CN108588075A - The cell line of people's Cx43 genes interference sequence, shRNA-Cx43 viruses and low expression Cx43 albumen - Google Patents
The cell line of people's Cx43 genes interference sequence, shRNA-Cx43 viruses and low expression Cx43 albumen Download PDFInfo
- Publication number
- CN108588075A CN108588075A CN201810412674.0A CN201810412674A CN108588075A CN 108588075 A CN108588075 A CN 108588075A CN 201810412674 A CN201810412674 A CN 201810412674A CN 108588075 A CN108588075 A CN 108588075A
- Authority
- CN
- China
- Prior art keywords
- shrna
- people
- viruses
- low expression
- cell
- 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
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
-
- 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
-
- 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
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0655—Chondrocytes; Cartilage
-
- 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
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/50—Physical structure
- C12N2310/53—Physical structure partially self-complementary or closed
- C12N2310/531—Stem-loop; Hairpin
-
- 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
- C12N2510/00—Genetically modified cells
-
- 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
- C12N2740/00—Reverse transcribing RNA viruses
- C12N2740/00011—Details
- C12N2740/10011—Retroviridae
- C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
- C12N2740/15041—Use of virus, viral particle or viral elements as a vector
- C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
Abstract
The invention discloses a kind of people Cx43 gene interference sequences, as shown in SEQ ID NO1.A kind of people shRNA Cx43 viruses are also disclosed, are in sequence construct to expression plasmid shown in SEQ ID NO1.A kind of cell line of low expression Cx43 albumen is also disclosed, for the cartilage cell infected by shRNA Cx43 viruses.People's Cx43 gene interference fragments are built into the U6 promoters downstream of slow virus carrier by molecular biology method by the present invention.Realize enhanced green fluorescent protein EGFP and puromycin resistant gene while interfering target gene, convenient for observation carrier working condition, the latter facilitates the stable cell line of screening target gene interference for the former.The plasmid of structure packs slow virus, the screening for stablizing strain.The stable cell lines of low expression connexin43 in cartilage cell can be used for studying the variation of the related inflammatory factor expression amount caused by low expression connexin43, to find the strategy of effective treatment of arthritis.
Description
Technical field
The present invention relates to people's Cx43 genes interference sequence, the cell lines of shRNA-Cx43 viruses and low expression Cx43 albumen.
Background technology
Osteoarthritis is clinical common joint disease, and one of the main reason for lead to deformity.However, bone closes so far
It is not yet completely clear to save scorching pathogenesis.In articular cartilage, connexin43 (Cx43) is to express connection egg the abundantest
In vain, it plays an important role in maintaining metabolism of articular cartilage stable state, is considered the generation close relation with osteoarthritis.Recently
Research find Human Osteoarthritis damaged cartilage region, the expression of Cx43 is changed, and Cx43 is non-in mid-deep strata
Damage zone occur distribution variation, Cx43 be distributed mainly on cytoplasm rather than on cell membrane (Human articular
chondrocytes express multiple gap junction proteins:differential expression
of connexins in normal and osteoarthritic cartilage,2013).Positions of the Cx43 in non-damaging area
The change for setting distribution is perhaps related with compensatory protective effect.But the missing of homotype connection albumen will produce exclusive pathology
Phenomenon, and cannot be by other kinds of connection albumen complete compensatory (Connexin43 hemichannels mediate
small molecule exchange between chondrocytes and matrix in biomechanically-
Stimulated temporomandibular joint cartilage, 2014).Cartilage cell in articular cartilage passes through seam
Gap connection is connected with each other to form the entirety of function coupling.The gap for participating in being formed by Cx43, which is connected to, maintains metabolism of articular cartilage to put down
With vital role in the stable state of weighing apparatus.Cx43 in addition to set up between flanking cell and cell and cellular matrix between
Outside mass exchange path, it can also be interacted by its c-terminus and intracellular multiple proteins and Signaling complex, activation
Different signal paths adjusts the expression of gene, influences existence and apoptosis (the Gap junctional regulation of cell
Of signal transduction in bone cells, 2010).
Invention content
The purpose of the present invention is to provide a kind of people Cx43 genes interference sequence, shRNA-Cx43 viruses and low expression Cx43
The cell line of albumen.
The purpose of the present invention is achieved through the following technical solutions：
A kind of people Cx43 gene interference sequences, as shown in SEQ ID NO1.
A kind of people shRNA-Cx43 viruses, for sequence construct shown in SEQ ID NO1 to pLKD-CMV-G＆PR-U6-
In shRNA plasmids, it is then packaged into virus.
Preferably, above-mentioned sequence construct is to the U6 promoters downstream of pLKD-CMV-G＆PR-U6-shRNA plasmids.
And a kind of cell line of low expression Cx43 albumen, for the cartilage cell infected by above-mentioned shRNA-Cx43 viruses.
The wherein described cartilage cell is SW1353 cell lines or Human Chondrocyte cell lines.
The study found that the formation of Cx43 and inflammatory conditions has close relationship (Reciprocal regulation
between proinflammatory cytokine-induced inducible NO synthase(iNOS)and
Connexin43 in bladder smooth muscle cells, 2011).The present invention is by molecular biology method by people
Cx43 gene interference fragments are built into the U6 promoters downstream of slow virus carrier (pLKD-CMV-G＆PR-U6-shRNA).The load
Enhanced green fluorescent protein EGFP and puromycin resistant gene, Qian Zhebian while interfering target gene may be implemented in body
In observation carrier working condition, the latter facilitates the stable cell line of screening target gene interference.The plasmid of structure packs slow virus,
Screening for stablizing strain.The stable cell lines of the present invention provides a kind of in cartilage cell low expression connexin43, can
Variation for studying the related inflammatory factor expression amount caused by low expression connexin43 is closed to find effectively treatment
Save scorching strategy, opposite zoopery can effectively shorten experimental period, conveniently carry out scientific research.
Description of the drawings
Fig. 1 is the structural schematic diagram of pLKD-CMV-G＆PR-U6-shRNA plasmids.
Fig. 2 is pLKD-CMV-G＆PR-U6-shRNA (Cx43) virus structure schematic diagram.
Specific implementation mode
Embodiment 1
1) according to people Cx43 (NCBI Reference Sequence:NM_000165.4) gene design interference effect site
CTGGCCTTGAATATCATTGAACTC.Primer synthesizes, and single-stranded primer is annealed into double-strand oligo sequences, connect into AgeI and
The pLKD-CMV-G＆PR-U6-shRNA (and first biotechnology (Shanghai) limited liability company) of EcoRI double digestions linearisation is dry
Carrier is disturbed, original ccdB virulent genes are replaced.Bacterium colony PCR screens transformant, and the positive colony of screening carries out sequence verification.
Sequence verification is correctly cloned, and high-purity plasmid extraction is carried out.
2) virus packaging.The plasmid built with first biotechnology (Shanghai) limited liability company by carrying out viral packaging：
By the high-purity purpose plasmid of extraction and packaging plasmid pLP1-gag/pol, pLP2-Rev and pLP/VSVG (and first biotechnology
(Shanghai) limited liability company) corotation enters 293T cells；Microscopically observation transfection efficiency；It is collected containing virus after transfecting 72h
Supernatant, Purification by filtration, -80 DEG C of preservations after titer determination.
3) 24 orifice plate culture Human Chondrocyte (HC) cell lines (wonderful logical (Shanghai) bio tech ltd),
37 DEG C, CO2Incubator overnight incubation；When cell confluency degree reaches 50%, from -80 ° of refrigerators take out shRNA-Cx43 viruses in
Melt on ice；
4) use nonreactive without blood DMEM with 1:10,1:100,1:1000 dilute the above virus respectively, and each 24 hole is separately added into
The virus 5 00ul of each dilution, in 37 °C of O2After incubator overnight incubation liquid is changed with 10%FBS+DMEM+ is dual anti-；
5) it changes after liquid 48h in fluorescence microscopy microscopic observation efficiency of infection, selects the hole that efficiency of infection is high and fluorescence is strong, pancreatin
It is terminated after digestion, 600 turns of the single cell suspension of preparation, centrifuges 5min；
6) dual anti-+ puro culture mediums of 10%FBS+DMEM+ are configured, are precipitated with the culture medium suspension cell, each 24 hole
Cell spreads 6 24 holes, replaces fresh above-mentioned culture medium within 3-5 days；(entire anti-virus operation process is strictly in accordance with slow virus manipulator
Volume)
7) the strong monoclonal hole of fluorescence, pancreatin digestion, after serum terminates, with the dual anti-+ puro of 10%FBS+DMEM+ are selected
Culture medium spreads cultivation, until 10cm dish are covered with, 1/3 cell freezing preservation, 1/3 receives cell precipitation for Western blot inspections
It surveys, 1/3 receipts cell precipitation adds 1ml trizol to be uniformly mixed for RT-PCR detections
8) positive clone strain is determined.Western Blot determine positive clone strain：Using GAPDH albumen as internal reference, compare sense
The variation of Cx43 expressing quantities in the cell and control cell of dye interference plasmid.Cx43 expressing quantities ratio in interference cell
Control cell is low for positive clone strain；RT-PCR determines positive clone strain：Using 18srRNA genes as internal reference, it is dry to compare infection
Disturb the variation of the cell and Cx43 gene expression amounts in control cell of plasmid.Cx43 gene expression amounts are thinner than compareing in interference cell
Born of the same parents are low for positive clone strain.Western Blot and RT-PCR detections are the cell strain of the positive, i.e. low expression Cx43 stablizes
Cell strain.
It also can be thin to substitute Human Chondrocyte with SW1353 cells (Beijing North Na Chuanlian research institutes) in step 3)
Born of the same parents system.
Sequence table
<110>Its moral is happy（Beijing）Biotechnology Co., Ltd
<120>The cell line of people's Cx43 genes interference sequence, shRNA-Cx43 viruses and low expression Cx43 albumen
<141> 2018-04-27
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 24
<212> DNA
<213> Homo sapiens
<400> 1
ctggccttga atatcattga actc 24
Claims (5)
1. a kind of people Cx43 gene interference sequences, as shown in SEQ ID NO1.
2. it is the sequence construct of claim 1 to pLKD-CMV-G＆PR-U6-shRNA plasmids a kind of people shRNA-Cx43 virus
In, then it is packaged into virus.
3. people shRNA-Cx43 viruses according to claim 2, it is characterised in that：The sequence construct of the claim 1
To the U6 promoters downstream of pLKD-CMV-G＆PR-U6-shRNA plasmids.
4. a kind of cell line of low expression Cx43 albumen, for the cartilage cell infected by the shRNA-Cx43 viruses of claim 2.
5. the cell line of low expression Cx43 albumen according to claim 4, it is characterised in that：The cartilage cell is
SW1353 cell lines or Human Chondrocyte cell lines.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810412674.0A CN108588075A (en) | 2018-05-03 | 2018-05-03 | The cell line of people's Cx43 genes interference sequence, shRNA-Cx43 viruses and low expression Cx43 albumen |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810412674.0A CN108588075A (en) | 2018-05-03 | 2018-05-03 | The cell line of people's Cx43 genes interference sequence, shRNA-Cx43 viruses and low expression Cx43 albumen |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN108588075A true CN108588075A (en) | 2018-09-28 |
Family
ID=63619521
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810412674.0A Pending CN108588075A (en) | 2018-05-03 | 2018-05-03 | The cell line of people's Cx43 genes interference sequence, shRNA-Cx43 viruses and low expression Cx43 albumen |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108588075A (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009102931A1 (en) * | 2008-02-15 | 2009-08-20 | Alcon Research, Ltd. | Rnai-mediated inhibition of connexin 43 for treatment of iop-related conditions |
| CN101573131A (en) * | 2005-02-03 | 2009-11-04 | 科达治疗有限公司 | Anti-connexin compounds and uses thereof |
| CN101835476A (en) * | 2006-12-11 | 2010-09-15 | 科达治疗公司 | Anti-connection albumen polynucleotide as impaired Wound healing compositions |
| CN102099475A (en) * | 2008-06-04 | 2011-06-15 | 科达治疗公司 | Treatment of pain with gap junction modulation compounds |
-
2018
- 2018-05-03 CN CN201810412674.0A patent/CN108588075A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101573131A (en) * | 2005-02-03 | 2009-11-04 | 科达治疗有限公司 | Anti-connexin compounds and uses thereof |
| CN101835476A (en) * | 2006-12-11 | 2010-09-15 | 科达治疗公司 | Anti-connection albumen polynucleotide as impaired Wound healing compositions |
| WO2009102931A1 (en) * | 2008-02-15 | 2009-08-20 | Alcon Research, Ltd. | Rnai-mediated inhibition of connexin 43 for treatment of iop-related conditions |
| CN102099475A (en) * | 2008-06-04 | 2011-06-15 | 科达治疗公司 | Treatment of pain with gap junction modulation compounds |
Non-Patent Citations (7)
| Title |
|---|
| DA XIE等: "Antitumor activity of resveratrol against human osteosarcoma cells: a key role of Cx43 and Wnt/β-catenin signaling pathway", 《ONCOTARGET》 * |
| JING ZHANG等: "Connexin43 Hemichannels Mediate Small Molecule Exchange between Chondrocytes and Matrix in Biomechanically-Stimulated Temporomandibular Joint Cartilage", 《OSTEOARTHRITIS CARTILAGE》 * |
| 胡银岗: "《植物基因工程》", 28 February 2006 * |
| 郑翠红等: "构建Cx43特异性shRNA真核表达载体及体外干扰效率的鉴定", 《中西医结合研究》 * |
| 陈玉祥: "《分子药剂学》", 31 January 2010 * |
| 陈立等: "Cx43基因shRNA慢病毒载体的构建及其对大鼠心肌细胞Cx43基因的作用", 《山东医药》 * |
| 陶永光: "《肿瘤分子生物学与细胞生物学实验手册》", 30 November 2014 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107828738A (en) | A kind of dnmt rna deficiency Chinese hamster ovary celI system and preparation method and application | |
| CN107513531A (en) | LncRNA XIST gRNA target sequences and its application are overexpressed for endogenous | |
| CN105985978B (en) | Construction and application of novel RNA cyclization expression vector | |
| CN106755089A (en) | Express cell line and its construction method and the application of goat lymphocyte activation molecule | |
| CN104805120A (en) | ShRNA-Ago2 coexpression lentivirus RNAi vector, recombinant plasmid and constructing method of recombinant plasmid | |
| CN109055374B (en) | shRNA for specifically inhibiting OCT1 gene expression and application thereof | |
| CN105039342B (en) | SiRNA and its application of MAT2A gene expressions can be suppressed | |
| CN103146752A (en) | Method of applying adenovirus vector mediation ribose nucleic acid (RNA) jamming technology to silence sterol regulatory element binding protein 1 | |
| CN108588075A (en) | The cell line of people's Cx43 genes interference sequence, shRNA-Cx43 viruses and low expression Cx43 albumen | |
| CN103352052A (en) | Construction and application of multi-cistron double-label expression lentivirus vector | |
| CN114672460B (en) | Preparation method and application of CD 44-targeted heterogeneous CIC cell model | |
| CN111378621B (en) | B lymphoma cell strain stably transfected by EB virus latent membrane protein 1, construction method and application thereof | |
| CN109266684B (en) | Method for constructing animal model with pathogen infection sensitivity | |
| CN104450781B (en) | A kind of cell line of overexpression CIAPIN1 albumen and its preparation method and application | |
| CN110964727A (en) | shRNA lentiviral expression vector construction method for specifically inhibiting c-myc gene expression and application thereof | |
| Zhao et al. | Construction of conditional lentivirus-mediated shRNA vector targeting the human Mirk gene and identification of RNAi efficiency in rhabdomyosarcoma RD cells | |
| CN105132413B (en) | It is used to prepare primer and preparation method, the carrier and construction method of shRNA | |
| CN110343700A (en) | A kind of pancreatic carcinoma and its construction method of XPO1 gene knockout | |
| CN111228292B (en) | Application of human TPT1/TCTP gene in preparation of antitumor drugs | |
| LU102533B1 (en) | miRNA (Mini Ribonucleic Acid) Precursor Polypeptide and Use thereof | |
| CN113388626B (en) | Application of novel coronavirus NSP13 gene | |
| CN110669791B (en) | Method for improving expression quantity of cell antibody | |
| CN113999816B (en) | Human umbilical cord mesenchymal stem cell line expressing HOXA10 and preparation method and application thereof | |
| CN109880828B (en) | siRNA for interfering Mroh7 gene expression and application, interference method and medicament thereof | |
| CN116948978A (en) | Human B lymphocytoma cell strain for stably expressing EBV (EBV-associated protein) lysate ZEBRA as well as construction method and application thereof |
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: 20180928 |
|
| RJ01 | Rejection of invention patent application after publication |