CN109182377A - A kind of ADAR1 is overexpressed viral vectors, its construction method and application - Google Patents
A kind of ADAR1 is overexpressed viral vectors, its construction method and application Download PDFInfo
- Publication number
- CN109182377A CN109182377A CN201811023833.4A CN201811023833A CN109182377A CN 109182377 A CN109182377 A CN 109182377A CN 201811023833 A CN201811023833 A CN 201811023833A CN 109182377 A CN109182377 A CN 109182377A
- Authority
- CN
- China
- Prior art keywords
- rna
- overexpressed
- viral vectors
- adenosine deaminase
- sequence
- 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
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
- 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
- A61K38/46—Hydrolases (3)
- A61K38/50—Hydrolases (3) acting on carbon-nitrogen bonds, other than peptide bonds (3.5), e.g. asparaginase
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
-
- 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
- C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
- C12N5/0645—Macrophages, e.g. Kuepfer cells in the liver; Monocytes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
- C12Y305/04—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in cyclic amidines (3.5.4)
- C12Y305/04004—Adenosine deaminase (3.5.4.4)
-
- 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
- C12N2710/10011—Adenoviridae
- C12N2710/10041—Use of virus, viral particle or viral elements as a vector
- C12N2710/10043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Medicinal Chemistry (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- General Chemical & Material Sciences (AREA)
- Plant Pathology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Gastroenterology & Hepatology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Oncology (AREA)
- Communicable Diseases (AREA)
- Epidemiology (AREA)
- Cell Biology (AREA)
- Hematology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Virology (AREA)
- Pain & Pain Management (AREA)
- Rheumatology (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The present invention provides the adenosine deaminase 1 of RNA a kind of be overexpressed viral vectors, preparation method and the RNA adenosine deaminase 1 be overexpressed viral vectors have protection small intestine structural integrity, maintain enteron aisle stable state；Improve the activity of enteron aisle macrophage；Inhibit the level of inflammatory cytokine and treats the application in pyemic product in preparation.The adenosine deaminase 1 of RNA of the present invention is overexpressed viral vectors and provides new potential therapy target for pyemic treatment.
Description
Technical field
The present invention relates to gene engineering technology fields, and in particular to a kind of ADAR1 overexpression viral vectors, it is especially a kind of
ADAR1 is overexpressed viral vectors, its construction method and in protection small intestine structural integrity, maintenance enteron aisle stable state；Improve enteron aisle macrophage
The activity of cell；Inhibit the application in the level and the pyemic product of preparation treatment of inflammatory cytokine.
Background technique
Pyemia (sepsis) is by infecting caused systemic inflammatory response syndrome, is severe trauma, burn and sense
The common complication of infectious diseases.When pyemia, which continues development, merges circulation function failure, disease progression is infectious shock
And multiple organ dysfunction syndrome.Pathogenesis of sepsis mechanism is complicated and case fatality rate is high, is wartime wound and Severe acute disease medicine
The protrusion problem faced.
Macrophage is a kind of multi-functional immunocyte, is primarily involved in body inherent immunity, while being also that connection is intrinsic
Immune and specific immune bridge, biological activity specifically include that phagocytosis is removed germ and non-viable non-apoptotic cell, antigen presentation and divided
Secrete inflammatory mediator.
Enteron aisle is both the organ that pyemia disease process is easily damaged and the pyemia initiating device that sb.'s illness took a turn for the worse
Official, gut barrier function obstacle caused by intestinal cell is impaired is recurred in pyemia has vital work in development
With.
Document Anti-cytokine therapies in response to systemic infection (J.Inve
Stigat.Dermatol.Sympos.Proc.6 (2001) 244-250), document Anti-cytokine and anti-
inflammatory therapies for the treatment of severe sepsis:progress and
Pitfalls (Proc.Nutr.Soc.63 (2004) 437-441) and document MiR-212-3p inhibits LPS-induced
inflammatory response through targeting HMGB1in murine macrophages(Exp.Cell
Res.350 (2017) 318-326) enteron aisle macrophages secrete a large amount of inflammatory factors when disclosing pyemia, cause enteron aisle stable state
It is unbalance, induce systemic injury.The overactivity of macrophage is the key that enteron aisle homeostasis effector cell, macrophage
Damage and activation be runaway inflammatory reaction occurs for body basic reason.
The adenosine deaminase 1 (ADAR1) of RNA is one of rna editing enzyme family member, is catalyzed double-strand by deamination
The upper adenosine of RNA becomes inosine, i.e. A-I conversion makes genetic code change, encoded protein knot
Structure and function change therewith.The adenosine deaminase 1 of RNA is a species specific modification enzyme, by the adenosine on special site
(A) it deaminizes and is changed into hypoxanthine (I), ribosomes pronounces guanine (G) when decoding, and leads to the replacement of double-stranded RNA nucleotide, phase
Answer Argine Monohydrochloride change, RNA structural instability, 50mRNA degradation, viral genetic unstability of dependenc RNA duplication etc..
ADAR1 is widely present in various tissues, participates in a variety of important physiology courses, including embryonic development, hemopoietic system development, maincenter
The transmitting of nervous system mediator and immunity of organism etc..Many research discovery rna editing enzymes play during tumor development
Critical function, such as document ADAR1prevents liver injury from inflammation and
suppresses interferon production in hepatocytes(Am.J.Pathol.185(2015)3224–
3237) disclosing rna editing leads to the change for participating in albumen in the process signal access.
Document ADAR1activation drives leukemia stem cell self-renewal
Byimpairing let-7biogenesis (Cell Stem Cell 19 (2016) 177-191), document ADAR1prevents
liver injury from inflammation and suppresses interferon production in
Hepatocytes (Am.J.Pathol.185 (2015) 3224-3237) and document Adenosine deaminase that
acts on RNA 1p150in alveolar macrophage is involved in LPS-induced lung
Individually disclosed in injury (Shock (Augusta, Ga) .31 (2009) 410-415) inflammatory activity of ADAR1 leukaemia,
Application in hepatic injury, injury of lungs, however currently without in hair existing literature or patent disclosure ADAR1 inflammatory activity and pyemia
The activation of macrophage reduces application in inflammatory factor expression.
Pyemic essence is runaway release and the inflammatory reaction Imbalance of inflammatory mediator, and body is caused self to continue
Property amplification inflammatory reaction so that cause body self destroy, be embodied in the inflammatory cell and excessive inflammatory mediator of activation
It is sent out by blood plasma to remote part and causes systemic inflammatory.Diagnosis of sepsis mainly passes through detection molecules marker RGL4
(CN106282355A), the level of LETMD1 (CN106367488A) or CYB561A3 (CN106119402A).For pyemia
Therapeutic agent, patent CN105878262A discloses therapeutic effect of the forsythin to rats with sepsis.Patent
CN104546832A, patent CN104784681A, patent CN105663137A, patent CN107998376, CN108025036A,
CN107496441A, CN107715105A, it CN108014119A, individually discloses containing Edaravone-
The pharmaceutical composition of 5- ketone or its pharmaceutically acceptable salt and borneol, protein kinase Stk38, pirenzepine, CD42b, Panx1
Hemichannel protein antagonist or a certain amount of Cx43 hemichannel protein antagonist, cyclodextrin, IL34, Phenylpropanoid Glycosides glucosides
Application of the Smiglaside A in preparation treatment medication for treating pyemia.CN106924559A,CN108272971A,
CN107669820A also discloses the pyemic Chinese medicine preparation for the treatment of.But ADAR1 over-express vector is not disclosed and is preparing
It is applied in treatment of sepsis drug.
Summary of the invention
Present inventors have surprisingly found that not only resulted in after the adenosine deaminase 1 (ADAR1) of the RNA in deletion macrophage
Cell quick death and inflammation is deteriorated.With that is, preparation ADAR1 is overexpressed viral vectors, regulation by creative work
The activity of body endogenous ADAR1 enzyme significantly improves the survival of pyemia mouse.ADAR1 of the present invention is overexpressed virus
Carrier is applied in treatment of sepsis product.The expression for reducing TNF-α, IL-6, IL-10 and IFN-β inflammatory factor is produced, it is special
It is not IL6 and TNF；It ensure that small intestine structural integrity, the improvement that enteron aisle homeostasis is unbalance；Improve the activity of enteron aisle macrophage；
And significantly improve the effect of the survival rate of pyemia mouse.The adenosine deaminase 1 of RNA of the present invention is overexpressed viral vectors
New potential therapy target is provided for pyemic treatment.Meanwhile ADAR1 of the present invention is overexpressed viral vectors
Preparation method is overexpressed the nucleic acid sequence in viral vectors to ADAR1 using the target sequence of RNAi and carries out same sense mutation, in target
The generation of miss target phenomenon is avoided into operating process.
The present invention provides the adenosine deaminases 1 of RNA a kind of to be overexpressed viral vectors, its construction method and application.ADAR1
Mainly by IFN signal and endoplasmic reticulum stress response, promote immunocyte infiltration, intestinal inflammation further promotes small
The expression of intestinal mucosa proinflammatory factor IL-1 α, IL-1 β, IL-6 and TNF-α etc., ADAR1 are the passes of enteron aisle macrophage immunity function
Key check point, ADAR1 can increase the rate that precursor microRNA is cut by Dicer digestion, and microRNA can be promoted to be loaded into
RNA induces processing editing microRNA on silencing complex.
The invention discloses the adenosine deaminases 1 of RNA a kind of to be overexpressed viral vectors, and the carrier includes the gland of RNA
Guanosine deaminase 1 is overexpressed nucleic acid sequence, and the adenosine deaminase 1 of the RNA is overexpressed nucleic acid sequence and is selected from NCBI accession number NM_
001146296.1 or with NM_001146296.1 sequence have at least 80%, at least 85%, at least 90%, at least 91%, at least
92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least
The sequence of 99.9% homology.
Preferably, the virus is adenovirus.
Preferably, the viral vectors is PCDH-CMV-MCS-EF1-copGFP.
The present invention also provides the construction method that the adenosine deaminase 1 of RNA a kind of is overexpressed viral vectors, the carriers
Adenosine deaminase 1 including RNA is overexpressed nucleic acid sequence, and the adenosine deaminase 1 of the RNA is overexpressed nucleic acid sequence and is selected from NCBI
Accession number NM_001146296.1 or with NM_001146296.1 sequence have at least 80%, at least 85%, at least 90%, at least
91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%
Or the sequence of at least 99.9% homology.
Preferably, the virus is adenovirus.
Preferably, described have at least 80% with NM_001146296.1 sequence, at least 85%, at least 90%, at least
91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%
Or the sequence of at least 99.9% homology is to be obtained by NM_001146296.1 series jump；Preferably, it is described sport it is same
Justice mutation.
Preferably, the viral vectors is PCDH-CMV-MCS-EF1-copGFP.
Preferably, the construction method, includes the following steps:
(1) nucleic acid sequence such as NCBI accession number for NM_001146296.1 is cloned；
(2) same sense mutation is carried out to the cloning nucleic acid sequences product that step (1) obtains according to the target sequence of RNAi；
(3) product after the mutation of step (2) acquisition is connected on virus particle PCDH-CMV-MCS-EF1-copGFP,
The adenosine deaminase 1 for obtaining RNA is overexpressed viral vectors.
It is further preferred that the same sense mutation is selected from conversion or transversion.
Invention further provides a kind of, and the adenosine deaminase 1 comprising above-mentioned RNA is overexpressed the cell of viral vectors.
Invention further provides a kind of tissues comprising above-mentioned cell.
Invention further provides a kind of organs comprising above-mentioned tissue.
Viral vectors, which is overexpressed, the present invention also provides the adenosine deaminase 1 of above-mentioned RNA a kind of is protecting small intestines structure
Completely, the application in enteron aisle stable state is maintained.
Preferably, the enteron aisle steady-state performance that maintains is the unbalance improvement of enteron aisle homeostasis.
Viral vectors, which is overexpressed, the present invention also provides the adenosine deaminase 1 of above-mentioned RNA a kind of is improving enteron aisle macrophage
Application in the activity of cell.
Viral vectors, which is overexpressed, the present invention also provides the adenosine deaminase 1 of above-mentioned RNA a kind of is inhibiting inflammatory cell
Application in the level of the factor.
Preferably, the inflammatory cytokine be selected from one or both of TNF-α, IL-6, IL-10 or IFN-β with
On combination.
Viral vectors, which is overexpressed, the present invention also provides the adenosine deaminase 1 of above-mentioned RNA a kind of treats septicopyemia in preparation
Application in the product of disease.
Preferably, the product is drug.
"and/or" of the present invention includes selecting the project and any amount of projects combo that one lists.
" comprising " of the present invention is open description, containing described specified ingredient or step, and will not
Other the specified ingredients or step substantially influenced.
" treatment (treating) " of the present invention (or " treatment (treat) " or " treatment (treatment) ") indicates to subtract
Progress or serious that is slow, interrupting, prevent, control, stopping, mitigating or reverse a kind of sign, symptom, imbalance, illness or disease
Property, but it is not necessarily referring to completely eliminating for all disease correlation signs, symptom, illness or imbalance.Term " treatment
Etc. (treating) " refer to that the treatment of sign, symptom for improving disease or pathological state after disease has started development etc. is dry
In advance.
" homology " of the present invention refers in terms of using protein sequence or nucleotide sequence, those skilled in the art
Member can need to be adjusted sequence according to real work, using sequence compared with the sequence that the prior art obtains, have (packet
Include but be not limited to) 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%,
16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%,
31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%,
46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%,
70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%,
99.7%, 99.8%, 99.9% homology.
Detailed description of the invention
Hereinafter, carrying out the embodiment that the present invention will be described in detail in conjunction with attached drawing, in which:
Fig. 1: the ADAR1 protective effect to pyemia mouse intestinal, wherein Sham is to inject PBS to false mould group mouse rat-tail
Group, CLP are that PBS group is injected to model mice rat-tail, and CLP+Ad-NC is not contain ADAR nucleic acid sequence to the injection of model mice rat-tail
The plasmid group of column, CLP+Ad-shADAR1 are to penetrate ADAR low expression viral vectors group, CLP+Ad+O/E- to model mouse endnote
ADAR1 is to penetrate ADAR1 to model mouse endnote to be overexpressed viral vectors group；
Fig. 2: the ADAR1 influence to pyemia mouse intestinal macrophage, wherein Sham is to infuse to false mould group mouse rat-tail
PBS group is penetrated, CLP is that PBS group is injected to model mice rat-tail, and CLP+Ad-NC is not contain ADAR to the injection of model mice rat-tail
The plasmid group of nucleic acid sequence, CLP+Ad-shADAR1 are to penetrate ADAR low expression viral vectors group, CLP+Ad+O/ to model mouse endnote
E-ADAR1 is to penetrate ADAR1 to model mouse endnote to be overexpressed viral vectors group；
Fig. 3: the ADAR1 influence to pyemia mice serum inflammatory factor, wherein Sham is to infuse to false mould group mouse rat-tail
PBS group is penetrated, CLP is that PBS group is injected to model mice rat-tail, and CLP+Ad-NC is not contain ADAR to the injection of model mice rat-tail
The plasmid group of nucleic acid sequence, CLP+Ad-shADAR1 are to penetrate ADAR low expression viral vectors group, CLP+Ad+O/ to model mouse endnote
E-ADAR1 is to penetrate ADAR1 to model mouse endnote to be overexpressed viral vectors group, and figure A is the change of serum inflammatory factors of senile TNF-α expression
Change, figure B is the variation of serum inflammatory factors of senile IL-6 expression, and figure C is the variation of serum inflammatory factors of senile IL-10 expression, and figure D is serum
The variation of inflammatory factor IFN-β expression；
Fig. 4: the ADAR1 influence to pyemia mouse survival rate, wherein Sham is to inject PBS to false mould group mouse rat-tail
Group, CLP are that PBS group is injected to model mice rat-tail, and CLP+Ad-NC is not contain ADAR nucleic acid sequence to the injection of model mice rat-tail
The plasmid group of column, CLP+Ad-shADAR1 are to penetrate ADAR low expression viral vectors group, CLP+Ad+O/E- to model mouse endnote
ADAR1 is to penetrate ADAR1 to model mouse endnote to be overexpressed viral vectors group.
Specific embodiment
Following will be combined with the drawings in the embodiments of the present invention, and technical solution in the embodiment of the present invention carries out clear, complete
Site preparation description, it is clear that described embodiment is only section Example of the invention, rather than all.Based in the present invention
Embodiment, every other embodiment obtained by those of ordinary skill in the art without making creative efforts, all
Belong to the scope of protection of the invention.
Experimental animal and material are as follows:
Experimental animal is purchased from The Fourth Military Medical University；
Macrophage is purchased from Chinese Academy of Medical Sciences's Cell Culture Center；
Bacteria lipopolysaccharide (LPS), #L2880 are purchased from Sigma-aldrich, Germany；
RNA extracts kit is purchased from Invitrogen, the U.S.；
PrimeScript RT Kit is purchased from TaKaRa, Japan；
Bio-Rad IQ5 is purchased from Bio-Rad, the U.S.；
SYBR Premix Ex Taq II kit is purchased from TaKaRa, Japan.
The building of 1 mouse ADAR1 expression vector of embodiment
Since, there may be " missing the target " phenomenon, we are according to the target sequence of RNAi to wild type in RNAi experiment
ADAR1 gene carries out same sense mutation, and specially respectively clone mouse ADAR1 is overexpressed the sequence (Ad+O/E-ADAR1) of adenovirus:
The sequence (Ad-shADAR1) of NM_001146296.1, ADAR1 low expression adenovirus: CCATGAACCTCGATTTAAA and feminine gender
Control sequence (Ad-NC): TTCTCCGAACGTGTCACGT carries out same sense mutation to the ADAR1 sequence for being overexpressed adenovirus, and
ADAR1 is overexpressed sequence, the sequence of ADAR1 low expression adenovirus and the negative control sequence of adenovirus respectively with slow virus
PCDH-CMV-MCS-EF1-copGFP plasmid is that skeleton constructs ADAR1 Lentiviral respectively.
Embodiment 2 establishes cecal legation perforation method (CLP) septicopyemia disease mouse model
One, experimental animal
The male C57BL/6 mouse of selection 6 weeks greatly, weight 20-25g are cultivated under standard laboratory conditions.
Two, experimental procedure is as follows:
(1) operation same day early morning mouse is fasted, and 4% hydration chloric acid anesthesia of intraperitoneal injection, supine position is grasped in experiment
Make on platform, after 75% alcohol disinfecting skin of abdomen, cut off the wound for being about 1cm along abdomen center, open abdominal cavity and find caecum,
And squeeze away distal ileum gas.
(2) it according to each group design requirement, uses 3-0 line at away from cecum 1cm respectively or the ligation of caecum basal part, avoids ligaturing
Mesenteric causes intestinal tube ischemic necrosis, and with No. 12 syringe needles, caecum is run through 2 times or 3 times in distal end at ligation, slightly firmly squeezes
Caecum sees if there is excrement extrusion, it is ensured that puncturing hole is unobstructed.
(3) caecum is put back into abdominal cavity, sutures abdomen muscle layer, last skin suture, marking pen label.
(4) postoperative every mouse peritoneal injecting normal saline 0.5mL carries out liquid resuscitation, puts back in each cage, keeps lying on the back
Position pays attention to heat preservation, obtains CLP septicopyemia disease mouse model, verifies for subsequent experimental.
3 ADAR1 of embodiment is overexpressed protective effect of the viral vectors to pyemia mouse
1, experimental animal: the CLP septicopyemia disease mouse model prepared in embodiment 2.
2, experimental program: being divided into five groups for mouse model, be placed in fixator, exposes tail, respectively disposable tail vein
Injection ADAR1 is overexpressed 100 microlitres of adenovirus vector and (includes 1 × 108PFU ADAR1 be overexpressed virus), with dosage low expression
Viral vectors, the plasmid group without containing ADAR nucleic acid sequence, with dosage PBS (false mould group, model group) --- CLP+Ad-NC group.Note
First day after penetrating and third day anesthetized mice collect small intestine sample and carry out histologic analysis.
3, experimental result
CLP inducing mouse pyemia is overexpressed respectively/interferes small intestine ADAR1, H&E dyeing assessment pyemia mouse small intestine
Tectology.Fig. 1 the result shows that: after interference pyemia small intestine ADAR1, small intestines structure is destroyed serious, and inflammatory infiltration increases.
Inflammatory infiltration reduces after being overexpressed pyemia small intestine ADAR1, and small intestines structure is more complete.
Influence of 4 ADAR1 of embodiment to pyemia mouse small intestine macrophage
Macrophage culture adds 10% fetal calf serum (FBS) in serum-free cell freezing media；Condition of culture
For 5%CO2,37 DEG C, passage in 2-3 days is once.With LPS activating macrophage, the outer sepsis model of construct is specially cultivated huge
Phagocyte injects the LPS of 100ng/mL thereto, carries out western blot analysis after cell cracking.Ad- is transfected in 24 hours
Then O/E-ADAR1, Ad-shADAR1 or Ad-NC collect cell.
CLP inducing mouse pyemia, is overexpressed respectively/interferes small intestine ADAR1, and F4/80 immunofluorescence dyeing assesses septicopyemia
The expression of disease mouse small intestine tissue macrophages.As shown in Fig. 2, interference small intestine ADAR1 significantly inhibits the number of enteron aisle macrophage
Amount is overexpressed the activity that small intestine ADAR1 then improves enteron aisle macrophage.
Influence of 5 ADAR1 of embodiment to pyemia mice serum inflammatory factor expression
With each blood inflammatory cytokines levels in non-diabetic of mouse first day and third day in ELISA method detection embodiment 2.
One, experimental procedure
1, the dilution step of the standard items of each serum inflammatory factors of senile is shown in Table 1
The dilution of 1 standard items of table
2, it is loaded: setting blank well (sample and enzyme marking reagent is not added in blank control wells, remaining each step operation is identical), mark respectively
Quasi- hole, sample to be tested hole.Standard items are accurately loaded 50 μ L on enzyme mark coating plate, first add sample diluting liquid 40 in sample to be tested hole
Then μ L adds sample to be tested (septicopyemia disease mouse model serum) 10 μ L again (the final dilution of sample is 5 times).Sample-adding adds sample
In ELISA Plate hole bottom, hole wall is not touched as far as possible, shakes gently mixing.
3, it incubates: being incubated 30 minutes with 37 DEG C of sealing plate film sealing plate postposition.
4, match liquid: will be spare after 30 times of concentrated cleaning solutions, 30 times of distilled water dilutions.
5, it washs: carefully taking sealing plate film off, discard liquid, dry, cleaning solution is filled it up in every hole, is discarded after standing 30 seconds, such as
This is repeated 5 times, and pats dry.
6, enzyme: every hole is added 50 μ L of enzyme marking reagent, except blank well.
7, incubate: operation is the same as 3.
8, wash: operation is the same as 5.
9, develop the color: 50 μ L of color developing agent A is first added in every hole, adds 50 μ L of color developing agent B, and gently concussion mixes, and 37 DEG C are kept away
Light develops the color 15 minutes.
10, terminate: every hole adds 50 μ L of terminate liquid, terminates reaction (blue is vertical at this time turns yellow).11, it measures: with blank sky
Zeroing, 450nm wavelength sequentially measure the absorbance (OD value) in each hole.Measurement should carry out within 15 minutes after adding terminate liquid.
Calculated result: the duplicate reading of average each standard and sample and the optical density (OD) for averagely subtracting zero standard.
Two, experimental result
As shown in Figure 3, interference/overexpression pyemia mouse ADAR1, ELISA detects serum levels of inflammatory cytokines TNF-α (figure
3A), the expression variation of IL-6 (Fig. 3 B), IL-10 (Fig. 3 C) and IFN-β (Fig. 3 D).Wherein
The result shows that after interference pyemia mouse ADAR1, on the expression of TNF-α, IL-6, IL-10 and IFN-β is significant
It adjusts；After being overexpressed pyemia mouse ADAR1, the expression of proinflammatory factor TNF-α and IL-6 are significantly lowered.
6 ADAR1 of embodiment improves pyemia mouse survival rate
As shown in figure 4, significantly improving the survival rate of pyemia mouse after being overexpressed pyemia mouse ADAR1；And interfere purulence
After toxication mouse ADAR1, mouse is all dead in four days.
In conclusion ADAR1 of the present invention be overexpressed viral vectors produce reduce TNF-α, IL-6, IL-10 and
The expression of IFN-β inflammatory factor, especially IL6 and TNF；It ensure that small intestine structural integrity, the improvement that enteron aisle homeostasis is unbalance；It mentions
The activity of high enteron aisle macrophage；And significantly improve the effect of the survival rate of pyemia mouse；It is provided for pyemic treatment
New potential therapy target.
The preferred embodiment of the present invention has been described above in detail, still, during present invention is not limited to the embodiments described above
Detail within the scope of the technical concept of the present invention can be with various simple variants of the technical solution of the present invention are made, this
A little simple variants all belong to the scope of protection of the present invention.
It is further to note that specific technical features described in the above specific embodiments, in not lance
In the case where shield, can be combined in any appropriate way, in order to avoid unnecessary repetition, the present invention to it is various can
No further explanation will be given for the combination of energy.
Claims (13)
1. the adenosine deaminase 1 of RNA a kind of is overexpressed viral vectors, which is characterized in that the carrier includes that the adenosine of RNA is de-
Adnosine deaminase 1 is overexpressed nucleic acid sequence, and the adenosine deaminase 1 of the RNA is overexpressed nucleic acid sequence and is selected from NCBI accession number NM_
001146296.1 or with NM_001146296.1 sequence have at least 80%, at least 85%, at least 90%, at least 91%, at least
92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least
The sequence of 99.9% homology.
2. carrier according to claim 1, which is characterized in that the viral vectors is PCDH-CMV-MCS-EF1-
copGFP。
3. the construction method that the adenosine deaminase 1 of RNA a kind of is overexpressed viral vectors, which is characterized in that the carrier includes
The adenosine deaminase 1 of RNA is overexpressed nucleic acid sequence, and the adenosine deaminase 1 of the RNA is overexpressed nucleic acid sequence and logs in selected from NCBI
Number NM_001146296.1 or have at least 80% with NM_001146296.1 sequence, at least 85%, at least 90%, at least
91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%
Or the sequence of at least 99.9% homology.
4. construction method according to claim 3, which is characterized in that described to have extremely with NM_001146296.1 sequence
Few 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least
96%, the sequence of at least 97%, at least 98%, at least 99% or at least 99.9% homology is by NM_001146296.1 sequence
Mutation obtains；Preferably, described to sport same sense mutation.
5. construction method according to claim 3, which is characterized in that the viral vectors is PCDH-CMV-MCS-
EF1-copGFP。
6. according to construction method as claimed in claim 3 to 5, which comprises the steps of:
(1) nucleic acid sequence such as NCBI accession number for NM_001146296.1 is cloned；
(2) same sense mutation is carried out to the cloning nucleic acid sequences product that step (1) obtains according to the target sequence of RNAi；
(3) product after the mutation of step (2) acquisition is connected on virus particle PCDH-CMV-MCS-EF1-copGFP, is obtained
The adenosine deaminase 1 of RNA is overexpressed viral vectors.
7. the cell that a kind of adenosine deaminase 1 comprising any RNA of claim 1-2 is overexpressed viral vectors.
8. a kind of tissue comprising cell described in claim 7.
9. a kind of organ comprising tissue according to any one of claims 8.
10. the adenosine deaminase 1 of any RNA of claim 1-2 a kind of is overexpressed viral vectors and is protecting small intestines structure
Completely, the application in enteron aisle stable state is maintained.
11. the adenosine deaminase 1 of any RNA of claim 1-2 a kind of is overexpressed viral vectors and is improving enteron aisle macrophage
Application in the activity of cell.
12. the adenosine deaminase 1 of any RNA of claim 1-2 a kind of, which is overexpressed viral vectors, is inhibiting inflammatory cell
Application in the level of the factor.
13. the adenosine deaminase 1 of any RNA of claim 1-2 a kind of, which is overexpressed viral vectors, treats septicopyemia in preparation
Application in the product of disease.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811023833.4A CN109182377A (en) | 2018-09-04 | 2018-09-04 | A kind of ADAR1 is overexpressed viral vectors, its construction method and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811023833.4A CN109182377A (en) | 2018-09-04 | 2018-09-04 | A kind of ADAR1 is overexpressed viral vectors, its construction method and application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN109182377A true CN109182377A (en) | 2019-01-11 |
Family
ID=64914335
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811023833.4A Pending CN109182377A (en) | 2018-09-04 | 2018-09-04 | A kind of ADAR1 is overexpressed viral vectors, its construction method and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109182377A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111549000A (en) * | 2020-06-18 | 2020-08-18 | 中国医学科学院整形外科医院 | Recombinant adipose-derived stem cell for over-expression of Hpgds, preparation method and application thereof |
| CN113439711A (en) * | 2021-07-01 | 2021-09-28 | 中国医科大学 | Modified macrophage and application thereof |
| CN114177298A (en) * | 2022-01-20 | 2022-03-15 | 中南大学湘雅三医院 | Application of ADAR1 as medicine for treating pulmonary hypertension disease |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102864172A (en) * | 2012-10-15 | 2013-01-09 | 中国人民解放军第二军医大学 | Leukemia mouse model based on gene co-transfection technology and preparation method thereof |
| CN107828736A (en) * | 2017-11-08 | 2018-03-23 | 重庆赛托斯创生物科技发展有限公司 | The enhanced mescenchymal stem cell of antibacterial functions and its application in pyemia |
-
2018
- 2018-09-04 CN CN201811023833.4A patent/CN109182377A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102864172A (en) * | 2012-10-15 | 2013-01-09 | 中国人民解放军第二军医大学 | Leukemia mouse model based on gene co-transfection technology and preparation method thereof |
| CN107828736A (en) * | 2017-11-08 | 2018-03-23 | 重庆赛托斯创生物科技发展有限公司 | The enhanced mescenchymal stem cell of antibacterial functions and its application in pyemia |
Non-Patent Citations (1)
| Title |
|---|
| SHANSHOU LIU等: "ADAR1 prevents small intestinal injury from inflammation in a murine model of sepsis", 《CYTOKINE》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111549000A (en) * | 2020-06-18 | 2020-08-18 | 中国医学科学院整形外科医院 | Recombinant adipose-derived stem cell for over-expression of Hpgds, preparation method and application thereof |
| CN113439711A (en) * | 2021-07-01 | 2021-09-28 | 中国医科大学 | Modified macrophage and application thereof |
| CN114177298A (en) * | 2022-01-20 | 2022-03-15 | 中南大学湘雅三医院 | Application of ADAR1 as medicine for treating pulmonary hypertension disease |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109182377A (en) | A kind of ADAR1 is overexpressed viral vectors, its construction method and application | |
| CN103372218B (en) | The microRNA that autoimmune disease is relevant and application thereof | |
| Han et al. | Involvement of a Stat3 binding site in inflammation-induced enteric apelin expression | |
| KR101815734B1 (en) | Pharmaceutical composition comprising miR-7, miR-18a or miR-18b for prevention or treatment of metabolic disease | |
| Zhongyin et al. | Isoliquiritin apioside relieves intestinal ischemia/reperfusion-induced acute lung injury by blocking Hif-1α-mediated ferroptosis | |
| EP4081642A1 (en) | Antagonist of pcsk9 | |
| CN111803634B (en) | Application of glycolysis inhibitor in preparation of medicine for preventing and treating porcine reproductive and respiratory syndrome virus infection | |
| CN112680445A (en) | shRNA expressed by target-directed knockdown Atf6 alpha gene, recombinant vector and application thereof | |
| CN103432592B (en) | Treatment of intestinal conditions by a compound which causes RNA interference | |
| Wang et al. | Mitochondrial fragmentation in liver cancer: emerging player and promising therapeutic opportunities | |
| Chen et al. | Shrimp antiviral mja-miR-35 targets CHI3L1 in human M2 macrophages and suppresses breast cancer metastasis | |
| CN102257140A (en) | Treating hepatitis c virus infection with over-expression of microrna-196 | |
| US8389711B2 (en) | Pharmaceutical composition for treatment of cancer and asthma | |
| CN108004310A (en) | The application of feritin (original) acceptor (P) RR genes and its inhibitor | |
| CN106906217A (en) | Suppress siRNA and its application of Kdm6a gene expressions | |
| CN108014129B (en) | Application of iron ions in RNA virus inhibition | |
| WO2021218802A1 (en) | Isolated recombinant oncolytic poxvirus capable of being regulated and controlled by microrna and use thereof | |
| JPWO2009069668A1 (en) | Malignant melanoma antigen expression increasing agent and use thereof | |
| Yu et al. | The effect of ghrelin on the fibrosis of chicken bursa of fabricius infected with infectious bursal disease virus | |
| Tian et al. | Adiponectin improves the therapeutic efficacy of mesenchymal stem cells by enhancing their engraftment and survival in the peri-infarct myocardium through the AMPK pathway | |
| CN113876937B (en) | Use of PZP and pharmaceutical composition for combating obesity comprising PZP | |
| CN106381297B (en) | The double-strand p-siRNA molecule and p-siRNA recombinant plasmid of inhibition SOX2 gene expression and its application | |
| CN111793686A (en) | Diagnostic and prognostic marker for luminal and HER2 breast cancers, and therapeutic PPAR γ inhibitor | |
| CN104491496B (en) | Application of the Gastrodin/Rhizoma Gastrodiae powder in anti-hepatic fibrosis medicines are prepared | |
| Yu et al. | Tissue distribution and developmental changes of interferon regulatory factors in chickens and effects of infectious bursal disease virus infection |
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: 20190111 |
|
| RJ01 | Rejection of invention patent application after publication |