CN102827867A - Establishment of ET2 stable expression cell line and its application in drug screening - Google Patents
Establishment of ET2 stable expression cell line and its application in drug screening Download PDFInfo
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- CN102827867A CN102827867A CN2011101608523A CN201110160852A CN102827867A CN 102827867 A CN102827867 A CN 102827867A CN 2011101608523 A CN2011101608523 A CN 2011101608523A CN 201110160852 A CN201110160852 A CN 201110160852A CN 102827867 A CN102827867 A CN 102827867A
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Abstract
The invention provides a preparation method of an ET2 stable expression cell line and then provides a cell model for screening drugs for preventing and/or treating hypertension and ischemic heart disease.
Description
Technical field
The invention belongs to bioengineering field and medical technical field, be specifically related to a kind of ET2 stable expression cell line and the application in drug screening thereof.
Background technology
Hypertension (hypertensive disease) is a kind of chronic disease that serves as main performance with the lasting rising of arteriotony, often causes the pathology of vitals such as the heart, brain, kidney and corresponding consequence occurs.At present, hypertension become cause in the world wide that cardiovascular event is most important one of can the change factor, its harm is on the rise.The existing Hypertensive Population of China has exceeded 200,000,000, and its treatment rate and inverse amplification factor are still not high.ET-1 (Endothelin; ET) be that separation of pure from the porcine aorta endotheliocyte of cultivating such as Japanese scholar Yanagisawa dissolves a kind of active polypeptide; It does not exist only in blood vessel endothelium; Also extensively being present in various tissues and the cell, is the important factor of regulating cardiovascular function, to keeping basal vascular tone and the cardiovascular systems stable state plays an important role.The polypeptide that it is made up of 21 amino acid, molecular weight are 2400D.ET2 is a member in the ET-1 family, can combine mediated cell inner cell path as part with endothelin receptor.ET-1 is to know the strongest vaso-excitor material so far, and its action time is lasting, is the long-acting vasoconstriction regulatory factor of a kind of endogenous.ET-1 also has powerful positive inotropic action, but and the blood vessel that contracts rise blood pressure effect also reflectivity cause that heart rate suppresses, and causes deficiency myocardial blood supply. but also can bring out the overload of myocardial cell's sugar, irregular pulse and energy metabolism of myocardial obstacle.At present big quantity research table hinders severe cardiac angina, AMI, myocardium 1/R damage, synthetic and release hinders to show through the body ET of bellows internal shaping art increases, or blood vessel is hyperfunction to the ET reactivity, all possibly promote the incidence and development of above-mentioned pathologic process.Use ET antibody or ET blocker and then can prevent and treat cardiovascular and cerebrovascular diseases such as hypertension.
Summary of the invention
The preparation method who the purpose of this invention is to provide a kind of ET2 stable expression cell line.
The preparation method of ET2 stable expression cell line provided by the invention may further comprise the steps:
1) encoding sequence of ET2 gene is inserted into the MCS of carrier for expression of eukaryon, obtains recombinant expression vector;
2) recombinant expression vector that step 1 is obtained imports in the host cell, and screening obtains the clone of stably express ET2.
Above-mentioned carrier for expression of eukaryon specifically can be pTagLite-Snap, pEGFP-N3 etc.
For the ease of the ET2 that expresses is carried out cellular localization, can in the host, express and produce the enzyme of colour-change or the gene of luminophor in adding on the carrier for expression of eukaryon, like SNAP, GFP, YFP etc.
Above-mentioned host cell can be Hela cell, MDA-MB-231 cell, MCF-7 cell etc.
Utilize the ET2 stable expression cell line of method for preparing also to belong to protection scope of the present invention.
Another object of the present invention provides the cell model that a kind of screening prevents and/or treats the medicine of hypertension, ischemic heart disease.
Experiment showed, that ET2 stable expression cell strain of the present invention is responsive to ET2 blocker and ET2 antibody, effect significantly.ET2 stable expression cell line of the present invention can be used for screening and prevents and/or treats hypertensive medicine for the effect of further investigation ET2 in hypertension provides the experimental technique platform.
Description of drawings
Fig. 1 is the structure schema of recombinant expression vector pTagLite-Snap-ET2
Fig. 2 is the ET2 expression amount experimental result of each stably express ET2 clone
Fig. 3 is that the ET2 stable expression cell line is to the sensitivity analysis experimental result to the ET2 suppressor factor
Embodiment
Below in conjunction with specific embodiment the present invention is described further, it is indicative that embodiment is merely, and never means to limit scope of the present invention by any way.
The concrete structure flow process of ET2 stable expression cell line of the present invention is as shown in Figure 1.
Embodiment 1.
The structure of recombinant eukaryon expression vector pTagLite-Snap-ET2
Dna primer used in the following experimentation is synthetic by the living worker in Shanghai Bioisystech Co., Ltd.
According to the cDNA sequence of ET2 encoding histone frame, design PCR primer is following: forward primer:
5 ' ACT
GATATCATGGTCTCCGTGCCTACCACCTGGT-3 ' (the underscore place is the recognition site of restriction enzyme); Reverse primer:
5 '-AAC
GAGCTCTCTCTTCCTCCACCTGGAATGTGTG-3 ' (the underscore place is the recognition site of restriction enzyme).
Getting Human umbilical vein endothelial cells HUVEC and extract total RNA, it is inverted to cDNA, is template with this cDNA, utilizes above-mentioned primer to carry out pcr amplification.Pcr amplification product is carried out agarose gel electrophoresis purifying and recovery; Carrying out enzyme with restriction enzyme EcoRV and the Xho I product after to purifying and recovering cuts; Enzyme is cut product and is carried out agarose gel electrophoresis purifying and recovery once more, obtains the cDNA sequence of ET2 encoding histone frame.Simultaneously, carrier for expression of eukaryon pTagLite-Snap (available from Cisbio company) is also carried out double digestion with EcoR V and Xho I, enzyme is cut product and is carried out agarose gel electrophoresis purifying and recovery.Product after cDNA sequence and the carrier for expression of eukaryon pTagLite-Snap enzyme that connects the ET2 encoding histone frame that above-mentioned amplification obtains with dna ligase cut; Connect product transformed into escherichia coli (E.coli Top 10) competent cell; And coat on the LB flat board that contains penbritin, the picking mono-clonal carries out PCR and detects.The PCR product is checked order, to detect the correct of insertion sequence in the positive colony.Sequencing result shows that the cDNA sequence of the ET2 encoding histone frame of the 534bp of insertion is consistent with the sequence from the 73rd the-the 606th at 5 ' end of GenBank Accession Number NM 001956.With the recombinant expression vector called after pTagLite-Snap-ET2 that obtains.
Embodiment 2.
The foundation of ET2 stable expression cell line
Recombination bacillus coli utilization
the Plus SVMinipreps (available from Promega) that above-mentioned steps one is obtained extracts plasmid; Obtain highly purified recombinant expression vector pTagLite-Snap-ET2; Recombinant expression vector pTagLite-Snap-ET2 is utilized Lipofectamine 2000 (available from Invitrogen) transfection Hela cell, and the Hela cell after the continuation cultivation transfection is more than 24 hours.With G418 (concentration is 1 μ g/ml) reorganization HeLa cell is screened, after repeatedly screening, obtain the stable expression cell line of ET2.Simultaneously with the cell that changes carrier for expression of eukaryon pTagLite-Snap over to as contrast.
Choose four ET2 stable expression cell strains, utilize the antibody (available from AbCam company) of anti-ET2 to carry out protein immunoblot experiment, the result is as shown in Figure 2.Wherein, A-D representes the ET2 expression amount of the stable expression cell strain of different ET2 respectively, and Blank is the blank cell that changes carrier for expression of eukaryon pTagLite-Snap over to, and anti-Tubulin representes reference.The result shows that the stable expression cell strain of each ET2 all has the ET2 without degree to express.
Embodiment 3.
The ET2 stable expression cell line is to the sensitivity analysis of ET2 suppressor factor
Select for use the ET2 stably express cell of the foregoing description 2, the cell that changes carrier for expression of eukaryon pTagLite-Snap over to carry out the sensitivity test of ET2 suppressor factor.With ET2 stably express cell, change carrier for expression of eukaryon pTagLite-Snap over to cell cultures in the substratum of DMEM+10% foetal calf serum; The cell (generally going down to posterity back about 20 hours) that will be in vigorous period is after membrane proteolytic enzyme digestion is resuspended; Carry out cell counting, with every hole 10
3To 10
4The amount of individual cell is inoculated in 96 orifice plates and increases, and every porocyte number is even.After 24 hours, add ET1 suppressor factor (Ambrisentan) respectively.The concentration of suppressor factor (Ambrisentan) is respectively 10,20,30,40 and 50nM.Then carry out HTRF (homogeneous phase time discrimination fluorescence) experiment according to ET cell-based Assay Kit (available from Cisbio) specification sheets; Add the anti-and two anti-of mark in the cell orifice plate after adding suppressor factor; Upward read light absorption value with the 620nm wavelength at sunrise ELIASA (TECAN Company products) after hatching 30min in 665; Carry out data analysis, concrete outcome is as shown in Figure 3.
Claims (6)
1. the preparation method of an ET2 stable expression cell line may further comprise the steps:
1) encoding sox of ET2 is inserted the MCS of carrier for expression of eukaryon, obtain recombinant expression vector;
2) recombinant expression vector that step 1 is obtained imports in the host cell, and screening obtains the clone of stably express ET2.
2. method according to claim 1 is characterized in that, said carrier for expression of eukaryon is pTagLite-Snap or pEGFP-N3.
3. method according to claim 1 is characterized in that, said host cell is HeLa cell, MDA-MB-231 cell or MCF-7 cell.
4. the ET2 stable expression cell line for preparing according to each described method among the claim 1-3.
5. ET2 stable expression cell line according to claim 4 is characterized in that, the HeLa clone that said ET2 stable expression cell line is the SNAP-ET2 stably express.
6. each described ET2 stable expression cell line prevents and/or treats to the application in the medicine of this target spot in screening among the claim 4-5.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001090118A2 (en) * | 2000-05-19 | 2001-11-29 | Genaissance Pharmaceuticals, Inc. | Haplotypes of the edn2 gene |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001090118A2 (en) * | 2000-05-19 | 2001-11-29 | Genaissance Pharmaceuticals, Inc. | Haplotypes of the edn2 gene |
Non-Patent Citations (3)
| Title |
|---|
| GIYOUN NA ET AL.: "Role of hypoxia in the regulation of periovulatory endothelin-2 (EDN2) expression in the mouse", 《CAN J PHYSIOL PHARMACOL.》 * |
| 林琳 等: "内皮素对人肝癌细胞增殖作用的探讨", 《江苏医药杂志》 * |
| 谭初兵 等: "内皮素受体拮抗剂的筛选", 《新药研发暨新药发现学术研讨会会议论文集》 * |
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Application publication date: 20121219 |