CN109943580A - Dual-gene recombination compound macrophage vaccine of one kind and its preparation method and application - Google Patents
Dual-gene recombination compound macrophage vaccine of one kind and its preparation method and application Download PDFInfo
- Publication number
- CN109943580A CN109943580A CN201910034389.4A CN201910034389A CN109943580A CN 109943580 A CN109943580 A CN 109943580A CN 201910034389 A CN201910034389 A CN 201910034389A CN 109943580 A CN109943580 A CN 109943580A
- Authority
- CN
- China
- Prior art keywords
- smurf1
- irak2
- rnai
- compound
- ires
- 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.)
- Pending
Links
- 210000002540 macrophage Anatomy 0.000 title claims abstract description 57
- 150000001875 compounds Chemical class 0.000 title claims abstract description 56
- 230000006798 recombination Effects 0.000 title claims abstract description 52
- 238000005215 recombination Methods 0.000 title claims abstract description 52
- 238000002360 preparation method Methods 0.000 title claims abstract description 27
- 229960005486 vaccine Drugs 0.000 title claims abstract description 23
- 206010008111 Cerebral haemorrhage Diseases 0.000 claims abstract description 28
- 101000852255 Homo sapiens Interleukin-1 receptor-associated kinase-like 2 Proteins 0.000 claims abstract description 21
- 238000011144 upstream manufacturing Methods 0.000 claims abstract description 15
- 230000014509 gene expression Effects 0.000 claims abstract description 14
- 229920001184 polypeptide Polymers 0.000 claims abstract description 14
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 14
- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 14
- 238000001890 transfection Methods 0.000 claims abstract description 13
- 206010061218 Inflammation Diseases 0.000 claims abstract description 10
- 230000004054 inflammatory process Effects 0.000 claims abstract description 10
- 230000004913 activation Effects 0.000 claims abstract description 8
- 102100039289 Glial fibrillary acidic protein Human genes 0.000 claims abstract description 6
- 101710193519 Glial fibrillary acidic protein Proteins 0.000 claims abstract description 6
- 210000005046 glial fibrillary acidic protein Anatomy 0.000 claims abstract description 6
- 102000020313 Cell-Penetrating Peptides Human genes 0.000 claims abstract description 5
- 108010051109 Cell-Penetrating Peptides Proteins 0.000 claims abstract description 5
- 101100149717 Homo sapiens SMURF1 gene Proteins 0.000 claims abstract description 3
- 210000004027 cell Anatomy 0.000 claims description 40
- 239000002299 complementary DNA Substances 0.000 claims description 24
- 102100038631 E3 ubiquitin-protein ligase SMURF1 Human genes 0.000 claims description 20
- 101000664993 Homo sapiens E3 ubiquitin-protein ligase SMURF1 Proteins 0.000 claims description 20
- 102100036433 Interleukin-1 receptor-associated kinase-like 2 Human genes 0.000 claims description 19
- 239000013598 vector Substances 0.000 claims description 17
- 108091008146 restriction endonucleases Proteins 0.000 claims description 15
- 238000012408 PCR amplification Methods 0.000 claims description 8
- 239000013605 shuttle vector Substances 0.000 claims description 8
- 101000911390 Homo sapiens Coagulation factor VIII Proteins 0.000 claims description 6
- 102000057593 human F8 Human genes 0.000 claims description 6
- 238000003780 insertion Methods 0.000 claims description 6
- 230000037431 insertion Effects 0.000 claims description 6
- 229940047431 recombinate Drugs 0.000 claims description 6
- 238000003757 reverse transcription PCR Methods 0.000 claims description 6
- 206010030113 Oedema Diseases 0.000 claims description 4
- 108091030071 RNAI Proteins 0.000 claims description 4
- 229940079593 drug Drugs 0.000 claims description 4
- 239000003814 drug Substances 0.000 claims description 4
- 230000006801 homologous recombination Effects 0.000 claims description 4
- 238000002744 homologous recombination Methods 0.000 claims description 4
- 241000894006 Bacteria Species 0.000 claims description 3
- 239000007864 aqueous solution Substances 0.000 claims description 3
- 239000000203 mixture Substances 0.000 claims description 3
- 238000003756 stirring Methods 0.000 claims description 3
- 108091028043 Nucleic acid sequence Proteins 0.000 claims 3
- 230000009368 gene silencing by RNA Effects 0.000 claims 3
- 150000007523 nucleic acids Chemical group 0.000 claims 3
- 125000003275 alpha amino acid group Chemical group 0.000 claims 2
- 206010048962 Brain oedema Diseases 0.000 abstract description 6
- 208000006752 brain edema Diseases 0.000 abstract description 6
- 230000006872 improvement Effects 0.000 abstract description 3
- 206010018852 Haematoma Diseases 0.000 abstract description 2
- 108020004684 Internal Ribosome Entry Sites Proteins 0.000 abstract description 2
- 238000000338 in vitro Methods 0.000 abstract description 2
- -1 terminator Proteins 0.000 abstract description 2
- 238000002560 therapeutic procedure Methods 0.000 abstract description 2
- 230000029087 digestion Effects 0.000 description 18
- 239000000047 product Substances 0.000 description 18
- 108090000623 proteins and genes Proteins 0.000 description 17
- 239000013612 plasmid Substances 0.000 description 15
- 241000699666 Mus <mouse, genus> Species 0.000 description 13
- 238000000034 method Methods 0.000 description 13
- 239000000499 gel Substances 0.000 description 12
- 241000700605 Viruses Species 0.000 description 9
- 102000004169 proteins and genes Human genes 0.000 description 8
- 101001047514 Bos taurus Lethal(2) giant larvae protein homolog 1 Proteins 0.000 description 7
- 241000588724 Escherichia coli Species 0.000 description 7
- 238000001514 detection method Methods 0.000 description 7
- 239000000284 extract Substances 0.000 description 7
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 6
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 description 6
- 238000000605 extraction Methods 0.000 description 6
- 230000006870 function Effects 0.000 description 6
- 238000000746 purification Methods 0.000 description 6
- 238000000246 agarose gel electrophoresis Methods 0.000 description 5
- 229960000723 ampicillin Drugs 0.000 description 5
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 5
- 238000005119 centrifugation Methods 0.000 description 5
- 230000036425 denaturation Effects 0.000 description 5
- 238000004925 denaturation Methods 0.000 description 5
- 230000003902 lesion Effects 0.000 description 5
- 238000012216 screening Methods 0.000 description 5
- 108090000790 Enzymes Proteins 0.000 description 4
- 102000004190 Enzymes Human genes 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 201000010099 disease Diseases 0.000 description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 238000012163 sequencing technique Methods 0.000 description 4
- 210000002966 serum Anatomy 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 230000009182 swimming Effects 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 238000005336 cracking Methods 0.000 description 3
- 230000001351 cycling effect Effects 0.000 description 3
- 238000001976 enzyme digestion Methods 0.000 description 3
- 210000003743 erythrocyte Anatomy 0.000 description 3
- 230000004927 fusion Effects 0.000 description 3
- 238000001727 in vivo Methods 0.000 description 3
- 230000001939 inductive effect Effects 0.000 description 3
- 208000015181 infectious disease Diseases 0.000 description 3
- 238000003259 recombinant expression Methods 0.000 description 3
- 230000028327 secretion Effects 0.000 description 3
- MZOFCQQQCNRIBI-VMXHOPILSA-N (3s)-4-[[(2s)-1-[[(2s)-1-[[(1s)-1-carboxy-2-hydroxyethyl]amino]-4-methyl-1-oxopentan-2-yl]amino]-5-(diaminomethylideneamino)-1-oxopentan-2-yl]amino]-3-[[2-[[(2s)-2,6-diaminohexanoyl]amino]acetyl]amino]-4-oxobutanoic acid Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@@H](N)CCCCN MZOFCQQQCNRIBI-VMXHOPILSA-N 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- 108090001007 Interleukin-8 Proteins 0.000 description 2
- 102000009073 Macrophage Migration-Inhibitory Factors Human genes 0.000 description 2
- 108010048043 Macrophage Migration-Inhibitory Factors Proteins 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 150000001413 amino acids Chemical group 0.000 description 2
- 210000004227 basal ganglia Anatomy 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 230000034994 death Effects 0.000 description 2
- 210000004443 dendritic cell Anatomy 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 230000028993 immune response Effects 0.000 description 2
- 230000006698 induction Effects 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 210000003734 kidney Anatomy 0.000 description 2
- 210000005228 liver tissue Anatomy 0.000 description 2
- 210000001161 mammalian embryo Anatomy 0.000 description 2
- 210000000496 pancreas Anatomy 0.000 description 2
- 230000010287 polarization Effects 0.000 description 2
- 230000000770 proinflammatory effect Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000010839 reverse transcription Methods 0.000 description 2
- 239000011550 stock solution Substances 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 230000001629 suppression Effects 0.000 description 2
- 230000009885 systemic effect Effects 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 102000010400 1-phosphatidylinositol-3-kinase activity proteins Human genes 0.000 description 1
- 241000208340 Araliaceae Species 0.000 description 1
- 208000032116 Autoimmune Experimental Encephalomyelitis Diseases 0.000 description 1
- 238000011740 C57BL/6 mouse Methods 0.000 description 1
- 102100021824 COP9 signalosome complex subunit 5 Human genes 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 108010012236 Chemokines Proteins 0.000 description 1
- 102000019034 Chemokines Human genes 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 108020004414 DNA Proteins 0.000 description 1
- 230000004568 DNA-binding Effects 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 241000305071 Enterobacterales Species 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 206010016654 Fibrosis Diseases 0.000 description 1
- 102000003688 G-Protein-Coupled Receptors Human genes 0.000 description 1
- 108090000045 G-Protein-Coupled Receptors Proteins 0.000 description 1
- 108700039691 Genetic Promoter Regions Proteins 0.000 description 1
- 101000896048 Homo sapiens COP9 signalosome complex subunit 5 Proteins 0.000 description 1
- 101001050288 Homo sapiens Transcription factor Jun Proteins 0.000 description 1
- 101000800807 Homo sapiens Tumor necrosis factor alpha-induced protein 8 Proteins 0.000 description 1
- 206010062016 Immunosuppression Diseases 0.000 description 1
- 102100037850 Interferon gamma Human genes 0.000 description 1
- 108010074328 Interferon-gamma Proteins 0.000 description 1
- 108090000978 Interleukin-4 Proteins 0.000 description 1
- 206010022773 Intracranial pressure increased Diseases 0.000 description 1
- 102000043136 MAP kinase family Human genes 0.000 description 1
- 108091054455 MAP kinase family Proteins 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 108010057466 NF-kappa B Proteins 0.000 description 1
- 102000003945 NF-kappa B Human genes 0.000 description 1
- 108091007960 PI3Ks Proteins 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 235000005035 Panax pseudoginseng ssp. pseudoginseng Nutrition 0.000 description 1
- 235000003140 Panax quinquefolius Nutrition 0.000 description 1
- 239000006146 Roswell Park Memorial Institute medium Substances 0.000 description 1
- 210000001744 T-lymphocyte Anatomy 0.000 description 1
- 108091023040 Transcription factor Proteins 0.000 description 1
- 102000040945 Transcription factor Human genes 0.000 description 1
- 102100023132 Transcription factor Jun Human genes 0.000 description 1
- 102100033649 Tumor necrosis factor alpha-induced protein 8 Human genes 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 210000001015 abdomen Anatomy 0.000 description 1
- 210000000683 abdominal cavity Anatomy 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 238000007792 addition Methods 0.000 description 1
- 238000013019 agitation Methods 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 210000000612 antigen-presenting cell Anatomy 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 210000003567 ascitic fluid Anatomy 0.000 description 1
- 208000006673 asthma Diseases 0.000 description 1
- 230000005784 autoimmunity Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 102000023732 binding proteins Human genes 0.000 description 1
- 108091008324 binding proteins Proteins 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 230000031018 biological processes and functions Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 208000029028 brain injury Diseases 0.000 description 1
- 210000005013 brain tissue Anatomy 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 230000000120 cytopathologic effect Effects 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 230000003828 downregulation Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000005611 electricity Effects 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 208000012997 experimental autoimmune encephalomyelitis Diseases 0.000 description 1
- 230000004761 fibrosis Effects 0.000 description 1
- 238000011010 flushing procedure Methods 0.000 description 1
- 238000012637 gene transfection Methods 0.000 description 1
- 235000008434 ginseng Nutrition 0.000 description 1
- 230000005934 immune activation Effects 0.000 description 1
- 210000002865 immune cell Anatomy 0.000 description 1
- 230000006058 immune tolerance Effects 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000003119 immunoblot Methods 0.000 description 1
- 230000001506 immunosuppresive effect Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000008595 infiltration Effects 0.000 description 1
- 238000001764 infiltration Methods 0.000 description 1
- 210000004969 inflammatory cell Anatomy 0.000 description 1
- 230000002757 inflammatory effect Effects 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 208000020658 intracerebral hemorrhage Diseases 0.000 description 1
- 229930027917 kanamycin Natural products 0.000 description 1
- 229960000318 kanamycin Drugs 0.000 description 1
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 1
- 229930182823 kanamycin A Natural products 0.000 description 1
- 230000002147 killing effect Effects 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 210000002751 lymph Anatomy 0.000 description 1
- 210000001165 lymph node Anatomy 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 210000004379 membrane Anatomy 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 210000004400 mucous membrane Anatomy 0.000 description 1
- 230000001537 neural effect Effects 0.000 description 1
- 230000016273 neuron death Effects 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000010837 receptor-mediated endocytosis Effects 0.000 description 1
- 102000005962 receptors Human genes 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000008439 repair process Effects 0.000 description 1
- 230000029058 respiratory gaseous exchange Effects 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 210000000813 small intestine Anatomy 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 210000001541 thymus gland Anatomy 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 238000002604 ultrasonography Methods 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
Abstract
The invention belongs to biomedicine fields, and in particular to the dual-gene recombination compound of one kind, vaccine of its preparation and its preparation method and application.The recombination compound is made of polypeptide complex and double gene expression box, and double gene expression box upstream to downstream is successively made of GFAP promoter, IRAK2 RNAi gene, terminator, internal ribosome entry site IRES, SMURF1 gene and terminator；The polypeptide complex is connected and composed by cell-penetrating peptide and positive charge polypeptide.It is provided by the invention it is described it is dual-gene recombination compound macrophage vaccine can in-vitro transfection cerebral hemorrhage Hematoma, it is able to suppress the activation of macrophage, effectively inhibit the inflammatory reaction and brain edema of cerebral hemorrhage, and improve Neuroscore, improvement for cerebral hemorrhage provides ideal strategy and preparation method is simple, it is at low cost, cerebral hemorrhage therapy field with good development and application prospects.
Description
Technical field
The invention belongs to biomedicine fields, and in particular to a kind of dual-gene recombination compound, its preparation vaccine and its
Preparation method and application.
Background technique
Cerebral hemorrhage (Intracerebral hemorrhage, ICH) is a kind of serious central lesion, tool
There are higher disability rate and case fatality rate.Cerebral hemorrhage Brain Injury After includes primary injury caused by hematoma enlargement and increased intracranial pressure
And secondary lesion.Inflammation plays an important role in secondary lesion after cerebral hemorrhage, and specific mechanism is related toThe work of macrophage Change, the infiltration of inflammatory cell and the release of cell factor and Chemokines, eventually lead to neuronal death.Macrophage is
Macrophage is usually divided into classical sharp by important innate immune cells and antigen presenting cell according to the phenotype of macrophage
The M1 type macrophage of approach (proinflammatory and killing) living and the M2 type macrophage of Alternative pathway (suppression is scorching and repairs).Macrophage It is the key pathological factor of the diseases such as panimmunity related disease such as autoimmunity disease, tumour that the polarization of cell is unbalance.
Macrophage migration inhibitory factor (macrophage migration inhibitory factor,IRAK2) be
The Pro-inflammatory mediator of a kind of pleiotropism can promote the secretion or expression of other a variety of anticusp inflammation factors.Its gene is big
There is 90% homology in most mammalian genomes.IRAK2 promoter region contains can be in conjunction with a variety of transcription factors
DNA binding site, simultaneously contain polymorphic site relevant to its expression.IRAK2 plays its biological function, a side
Face can realize the phase of IRAK2 and c-Jun activation structure domain binding protein -1 (JAB1) by non-receptor mediated endocytosis
Interaction；On the other hand, the IRAK2 of receptor-independent type can be activated related to g protein coupled receptor including PI3K/AKT, MAPK
Signal transduction path etc..IRAK2 is it is verified that participate in reconciling the biological processes such as inflammation, tumour generation and fibrosis.
8 sample molecule of tumor necrosis factor α inducible protein, 2 (tumor necrosis factor- α-induced protein
8-like 2,SMURF1) it is (the tumor necrosis factor- α-induced of tumor necrosis factor α inducible protein 8
One of protein 8, TNFAIP8) family member, most sent out early in mice with experimental autoimmune encephalomyelitis animal model
Existing, SMURF1 is mainly expressed in thymus gland, lymph node, spleen and mucous membrane of small intestine etc.,In macrophage, T lymphocyte and B lymph High expression in the immunocytes such as cell plays important negative regulation effect in immune response.SMURF1 participates in a plurality of signal
The regulation of access, such as JNK, NF-kB.Research finds SMURF1 in systemic loupus erythematosus, chronic hbv-infection and heavy breathing
Expressing in asthma has change.SMURF1 expresses downward in Patients with SLE, studies have found that SMURF1 can lead to
It crosses the polarization of induction M2 type macrophage and mitigates systemic loupus erythematosus.
There are mainly two types of the modes of gene association treatment: first is that by a variety of recombinant expression carriers for carrying different genes respectively
Transfected target cells, this mode can freely adjust the ratio of each recombinant expression carrier simultaneously, but need to construct carrying respectively not
Isogenic recombinant expression carrier, construction work is cumbersome, time-consuming, and influence factor is more；Further, since transfection process have centainly with
Machine, the cell after transfection have that gene copy is inhomogenous, have not only been unfavorable for expression and the successive treatment of target gene, but also
Experimental result is caused to be difficult to analysis and assessment, therefore disadvantages mentioned above limits further applying for such mode.Second is thatBy two or More than two genes carry expression by a carrier,This mode can by multiple promoter carrier, polycistronic vector or
The control measures such as fusion improve efficiency gene transfection, increase expression intensity, and maintain opposite expression ratio, to overcome
The deficiency of former mode, is increasingly subject to the attention of researcher in recent years.
Macrophage is intracorporal immunity regulatory cell, in vivo under the action of microenvironment, can be divided into promotion or suppression
The immunocyte of immune response processed.External structure carries the recombinant virus of immunosuppression molecule gene and to transfect macrophage thin
Born of the same parents, then fed back in vivo, it is widely used to the treatment of inducing immune tolerance etc..Therefore, exempt from for after cerebral hemorrhage Epidemic disease activation, inflammation and some secondary lesions, the invention proposes the recombination compound macrophage of IRAK2 and SMURF1 a kind of is thin Born of the same parents' vaccine and its application in cerebral hemorrhage treatment.
Summary of the invention
One of the objects of the present invention is to provide a kind of dual-gene recombination compound, the dual-gene recombination compound, institute
Stating dual-gene recombination compound may participate in induction inhibitory macrophages, effectively inhibit the immune activation after cerebral hemorrhage, and improve
Nervous function.
To achieve the above object, the present invention uses following scheme:
A kind of dual-gene recombination compound, is made of polypeptide complex and double gene expression box, the double gene expression box
Upstream to downstream is successively by GFAP promoter, IRAK2RNAi gene, terminator, internal ribosome entry site IRES, SMURF1
Gene and terminator composition；The polypeptide complex is connected and composed by cell-penetrating peptide and positive charge polypeptide.
Further, the amino acid sequence of the cell-penetrating peptide is as shown in SEQ ID No.1, the amino acid of the positive charge polypeptide
Sequence is as shown in SEQ ID No.2.
The second object of the present invention is to provide a kind of preparation method of dual-gene recombination compound, passes through the preparation
Method can efficiently and accurately prepare the dual-gene recombination compound.
To achieve the above object, the present invention uses following scheme:
The preparation method of the dual-gene recombination compound, comprising the following steps:
1) RT-PCR expands IRAK2RNAi cDNA overall length and SMURF1cDNA overall length；
2) by the upstream IRES of the insertion pCMV carrier of IRAK2RNAi cDNA overall length obtained by step 1), gained SMURF1cDNA
Overall length is inserted into the downstream IRES of pCMV carrier, obtains recombinant vector pCMV-IRAK2RNAi-IRES-SMURF1；
3) using recombinant vector pCMV-IRAK2RNAi-IRES-SMURF1 obtained by step 2) as template, PCR amplification
IRAK2RNAi-IRES-SMURF1 segment and the restriction enzyme site for replacing the segment both ends；
4) IRAK2RNAi-IRES-SMURF1 segment obtained by step 3) is inserted into compound shuttle vector pShuttle-
The GFAP promoter downstream of GFAP, must recombinate compound shuttle vector pShuttle-GFAP-IRAK2RNAi-IRES-SMURF1；
5) will recombination compound shuttle vector pShuttle-GFAP-IRAK2RNAi-IRES-SMURF1 obtained by step 4) with
Carrier pAdEasy-1 connection must recombinate complexes carrier pAd-IRAK2RNAi-MURF1 after homologous recombination in inverted, bacterium；
6) the recombination complexes carrier pAd-IRAK2RNAi-MURF1 that step 5) obtains is packaged into recombination again in cell
Close object Ad-IRAK2RNAi-MURF1.
Further, RT-PCR expands IRAK2RNAi cDNA overall length method particularly includes: human liver tissue total serum IgE is extracted, it is inverse
Transcription obtains cDNA, then using the cDNA as template, using sequence shown in SEQ ID No.3 and SEQ ID No.4 as upstream and downstream primer,
PCR amplification both ends are respectively provided with the IRAK2RNAi overall length of Bgl1 and Apo1 restriction enzyme site, PCR condition are as follows: 94 DEG C of initial denaturations 5 are divided
Clock；Then it is denaturalized 50 seconds for 94 DEG C, 58 DEG C are annealed 50 seconds, and 72 DEG C extend 2 minutes, and totally 30 recycle；Last 72 DEG C extend 10 minutes；
The purified rear clone of PCR product enters pT-Easy carrier, obtains recombinant vector pT-IRAK2.
Further, RT-PCR expands SMURF1cDNA overall length method particularly includes: extracts Human Pancreas total serum IgE, reverses
Record obtains cDNA, then using the cDNA as template, using sequence shown in SEQ ID No.5 and SEQ ID No.6 as upstream and downstream primer,
PCR amplification both ends are respectively provided with the SMURF1cDNA overall length of BamH I and NotI restriction enzyme site, P PCR condition are as follows: 94 DEG C of initial denaturations
4 minutes；Then it is denaturalized 45 seconds for 94 DEG C, 58 DEG C are annealed 60 seconds, and 72 DEG C extend 1 minute, and totally 30 recycle；Last 72 DEG C extend 5 points
Clock；The purified rear clone of PCR product enters pT-Easy carrier, obtains recombinant vector pT-SMURF1.
Further, step 2) method particularly includes: gone out with Bgl1 and Apo1 double digestion from recombinant vector pT-IRAK2
IRAK2cDNA overall length is connect with the pCMV carrier of Bgl1 and Apo1 double digestion with same after purified, obtains recombinant vector pCMV-
IRAK2；Go out SMURF1cDNA overall length with BamH I and NotI double digestion from recombinant vector pT-SMURF1 again, it is after purified and same
Sample is connected with BamH I with the recombinant vector pCMV-IRAK2 of NotI double digestion, obtains recombinant vector pCMV-IRAK2-IRES-
SMURF1。
Further, step 3) method particularly includes: using recombinant vector pCMV-IRAK2-IRES-SMURF1 as template, SEQ
Sequence shown in ID No.7 and SEQ ID No.8 is that upstream and downstream primer carries out PCR, by IRAK2-IRES-SMURF1 segment both ends
Restriction enzyme site is changed to NotI restriction enzyme site, PCR condition are as follows: 94 DEG C initial denaturation 4 minutes；Then it is denaturalized 45 seconds, 58 DEG C for 94 DEG C
Annealing 60 seconds, 72 DEG C extend 1 minute, and totally 30 recycle；Last 72 DEG C extend 5 minutes；The purified rear clone of PCR product enters pT-
Easy carrier obtains recombinant vector pT-IRAK2-IRES-SMURF1.
Further, step 4) method particularly includes: NotI and Not is used from recombinant vector pT-IRAK2-IRES-SMURF1
I double digestion goes out IRAK2-IRES-SMURF1 segment, shuttles and carries with the same compound with I double digestion of NotI and Not after purified
Body pShuttle-GFAP connection, must recombinate compound shuttle vector pShuttle-GFAP-IRAK2-IRES-SMURF1.
Further, step 5) method particularly includes: compound shuttle vector pShuttle-GFAP-IRAK2- will be recombinated
IRES-SMURF1 I linearization for enzyme restriction of Pme, then with Composite Scaffolds carrier pAdEasy-1 electrotransformation e. coli O157 simultaneously
Competent cell carries out homologous recombination intracellular, must recombinate complexes carrier pAd-IRAK2-SMURF1.
Further, step 6) method particularly includes: be inoculated with human embryo kidney 293T cell with the cell density of 40%-60%
Into culture bottle, when cell grows to 30%-50% fusion, compound will be recombinated using 2000 reagent of Lipofectamine
Carrier pAd-IRAK2-SMURF1 transfects 293T cell, and cell is collected by centrifugation when obvious lesion occurs in cell, slow with phosphate
After fliud flushing is resuspended, multigelation makes cell cracking, and it is multiple to get recombination to collect the supernatant containing virus for centrifugation removal cell fragment
Close object Ad-IRAK2-SMURF1.
Further, the step further includes that the recombination compound Ad-IRAK2RNAi-SMURF1 for obtaining step 6) is used
0.9% Nacl solution dissolution, and the aqueous solution for being dissolved with polypeptide complex is added dropwise, continue stirring 10 minutes after being added dropwise,
Stand 10 minutes again to get the dual-gene recombination compound.
The third object of the present invention is to provide a kind of macrophage vaccine, the specially dual-gene recombination compound
The Ad-IRAK2-SMURF1 macrophage vaccine of preparation.
The fourth object of the present invention is the provision of a kind of preparation method of macrophage vaccine, specially described
The preparation method of Ad-IRAK2-SMURF1 macrophage vaccine.
To achieve the above object, the present invention uses following scheme:
The Ad-IRAK2-SMURF1 macrophage vaccine the preparation method comprises the following steps: the dual-gene recombination by forth generation is compound
Object Transfection of macrophages is up to the Ad-IRAK2-SMURF1 macrophage vaccine.
The fifth object of the present invention is the provision of a kind of application of dual-gene recombination compound, is specially preparing
Inhibit macrophage activation, inhibits the application in the cerebral hemorrhage treatment drug of cerebral hemorrhage inflammation and oedema.
The sixth object of the present invention is the provision of answering for Ad-IRAK2-SMURF1 macrophage vaccine described in one kind
With specially inhibiting macrophage activation in preparation, inhibit the application in the cerebral hemorrhage treatment drug of cerebral hemorrhage inflammation and oedema.
The beneficial effects of the present invention are:
1) it is provided by the invention it is described it is dual-gene recombination compound macrophage vaccine can in-vitro transfection cerebral hemorrhage hemotoncus
Surrounding tissue is able to suppress the activation of macrophage, effectively inhibits the inflammatory reaction and brain edema of cerebral hemorrhage, and improves neural function
It can score, provide ideal strategy for the improvement of cerebral hemorrhage；
2) preparation method provided by the invention is simple, at low cost, in the therapy field of cerebral hemorrhage there is good exploitation to answer
Use prospect.
Detailed description of the invention
Fig. 1 is the agarose gel electrophoresis figure of the SMURF1 full-length cDNA PCR product of embodiment 1.2.
Fig. 2 is the protein immunoblot result figure of the dual-gene recombination compound Transfection of macrophages of embodiment 2.2.
Fig. 3 is the cytokine secretion histogram for the macrophage that the dual-gene recombination compound of embodiment 2.3 is incubated for.
Fig. 4 is the brain edema testing result histogram of 3 cerebral hemorrhage mouse of embodiment.
Fig. 5 is the nervous function testing result histogram of 4 cerebral hemorrhage mouse of embodiment.
Specific embodiment
Illustrated embodiment is in order to which preferably the present invention will be described, but is not that the contents of the present invention are limited only to institute
For embodiment.So those skilled in the art according to foregoing invention content to embodiment carry out it is nonessential improvement and
Adjustment, still falls within protection scope of the present invention.
The preparation of the dual-gene recombination compound of embodiment 1
The clone of 1.1IRAK2RNAi
Human liver tissue total serum IgE is extracted by Trizol kit specification, reverse transcription obtains total cDNA, then is with total cDNA
Template, with F1:5 '-aatctgcag(SEQ ID No.3, underscore part are Bgl1 to acttttcgcccgcacagagcgg-3 '
Restriction enzyme site) and R1:5 '-tacgaattcAagcgcgtgcacgaggaggtcacg-3 ' (SEQ ID No.4, underscore part
For Apo1 restriction enzyme site) it is upstream and downstream primer, PCR amplification IRAK2 full-length RNA i, PCR reaction system is 50 μ l, cycling condition ginseng
Number are as follows: 94 DEG C initial denaturation 4 minutes；Then it is denaturalized 45 seconds for 94 DEG C, 58 DEG C are annealed 60 seconds, and 72 DEG C extend 1 minute, and totally 30 recycle；
Last 72 DEG C extend 5 points.PCR product through agarose gel electrophoresis identify, gel reclaims kit gel extraction after purification, with
The connection of pT-Easy carrier, connection product converts bacillus coli DH 5 alpha competent cell, with the LB flat screen containing ampicillin
Positive colony is selected, plasmid is extracted, positive colony plasmid is named as pT-IRAK2RNAi by sequencing identification.
The clone of 1.2SMURF1 full-length cDNA
Human Pancreas total serum IgE is extracted by Trizol kit specification, reverse transcription obtains total cDNA, then with total cDNA
For template, with F2:5 '-gatcggatcc(SEQ ID No.5, underscore part are I digestion of BamH to gtagacaggtacccc-3 '
Site) and R2:5 '-acgcgtcgac(SEQ ID No.6, underscore part are NotI enzyme to caaatttagaataggttcc-3 '
Enzyme site) it is upstream and downstream primer, PCR amplification SMURF1 full-length cDNA, PCR reaction system is 50 μ l, cycling condition parameter are as follows: 94
DEG C initial denaturation 4 minutes；Then it is denaturalized 45 seconds for 94 DEG C, 58 DEG C are annealed 60 seconds, and 72 DEG C extend 1 minute, and totally 30 recycle；Last 72 DEG C
Extend 5 points.PCR product after purification, is carried through agarose gel electrophoresis identification, gel reclaims kit gel extraction with pT-Easy
Body connection, connection product convert bacillus coli DH 5 alpha competent cell, with positive gram of the LB plate screening containing ampicillin
It is grand, plasmid is extracted, positive colony plasmid is named as pT-SMURF1 by sequencing identification.
Agarose gel electrophoresis results show that PCR product is in single specificity band (Fig. 1) at about 1000bp, with expection
As a result it is consistent；Sequencing result shows the insertion gene order and GenBank (nm_001199847.1) of positive colony plasmid
SMURF1 full length cDNA sequence is consistent (1 swimming lane is DNA molecular amount standard, and 2 swimming lanes are PCR product).
The building of 1.3 recombinant vector pCMV-IRAK2RNAi-SMURF1
According to restriction enzyme site designed by IRAK2 and SMURF1 full-length cDNA both ends, IRAK2 full-length cDNA is inserted into first
The upstream IRES of pCMV carrier, then by SMURF1 full-length cDNA insertion pCMV carrier the downstream IRES.Method particularly includes: first will
PT-IRAK2 carrier Bgl1 and Apo1 double digestion, double enzyme digestion product through gel reclaims kit gel extraction after purification, and it is same
Sample is connected through the pCMV carrier of Bgl1 with Apo1 double digestion, and connection product converts bacillus coli DH 5 alpha competent cell, with containing
The LB plate screening positive colony of ampicillin, extracts plasmid, and positive colony plasmid is ordered in the identification of Bgl1 and Apo1 double digestion
Entitled pCMV-IRAK2；Again by pT-SMURF1 carrier BamH I and NotI double digestion, double enzyme digestion product is through gel reclaim reagent
Box gel extraction after purification, is connect with the pCMV-IRAK2 carrier equally through BamH I and NotI double digestion, and connection product conversion is big
Enterobacteria DH5 α competent cell extracts plasmid, I He of BamH with the LB plate screening positive colony containing ampicillin
The identification of NotI double digestion, is named as pCMV-IRAK2RNAi-IRES-SMURF1 for positive colony plasmid.
The building of 1.4 recombinant vector pShuttle-GFAP-IRAK2RNAi-IRES-SMURF1
Using pCMV-IRAK2RNAi-IRES-SMURF1 carrier as template, with F3:5 '-acgcgtcgaccacatcgcggc
Cggagcacttt-3 ' (SEQ ID No.7, underscore part are NotI restriction enzyme site) and R3:5 '-agaatgcggccgcaga
Acaaatatttggttcc-3 ' (SEQ ID No.8, underscore part are I restriction enzyme site of Not) is upstream and downstream primer, PCR amplification
The restriction enzyme site at the segment both ends is simultaneously changed to I digestion of NotI restriction enzyme site and Not by IRAK2RNAi-IRES-SMURF1 segment
Site, PCR reaction system and cycling condition parameter are the same.PCR product is through agarose gel electrophoresis identification, gel reclaims kit
Gel extraction after purification, is connect with pT-Easy carrier, and connection product converts bacillus coli DH 5 alpha competent cell, with containing ammonia
The LB plate screening positive colony of parasiticin, extracts plasmid, and positive colony plasmid is named as pT- by sequencing identification
IRAK2RNAi-IRES-SMURF1。
By pT-IRAK2RNAi-IRES-SMURF1 carrier I double digestion of NotI and Not, double enzyme digestion product is recycled through gel
Kit gel extraction after purification, is connect, connection product with the pShuttle-GFAP carrier equally through I double digestion of NotI and Not
Bacillus coli DH 5 alpha competent cell is converted, with the LB plate screening positive colony containing ampicillin, extracts plasmid, NotI
It is identified with I double digestion of Not, positive colony plasmid is named as pShuttle-GFAP-IRAK2RNAi-IRES-SMURF1.
The building of 1.5 recombination complexes carrier pAd-IRAK2RNAi-SMURF1
By pShuttle-GFAP-IRAK2RNAi-IRES-SMURF1 carrier I linearization for enzyme restriction of Pme, then with
Electrotransformation e. coli BJ5183 competent cell, progress bacterium In vivo homologous recombination block that with containing to pAdEasy-1 carrier simultaneously
The LB plate culture of mycin, picking extract plasmid with LB liquid medium overnight incubation containing kanamycin compared with petite,
I digestion of Pac identification, is named as pAd-IRAK2RNAi-SMURF1 for positive plasmid.
The packaging of 1.6 recombination complexes carrier pAd-IRAK2RNAi-SMURF1
Human embryo kidney 293T cell is seeded in T-25 culture bottle with 40%~60% cell density, is grown to cell
To culture solution is discarded when 30%~50% fusion, pAd-IRAK2RNAi-SMURF1 is carried with 2000 reagent of Lipofectamine
Body transfects 293T cell, is changed by micro- sem observation cell pathology, and when obvious cytopathic effect to appear is collected by centrifugation thin
Born of the same parents after cell precipitation is resuspended with PBS, make cell cracking for multigelation 4 times, and centrifugation removal cell fragment is collected containing the upper of virus
Clear liquid is to get Ad-IRAK2RNAi-SMURF1 virus stock solution used；Virus stock solution used is re-infected into 293T cell in the ratio of 1:10,
The supernatant containing virus is collected, is repeated aforesaid operations 3 times, forth generation recombination compound Ad-IRAK2RNAi-SMURF1 is obtained.
The preparation of 1.7 dual-gene recombination compounds
Under agitation, it is added dropwise and is dissolved with into the 0.9% Nacl solution dissolved with Ad-IRAK2RNAi-SMURF1
The aqueous solution of polypeptide complex, continue after being added dropwise stirring 10 minutes, then stand 10 minutes to get.
The preparation of the dual-gene recombination compound Transfection of macrophages vaccine of embodiment 2
2.1. the sorting of macrophage
C57BL/6 normal mouse is taken, neck is taken off and puts to death, through 75% alcohol routine disinfection.Serum-free is injected into mouse peritoneal
1640 culture medium 5m L of RPMI rubs abdomen 5min repeatedly, opens abdominal cavity after standing 3min, extracts peritoneal fluid centrifugation, detection
Kind is cultured in 6 orifice plates 3h after cell density, removes non-attached cell, rejoins culture medium and cell factor: IFN-γ
10ng/m L,LPS 100ng/m L；M2 type macrophage: IL-4 10ng/m L) stimulation culture is for 24 hours.
2.2. dual-gene recombination compound Transfection of macrophages
Macrophage is inoculated in 6 orifice plates with cell concentration for 1 × 106/ml, is 100 additions by infection multiplicity (MOI)
The dual-gene recombination compound of forth generation, 48 hours collection cells after infection are washed and are resuspended with PBS and is multiple to get dual-gene recombination
Close object Transfection of macrophages.
Western Blot detection: the macrophage of the dual-gene recombination compound transfection of ultrasound cracking and empty virus turn respectively
The macrophage of dye collects total protein of cell and carries out SDS-PAGE, and electricity transfer pvdf membrane, washes film after electrophoresis, closes, and is added
Rabbit-anti people's SMURF1 polyclonal antibody, 37 DEG C are incubated for 1 hour, wash film, add goat anti-rabbit igg, and 37 DEG C are incubated for 1 hour, wash film,
Colour developing.As a result see Fig. 2, dual-gene recombination compound can express SMURF1 albumen in macrophage, and (1 swimming lane is Ad-
The total protein of IRAK2/SMURF1 virus transfection Dendritic Cells, 2 swimming lanes are the total protein of empty virus transfection Dendritic Cells).
2.3. the detection of the inflammatory factor of macrophage after erythrocyte splitting object is handled,
The macrophage or control macrophage that the dual-gene recombination compound that 0.1mg is added in 10 μ l erythrocyte cracked liquids is incubated for
Cell, 37 DEG C, for 24 hours, with the ability of ELISA method detection erythrocyte cracked liquid stimulating expression of macrophage secretion IL-8 and TNF-α.Research
As a result as shown in figure 3, IL-8 secreted by the macrophage that dual-gene recombination compound is incubated for and TNF-α are less than control macrophage
Cell.
The detection of the brain edema of 3 cerebral hemorrhage mouse of embodiment
The 25 self whole bloods of μ l are injected into mouse Basal ganglia, after 10min, and in the dual-gene heavy of same area injection 1mg
Group compound macrophage vaccine or control (PBS).After 3 days, mouse is put to death, obtains brain tissue, and small with dry and wet weight method detection
The brain edema situation of mouse.Result of study is as shown in figure 4, compared with control (PBS), dual-gene recombination compound macrophage vaccine
The brain edema of the substantially reduced cerebral hemorrhage mouse of energy.
The detection of the nervous function of 4 cerebral hemorrhage mouse of embodiment
The 25 self whole bloods of μ l are injected into mouse Basal ganglia, after 10min, and in the dual-gene heavy of same area injection 1mg
Group compound macrophage vaccine or control (PBS).After 3 days, with Neuroscore method, the nervous function of mouse is observed.It grinds
Result is studied carefully as shown in figure 5, dual-gene recombination compound macrophage vaccine can significantly improve with control compared with compareing (PBS)
The nervous function of mouse.
Finally, it is stated that the above examples are only used to illustrate the technical scheme of the present invention and are not limiting, although referring to compared with
Good embodiment describes the invention in detail, those skilled in the art should understand that, it can be to skill of the invention
Art scheme is modified or replaced equivalently, and without departing from the objective and range of technical solution of the present invention, should all be covered at this
In the scope of the claims of invention.
Claims (10)
1. a kind of dual-gene recombination compound, which is characterized in that the recombination compound is by polypeptide complex and double gene expression
Box composition, double gene expression box upstream to downstream is successively by GFAP promoter, IRAK2 RNAi gene, terminator, inside
Ribosome entry site IRES, SMURF1 gene and terminator composition；The polypeptide complex is by cell-penetrating peptide and positive charge polypeptide
It connects and composes.
2. dual-gene recombination compound according to claim 1, which is characterized in that the amino acid sequence of the cell-penetrating peptide is such as
Shown in SEQ ID No.1, the amino acid sequence of the positive charge polypeptide is as shown in SEQ ID No.2.
3. the preparation method of dual-gene recombination compound described in claim 1-3, comprising the following steps:
1) RT-PCR expands IRAK2 RNAi cDNA overall length and SMURF1 cDNA overall length；
2) by the upstream IRES of the insertion pCMV carrier of IRAK2 RNAi cDNA overall length obtained by step 1), gained SMURF1 cDNA is complete
The downstream IRES of long insertion pCMV carrier, obtains recombinant vector pCMV-IRAK2 RNAi-IRES-SMURF1；
3) using recombinant vector pCMV-IRAK2 RNAi-IRES-SMURF1 obtained by step 2) as template, PCR amplification IRAK2
RNAi-IRES-SMURF1 segment and the restriction enzyme site for replacing the segment both ends；
4) by the insertion of IRAK2 RNAi-IRES-SMURF1 segment obtained by step 3) compound shuttle vector pShuttle-GFAP's
GFAP promoter downstream must recombinate compound shuttle vector pShuttle-GFAP-IRAK2 RNAi-IRES-SMURF1；
5) by recombination compound shuttle vector pShuttle-GFAP-IRAK2 RNAi-IRES-SMURF1 obtained by step 4) and load
Body pAdEasy-1 connection must recombinate complexes carrier pAd-IRAK2 RNAi-MURF1 after homologous recombination in inverted, bacterium；
6) by the recombination complexes carrier pAd-IRAK2 RNAi-MURF1 that step 5) obtains be packaged into cell recombination it is compound
Object Ad-IRAK2 RNAi-MURF1.
4. the preparation method according to claim 4, which is characterized in that step 1) RT-PCR expands IRAK2 RNAi cDNA
The nucleic acid sequence of the upstream and downstream primer of overall length is as shown in SEQ ID No.3 and SEQ ID No.4；RT-PCR expands SMURF1
The nucleic acid sequence of the upstream and downstream primer of cDNA overall length is as shown in SEQ ID No.5 and SEQ ID No.6.
5. the preparation method according to claim 4, which is characterized in that step 3) PCR amplification IRAK2 RNAi-IRES-
The nucleic acid sequence of the upstream and downstream primer of SMURF1 segment is as shown in SEQ ID No.7 and SEQ ID No.8, IRAK2 RNAi-
The restriction enzyme site at IRES-SMURF1 segment both ends is changed to NotI restriction enzyme site.
6. the preparation method according to claim 4, which is characterized in that the step further includes the recombination for obtaining step 6)
Compound Ad-IRAK2 RNAi-SMURF1 dissolves and is added dropwise the aqueous solution of polypeptide complex, and stirring stands to get described biradical
Because recombinating compound.
7. the Ad-IRAK2-SMURF1 macrophage vaccine of dual-gene recombination compound preparation claimed in claims 1-2.
8. the preparation method of Ad-IRAK2-SMURF1 macrophage vaccine described in claim 7, which is characterized in that by forth generation
Dual-gene recombination compound Transfection of macrophages up to the Ad-IRAK2-SMURF1 macrophage vaccine.
9. dual-gene recombination compound claimed in claims 1-2 inhibits macrophage activation in preparation, inhibit cerebral hemorrhage inflammation
With the application in the cerebral hemorrhage treatment drug of oedema.
10. Ad-IRAK2-SMURF1 macrophage vaccine as claimed in claim 7 inhibits macrophage activation in preparation, inhibit
Application in the cerebral hemorrhage treatment drug of cerebral hemorrhage inflammation and oedema.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910034389.4A CN109943580A (en) | 2019-01-15 | 2019-01-15 | Dual-gene recombination compound macrophage vaccine of one kind and its preparation method and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910034389.4A CN109943580A (en) | 2019-01-15 | 2019-01-15 | Dual-gene recombination compound macrophage vaccine of one kind and its preparation method and application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN109943580A true CN109943580A (en) | 2019-06-28 |
Family
ID=67007170
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910034389.4A Pending CN109943580A (en) | 2019-01-15 | 2019-01-15 | Dual-gene recombination compound macrophage vaccine of one kind and its preparation method and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109943580A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111494616A (en) * | 2020-04-27 | 2020-08-07 | 重庆医科大学附属永川医院 | New coronavirus immune enhanced gene vaccine and preparation method thereof |
| CN111840576A (en) * | 2020-07-27 | 2020-10-30 | 重庆医科大学附属永川医院 | TIPE-2 and NRDP1 recombinant complex and preparation method and application thereof |
-
2019
- 2019-01-15 CN CN201910034389.4A patent/CN109943580A/en active Pending
Non-Patent Citations (7)
| Title |
|---|
| HAN D等: "《NCBI Reference Sequence: NM_001199847.2》", 《HOMO SAPIENS SMAD SPECIFIC E3 UBIQUITIN PROTEIN LIGASE 1 (SMURF1),TRANSCRIPT VARIANT 3, MRNA》 * |
| LI T等: "《Homo sapiens macrophage migration inhibitory factor (MIF), mRNA》", 《NCBI REFERENCE SEQUENCE: NM_002415.2》 * |
| MING‑WEI LIU等: "《Atorvastatin increases lipopolysaccharide‑induced expression of tumour necrosis factor‑α‑induced protein 8‑like 2 in RAW264.7 cells》", 《EXPERIMENTAL AND THERAPEUTIC MEDICINE》 * |
| SHAO S, TSOI LC等: "《Homo sapiens interleukin 1 receptor associated kinase 2 (IRAK2), mRNA》", 《NCBI REFERENCE SEQUENCE: NM_001570.4》 * |
| SHI B等: "《Homo sapiens TNF alpha induced protein 8 like 2 (TNFAIP8L2), mRNA》", 《NCBI REFERENCE SEQUENCE: NM_024575.5》 * |
| 丁兆军等主编: "《大肠癌基础与临床》", 31 August 2007, 山东科学技术出版社 * |
| 李颖著: "《细胞抗病毒天然免疫信号转导的调控机制》", 31 March 2015, 武汉大学出版社 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111494616A (en) * | 2020-04-27 | 2020-08-07 | 重庆医科大学附属永川医院 | New coronavirus immune enhanced gene vaccine and preparation method thereof |
| CN111840576A (en) * | 2020-07-27 | 2020-10-30 | 重庆医科大学附属永川医院 | TIPE-2 and NRDP1 recombinant complex and preparation method and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110128550B (en) | Novel replicative oncolytic adenovirus capable of simultaneously blocking immune check points PD-L1 and TIGIT and application | |
| CN112626090B (en) | Nucleotide sequence for coding novel coronavirus antigen and application thereof | |
| Zullo et al. | Regulation of c-myc and c-fos mRNA levels by polyomavirus: distinct roles for the capsid protein VP1 and the viral early proteins. | |
| WO2024002149A1 (en) | Recombinant type-iii collagen and method for preparing same | |
| WO2022007742A1 (en) | Recombinant pseudorabies virus and vaccine composition thereof | |
| CN110484615A (en) | LncRNA regulates and controls the polarized application of macrophage in vital myocarditis | |
| WO2022100746A1 (en) | Bispecific single-chain antibody, antibody-loaded recombinant oncolytic virus, and virus combination | |
| CN111606999B (en) | Replicative oncolytic adenovirus with functions of activating immune co-stimulatory signaling pathway and blocking immune checkpoint and application thereof | |
| CN109943580A (en) | Dual-gene recombination compound macrophage vaccine of one kind and its preparation method and application | |
| CN102115787B (en) | MicroRNA (Ribose Nucleic Acid) and application of antisensenucleic acid of microRNA in diagnosis, prevention, treatment and/or prognostic evaluation of heart disease | |
| CN108588026A (en) | The preparation method and its usage of the clinical grade mescenchymal stem cell of height expression IL10 | |
| CN114395574B (en) | Porcine epidemic diarrhea virus fusion protein, and encoding gene and application thereof | |
| CN110157686B (en) | Replication type oncolytic adenovirus activated by immune checkpoint and immune co-stimulation and construction method and application thereof | |
| CN111607571B (en) | Replicative oncolytic adenovirus for specifically activating immune co-stimulation pathway and preparation method and application thereof | |
| CN113249408B (en) | Construction and application of nucleic acid vaccine vector for targeting activation of humoral immunity and cellular immunity | |
| CN109456948A (en) | MIF and TIPE2 double gene coexpression recombined adhenovirus and its preparation method and application | |
| CN116970058A (en) | Tumor neoantigen polypeptide aiming at TP53 gene R249S mutation and application thereof | |
| CN110423812B (en) | Use of Skiv2l2 (MTR 4) gene in tumor treatment | |
| CN102660579B (en) | HBx and human IL-12 double-gene recombinant vector and liver caner-resistant vaccine | |
| CN113248577B (en) | Coronavirus vaccine using adenovirus as carrier and its preparing method | |
| CN110628728A (en) | Recombinant oncolytic gene-adenovirus of targeted cancer and construction method and application thereof | |
| CN109593761B (en) | Small RNA related to Brucella virulence and application thereof in preparation of attenuated Brucella | |
| CN113373176A (en) | Construction method of gene therapy vector and application of gene therapy vector in Alzheimer disease drugs | |
| CN113699123B (en) | Preparation and application of targeted hypersensitive broad-spectrum oncolytic virus | |
| CN104830905B (en) | Iba1 and the double gene coexpression recombinant adenoviral vectors of LAG 3 and its preparation method and application |
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 | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20190628 |
|
| RJ01 | Rejection of invention patent application after publication |