再用等体积的0.1mol/L的Na 2CO 3-NaHCO 3在28℃裂解25分钟。上述样品经SDS-PAGE分离后，用多角体蛋白的抗体、绿色荧光蛋白的抗体进行Western blot检测。与裂解后的多角体放在-20℃保存14天的样本相比，裂解后的多角体放在25℃保存14天，基本上观察不到代表多角体蛋白、绿色荧光蛋白的信号条带，说明多角体蛋白、绿色荧光蛋白在保存过程发生明显的降解，而没有裂解的多角体在25℃或-20℃保存14天仍可观察到代表多角体蛋白、绿色荧光蛋白的信号条带，说明多角体对包埋的绿色荧光蛋白有保护作用（图5）。 (1) Routinely extract the total RNA from the midgut tissue infected with silkworm plasmopolyhedrosis virus, use RNA PCR KitVer. The coding sequence of viral polyhedrin gene (GenBank accession number: GQ924589) designed primer polh-EI (GAATTCATGGCAGACGTAGCAGGAACA, with Eco R
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site) and polh-XB (TCTAGATCACTGACGGTTACTCAGAGC with Xba
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Restriction site), by PCR amplification of 5'- and 3'-ends with Eco R
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and Xba
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The coding sequence of the plastid polyhedrin gene at the locus was cloned into the T-vector, and after sequencing verification, Eco R
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and Xba
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Double-enzyme-digested and cloned into pFastBac TM Dual that was also digested to obtain the recombinant transfer plasmid pFastBac TM Dual-Polh; (2) Chemically synthesize the MCS-TCS-VP5 sequence with a Kpn I site at the 3' end according to SEQ ID NO:1 , wherein the MCS sequence contains Xho I, Sph I, Nco I, Nhe I, Pvu II, Nsi II restriction sites; SEQ ID NO: 1: CTCGAGGCATGCCCATGGGCTAGCAGCTGTATGCATCTCGTGCCTAGAGGTTCAGGACTGTCTCCAATAGCTTTGGCCCAGAAAAAACACGAAATGATGCTGCACACACACGAAATCCACAGCATGGACATCGATG GCTCAATTTAA; (3) The sequence MCS-TCS-VP5 synthesized in step (2) After sequencing verification (Figure 1), clone into pFastBac TM Dual-Polh between Xho I and Kpn I to obtain pFast-VP5-Polh; (4) Artificially synthesize and optimize the green fluorescent protein gene sequence according to the sequence shown in SEQ ID NO:2 , and add Xho I and Sph I sites at the 5' and 3' ends respectively, after sequencing verification, clone into the Xho I and Sph I sites of pFast-VP5-Polh to obtain pFast-EGFP-VP5-Polh; SEQ ID NO: 2: ; (5) pFast-EGFP-VP5-Polh transformed DH10/Bac competent cells, coated on tetracycline (10 µg/ml), kanamycin (50 µg/ml), Genda Mycin (7 µg/ml), IPTG (40 µg/ml), X-gal (100 µg/ml) LB agar culture plate, cultured at 37°C for 48 hours, picked white colonies for culture, and extracted recombinant Bacmid genomic DNA , use M13 forward primer (5'-CCCAGTCACGACGTTGTAAAACG-3') and M13 reverse primer (5'-AGCGGATAACAATTTCACACAGG-3') to perform PCR on recombinant Bacmid genomic DNA, which can be amplified from recombinant Bacmid DNA with theoretical molecular weight ( Target gene +2650bp) consistent target band (Figure 2), indicating that the recombinant Bacmid DNA has been correctly constructed as required, named Bacmid-EGFP-VP5-Polh; (6) Bacmid-EGFP-VP5-Polh (4 μg) Under the mediation of liposomes (FuGENE HD transfection reagent, Roche Company), 1× 106 cultured silkworm cells were transfected, cultured at 27°C for 5 days, and the supernatant of diseased cultured cells was collected to obtain the P1 generation recombinant virus BmNPV-EGFP-VP5 -Polh. Take the P1 virus-infected silkworm cultured cells, and after 5 days of culture, collect the supernatant of the virus-infected cultured cells to obtain the P2-generation recombinant virus and store it in the dark at 4°C; (7) Infect the silkworm with the P2-generation recombinant virus with a multiplicity of infection of 5 and culture The cells were cultured at 27°C for 4 days, and the cultured cells were collected. After the cells were broken, they were subjected to differential centrifugation at 1000 rpm and 3000 rpm (repeated three times) to obtain purified polyhedron EGFP-Polyhedra. The green fluorescence of the polyhedron can be observed under the fluorescence microscope (Figure 3), indicating that the green fluorescent protein was embedded into the polyhedron under the guidance of the VP5-tag; The intersegmental membrane was punctured to inoculate the 5th instar silkworm, raised at 25°C, and the hemolymph of the infected silkworm was collected after 7 days. The hemolymph of the infected silkworm was subjected to differential centrifugation at 1000 rpm and 3000 rpm (repeated three times). Can be purified to polyhedrons. After the purified polyhedron was separated by SDS-PAGE, the protein on the PAGE gel was transferred to PVDF membrane (Roche Company), and Western blot detection was performed with the antibody of green fluorescent protein, and the hybridization signal representing the recombinant green protein could be detected (Fig. 4), indicating that the recombinant green protein was embedded into polyhedra; (9) Take 2×10 7 purified polyhedron EGFP-Polyhedra (100 µL) in 4 parts, 2 of which were treated with an equal volume of 0.1mol/L Na 2 CO 3 -NaHCO 3 was cracked at 28°C for 25 minutes, and stored at 25°C and -20°C for 14 days respectively; L of Na 2 CO 3 -NaHCO 3 was cleaved at 28 °C for 25 min. After the above samples were separated by SDS-PAGE, they were detected by Western blot with antibodies against polyhedrin and green fluorescent protein. Compared with the sample in which the lysed polyhedron was stored at -20°C for 14 days, the lysed polyhedron was stored at 25°C for 14 days, and the signal bands representing polyhedrin and green fluorescent protein were basically not observed. It shows that the polyhedrin and green fluorescent protein are obviously degraded during the preservation process, and the polyhedrin without cracking can still be observed for 14 days at 25°C or -20°C. The signal bands representing the polyhedrin and green fluorescent protein indicate that Polyhedrons protect the embedded GFP (Figure 5).
实施例二包埋碱性成纤维细胞生长因子多角体的构建。Example 2 Construction of Polyhedrons Encapsulating Basic Fibroblast Growth Factor.
(1)家蚕质型多角体用0.1mol/L的Na 2CO 3-NaHCO 3在28℃裂解25分钟，用盐酸调pH至多角体蛋白的等电点，12000转/分离心10分钟，上清依次用酚、酚/氯仿（1∶1体积比）、氯仿抽提后，用乙醇沉淀RNA，尔后用RNA PCR KitVer.3.0 (Qiagen公司) 按产品录说明书进行反转录，获cDNA，用实施例一步骤（1）中的引物（polh-EI、polh-XB）通过PCR扩增质型多角体蛋白基因的编码序列，克隆进T-载体，经过测序验证后，用 EcoR
Figure dest_path_image001aaaaaaaaaaaaaaaaaaaaaaa
和 Xba
Figure dest_path_image001aaaaaaaaaaaaaaaaaaaaaaaa
双酶切克隆进同样酶切的pFastBac TMDual，获重组转移质粒pFastBac TM Dual-Polh；(2)同实施例一中的步骤（2）。 (1) The silkworm plastopolyhedron was cracked with 0.1mol/L Na 2 CO 3 -NaHCO 3 at 28°C for 25 minutes, adjusted to the isoelectric point of the polyhedrin with hydrochloric acid, centrifuged at 12,000 rpm for 10 minutes, and After the clear was extracted with phenol, phenol/chloroform (1:1 volume ratio), and chloroform successively, the RNA was precipitated with ethanol, and then reverse-transcribed with RNA PCR KitVer. The primers (polh-EI, polh-XB) in step (1) of Example 1 amplified the coding sequence of the plasmopolyhedrin gene by PCR, cloned into T-vector, and after sequencing verification, used Eco R
Figure dest_path_image001aaaaaaaaaaaaaaaaaaaaaaa
and Xba
Figure dest_path_image001aaaaaaaaaaaaaaaaaaaaaaaa
Double-enzyme digestion cloned into pFastBac TM Dual that was also digested to obtain the recombinant transfer plasmid pFastBac TM Dual-Polh; (2) Same as step (2) in Example 1.
(3)同实施例一中的步骤（3）。(3) Same as step (3) in Example 1.
(4)根据SEQ ID NO:3所示序列人工合成优化碱性成纤维细胞生长因子序列，并在5’端、3’端分别添加 XhoI、 SphI的位点，经过测序验证后(图6)，克隆至pFast-VP5-Polh的 XhoI、 SphI的位点获pFast-bFGF-VP5-Polh；SEQ ID NO:3：ATGGCTGCCGGCTCGATAACAACTCTGCCGGCTTTGCCCGAAGACGGTGGAAGTGGCGCCTTCCCTCCAGGTCACTTCAAAGATCCTAAGAGATTGTACTGCAAAAACGGCGGTTTCTTCCTCAGAATCCACCCGGACGGTAGAGTCGATGGAGTGAGAGAAAAATCCGACCCCCACATCAAGCTCCAACTGCAGGCTGAAGAAAGAGGTGTGGTTTCAATTAAGGGAGTTTGTGCTAACAGATACCTGGCCATGAAAGAAGACGGAAGACTGTTGGCCTCAAAGTGCGTCACAGATGAATGCTTCTTCTTCGAAAGATTGGAATCTAACAACTACAACACCTACAGAAGCAGAAAATACACATCCTGGTACGTGGCTCTCAAGAGAACTGGACAATACAAACTGGGCAGCAAGACCGGACCTGGCCAGAAAGCTATCTTGTTCCTCCCAATGTCAGCCAAGTCT；(5) 用pFast-bFGF-VP5-Polh替代实施例一步骤（5）的pFast-EGFP-VP5-Polh，按实施例一步骤（5）的方法构建重组Bacmid，并用M13正反向引物进行PCR鉴定。可从重组Bacmid DNA中扩增出与理论分子量（目的基因+2650bp）一致的目的条带(图7)，说明已按要求正确构建了重组Bacmid，命名为Bacmid-bFGF-VP5-Polh；(6)用Bacmid-bFGF-VP5-Polh替代实施例一步骤（6）中Bacmid-EGFP-VP5-Polh，按实施例一步骤（6）的方案制备P1代重组病毒BmNPV-bFGF-VP5-Polh；取P1代病毒感染家蚕培养细胞，培养5天后，收取病毒感染的培养细胞上清，获P2代重组病毒，-80℃保存； (7) 用感染复数为5的P2代重组病毒感染家蚕培养细胞，27℃培养4天，收集培养细胞，细胞破碎后，经1000转/分和3000转/分的反复差速离心（五次），获得纯化的多角体bFGF-Polyhedra；(8) 用昆虫针醮取P2代重组病毒，从节间膜处穿刺接种5龄起蚕，25℃饲养，7天后收集发病蚕的血淋巴，感染发病的蚕的血淋巴通过1000转/分和3000转/分的反复差速离心（五次），可纯化得到多角体bFGF-Polyhedra。纯化的多角体经SDS-PAGE分离后，将PAGE胶上的蛋白转移到PVDF膜（Roche公司）上，用碱性成纤维细胞生长因子的抗体进行Western blot检测，可检测到代表碱性成纤维细胞生长因子的杂交信号（图8），说明重组碱性成纤维细胞生长因子被包埋进行多角体；(9)取步骤（8）中的血淋巴，破碎细胞后，3000转/分离心10分钟，得上清和沉淀（多角体），SDS-PAGE分离后，用兔抗bFGF单克隆抗体通过Western blot检测上清和沉淀中的碱性成纤维细胞生长因子（图9），通过灰度扫描分析测得在家蚕血淋巴中表达的重组碱性成纤维细胞生长因子有51%被包埋进多角体中； (10) 在24孔板中选取4个孔，每孔添加500μL高糖培养基，尔后分别添加用0.1mol/L的Na 2CO 3-NaHCO 3裂解的5×10 5个bFGF-polyhedra的裂解液（2.5μL）、5×10 5个bFGF-polyhedra （2.5μL）、野生性多角体5×10 5个（2.5μL），并同设空白对照。将L-929细胞接种在Transwell小室中，5×10 3个/孔，小室内细胞生长于100μL高糖培养基。将接种细胞的小室放于上述含有多角体或多角体裂解液的24孔板的孔中，37℃培养48小时，尔后作吉姆萨染色（图10），结果显示，小室中的细胞数量按bFGF-polyhedra的裂解液组、bFGF-polyhedra组、野生型多角体组依次减少，野生型多角体组的细胞数量与空白对照无明显区别。说明从bFGF-polyhedra经Na 2CO 3-NaHCO 3裂解处理释放出的重组成纤维细胞生长因子具有生物学活性，促进细胞生长；包埋在多角体中的重组成纤维细胞生长因子在细胞培养条件下可缓慢释放。 (4) Artificially synthesize and optimize the basic fibroblast growth factor sequence according to the sequence shown in SEQ ID NO:3, and add Xho I and Sph I sites at the 5' end and the 3' end respectively, after sequencing verification (Fig. 6)，克隆至pFast-VP5-Polh的Xho I、 Sph I的位点获pFast-bFGF-VP5-Polh；SEQ ID NO:3：ATGGCTGCCGGCTCGATAACAACTCTGCCGGCTTTGCCCGAAGACGGTGGAAGTGGCGCCTTCCCTCCAGGTCACTTCAAAGATCCTAAGAGATTGTACTGCAAAAACGGCGGTTTCTTCCTCAGAATCCACCCGGACGGTAGAGTCGATGGAGTGAGAGAAAAATCCGACCCCCACATCAAGCTCCAACTGCAGGCTGAAGAAAGAGGTGTGGTTTCAATTAAGGGAGTTTGTGCTAACAGATACCTGGCCATGAAAGAAGACGGAAGACTGTTGGCCTCAAAGTGCGTCACAGATGAATGCTTCTTCTTCGAAAGATTGGAATCTAACAACTACAACACCTACAGAAGCAGAAAATACACATCCTGGTACGTGGCTCTCAAGAGAACTGGACAATACAAACTGGGCAGCAAGACCGGACCTGGCCAGAAAGCTATCTTGTTCCTCCCAATGTCAGCCAAGTCT；(5) 用pFast-bFGF-VP5-Polh替代实施例For the pFast-EGFP-VP5-Polh in the first step (5), construct the recombinant Bacmid according to the method in the first step (5) of Example 1, and use M13 forward and reverse primers for PCR identification. A target band consistent with the theoretical molecular weight (target gene + 2650bp) can be amplified from the recombinant Bacmid DNA (Figure 7), indicating that the recombinant Bacmid has been correctly constructed as required, named Bacmid-bFGF-VP5-Polh; (6 ) use Bacmid-bFGF-VP5-Polh to replace Bacmid-EGFP-VP5-Polh in step (6) of Example 1, and prepare the P1 generation recombinant virus BmNPV-bFGF-VP5-Polh according to the scheme of step (6) of Example 1; The P1 generation virus infected silkworm cultured cells, and after culturing for 5 days, the supernatant of the virus-infected cultured cells was collected to obtain the P2 generation recombinant virus, and stored at -80°C; (7) Infect the silkworm cultured cells with the P2 generation recombinant virus with a multiplicity of infection of 5, Cultivate at 27°C for 4 days, collect the cultured cells, and after the cells are broken, undergo repeated differential centrifugation at 1000 rpm and 3000 rpm (five times) to obtain the purified polyhedron bFGF-Polyhedra; (8) use insect needles Take the P2 generation recombinant virus, inoculate silkworms from the 5th instar from the intersegmental membrane, raise them at 25°C, and collect the hemolymph of infected silkworms after 7 days. Differential centrifugation (five times), the polyhedron bFGF-Polyhedra can be purified. After the purified polyhedron was separated by SDS-PAGE, the protein on the PAGE gel was transferred to PVDF membrane (Roche Company), and Western blot detection was performed with the antibody of basic fibroblast growth factor, and the protein representing basic fibroblast growth factor could be detected. The hybridization signal of cell growth factor (Figure 8) shows that the recombinant basic fibroblast growth factor is embedded into polyhedron; (9) take the hemolymph in step (8), break the cells, and centrifuge at 3000 rpm for 10 Minutes, get supernatant and precipitate (polyhedron), after SDS-PAGE separation, use rabbit anti-bFGF monoclonal antibody to detect basic fibroblast growth factor in supernatant and precipitate by Western blot (Figure 9), and analyze by grayscale scanning It was measured that 51% of the recombinant basic fibroblast growth factor expressed in silkworm hemolymph was embedded in polyhedrons; (10) select 4 wells in a 24-well plate, and add 500 μL of high-glucose medium to each well, Then add the lysate (2.5 μL) of 5×10 5 bFGF-polyhedra lysed with 0.1 mol/L Na 2 CO 3 -NaHCO 3 , 5×10 5 bFGF-polyhedra (2.5 μL), wild polyhedra 5×10 5 cells (2.5 μL), and a blank control was also set up. L-929 cells were seeded in Transwell chambers, 5×10 3 cells/well, and the cells in the chambers were grown in 100 μL of high-glucose medium. Put the chamber inoculated with cells into the wells of the above-mentioned 24-well plate containing polyhedron or polyhedron lysate, incubate at 37°C for 48 hours, and then perform Giemsa staining (Figure 10). The results show that the number of cells in the chamber is determined by bFGF -polyhedra lysate group, bFGF-polyhedra group, and wild-type polyhedron group decreased sequentially, and the number of cells in the wild-type polyhedron group had no significant difference from the blank control. It shows that the recombinant fibroblast growth factor released from bFGF-polyhedra after Na 2 CO 3 -NaHCO 3 cleavage treatment has biological activity and promotes cell growth; Press down for a slow release.

Claims (10)

  1. 一种制备包裹外源蛋白的多角体的方法，其特征在于，包括下列步骤：A method for preparing polyhedrons wrapping foreign proteins, characterized in that it comprises the following steps:
    (1) 将家蚕质型多角体病毒多角体蛋白基因的编码序列克隆进Bac-to-Bac表达系统载体的多角体基因启动子下游，构建重组转移质粒；(1) Cloning the coding sequence of the polyhedrin gene of the silkworm plasmopolyhedrosis virus into the downstream of the polyhedrin gene promoter of the Bac-to-Bac expression system vector to construct a recombinant transfer plasmid;
    （2）合成由多克隆位点、蛋白酶酶切位点、家蚕质型多角体病毒VP5序列、终止密码子依次串联的MCS-VP5序列；所述家蚕质型多角体病毒VP5序列为：GGACTGTCTCCAATAGCTTTGGCCCAGAAAAAACACGAAATGATGCTGCACACACACGAAATCCACAGCATGGACATCGATGGCTCAATT(2) Synthesizing the MCS-VP5 sequence consisting of the multiple cloning site, the protease cleavage site, the VP5 sequence of the silkworm plasmopolyhedrosis virus, and the stop codon in sequence; the VP5 sequence of the silkworm plasmopolyhedrosis virus is: GGACTGTCTCCAATAGCTTTGGCCCAGAAAAAACACGAAATGATGCTGCACACACACGAAATCCAGCATGGACATCGATGGCTCAATT
    （3）将MCS-VP5序列克隆至步骤（1）的重组转移质粒的P10启动子的下游获得pFast-VP5-Polh；(3) Cloning the MCS-VP5 sequence to the downstream of the P10 promoter of the recombinant transfer plasmid in step (1) to obtain pFast-VP5-Polh;
    （4）将去除终止密码的编码目的蛋白的序列克隆在pFast-VP5-Polh质粒中的MCS-VP5序列的上游或MCS-VP5序列中的多克隆位点区域，获得pFast-Pr-VP5-Polh；(4) Cloning the sequence encoding the target protein without the stop codon into the upstream of the MCS-VP5 sequence in the pFast-VP5-Polh plasmid or the multiple cloning site region in the MCS-VP5 sequence to obtain pFast-Pr-VP5-Polh ;
    (5)将pFast-Pr-VP5-Polh转化感受态细胞，涂布于LB琼脂平板上，培养后挑取白色菌落，提取重组Bacmid-Pr-VP5-Polh DNA；(5) transform pFast-Pr-VP5-Polh into competent cells, smear them on LB agar plates, pick white colonies after cultivation, and extract recombinant Bacmid-Pr-VP5-Polh DNA;
    (6) 将重组Bacmid-Pr-VP5-Polh DNA转染家蚕培养细胞，培养至细胞发病后，收集细胞培养上清，获重组病毒BmNPV-Pr-VP5-Polh；(6) The recombinant Bacmid-Pr-VP5-Polh DNA was transfected into cultured silkworm cells, and cultured until the cells became onset, the cell culture supernatant was collected to obtain the recombinant virus BmNPV-Pr-VP5-Polh;
    (7) 重组病毒BmNPV-Pr-VP5-Polh接种家蚕培养细胞，培养后，收集细胞，破碎细胞后通过差速离心获包裹外源蛋白的多角体；(7) The recombinant virus BmNPV-Pr-VP5-Polh was inoculated into silkworm cultured cells, after culturing, the cells were collected, and after the cells were broken, polyhedrons wrapped with foreign proteins were obtained by differential centrifugation;
    或or
    (8)用步骤（6）获得的重组病毒BmNPV-Pr-VP5-Polh接种家蚕，发病后，收集家蚕，匀浆后通过差速离心纯化多角体，获包裹外源蛋白的多角体。(8) The recombinant virus BmNPV-Pr-VP5-Polh obtained in step (6) was used to inoculate silkworms. After the onset of the disease, the silkworms were collected, homogenized and purified by differential centrifugation to obtain polyhedrons encapsulating foreign proteins.
  2. 根据权利要求1所述制备包裹外源蛋白的多角体的方法，其特征在于，步骤（1）中，家蚕质型多角体病毒多角体蛋白基因的编码序列通过PCR扩增获得；或者化学合成获得；Bac-to-Bac表达系统载体包括pFastBac TMDual载体。 The method for preparing polyhedrons encapsulating foreign proteins according to claim 1, characterized in that, in step (1), the coding sequence of the polyhedrin gene of silkworm plastopolyhedrosis virus is obtained by PCR amplification; or obtained by chemical synthesis ; Bac-to-Bac expression system vectors include pFastBac TM Dual vectors.
  3. 根据权利要求1所述制备包裹外源蛋白的多角体的方法，其特征在于，步骤（2）中，蛋白酶酶切位点为凝血酶酶切位点、Xa因子酶切位点或者肠激酶酶切位点；终止密码子为TAA或TAG或TGA。The method for preparing polyhedrons encapsulating foreign proteins according to claim 1, wherein in step (2), the protease cleavage site is a thrombin cleavage site, factor Xa cleavage site or enterokinase enzyme cutting site; the stop codon is TAA or TAG or TGA.
  4. 根据权利要求1所述制备包裹外源蛋白的多角体的方法，其特征在于，步骤（3）中，克隆为酶切连接克隆或者无缝克隆。The method for preparing polyhedrons encapsulating foreign proteins according to claim 1, characterized in that, in step (3), the cloning is enzyme-cut junction cloning or seamless cloning.
  5. 根据权利要求1所述制备包裹外源蛋白的多角体的方法，其特征在于，步骤（4）中，目的基因的读码框与蛋白酶酶切位点、家蚕质型多角体病毒VP5序列的读码框融合获得pFast-Pr-VP5-Polh。The method for preparing a polyhedron encapsulating an exogenous protein according to claim 1, characterized in that, in step (4), the reading frame of the target gene and the protease cleavage site, the reading of the Bombyx mori plasmopolyhedrosis virus VP5 sequence Code frame fusion obtained pFast-Pr-VP5-Polh.
  6. 根据权利要求1所述制备包裹外源蛋白的多角体的方法，其特征在于，步骤（5）中，感受态细胞为DH10/Bac感受态细胞；LB琼脂平板含四环素、卡那霉素、庆大霉素、X-gal、IPTG。The method for preparing polyhedrons encapsulating foreign proteins according to claim 1, characterized in that, in step (5), the competent cells are DH10/Bac competent cells; LB agar plates contain tetracycline, kanamycin, Amamicin, X-gal, IPTG.
  7. 根据权利要求1所述制备包裹外源蛋白的多角体的方法，其特征在于，步骤（6）中，培养温度为26～27℃；步骤（7）中，培养为26～27℃培养3～5天。The method for preparing polyhedrons encapsulating foreign proteins according to claim 1, characterized in that in step (6), the culture temperature is 26-27°C; in step (7), the culture is 26-27°C for 3-3 5 days.
  8. 根据权利要求1所述制备包裹外源蛋白的多角体的方法制备的包裹外源蛋白的多角体。The polyhedron encapsulating exogenous protein prepared by the method for preparing polyhedron encapsulating exogenous protein according to claim 1.
  9. 家蚕质型多角体病毒VP5序列在制备蛋白载体或者包裹外源蛋白的多角体中的应用，或者在作为蛋白载体中的应用。The use of the Bombyx mori plasmopolyhedrosis virus VP5 sequence in the preparation of protein carriers or polyhedrons wrapping foreign proteins, or as a protein carrier.
  10. 权利要求8所述包裹外源蛋白的多角体在制备蛋白缓释体系或者作为蛋白缓释体系中的应用。The application of the polyhedron encapsulating foreign protein according to claim 8 in the preparation of a protein sustained-release system or as a protein sustained-release system.
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