CN113215198A - Recombinant baculovirus and preparation method and application thereof - Google Patents
Recombinant baculovirus and preparation method and application thereof Download PDFInfo
- Publication number
- CN113215198A CN113215198A CN202110370474.5A CN202110370474A CN113215198A CN 113215198 A CN113215198 A CN 113215198A CN 202110370474 A CN202110370474 A CN 202110370474A CN 113215198 A CN113215198 A CN 113215198A
- Authority
- CN
- China
- Prior art keywords
- silkworm
- recombinant
- sod
- seq
- baculovirus
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 241000701447 unidentified baculovirus Species 0.000 title claims abstract description 48
- 238000002360 preparation method Methods 0.000 title abstract description 9
- 241000255789 Bombyx mori Species 0.000 claims abstract description 110
- 108010012715 Superoxide dismutase Proteins 0.000 claims abstract description 65
- 102000019197 Superoxide Dismutase Human genes 0.000 claims abstract description 61
- 108010064862 Nicotinamide phosphoribosyltransferase Proteins 0.000 claims abstract description 40
- 102000015532 Nicotinamide phosphoribosyltransferase Human genes 0.000 claims abstract description 38
- 239000000843 powder Substances 0.000 claims abstract description 27
- 241000700605 Viruses Species 0.000 claims abstract description 25
- 239000013598 vector Substances 0.000 claims abstract description 18
- 101150017120 sod gene Proteins 0.000 claims abstract description 16
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 claims abstract description 14
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 claims abstract description 14
- 230000003712 anti-aging effect Effects 0.000 claims abstract description 13
- 238000000034 method Methods 0.000 claims abstract description 13
- 239000003814 drug Substances 0.000 claims abstract description 12
- 239000002245 particle Substances 0.000 claims abstract description 9
- 239000006228 supernatant Substances 0.000 claims abstract description 8
- 101150086808 NAMPT gene Proteins 0.000 claims abstract description 6
- 229940079593 drug Drugs 0.000 claims abstract description 6
- 241000894006 Bacteria Species 0.000 claims abstract description 5
- 238000001976 enzyme digestion Methods 0.000 claims abstract description 5
- 238000004108 freeze drying Methods 0.000 claims abstract description 4
- 238000010369 molecular cloning Methods 0.000 claims abstract description 3
- 101100366137 Mesembryanthemum crystallinum SODCC.1 gene Proteins 0.000 claims abstract 2
- 101100096142 Panax ginseng SODCC gene Proteins 0.000 claims abstract 2
- 238000011144 upstream manufacturing Methods 0.000 claims description 9
- 239000004480 active ingredient Substances 0.000 claims description 6
- 238000004519 manufacturing process Methods 0.000 claims description 3
- 210000002845 virion Anatomy 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 44
- 238000005119 centrifugation Methods 0.000 abstract description 2
- 102000004190 Enzymes Human genes 0.000 description 34
- 108090000790 Enzymes Proteins 0.000 description 34
- 108090000623 proteins and genes Proteins 0.000 description 22
- 210000000087 hemolymph Anatomy 0.000 description 19
- 241000382353 Pupa Species 0.000 description 15
- 102000004169 proteins and genes Human genes 0.000 description 15
- 241000700159 Rattus Species 0.000 description 12
- 238000002835 absorbance Methods 0.000 description 9
- 210000004027 cell Anatomy 0.000 description 8
- 238000005520 cutting process Methods 0.000 description 8
- 201000010099 disease Diseases 0.000 description 8
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 8
- 239000013605 shuttle vector Substances 0.000 description 7
- 239000012634 fragment Substances 0.000 description 6
- 101000702488 Rattus norvegicus High affinity cationic amino acid transporter 1 Proteins 0.000 description 5
- 101710119418 Superoxide dismutase [Mn] Proteins 0.000 description 5
- 101710202572 Superoxide dismutase [Mn], mitochondrial Proteins 0.000 description 5
- 238000010276 construction Methods 0.000 description 5
- 210000002966 serum Anatomy 0.000 description 5
- 239000000126 substance Substances 0.000 description 5
- 102100032891 Superoxide dismutase [Mn], mitochondrial Human genes 0.000 description 4
- 210000004369 blood Anatomy 0.000 description 4
- 239000008280 blood Substances 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 238000003776 cleavage reaction Methods 0.000 description 4
- 238000003304 gavage Methods 0.000 description 4
- 230000007017 scission Effects 0.000 description 4
- 239000005723 virus inoculator Substances 0.000 description 4
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 3
- 230000003203 everyday effect Effects 0.000 description 3
- 229910052760 oxygen Inorganic materials 0.000 description 3
- 239000001301 oxygen Substances 0.000 description 3
- 238000012408 PCR amplification Methods 0.000 description 2
- 208000005374 Poisoning Diseases 0.000 description 2
- 239000013543 active substance Substances 0.000 description 2
- 230000036528 appetite Effects 0.000 description 2
- 235000019789 appetite Nutrition 0.000 description 2
- 230000033228 biological regulation Effects 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000003018 immunoassay Methods 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 238000011081 inoculation Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000013612 plasmid Substances 0.000 description 2
- 231100000572 poisoning Toxicity 0.000 description 2
- 230000000607 poisoning effect Effects 0.000 description 2
- 230000005855 radiation Effects 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 206010002091 Anaesthesia Diseases 0.000 description 1
- 208000023275 Autoimmune disease Diseases 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 208000002177 Cataract Diseases 0.000 description 1
- 108091026890 Coding region Proteins 0.000 description 1
- 108020004414 DNA Proteins 0.000 description 1
- 206010014561 Emphysema Diseases 0.000 description 1
- JOYRKODLDBILNP-UHFFFAOYSA-N Ethyl urethane Chemical compound CCOC(N)=O JOYRKODLDBILNP-UHFFFAOYSA-N 0.000 description 1
- 102000006587 Glutathione peroxidase Human genes 0.000 description 1
- 108700016172 Glutathione peroxidases Proteins 0.000 description 1
- 101000998855 Homo sapiens Nicotinamide phosphoribosyltransferase Proteins 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 206010030113 Oedema Diseases 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 208000038016 acute inflammation Diseases 0.000 description 1
- 230000006022 acute inflammation Effects 0.000 description 1
- 210000000577 adipose tissue Anatomy 0.000 description 1
- COAIBTZEXXESAS-TZSLYBJXSA-N adiposin Chemical compound OC[C@H]1O[C@H](O)[C@H](O)[C@@H](O)[C@@H]1N[C@@H]1[C@H](O)[C@@H](O)[C@H](O)C(CO)=C1 COAIBTZEXXESAS-TZSLYBJXSA-N 0.000 description 1
- 230000037005 anaesthesia Effects 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 210000000702 aorta abdominal Anatomy 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 238000003149 assay kit Methods 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 238000007598 dipping method Methods 0.000 description 1
- 230000009977 dual effect Effects 0.000 description 1
- 210000003743 erythrocyte Anatomy 0.000 description 1
- 210000004744 fore-foot Anatomy 0.000 description 1
- 230000002496 gastric effect Effects 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- 208000019622 heart disease Diseases 0.000 description 1
- 230000002440 hepatic effect Effects 0.000 description 1
- 102000000418 human nicotinamide phosphoribosyltransferase Human genes 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 210000005229 liver cell Anatomy 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000002107 myocardial effect Effects 0.000 description 1
- 238000001543 one-way ANOVA Methods 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 230000003449 preventive effect Effects 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 235000019624 protein content Nutrition 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 201000008525 senile cataract Diseases 0.000 description 1
- VSIVTUIKYVGDCX-UHFFFAOYSA-M sodium;4-[2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)tetrazol-2-ium-5-yl]benzene-1,3-disulfonate Chemical compound [Na+].COC1=CC([N+]([O-])=O)=CC=C1[N+]1=NC(C=2C(=CC(=CC=2)S([O-])(=O)=O)S([O-])(=O)=O)=NN1C1=CC=C([N+]([O-])=O)C=C1 VSIVTUIKYVGDCX-UHFFFAOYSA-M 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 239000012096 transfection reagent Substances 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 230000009278 visceral effect Effects 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Images
Classifications
-
- 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
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/033—Rearing or breeding invertebrates; New breeds of invertebrates
- A01K67/0333—Genetically modified invertebrates, e.g. transgenic, polyploid
- A01K67/0337—Genetically modified Arthropods
- A01K67/0339—Genetically modified insects, e.g. Drosophila melanogaster, medfly
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/56—Materials from animals other than mammals
- A61K35/63—Arthropods
- A61K35/64—Insects, e.g. bees, wasps or fleas
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/43—Enzymes; Proenzymes; Derivatives thereof
- A61K38/44—Oxidoreductases (1)
- A61K38/446—Superoxide dismutase (1.15)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/43—Enzymes; Proenzymes; Derivatives thereof
- A61K38/45—Transferases (2)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P39/00—General protective or antinoxious agents
- A61P39/06—Free radical scavengers or antioxidants
-
- 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0089—Oxidoreductases (1.) acting on superoxide as acceptor (1.15)
-
- 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1048—Glycosyltransferases (2.4)
- C12N9/1077—Pentosyltransferases (2.4.2)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y115/00—Oxidoreductases acting on superoxide as acceptor (1.15)
- C12Y115/01—Oxidoreductases acting on superoxide as acceptor (1.15) with NAD or NADP as acceptor (1.15.1)
- C12Y115/01001—Superoxide dismutase (1.15.1.1)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y204/00—Glycosyltransferases (2.4)
- C12Y204/02—Pentosyltransferases (2.4.2)
- C12Y204/02012—Nicotinamide phosphoribosyltransferase (2.4.2.12), i.e. visfatin
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2207/00—Modified animals
- A01K2207/05—Animals modified by non-integrating nucleic acids, e.g. antisense, RNAi, morpholino, episomal vector, for non-therapeutic purpose
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2227/00—Animals characterised by species
- A01K2227/70—Invertebrates
- A01K2227/706—Insects, e.g. Drosophila melanogaster, medfly
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2267/00—Animals characterised by purpose
- A01K2267/01—Animal expressing industrially exogenous 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
- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/14011—Baculoviridae
- C12N2710/14111—Nucleopolyhedrovirus, e.g. autographa californica nucleopolyhedrovirus
- C12N2710/14141—Use of virus, viral particle or viral elements as a vector
- C12N2710/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
-
- 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
- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/103—Plasmid DNA for invertebrates
- C12N2800/105—Plasmid DNA for invertebrates for insects
-
- 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
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- General Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Medicinal Chemistry (AREA)
- Animal Behavior & Ethology (AREA)
- Biomedical Technology (AREA)
- Veterinary Medicine (AREA)
- Pharmacology & Pharmacy (AREA)
- Public Health (AREA)
- Molecular Biology (AREA)
- Epidemiology (AREA)
- Microbiology (AREA)
- Environmental Sciences (AREA)
- Insects & Arthropods (AREA)
- Animal Husbandry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Immunology (AREA)
- Gastroenterology & Hepatology (AREA)
- Physics & Mathematics (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Toxicology (AREA)
- Virology (AREA)
- Plant Pathology (AREA)
- Biodiversity & Conservation Biology (AREA)
- Biophysics (AREA)
- Enzymes And Modification Thereof (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The invention discloses a recombinant baculovirus and a preparation method and application thereof, the recombinant baculovirus comprises a superoxide dismutase SOD gene and a nicotinamide phosphoribosyl transferase NAMPT gene, the SOD gene is a thermophilic bacteria superoxide dismutase SOD gene, primers are respectively designed according to the OFR of the SOD gene and the NAMPT gene, the method of double enzyme digestion and molecular cloning is utilized to connect the ORF of the SOD and the NAMPT to the same baculovirus vector, a recombinant vector is constructed, and the preparation method of silkworm chrysalis powder comprises the following steps: infecting silkworm cells with the recombinant baculovirus to obtain virus particles; the virus particles are inoculated to the silkworm chrysalis, juicing is carried out after 5-6 days, supernatant is taken out after centrifugation at 8000rpm and freeze-drying is carried out, the silkworm chrysalis freeze-dried powder is obtained, and the recombinant baculovirus and the recombinant protein expressed by the recombinant baculovirus are applied to the anti-aging drugs, and have the technical characteristics of anti-aging, low cost, remarkable effect and the like.
Description
Technical Field
The invention relates to a virus, a preparation method and application thereof, in particular to a recombinant baculovirus, a preparation method and application thereof, and relates to the technical field of polypeptide drug production by genetic engineering in biotechnology pharmaceutical engineering.
Background
Superoxide Dismutase (SOD), also known as hepatic protein, is called SOD for short, and is an active substance from living bodies, can eliminate harmful substances generated in the metabolism process of organisms, and has a special anti-aging effect if a human body is continuously supplemented with SOD. Superoxide dismutase is firstly separated from bovine red blood cells in 1938, so far, the research on SOD has been for many years, the SOD is formally named as superoxide dismutase in 1969, and the SOD is one of the most important enzymes in human body, and the effect of the SOD is not small. It can be used for clinically treating and preventing acute inflammation, edema and oxygen poisoning (preventive measures, working personnel entering a hyperbaric oxygen chamber can inject SOD in advance, oxygen poisoning treatment, autoimmune diseases (early treatment), emphysema, radiation diseases, radiation protection, senile cataract and the like, and has an anti-aging function, NAMPT (nicotinamide phosphoribosyltransferase), which is also called visceral adiposin, is a multifunctional protein widely existing in tissues such as adipose tissue, liver, spleen, kidney and the like in vivo, participates in regulation and control of various physiological processes in the body, and also participates in regulation and control of the NAD level of myocardial cells In this state, the high-efficiency, healthy and safe NAMPT medicine is developed, and can be well applied in the fields of heart diseases and the like. The combined expression of SOD and NAMPT is a necessary trend and a feasible path, and can be used for preparing effective anti-aging medicaments.
Disclosure of Invention
The invention aims to introduce a target gene of NAMPT and a high-temperature resistant SOD target gene into silkworm baculovirus together, transfect silkworm and larva thereof with the silkworm baculovirus, and jointly express the target genes of the silkworm baculovirus and the larva thereof in silkworm to generate novel combined protein, thereby realizing a novel approach for producing SOD and NAMPT.
In order to achieve the purpose, the invention is realized by the following technical scheme:
a baculovirus of a recombinant superoxide dismutase gene comprises a superoxide dismutase SOD gene and a nicotinamide phosphoribosyl transferase NAMPT gene, wherein the SOD gene is a thermophilic bacteria superoxide dismutase SOD gene.
Preferably, the OFR sequence of the SOD gene is SEQ ID NO.1, and the SEQ ID NO.1 is
ATGCCATTTGAATTGCCAGCATTGCCGTATCCGTATGATGCGCTTGAGCCGCACATCGACAAAG AAACGATGAACATTCACCACACGAAGCACCATAACACATACGTTACAAATTTGAATGCGGCGCTTGAA GGGCATCCGGATTTGCAAAACAAATCGCTCGAAGAATTGCTCAGCAATTTGGAAGCCCTTCCGGAAAG CATTCGCACGGCGGTGCGCAACAACGGCGGCGGTCATGCAAACCACTCGCTTTTCTGGACGATTTTGT CGCCAAATGGCGGCGGTGAGCCGACGGGTGAGCTGGCTGAGGCGATCAACAAAAAATTCGGCAGCTTC ACCGCGTTTAAAGACGAGTTTTCGAAAGCAGCGGCCGGCCGTTTCGGTTCTGGCTGGGCATGGCTTGT CGTGAACAACGGCGAGCTGGAAATTACGAGCACGCCGAACCAAGACTCGCCGATCATGGAAGGCAAAA CGCCGATTCTCGGCTTGGACGTTTGGGAGCATGCGTACTACTTGAAATACCAAAACCGCCGTCCGGAA TACATTGCCGCATTCTGGAACATTGTCAACTGGGACGAAGTGGCGAAACGGTACAGCGAAGCGAAAGC GAAGTAA。
Preferably, the OFR sequence of the NAMPT gene is SEQ ID NO.2, and the SEQ ID NO.2 is
ATGAATCCTGCGGCAGAAGCCGAGTTCAACATCCTCCTGGCCACCGACTCCTACAAGGTTACTC ACTATAAACAATATCCACCCAACACAAGCAAAGTTTATTCCTACTTTGAATGCCGTGAAAAGAAGACA GAAAACTCCAAATTAAGGAAGGTGAAATATGAGGAAACAGTATTTTATGGGTTGCAGTACATTCTTAA TAAGTACTTAAAAGGTAAAGTAGTAACCAAAGAGAAAATCCAGGAAGCCAAAGATGTCTACAAAGAAC ATTTCCAAGATGATGTCTTTAATGAAAAGGGATGGAACTACATTCTTGAGAAGTATGATGGGCATCTT CCAATAGAAATAAAAGCTGTTCCTGAGGGCTTTGTCATTCCCAGAGGAAATGTTCTCTTCACGGTGGA AAACACAGATCCAGAGTGTTACTGGCTTACAAATTGGATTGAGACTATTCTTGTTCAGTCCTGGTATC CAATCACAGTGGCCACAAATTCTAGAGAGCAGAAGAAAATATTGGCCAAATATTTGTTAGAAACTTCT GGTAACTTAGATGGTCTGGAATACAAGTTACATGATTTTGGCTACAGAGGAGTCTCTTCCCAAGAGAC TGCTGGCATAGGAGCATCTGCTCACTTGGTTAACTTCAAAGGAACAGATACAGTAGCAGGACTTGCTC TAATTAAAAAATATTATGGAACGAAAGATCCTGTTCCAGGCTATTCTGTTCCAGCAGCAGAACACAGT ACCATAACAGCTTGGGGGAAAGACCATGAAAAAGATGCTTTTGAACATATTGTAACACAGTTTTCATC AGTGCCTGTATCTGTGGTCAGCGATAGCTATGACATTTATAATGCGTGTGAGAAAATATGGGGTGAAG ATCTAAGACATTTAATAGTATCAAGAAGTACACAGGCACCACTAATAATCAGACCTGATTCTGGAAAC CCTCTTGACACTGTGTTAAAGGTTTTGGAGATTTTAGGTAAGAAGTTTCCTGTTACTGAGAACTCAAA GGGTTACAAGTTGCTGCCACCTTATCTTAGAGTTATTCAAGGGGATGGAGTAGATATTAATACCTTAC AAGAGATTGTAGAAGGCATGAAACAAAAAATGTGGAGTATTGAAAATATTGCCTTCGGTTCTGGTGGA GGTTTGCTACAGAAGTTGACAAGAGATCTCTTGAATTGTTCCTTCAAGTGTAGCTATGTTGTAACTAA TGGCCTTGGGATTAACGTCTTCAAGGACCCAGTTGCTGATCCCAACAAAAGGTCCAAAAAGGGCCGAT TATCTTTACATAGGACGCCAGCAGGGAATTTTGTTACACTGGAGGAAGGAAAAGGAGACCTTGAGGAA TATGGTCAGGATCTTCTCCATACTGTCTTCAAGAATGGCAAGGTGACAAAAAGCTATTCATTTGATGA AATAAGAAAAAATGCACAGCTGAATATTGAACTGGAAGCAGCACATCATTAG。
A method for preparing baculovirus of a recombinant superoxide dismutase gene comprises the following steps: corresponding primers are designed according to OFR of SEQ ID NO.1 and OFR of SEQ ID NO.2 respectively, and the ORFs of SOD and NAMPT are connected to the same baculovirus vector by using methods of double enzyme digestion and molecular cloning to construct a recombinant vector, wherein the baculovirus vector is pFastBacDual, and the baculovirus is silkworm baculovirus.
Preferably, the primer of SEQ ID NO.1 comprises an upstream primer SEQ ID NO.3 and a downstream primer SEQ ID NO. 4; the SEQ ID NO.2 comprises an upstream primer SEQ ID NO.5 and a downstream primer SEQ ID NO. 6.
A recombinant protein obtained by infecting silkworm pupa or silkworm cell with baculovirus of recombinant superoxide dismutase gene.
A pharmaceutical characterized in that a pharmaceutically active ingredient is the recombinant protein according to claim 6.
A silkworm chrysalis powder, wherein the silkworm chrysalis powder active ingredient is the recombinant protein of claim 6.
The preparation method of the silkworm chrysalis powder is characterized by comprising the following steps:
step 1): infecting silkworm cells with baculovirus to obtain virus particles;
step 2): inoculating the virus particles in the step 1) to the silkworm chrysalis, juicing after 5-6 days, centrifuging at 8000rpm, taking supernatant, and freeze-drying to obtain the silkworm chrysalis freeze-dried powder.
A baculovirus of a recombinant superoxide dismutase gene and application of a recombinant protein expressed by the baculovirus in anti-aging drugs.
Has the advantages that: SOD and NAMPT proteins are expressed in a combined manner, and the enzyme activities of the SOD protein and the NAMPT protein expressed by silkworm pupas infected and diseased by inoculated viruses are higher than the enzyme activities of the protease contained in the silkworm pupas not inoculated with the viruses, so that the SOD and NAMPT expressed by the silkworm pupas in a combined manner is a feasible path and can be used for preparing effective anti-aging medicaments; the anti-aging medicine is wrapped by the silkworm chrysalis powder, has better oral effect, does not need to add other auxiliary materials, and has simple preparation process;
the anti-aging medicament provided by the invention has low active ingredient content, low cost and obvious effect. Compared with the prior art that the SOD activity in 50000U/kg SOD blood orally administered by rats has no obvious difference, the drug provided by the invention has obvious effect when the administration amount of SOD is 8626U/kg;
the provided baculovirus infects silkworm larvae and pupae, the SOD specific activity is obviously improved, wherein the SOD specific activity in haemolymph is improved by 14 times, which is far higher than that in the prior art and is improved by 5 times compared with that in the prior art;
the action mechanisms of SOD and NAMPT in vivo are not related, but the silkworm chrysalis powder and the medicament test result provided by the application show that the antioxidant activity of SOD co-expressed by the two proteins is obviously improved, and the anti-aging effect is obvious.
Drawings
FIG. 1: PCR amplification of SOD;
FIG. 2: PCR amplification of NAMPT;
FIG. 3: enzyme digestion identification of the recombinant vector pFastBacDual-SOD-NAMPT;
FIG. 4: double enzyme digestion identification of the recombinant vector pFastBacDual-SOD-NAMPT;
FIG. 5: PCR identification of the recombinant vector pFastBacDual-SOD-NAMPT;
FIG. 6: the cross PCR result of the recombinant shuttle vector Bacmid-SOD-NAMPT;
FIG. 7: normal bombyx mori bnn;
FIG. 8: diseased bombyx mori bnn;
FIG. 9: and (3) carrying out PCR identification on the recombinant virus particles.
Detailed Description
The present invention will be further described with reference to the drawings attached to the specification, but the present invention is not limited to the following examples.
The virus particles prepared by the recombinant baculovirus can express SOD and NAMPT proteins in silkworm larvae and silkworm pupae simultaneously, the silkworm pupae powder prepared by the baculovirus can obviously improve the enzyme activity levels of SOD and GSH-Px in the blood of old rats, and although the protein contents of SOD and NAMPT in the obtained silkworm pupae powder are limited, the effect is obvious.
pET-28a-MnSOD plasmid (provided by Zhejiang university silkworm bioreactor laboratory), BmDH10Bac strain (provided by Zhejiang university silkworm bioreactor laboratory), pFastBac-Dual vector, Xho I, Kpn I, EcoR I, Hind III and BamH I, antibiotic, transfection reagent FuGene 6 and serum. Reagents, carriers and strains required by the experiment can be obtained by market without special description.
Example 1
Construction of recombinant shuttle vector pFastBac-Dual-MnSOD
Design of MnSOD coding region primer
1. Selecting high-temperature-resistant MnSOD as an expression target gene, wherein the ORF sequence is as follows:
>MnSOD
ATGCCATTTGAATTGCCAGCATTGCCGTATCCGTATGATGCGCTTGAGCCGCACATCGACAAAGAAAC GATGAACATTCACCACACGAAGCACCATAACACATACGTTACAAATTTGAATGCGGCGCTTGAAGGGC ATCCGGATTTGCAAAACAAATCGCTCGAAGAATTGCTCAGCAATTTGGAAGCCCTTCCGGAAAGCATT CGCACGGCGGTGCGCAACAACGGCGGCGGTCATGCAAACCACTCGCTTTTCTGGACGATTTTGTCGCC AAATGGCGGCGGTGAGCCGACGGGTGAGCTGGCTGAGGCGATCAACAAAAAATTCGGCAGCTTCACCG CGTTTAAAGACGAGTTTTCGAAAGCAGCGGCCGGCCGTTTCGGTTCTGGCTGGGCATGGCTTGTCGTG AACAACGGCGAGCTGGAAATTACGAGCACGCCGAACCAAGACTCGCCGATCATGGAAGGCAAAACGCC GATTCTCGGCTTGGACGTTTGGGAGCATGCGTACTACTTGAAATACCAAAACCGCCGTCCGGAATACA TTGCCGCATTCTGGAACATTGTCAACTGGGACGAAGTGGCGAAACGGTACAGCGAAGCGAAAGCGAAG TAA；
designing primers according to the ORF of the MnSOD gene, and respectively introducing XhoI enzyme cutting sites and Kpn I enzyme cutting sites as follows:
upstream primer F (SEQ ID NO. 3): cCTCGAGATGCCATTTGAATTGCCAG (Xho I cleavage sites underlined);
downstream primer R (SEQ ID NO. 4): gGGTACCTTACTTCGCTTTCGCTTCGC (underlined Kpn I cleavage site).
2. The ORF sequence of the expressed target gene NAMPT is as follows:
>NAMPT
ATGAATCCTGCGGCAGAAGCCGAGTTCAACATCCTCCTGGCCACCGACTCCTACAAGGTTACTCACTA TAAACAATATCCACCCAACACAAGCAAAGTTTATTCCTACTTTGAATGCCGTGAAAAGAAGACAGAAA ACTCCAAATTAAGGAAGGTGAAATATGAGGAAACAGTATTTTATGGGTTGCAGTACATTCTTAATAAG TACTTAAAAGGTAAAGTAGTAACCAAAGAGAAAATCCAGGAAGCCAAAGATGTCTACAAAGAACATTT CCAAGATGATGTCTTTAATGAAAAGGGATGGAACTACATTCTTGAGAAGTATGATGGGCATCTTCCAA TAGAAATAAAAGCTGTTCCTGAGGGCTTTGTCATTCCCAGAGGAAATGTTCTCTTCACGGTGGAAAAC ACAGATCCAGAGTGTTACTGGCTTACAAATTGGATTGAGACTATTCTTGTTCAGTCCTGGTATCCAAT CACAGTGGCCACAAATTCTAGAGAGCAGAAGAAAATATTGGCCAAATATTTGTTAGAAACTTCTGGTA ACTTAGATGGTCTGGAATACAAGTTACATGATTTTGGCTACAGAGGAGTCTCTTCCCAAGAGACTGCT GGCATAGGAGCATCTGCTCACTTGGTTAACTTCAAAGGAACAGATACAGTAGCAGGACTTGCTCTAAT TAAAAAATATTATGGAACGAAAGATCCTGTTCCAGGCTATTCTGTTCCAGCAGCAGAACACAGTACCA TAACAGCTTGGGGGAAAGACCATGAAAAAGATGCTTTTGAACATATTGTAACACAGTTTTCATCAGTG CCTGTATCTGTGGTCAGCGATAGCTATGACATTTATAATGCGTGTGAGAAAATATGGGGTGAAGATCT AAGACATTTAATAGTATCAAGAAGTACACAGGCACCACTAATAATCAGACCTGATTCTGGAAACCCTC TTGACACTGTGTTAAAGGTTTTGGAGATTTTAGGTAAGAAGTTTCCTGTTACTGAGAACTCAAAGGGT TACAAGTTGCTGCCACCTTATCTTAGAGTTATTCAAGGGGATGGAGTAGATATTAATACCTTACAAGA GATTGTAGAAGGCATGAAACAAAAAATGTGGAGTATTGAAAATATTGCCTTCGGTTCTGGTGGAGGTT TGCTACAGAAGTTGACAAGAGATCTCTTGAATTGTTCCTTCAAGTGTAGCTATGTTGTAACTAATGGC CTTGGGATTAACGTCTTCAAGGACCCAGTTGCTGATCCCAACAAAAGGTCCAAAAAGGGCCGATTATC TTTACATAGGACGCCAGCAGGGAATTTTGTTACACTGGAGGAAGGAAAAGGAGACCTTGAGGAATATG GTCAGGATCTTCTCCATACTGTCTTCAAGAATGGCAAGGTGACAAAAAGCTATTCATTTGATGAAATA AGAAAAAATGCACAGCTGAATATTGAACTGGAAGCAGCACATCATTAG
designing primers according to ORF of the target gene NAMPT, and respectively introducing EcoR I and HindIII enzyme cutting sites as follows:
upstream primer F (SEQ ID NO. 5): gGAATTCATGAATCCTGCGGCAGAAG (the EcoR I site is underlined);
a downstream primer R: (SEQ ID NO.6) CCAAGCTTCTAATGATGTGCTGCTTCCAG (HindIII sites underlined).
3. Construction of recombinant vector pFastBacDual-SOD-NAMPT
PCR technology is used for amplifying pET-28a-MnSOD plasmid (provided by silkworm bioreactor laboratories of Zhejiang university of physiology) to obtain a high temperature resistant SOD gene ORF fragment (shown in figure 1), Xho I enzyme cutting sites and Kpn I enzyme cutting sites are respectively introduced into the tail ends of the fragment, and the fragment is subjected to double enzyme cutting to connect a baculovirus shuttle vector pFastBacDual to construct a recombinant vector pFastBacDual-SOD.
The PCR technology is used for amplifying NAMPT enzyme target gene ORF fragment from human liver cell cDNA, EcoR I and BamH I enzyme cutting sites are respectively introduced at two ends of the fragment, and the recombinant vector pFastBacDual-SOD-NAMPT is constructed by double enzyme cutting connection. As can be seen from FIGS. 1 and 2, the objective genes of SOD and NAMPT were successfully amplified by PCR, and the constructed recombinant vector pFastBacDual-SOD-NAMPT was subjected to double digestion and PCR identification, as shown in FIGS. 3, 4 and 5, the recombinant vector pFastBacDual-SOD-NAMPT was successfully constructed.
4. Construction and identification of recombinant baculovirus shuttle vector BmBacmid-MnSOD-NAMPT
The recombinant vector pFastBacDual-SOD-NAMPT adopts a bombyx mori baculovirus shuttle vector BmBacmid in patent document CN201210037290.8 to construct a recombinant virus shuttle vector BmBacmid-MnSOD-NAMPT (the construction method is shown in CN 201210037290.8), cross PCR verification is carried out, as shown in figure 6, the recombinant virus BmBacmid-MnSOD-NAMPT DNA is extracted as a template, different primer pairs (SEQ ID NO.3+ SEQ ID NO.4 and SEQ ID NO.5+ SEQ ID NO.6) are used to obtain fragments which are consistent with an expected result, and the construction success of the recombinant baculovirus shuttle vector BmBacmid-MnSOD-NAMPT is demonstrated.
Example 2
Recombinant BmBacmind-SOD-NAMPT transfects silkworm cells to obtain virus particles and identification
Silkworm BmN cells were plated (as shown in FIG. 7). The recombinant BmBacmined-SOD-NAMPT is transfected into the BmN cells of the silkworms by Fugene 6, and the cells float after 5 to 7 days and become large and round (as shown in figure 8), which indicates that the cells are attacked and successfully obtains the recombinant baculovirus. Centrifuging to obtain supernatant, adding upstream and downstream primers F and R of MnSOD and NAMPT for PCR identification, and successfully amplifying bands of target genes ORF of SOD and NAMPT, wherein the sizes are consistent with theory, thereby indicating that the recombinant virus vBmBacmed-MnSOD-NAMPT is successfully constructed, and the method is shown in figure 9.
Example 3
SOD and NAMPT dual recombinant protein prepared by infecting vBmBacmind-MnSOD-NAMPT silkworm with recombinant baculovirus
1. Virus inoculation of silkworm larva
The recombinant baculovirus liquid obtained in example 2 is dipped by a needle and inoculated to the second section of the tail of the silkworm larva, the state of the silkworm inoculated with the virus is recorded every day, the volume of the silkworm becomes large and the appetite of the silkworm increases after the silkworm is inoculated with the virus for one day, the appetite of the silkworm decreases along with the increase of time, the tail becomes yellow, the silkworm becomes "violent" and is favored to climb around, the virus is shown to infect the silkworm larva, and the forefoot of the larva is cut off after the larva is attacked for three days to collect hemolymph.
2. Virus inoculation of silkworm chrysalis
The same is inoculated on the second section of the tail part of the silkworm pupae, and the state of the silkworm pupae after virus inoculation is recorded every day. After the toxicity is caused, no obvious change is caused in the first two days, the silkworm pupae gradually become soft along with the time extension after the third day, the disease onset starts, the silkworm pupae three days after the disease onset (generally, 5-6 days after inoculation) are ground and homogenized, supernatant is taken after centrifugation at 8000rpm and freeze-dried, and the freeze-dried powder is the silkworm pupae freeze-dried powder containing SOD and NAMPT double recombinant protein.
Example 4
Detection of specific enzyme activity of silkworm larva expression duplex recombinant protein SOD and NAMPT
After three days of larvae disease, the forefeet are cut off to collect hemolymph. Detecting the total protein concentration in the silkworm hemolymph by using a Bradford protein concentration kit of Biyuntian company, and drawing a standard curve according to an experimental method to obtain an equation: y is 0.4697x +0.1219, the haemolymph which needs to detect the protein concentration is diluted by 500 times, and the absorbance values of the protein in the haemolymph of four groups of silkworms are measured under the wavelength of 570nm respectively as follows: the normal haemolymph 1OD value is 0.143, the normal haemolymph 2OD value is 0.142, the silkworm haemolymph 1OD value is 0.261, the silkworm haemolymph 2OD value is 0.242, and the normal larva haemolymph protein concentration and the pus larva haemolymph protein concentration are calculated. After diluting 500 times, the activity unit of SOD was measured according to the total SOD active agent assay kit (WST-8 method), and absorbance at a wavelength of 450nm was measured as shown in Table 1:
TABLE 1 SOD Activity measurement Absorbance value at 450nm wavelength
Calculating the percent inhibition [ (A) according to the formula of the kitBlank control 1-ABlank control 2)-(ASample (I)- ABlank control 3)]/(ABlank control 1-ABlank control 2) X is 100%; b. the SOD enzyme activity unit in the sample to be tested is inhibition percentage/(1-inhibition percentage) units. Calculating to obtain the normal silkworm hemolymph enzyme activity unit: 348.380U/mL, pus-forming silkworm hemolymph enzyme activity unit: 808.929U/mL. Normal silkworm hemolymph specific enzyme activity: 2.524U/mg, the hemolymph specific enzyme activity of the silkworm with the pus: 36.77U/mg. The result shows that the specific SOD activity of the pus-forming silkworm is 14 times higher than that of the normal silkworm.
The specific activity of NAMPT in silkworm hemolymph is detected according to a human NAMPT enzyme immunoassay kit (enzyme immunoassay company), and the absorbance value of the standard substance is measured as shown in table 2, wherein the OD value of a blank hole is 0.055:
TABLE 2 Absorbance value of Standard substance
Respectively drawing a linear relation between the absorbance of the standard substance and the concentration (U/L) according to the absorbance value of the standard substance after zero setting: y 234.96x + 29.704; linear relationship of standard absorbance to concentration (ng/mL): y is 4.895x + 0.6188. Silkworm hemolymph data (as shown in table 3) were measured at a wavelength of 450nm, where the blank well OD value was 0.057:
TABLE 3 absorbance at 450nm wavelength of hemolymph of Bombyx mori
Example 5
Enzyme activity detection of silkworm pupa freeze-dried powder expressing duplex recombinant protein SOD and NAMPT
Grinding and homogenizing silkworm pupa three days after disease attack (generally 5-6 days after inoculation), centrifuging at 8000rpm, taking supernatant, freeze-drying, dissolving, centrifuging, measuring total protein concentration and enzyme activity of the supernatant by the same method, and calculating to obtain specific enzyme activity. And (3) measuring the total protein concentration of the supernatant of the normal silkworm pupa sample: 50.500mg/mL, concentration of diseased silkworm pupa protein: 26.500 mg/mL. The SOD activity is measured by the same method to obtain the normal silkworm chrysalis enzyme activity unit: 0.677U, activity unit of the disease-causing silkworm chrysalis enzyme: 2.286U, calculating the specific enzyme activity of the normal silkworm chrysalis: 6.703U/mg, specific enzyme activity of the disease-induced silkworm chrysalis: 43.132U/mg. The results show that the specific enzyme activity of SOD of the disease silkworm pupae is 6.4 times of that of the normal silkworm pupae, the silkworm pupae are used as a bioreactor and are more suitable for the expression of SOD, and the specific enzyme activity of NAMPT protein in the silkworm pupae is measured by the same method as shown in the table 4:
TABLE 4 determination of specific enzyme Activity of silkworm pupa NAMPT protein
As can be seen from the enzyme activity and concentration value of NAMPT in silkworm pupae, the NAMPT expressed by diseased silkworm pupae is higher than that expressed by non-diseased silkworm pupae.
The vBmBacmid-MnSOD-NAMPT virus is inoculated to silkworm pupae in large batch to express the MnSOD-NAMPT duplex protein in large scale. Dipping the virus with a needle to inoculate the virus at the second section of the tail of the silkworm chrysalis, and recording the state of the silkworm chrysalis after virus inoculation every day. After the silkworm pupae are inoculated with the virus, no obvious change exists in the first two days, and the silkworm pupae gradually become soft along with the time extension after the third day. The black silkworm pupa is infected by bacteria and will not become black when infected by virus. And (3) removing the black silkworm pupae infected by bacteria in time, and inoculating silkworm pupae freeze-dried powder SOD and NAMPT protein on a large scale for detecting the specific enzyme activity.
After silkworm pupas are inoculated with vBmBacmid-MnSOD-NAMPT virus in a large scale and are juiced and freeze-dried into dry powder, 10mg of silkworm pupa freeze-dried powder is dissolved in 1mLPBS solution, the specific enzyme activity of recombinant SOD protein and NAMPT protein is measured by the same method, and the specificity of the expression of recombinant protein of different silkworm pupa individuals is detected. The result shows that the expressed protein has little difference in enzyme activity compared with the enzyme activity after the silkworm pupa individuals of different batches are inoculated with the virus for the same time.
Example 6
Anti-aging test
Materials: 36 female old rats with the weight of 400-; 12 female rats with the age of 4 months with the weight of 200-.
Drugs and reagents: the silkworm chrysalis powder containing SOD and NAMPT (expressed SOD and NAMPT) obtained in example 3 (hereinafter referred to as "twin silkworm chrysalis powder") and commercially available normal silkworm chrysalis powder are prepared with physiological saline when used. Superoxide dismutase SOD reagent kit and glutathione peroxidase GSH-Px reagent kit.
The method comprises the following steps: old rats were randomly divided into a duplex high-dose group, a duplex low-dose group and an old control group, each group containing 12 rats. The 4-month rats were additionally selected as young control groups. The gavage of the rats is calculated according to the surface area of human bodies and animal bodies, the gavage is 200 mg/kg and 500mg/kg once a day, the gavage is continuously performed for 4 weeks, and the control group is given with common silkworm chrysalis powder. Fasting was not followed by water deprivation at the end of week 4 after the last gavage. After 10% urethane anesthesia, abdominal aorta is bled, after standing for 4 hours at room temperature, the blood is centrifuged for 10 minutes at 4000 rpm, and serum is separated for detecting SOD and GSH-Px.
Statistical analysis experimental data were statistically analyzed using SPSS17.0 software and comparisons between groups were analyzed using one-way anova.
The effects on the activities of SOD and GSH-Px in serum of an old rat are obviously reduced; after 4 weeks of gastric lavage, the activity of SOD and GSH-Px in rat serum was significantly enhanced (P <0.05) in the treated group compared with the aged control group, as shown in Table 5.
TABLE 5 Effect of recombinant SOD and-NAMPT double pupa Bombycis powder on serum SOD and GSH-Px activity in aged rats
The virus particles prepared by the recombinant baculovirus provided by the invention can simultaneously express SOD and NAMPT proteins in silkworm larvae and silkworm pupae. The silkworm chrysalis powder prepared by the baculovirus can obviously improve the activity levels of SOD and GSH-Px enzymes in the blood of old rats, and although the silkworm chrysalis powder needs less active ingredients compared with the prior art, the anti-aging effect is obviously improved.
Finally, it should be noted that the present invention is not limited to the above embodiments, and many variations are possible. All modifications which can be derived or suggested by a person skilled in the art from the disclosure of the present invention are to be considered within the scope of the invention.
The invention name is as follows:
recombinant baculovirus and preparation method and application thereof
The applicant: zhejiang An Luo Biotechnology Ltd
SEQ ID NO.1 is:
ATGCCATTTGAATTGCCAGCATTGCCGTATCCGTATGATGCGCTTGAGCCGCACATCGACAAAGAAACGATGAACATTCACCACACGAAGCACCATAACACATACGTTACAAATTTGAATGCGGCGCTTGAAGGGCATCCGGATTTGCAAAACAAATCGCTCGAAGAATTGCTCAGCAATTTGGAAGCCCTTCCGGAAAGCATTCGCACGGCGGTGCGCAACAACGGCGGCGGTCATGCAAACCACTCGCTTTTCTGGACGATTTTGTCGCCAAATGGCGGCGGTGAGCCGACGGGTGAGCTGGCTGAGGCGATCAACAAAAAATTCGGCAGCTTCACCGCGTTTAAAGACGAGTTTTCGAAAGCAGCGGCCGGCCGTTTCGGTTCTGGCTGGGCATGGCTTGTCGTGAACAACGGCGAGCTGGAAATTACGAGCACGCCGAACCAAGACTCGCCGATCATGGAAGGCAAAACGCCGATTCTCGGCTTGGACGTTTGGGAGCATGCGTACTACTTGAAATACCAAAACCGCCGTCCGGAATACATTGCCGCATTCTGGAACATTGTCAACTGGGACGAAGTGGCGAAACGGTACAGCGAAGCGAAAGCGAAGTAA。
SEQ ID NO.2 is:
ATGAATCCTGCGGCAGAAGCCGAGTTCAACATCCTCCTGGCCACCGACTCCTACAAGGTTACTCACTATAAACAATATCCACCCAACACAAGCAAAGTTTATTCCTACTTTGAATGCCGTGAAAAGAAGACAGAAAACTCCAAATTAAGGAAGGTGAAATATGAGGAAACAGTATTTTATGGGTTGCAGTACATTCTTAATAAGTACTTAAAAGGTAAAGTAGTAACCAAAGAGAAAATCCAGGAAGCCAAAGATGTCTACAAAGAACATTTCCAAGATGATGTCTTTAATGAAAAGGGATGGAACTACATTCTTGAGAAGTATGATGGGCATCTTCCAATAGAAATAAAAGCTGTTCCTGAGGGCTTTGTCATTCCCAGAGGAAATGTTCTCTTCACGGTGGAAAACACAGATCCAGAGTGTTACTGGCTTACAAATTGGATTGAGACTATTCTTGTTCAGTCCTGGTATCCAATCACAGTGGCCACAAATTCTAGAGAGCAGAAGAAAATATTGGCCAAATATTTGTTAGAAACTTCTGGTAACTTAGATGGTCTGGAATACAAGTTACATGATTTTGGCTACAGAGGAGTCTCTTCCCAAGAGACTGCTGGCATAGGAGCATCTGCTCACTTGGTTAACTTCAAAGGAACAGATACAGTAGCAGGACTTGCTCTAATTAAAAAATATTATGGAACGAAAGATCCTGTTCCAGGCTATTCTGTTCCAGCAGCAGAACACAGTACCATAACAGCTTGGGGGAAAGACCATGAAAAAGATGCTTTTGAACATATTGTAACACAGTTTTCATCAGTGCCTGTATCTGTGGTCAGCGATAGCTATGACATTTATAATGCGTGTGAGAAAATATGGGGTGAAGATCTAAGACATTTAATAGTATCAAGAAGTACACAGGCACCACTAATAATCAGACCTGATTCTGGAAACCCTCTTGACACTGTGTTAAAGGTTTTGGAGATTTTAGGTAAGAAGTTTCCTGTTACTGAGAACTCAAAGGGTTACAAGTTGCTGCCACCTTATCTTAGAGTTATTCAAGGGGATGGAGTAGATATTAATACCTTACAAGAGATTGTAGAAGGCATGAAACAAAAAATGTGGAGTATTGAAAATATTGCCTTCGGTTCTGGTGGAGGTTTGCTACAGAAGTTGACAAGAGATCTCTTGAATTGTTCCTTCAAGTGTAGCTATGTTGTAACTAATGGCCTTGGGATTAACGTCTTCAAGGACCCAGTTGCTGATCCCAACAAAAGGTCCAAAAAGGGCCGATTATCTTTACATAGGACGCCAGCAGGGAATTTTGTTACACTGGAGGAAGGAAAAGGAGACCTTGAGGAATATGGTCAGGATCTTCTCCATACTGTCTTCAAGAATGGCAAGGTGACAAAAAGCTATTCATTTGATGAAATAAGAAAAAATGCACAGCTGAATATTGAACTGGAAGCAGCACATCATTAG。
upstream primer F (SEQ ID NO. 3): cCTCGAGATGCCATTTGAATTGCCAG (underlined)XhoI cleavage site).
Downstream primer R (SEQ ID NO. 4): gGGTACCTTACTTCGCTTTCGCTTCGC (underlined)KpnI cleavage site).
Upstream primer F (SEQ ID NO. 5): gGAATTCATGAATCCTGCGGCAGAAG (underlined)EcoR I position).
A downstream primer R: (SEQ ID NO.6) CCAAGCTTCTAATGATGTGCTGCTTCCAG (underlined)HinA diiii site).
Claims (10)
1. A recombinant baculovirus, characterized in that the recombinant baculovirus comprises a superoxide dismutase SOD gene and a nicotinamide phosphoribosyltransferase NAMPT gene, wherein the SOD gene is a thermophilic bacteria superoxide dismutase SOD gene.
2. The recombinant baculovirus as claimed in claim 1, wherein the OFR sequence of the SOD gene is SEQ ID No.1, and the OFR sequence of the SOD gene is ATGCCATTTGAATTGCCAGCATTGCCGTATCCGTATGATGCGCTTGAGCCGCACATCGACAAAGAAACGATGAACATTCACCACACGAAGCACCATAACACATACGTTACAAATTTGAATGCGGCGCTTGAAGGGCATCCGGATTTGCAAAACAAATCGCTCGAAGAATTGCTCAGCAATTTGGAAGCCCTTCCGGAAAGCATTCGCACGGCGGTGCGCAACAACGGCGGCGGTCATGCAAACCACTCGCTTTTCTGGACGATTTTGTCGCCAAATGGCGGCGGTGAGCCGACGGGTGAGCTGGCTGAGGCGATCAACAAAAAATTCGGCAGCTTCACCGCGTTTAAAGACGAGTTTTCGAAAGCAGCGGCCGGCCGTTTCGGTTCTGGCTGGGCATGGCTTGTCGTGAACAACGGCGAGCTGGAAATTACGAGCACGCCGAACCAAGACTCGCCGATCATGGAAGGCAAAACGCCGATTCTCGGCTTGGACGTTTGGGAGCATGCGTACTACTTGAAATACCAAAACCGCCGTCCGGAATACATTGCCGCATTCTGGAACATTGTCAACTGGGACGAAGTGGCGAAACGGTACAGCGAAGCGAAAGCGAAGTAA in the SEQ ID No. 1.
3. The recombinant baculovirus as claimed in claim 1, wherein the OFR sequence of the NAMPT gene is SEQ ID No.2, and the SEQ ID No.2 is ATGAATCCTGCGGCAGAAGCCGAGTTCAACATCCTCCTGGCCACCGACTCCTACAAGGTTACTCACTATAAACAATATCCACCCAACACAAGCAAAGTTTATTCCTACTTTGAATGCCGTGAAAAGAAGACAGAAAACTCCAAATTAAGGAAGGTGAAATATGAGGAAACAGTATTTTATGGGTTGCAGTACATTCTTAATAAGTACTTAAAAGGTAAAGTAGTAACCAAAGAGAAAATCCAGGAAGCCAAAGATGTCTACAAAGAACATTTCCAAGATGATGTCTTTAATGAAAAGGGATGGAACTACATTCTTGAGAAGTATGATGGGCATCTTCCAATAGAAATAAAAGCTGTTCCTGAGGGCTTTGTCATTCCCAGAGGAAATGTTCTCTTCACGGTGGAAAACACAGATCCAGAGTGTTACTGGCTTACAAATTGGATTGAGACTATTCTTGTTCAGTCCTGGTATCCAATCACAGTGGCCACAAATTCTAGAGAGCAGAAGAAAATATTGGCCAAATATTTGTTAGAAACTTCTGGTAACTTAGATGGTCTGGAATACAAGTTACATGATTTTGGCTACAGAGGAGTCTCTTCCCAAGAGACTGCTGGCATAGGAGCATCTGCTCACTTGGTTAACTTCAAAGGAACAGATACAGTAGCAGGACTTGCTCTAATTAAAAAATATTATGGAACGAAAGATCCTGTTCCAGGCTATTCTGTTCCAGCAGCAGAACACAGTACCATAACAGCTTGGGGGAAAGACCATGAAAAAGATGCTTTTGAACATATTGTAACACAGTTTTCATCAGTGCCTGTATCTGTGGTCAGCGATAGCTATGACATTTATAATGCGTGTGAGAAAATATGGGGTGAAGATCTAAGACATTTAATAGTATCAAGAAGTACACAGGCACCACTAATAATCAGACCTGATTCTGGAAACCCTCTTGACACTGTGTTAAAGGTTTTGGAGATTTTAGGTAAGAAGTTTCCTGTTACTGAGAACTCAAAGGGTTACAAGTTGCTGCCACCTTATCTTAGAGTTATTCAAGGGGATGGAGTAGATATTAATACCTTACAAGAGATTGTAGAAGGCATGAAACAAAAAATGTGGAGTATTGAAAATATTGCCTTCGGTTCTGGTGGAGGTTTGCTACAGAAGTTGACAAGAGATCTCTTGAATTGTTCCTTCAAGTGTAGCTATGTTGTAACTAATGGCCTTGGGATTAACGTCTTCAAGGACCCAGTTGCTGATCCCAACAAAAGGTCCAAAAAGGGCCGATTATCTTTACATAGGACGCCAGCAGGGAATTTTGTTACACTGGAGGAAGGAAAAGGAGACCTTGAGGAATATGGTCAGGATCTTCTCCATACTGTCTTCAAGAATGGCAAGGTGACAAAAAGCTATTCATTTGATGAAATAAGAAAAAATGCACAGCTGAATATTGAACTGGAAGCAGCACATCATTAG.
4. A method for producing a recombinant baculovirus as claimed in claim 1, 2 or 3, which is obtained by: corresponding primers are designed according to OFR of SEQ ID NO.1 and OFR of SEQ ID NO.2 respectively, and the ORFs of SOD and NAMPT are connected to the same baculovirus vector by using methods of double enzyme digestion and molecular cloning to construct a recombinant vector, wherein the baculovirus vector is pFastBacDual, and the recombinant baculovirus is bombyx mori baculovirus.
5. The method for producing a recombinant baculovirus according to claim 4, wherein: the primer of SEQ ID NO.1 comprises an upstream primer SEQ ID NO.3 and a downstream primer SEQ ID NO. 4; the SEQ ID NO.2 comprises an upstream primer SEQ ID NO.5 and a downstream primer SEQ ID NO. 6.
6. A recombinant protein obtained by infecting silkworm pupae or silkworm cell with the recombinant baculovirus of claim 1, 2 or 3.
7. A pharmaceutical characterized in that a pharmaceutically active ingredient is the recombinant protein according to claim 6.
8. A silkworm chrysalis powder, wherein the silkworm chrysalis powder active ingredient is the recombinant protein of claim 6.
9. A method for preparing the silkworm chrysalis powder of claim 8, wherein the method comprises the following steps:
step 1): infecting silkworm cells with the recombinant baculovirus of any one of claims 1 or 2 or 3 or 4 to obtain virions;
step 2): inoculating the virus particles in the step 1) to the silkworm chrysalis, juicing after 5-6 days, centrifuging at 8000rpm, taking supernatant, and freeze-drying to obtain the silkworm chrysalis freeze-dried powder.
10. The recombinant baculovirus as defined in claim 1 and the recombinant protein expressed by the same are used for anti-aging drugs.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110370474.5A CN113215198B (en) | 2021-04-07 | 2021-04-07 | Recombinant baculovirus and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110370474.5A CN113215198B (en) | 2021-04-07 | 2021-04-07 | Recombinant baculovirus and preparation method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113215198A true CN113215198A (en) | 2021-08-06 |
| CN113215198B CN113215198B (en) | 2023-09-22 |
Family
ID=77086465
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110370474.5A Active CN113215198B (en) | 2021-04-07 | 2021-04-07 | Recombinant baculovirus and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113215198B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114277004A (en) * | 2021-12-01 | 2022-04-05 | 浙江安各洛生物技术有限公司 | High-temperature-resistant recombinant mutant SOD (superoxide dismutase), and coding gene and application thereof |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101015569A (en) * | 2007-01-10 | 2007-08-15 | 浙江大学 | Silkworm powder expressing SOD and preparing method thereof |
| CN101182549A (en) * | 2007-12-05 | 2008-05-21 | 浙江大学 | Construction method of recombination double expressions cultivated silkworm polyhedrosis baculovirus containing SOD gene |
| CN101613679A (en) * | 2008-06-25 | 2009-12-30 | 浙江中奇生物药业股份有限公司 | A kind of baculovirus of recombination superoxide dismutase gene and preparation thereof and application |
| CN103898147A (en) * | 2014-02-27 | 2014-07-02 | 天津耀宇生物技术有限公司 | Expression method of MnSOD fusion bombyx mori nuclear polyhydrosis virus p10 protein |
| CN105223357A (en) * | 2010-09-16 | 2016-01-06 | Cbs生物科学有限公司 | The composition of predicting liver cancer prognosis or kit |
| US20160038576A1 (en) * | 2012-12-03 | 2016-02-11 | Unity Biotechnolgoy, Inc | Immunogenic compositions for inducing an immune response for elimination of senescent cells |
| WO2020129997A1 (en) * | 2018-12-18 | 2020-06-25 | 帝人株式会社 | Genetically modified microorganism and method both for producing nicotinamide derivative, and vector for use in same |
-
2021
- 2021-04-07 CN CN202110370474.5A patent/CN113215198B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101015569A (en) * | 2007-01-10 | 2007-08-15 | 浙江大学 | Silkworm powder expressing SOD and preparing method thereof |
| CN101182549A (en) * | 2007-12-05 | 2008-05-21 | 浙江大学 | Construction method of recombination double expressions cultivated silkworm polyhedrosis baculovirus containing SOD gene |
| CN101613679A (en) * | 2008-06-25 | 2009-12-30 | 浙江中奇生物药业股份有限公司 | A kind of baculovirus of recombination superoxide dismutase gene and preparation thereof and application |
| CN105223357A (en) * | 2010-09-16 | 2016-01-06 | Cbs生物科学有限公司 | The composition of predicting liver cancer prognosis or kit |
| US20160038576A1 (en) * | 2012-12-03 | 2016-02-11 | Unity Biotechnolgoy, Inc | Immunogenic compositions for inducing an immune response for elimination of senescent cells |
| CN103898147A (en) * | 2014-02-27 | 2014-07-02 | 天津耀宇生物技术有限公司 | Expression method of MnSOD fusion bombyx mori nuclear polyhydrosis virus p10 protein |
| WO2020129997A1 (en) * | 2018-12-18 | 2020-06-25 | 帝人株式会社 | Genetically modified microorganism and method both for producing nicotinamide derivative, and vector for use in same |
Non-Patent Citations (6)
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114277004A (en) * | 2021-12-01 | 2022-04-05 | 浙江安各洛生物技术有限公司 | High-temperature-resistant recombinant mutant SOD (superoxide dismutase), and coding gene and application thereof |
| CN114277004B (en) * | 2021-12-01 | 2023-10-17 | 浙江安各洛生物技术有限公司 | High-temperature-resistant recombinant mutant SOD and encoding gene and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113215198B (en) | 2023-09-22 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| KR102649950B1 (en) | Methods of detection and removal of rhabdoviruses from cell lines | |
| CN105209617B (en) | Microcapsule bubble and its manufacturing method | |
| US20200155652A1 (en) | Use of manganese superoxide dismutase with high stability | |
| EA024878B1 (en) | Gene encoding human glucokinase mutant characterized by enhanced stability, and use thereof for controlling blood glucose or for preventing and treating disturbances of carbonydrate metabolism | |
| CN106659805A (en) | Method for inhibiting ebola virus via miRNA | |
| CN102886050A (en) | Application of miRNA-489 and medicinal composition | |
| CN113215198A (en) | Recombinant baculovirus and preparation method and application thereof | |
| CN114214404A (en) | Application of exosome microRNA and mRNA and kit thereof | |
| WO2024012540A1 (en) | Anti-tumor effect, preparation and use of schistosoma japonicum eggs and secreted and excreted proteins thereof | |
| Seah et al. | Variation in ORF3 of genogroup 2 Norwalk-like viruses | |
| CN108034640A (en) | A kind of recombinant Newcastle disease virus for expressing new Duck parvovirus VP3 genes and its application | |
| CN110331152B (en) | Isaria farinosa Cyanovirin-N gene, recombinant protein and application | |
| CN117224669A (en) | Application of African swine fever virus MGF360-21R protein as immunity inducer or adjuvant | |
| WO2020107565A1 (en) | Small molecule protein for eliminating oxygen free radical and use thereof | |
| CN106011088A (en) | Establishment method of cLYZ/hTLF double-antibacterial gene recombinant adenovirus, and recombinant adenovirus and application thereof | |
| US20220403412A1 (en) | Baculovirus Of Recombinant Superoxide Dismutase Gene, Preparation And Application Thereof | |
| CN101906423A (en) | Preparation method of recombined human nerve growth factor based on insect baculovirus expression system | |
| KR20050046754A (en) | Hollow nanoparticles of protein and drug using the same | |
| CN112807375B (en) | Application of Wangshi Baochi pill in preparation of medicine for preventing and/or treating alcoholic liver injury | |
| CN110938134A (en) | Preparation method and application of recombinant human erythropoietin | |
| US5288616A (en) | Process for producing proteins using silkworms infected with recombinant virus | |
| WO2007111534A1 (en) | Method for producing an interleukin-2 preparation and the thus obtainable preparation | |
| CN111171144A (en) | Preparation and application of antibody for resisting porcine epidemic diarrhea virus | |
| CN109824758B (en) | Polypeptide for inhibiting nitric oxide synthase degradation and hypertension and application thereof | |
| CN104491496B (en) | Application of the Gastrodin/Rhizoma Gastrodiae powder in anti-hepatic fibrosis medicines are prepared |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |