CN1219062C - Mouse cholesterol ester synthetase-2 gene promotor - Google Patents
Mouse cholesterol ester synthetase-2 gene promotor Download PDFInfo
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Abstract
The present invention provides a mouse cholesterol ester synthetase-2 promoter sequence, a main cis-acting element thereof, a structure containing the sequence, a vector containing the sequence and application thereof. The cholesterol ester synthetase-2 promoter of the present invention can be used for developing and screening medicine for curing or auxiliary curing atherosclerosis and expressing heterologous protein in mammalian cells.
Description
Invention field
The present invention relates to molecular biology and DNA recombinant technology field.More specifically, the present invention relates to the promotor of mouse cholesteryl ester synthetic enzyme-2, and their purposes.
Background technology
Mouse cholesteryl ester synthetic enzyme-2 gene promoter is the mouse acyl-CoA: cholesterol acyltransferase-2 promotor (hereinafter to be referred as mouse ACAT-2 gene promoter), its regulation and control mouse cholesteryl ester synthetic enzyme-2 expression of gene.
Cholesterol is biomembranous important component, also is the precursor of synthetic bile acid, steroid hormone and lipoprotein, and it has also participated in the signal conductive process recent findings, is indispensable in the Mammals vital process therefore.All will influence its normal vital process but the animal body inner cholesterol is too high or too low, even produce serious pathology.Atherosclerosis or gallbladdergallstonecholetithiasis are brought out in too high meeting; Cross lowly, then cause cerebral nerve underdevelopment, produce dementia, recent research shows that also hypercholesterolemia is the Hazard Factor of Alzheimer's disease.The cholesterol molecule is extremely hydrophobic, can not utilize it as the carbon source and the energy in the body, and therefore, organism inner cholesterol and ester class thereof must be among the strict balance.
Acyl-CoA: cholesterol acyltransferase (ACAT) (EC2.3.1.26) is is the endoplasmic reticulum albumen that substrate catalysis forms cholesteryl ester with lipid acid and cholesterol in the cell.ACAT is by changing cholesterol into cholesteryl ester, avoided the toxicity of too high free cholesterol pair cell.
People just propose the existence of ACAT enzyme and recognize its importance as far back as the fifties in last century, but up to 1993, have just at first been cloned the cDNA of people ACAT-1 by the TY Chang professor laboratory of U.S. Dartmouth medical college.On this basis, the ACAT-1 cDNA of other species is cloned in succession.The ACAT-2 cDNA of different genera has been cloned in 1998 three tame laboratories respectively, and they are people (Oelkers P, Behari A, CromleyD et al.1998, J.Biol.Chem., 273:26765-26771), ape (Anderson RA, JoyceC, Davis M, Reagan JW et al.1998, J.Biol.Chem., 273:26747-26754.) and mouse (Cases S, Novak S, Zheng Y et al.1998, J.Biol.Chem., 273:26755-26764).
Up to now, in Mammals, found the ACAT of two kinds of forms.ACAT-1 is distributed widely in the various histocytes, and mainly participate in intravital cholesterol metabolic balance: ACAT-2 then mainly expresses in the liver sausage cell, participates in the absorption of external source cholesterol and the assembling of lipoprotein.
The mouse ACAT-2 assignment of genes gene mapping is on the ten No. five karyomit(e), and is identical in the position on the ten No. two karyomit(e) with the people ACAT-2 assignment of genes gene mapping.Some higher fatty acid with the induction low-density lipoprotein, that the hypercholesterolemia absorption is relevant genes have been concentrated on this zone with vldl.525 amino acid of ACAT-2 cDNA coding of mouse have some height hydrophobic sequences, are indicating that ACAT-2 has a plurality of film districts of striding.
Recently, the ACAT-2 gene knockout of mouse success (ACAT-2
-/-).ACAT-2
-/-Mouse has completely lost the ACAT activity in liver and small intestine.This has further confirmed the vital role of ACAT-2 the mouse cholesterol absorption from genomic level.
Along with the raising of living standards of the people and the change of dietary structure, cardiovascular disorder and neural transformation disease more and more become the important killer who threatens people's life and health, and the research of massive epidemiology and molecular level has proved that these diseases and organism inner cholesterol level are closely related.Current paper is thought, the acat-2 of liver sausage cell specific expression participates in the absorption of external source cholesterol and the assembling of lipoprotein, directly influence cholesterol levels in the blood, and be the medicine of effect target protein with acat-2, can in control body's cholesterol level, not destroy intracellular cholesterol balance again, so the research of acat-2 more and more is subject to people's attention.
Have the ACAT of two kinds of forms in knowing human body after, the researchdevelopment of medicinal ACAT inhibitor is very fast.The special inhibitor of screening ACAT becomes the focus that some drugmakers are competitively studied.Screen at present the inhibitor of a large amount of ACAT by measuring method for activity in several external, the body of setting up, these inhibitor mechanisms of action are: 1, increase intracellular cholesterol outflow, cause in the endoplasmic reticulum the available cholesterol of ACAT to strip off; 2, some inhibitor is the analogue of cholesterol or mono-unsaturated fat acid, can compete and suppress combining of ACAT and its natural substrate; 3, the activity that directly suppresses ACAT in the arterial wall.But that present most inhibitor also belongs to is nonspecific (Cases S, Novak S, Zheng Y et al.1998, J.Biol.Chem., 273:26755-26764).And because the ACAT albumen of natural form does not also have the purifying success, it is also very difficult therefore to seek the special effective inhibitors of ACAT.Now, though many researchs about people ACAT inhibitor aspect are arranged, mouse ACAT-1 knocks out experimental study and shows, only optionally suppresses ACAT-1, can form hyperlipidaemia.Whether suppress ACAT-2 can be favourable, still unknown at present to treatment hyperlipidaemia and atherosclerosis.Because acat-2 is specifically expressing in the maincenter organ-liver of cholesterol metabolic and small intestine only, therefore makees model with mouse, research and development will make it have minimum toxic side effect in control organism inner cholesterol level at the medicine of acat-2; The many aspects of acat-2 expression regulation being carried out the research of molecular level explores significant.
Promotor plays a crucial role in the expression of gene regulation and control, yet, before the application, also do not obtain mouse cholesteryl ester synthetic enzyme-2 promotor and relevant regulating and controlling sequence.
Therefore, this area presses for cholesteryl ester synthetic enzyme-2 promoter sequence of exploitation mouse and relevant regulating and controlling sequence.
Summary of the invention
Purpose of the present invention just provides a kind of new mouse cholesteryl ester synthetic enzyme-2 promoter sequence and relevant regulating and controlling sequence.
Another object of the present invention provides the method for these regulating and controlling sequences of production and the purposes of described regulating and controlling sequence.
In a first aspect of the present invention, a kind of promotor is provided, it comprises the nucleotide sequence of mouse cholesteryl ester synthetic enzyme-2 promotor.
In a preference, described promotor comprises the nucleotide sequence that is selected from down group:
(a) nucleotide sequence shown in the 47-1137 position (corresponding to-1080 to-1 among Fig. 1) among the SEQ ID NO:1; Or
(b) nucleotide sequence shown in the 98-558 position among the SEQ ID NO:1 (corresponding to-1030 bp among Fig. 1 to-569).
In a second aspect of the present invention, a kind of carrier is provided, it contains the above-mentioned promotor of the present invention.
In a preference, described carrier also contains the foreign gene that described promotor is operably connected.
In a third aspect of the present invention, a kind of host cell is provided, it contains the above-mentioned carrier of the present invention.Preferably, described host cell is a mammalian cell.More preferably, described host cell is mouse, rat and people's a cell.
In a fourth aspect of the present invention, the purposes of promotor of the present invention is provided, it is used to screen the medicine of treatment cholesterol related diseases.
In a fifth aspect of the present invention, a kind of method of screening the medicine of treatment cholesterol related diseases is provided, comprise step:
(a) under the condition that is fit to growth, cultivate host cell, described host cell contains an expression vector, described expression vector contains promotor of the present invention and the reporter gene that links to each other with this promotor operability, wherein in the substratum of first group of host cell, add candidate substances, in the substratum of second group of host cell, do not add candidate substances;
(b) measure the expression amount of reporter gene in first group and the second group of host cell, wherein the expression amount of reporter gene is higher than second group and just represents that this candidate substances is the medicine of treatment cholesterol related diseases in first group of host cell.
Others of the present invention are because disclosing of the technology of this paper is conspicuous to those skilled in the art.
Description of drawings
Following accompanying drawing is used to illustrate specific embodiments of the present invention, and is not used in qualification by the scope of the invention that claims defined.
Fig. 1 has shown the sequence of mouse cholesteryl ester synthetic enzyme-2 promotor.
Fig. 2 has shown that 5 '-RACE identifies the electrophorogram and the correlated series of mouse ACAT-2 gene promoter transcription initiation site.Wherein transcription initiation site marks with " * ".
Fig. 3 has shown determining the maximum transcriptional activity section of mouse ACAT-2 gene promoter.
Embodiment
The inventor is through extensive and deep research, separating clone obtains the promoter sequence of ACAT-2 gene first, and it is analyzed, determine the transcription initiation site of this promotor, make up the expression vector of the mouse ACAT-2 gene promoter of different lengths respectively, the transfection mammalian cell transient expression in order to the transcriptional activity of analysis mouse acat-2 promotor and the section at main transcriptional activity place, is found out the transcription factor binding member that plays a major role simultaneously.Finished the present invention on this basis.
As used herein, term " cholesteryl ester synthetic enzyme-2 promotor ", " ACAT-2 gene promoter ", " acat-2 promotor " " be used interchangeably, refer to have the acat-2 promoter sequence (SEQ ID NO:1) or its starts the polynucleotide of active fragments.
The invention still further relates to the polynucleotide varient of above-mentioned promotor.The varient that allelic variant that these polynucleotide varients can be natural generations or non-natural take place.These nucleotide diversity bodies comprise and replace varient, deletion mutation body and insert varient.As known in the art, allelic variant is the replacement form of polynucleotide, and it may be replacement, disappearance or the insertion of one or more Nucleotide, but can be from not changing the function of this promotor in fact.
The invention still further relates to nucleic acid fragment with above-mentioned sequence hybridization.As used herein, the length of " nucleic acid fragment " contains 50 Nucleotide at least, better is at least 100 Nucleotide, is more preferably at least 150 Nucleotide, preferably at least 200 above total lengths to cholesteryl ester synthetic enzyme-2 promotor of Nucleotide.Nucleic acid fragment can be used for the amplification technique (as PCR) of nucleic acid to determine and/or to clone the nucleotide sequence of promotor of the present invention.
The Nucleotide full length sequence of people acat-2 promotor of the present invention or its fragment can obtain with the method for pcr amplification method, recombination method or synthetic usually.For the pcr amplification method, can be disclosed according to the present invention relevant nucleotide sequence design primer, and with mouse gene group DNA or the artificial chromosome clone that contains mouse chromosome as template, amplification and must relevant sequence.
In case obtained relevant sequence, just can obtain relevant sequence in large quantity with recombination method.This normally is cloned into carrier with it, changes cell again over to, separates obtaining relevant sequence then from the host cell after the propagation by ordinary method.
In addition, also the method for available synthetic is synthesized relevant sequence, especially fragment length more in short-term.Usually, by first synthetic a plurality of small segments, and then connect and to obtain the very long fragment of sequence.At present, can be fully obtain the dna sequence dna of promotor of the present invention (or its fragment, or derivatives thereof) by chemosynthesis.This dna sequence dna can be introduced in various existing dna moleculars as known in the art (or as carrier) and the cell then.
Use method (Saiki, the et al. Science1985 of round pcr DNA amplification/RNA; 230:1350-1354) be optimized for acquisition promotor of the present invention.
The present invention also relates to comprise the construction or the carrier of promotor of the present invention, and transform or the host cell of transduction with described construction or carrier, and the method that produces foreign protein through recombinant technology.
Be applicable to that foreign gene of the present invention is not particularly limited, nearly all foreign gene all can be used for the present invention.The example of representational foreign gene comprises (but being not limited to): the various report gene, and as GFP, luciferase reporter gene and various functional genes etc.It should be noted that foreign gene also comprises the gene from host itself.For example, for some inherited disease (for example because of certain protein expression not enough or do not express the disease that is caused), can separate from patient itself and obtain related gene, then it is linked to each other with cholesteryl ester synthetic enzyme-2 promoter element of the present invention, formation contains the construction of foreign gene, then described construction is used for gene therapy.
A kind of example of construction is exactly the foreign gene that links to each other with cholesteryl ester synthetic enzyme-2 promotor.As for the position relation of cholesteryl ester synthetic enzyme-2 promotor and foreign gene, promotor should be positioned at the upstream of foreign gene
Among the present invention, the nucleotide sequence that contains the acat-2 promoter element can preferably be inserted into carrier, for example in the expression vector.Term " carrier " refers to that bacterial plasmid well known in the art, phage, yeast plasmid, vegetable cell virus, mammalian cell virus are as adenovirus, retrovirus or other carriers.The carrier of Shi Yonging includes but not limited in the present invention: expression vector, cloning vector for example are applicable to the carrier in protokaryon (as bacteriums such as intestinal bacteria), eucaryon (as yeast), insect cell, vegetable cell, the mammalian cell.In a word, as long as can duplicate in host and stablize, any plasmid and carrier can be used.In expression vector, except containing replication orgin, cholesteryl ester synthetic enzyme-2 promotor, also can contain marker gene and other translation controlling elementss.
Method well-known to those having ordinary skill in the art can be used to make up and contains cholesteryl ester synthetic enzyme-2 promotor and suitable transcribing/the translate expression vector of control signal.These methods comprise (Sambroook, et al.Molecular Cloning, a LaboratoryManual, Cold Spring Harbor Laboratory.New York, 1989) such as extracorporeal recombinant DNA technology, DNA synthetic technology, the interior recombinant technologys of body.Described dna sequence dna can be connected with treating expressed exogenous gene effectively, and is synthetic to instruct described foreign gene mRNA.
In addition, expression vector preferably comprises one or more selected markers, to be provided for selecting the phenotypic character of transformed host cells, cultivate Tetrahydrofolate dehydrogenase, neomycin resistance and the green fluorescent protein (GFP) of usefulness as eukaryotic cell, or be used for colibacillary tsiklomitsin or amicillin resistance.
Comprise the carrier of above-mentioned suitable dna sequence dna and suitable promotor or control sequence, can be used to transform appropriate host cell, so that it can express exogenous protein.
Host cell can be a prokaryotic cell prokaryocyte, as bacterial cell; Or eukaryotic cell such as low, as yeast cell; Or higher eucaryotic cells, as mammalian cell.Representative example has: intestinal bacteria, streptomyces; The bacterial cell of Salmonella typhimurium; Fungal cell such as yeast; Vegetable cell; The insect cell of fruit bat S2 or Sf9; The zooblast of CHO, COS, 293 cells or Bowes melanoma cells etc.The preferred mammal cell, as mouse, rat and people's cell.
Can carry out with routine techniques well known to those skilled in the art with the recombinant DNA transformed host cell.The method for transformation of some employings includes, but are not limited to: coprecipitation of calcium phosphate method, conventional mechanical method such as microinjection, electroporation, liposome packing etc.
The transformant that obtains can be cultivated with ordinary method, with the expression alien gene encoded polypeptide.According to used host cell, used substratum can be selected from various conventional substratum in the cultivation.Under the condition that is suitable for the host cell growth and expresses, cultivate.
In one embodiment of the invention, obtained mouse ACAT-2 gene promoter sequence by pcr amplification, find that simultaneously plain like growth factor conjugated protein (IGFBP-6) gene of ACAT-2 gene and mouse islets is from beginning to end, the about 1.1Kb of distance between the two, and then the The sequencing results of mouse ACAT-2 gene promoter shown, though do not have typical TATA Box and CCAAT Box in this section sequence, but many binding sites to various tissue-specific potential transcription factors are arranged, comprise a HNF-1 α, two Cdx-2, two GATA-1; A reverse P300; A C/EBP; DeltaE etc. illustrate that this section sequence is typical transcripting starting subarea.
The present invention also uses 5 '-method of RACE (Rapid amplication of the 5 '-cDNA end), identified that the transcription initiation site of mouse ACAT-2 gene promoter is VITAMIN B4 A.
In another embodiment, the present invention is respectively with the expression vector of the mouse ACAT-2 gene promoter of different lengths, the transfection mammalian cell transient expression, analyze the transcriptional activity of mouse acat-2 promotor and the section at main transcriptional activity place, found the transcription factor binding member that plays a major role simultaneously.
Cholesteryl ester synthetic enzyme-2 promotor of the present invention has broad application prospects.The ACAT-2 promotor is still all significant to diagnosis, treatment and the drug screening of cholesterol related diseases (comprising atherosclerosis, Alzheimer's disease) to fundamental research; Especially utilizing mouse aspect medicaments sifting model, to have very big applied value.For example,, in specific period, particular organization, can change the expression level of acat-2, for treatment cholesterol related diseases (as atherosclerosis and Alzheimer's disease etc.) provides a possible approach by acting on this promotor.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, people such as Sambrook for example, molecular cloning: laboratory manual (New York:ColdSpring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
Clone, the evaluation of mouse ACAT-2 gene promoter
For obtaining mouse ACAT-2 gene promoter, with reference to Mouse GenomeWalkerTM Kits (available from CLONTECH company), design and synthesize the primer GSP1 and the GSP2 of two mouse ACAT-2 gene specifics, primer sequence is as follows:
GSP1：5’-CCCTTCTCTCCTCCGAAGCTG-3’(SEQ?ID?NO：2)
GSP2：5’-TGCATGGTGCCAGTGTAGAGC-3’(SEQ?ID?NO：3)
With the joint primer that provides among GSP1 and the Kit (Adaport primer) AP1, be template with MouseGenomeWalker Libraaries (available from CLONTECH company), carry out first run polymerase chain reaction (PCR).The primer of nest-type PRC is GSP2 and AP2, and loop parameter is consulted product description, and the PCR product that obtains is mouse ACAT-2 gene promoter, and its length is about 1.1Kb.This product cloning to T-EasyVector (available from Promega), is checked order and analyzes the transcription factor binding site point of this sequence.
The sequence of the ACAT-2 promotor that records is shown in Fig. 1 and SEQ ID NO:1.
Determining of mouse ACAT-2 gene promoter transcription initiation site
Be to determine ACAT-2 gene 5 ' end transcription initiation site, adopt 5 '-method of RACE, be template with mouse ACAT-2cDNA, design primer mA2-1 and mA2-2, their sequence is as follows:
mA2-1：5’-CCCTTCTCTCCTCCGAAGCTG-3’(SEQ?ID?NO：4)
mA2-2：5’-TGCATGGTGCCAGTGTAGAGC-3’(SEQ?ID?NO：5)
Total RNA with 2 μ g mouse intestinal tissues is a template, with reference to the method described in BOEHRINGER company 5 '/3 ' RACE Kit, carries out reverse transcription reaction, adds end reaction and two-wheeled PCR reaction, obtains the product (Fig. 2, top) of a treaty 130bp.This product cloning in T-Easy Vector (available from Promega), is selected 4 strain positive colony sequencing analysis then, determine that VITAMIN B4 A is the transcription initiation site (seeing Fig. 2, the below) of mouse ACAT-2 gene promoter.
The sequential analysis of mouse ACAT-2 gene promoter
Though there is not typical TATA Box in this section sequence, there is not CCAAT Box yet, detailed analysis is found, has concentrated the characteristic sequence of some transcription factor binding members in this section sequence:
-770～-there is the characteristic sequence of a HNF-1 α binding site at the 757bp place;
Respectively there is a Cdx-2 binding site at-64～-660 places and-897～-893 places;
At-421～-410 and-249～-236 places two GATA-1 binding sites are arranged;
There is a reverse P300 binding site at-206～-188 places;
There is a C/EBP binding site at-721～-710 places;
There is a DeltaE binding site at-642～-614 places, illustrate that this section sequence is typical transcripting starting subarea (referring to Fig. 1).
Embodiment 4
The structure of the eukaryon expression plasmid of mouse ACAT-2 gene promoter
For determining the main transcriptional activity section of mouse ACAT-2 gene promoter, utilize highly sensitive luciferase reporter gene carrier pGL3-Basic (available from Promega company), mouse ACAT-2 gene promoter section with different lengths is connected on the reporter gene upstream respectively, makes up a series of recombinant expression plasmids.(referring to Fig. 3)
Determining of main transcriptional activity section of cell cultures, DNA transfection and mouse ACAT-2 gene promoter and main transcription factor binding member.
In the present embodiment, mouse ACAT-2 gene promoter with different lengths inserts luciferase reporter gene carrier pGL3-Basic upstream respectively, with calcium phosphate method difference transfection rat hepatocytes (LW-13), people's liver cancer blastoma cell (HepG2) and human colon cancer cell (Caco-2), measure the plain enzymic activity of relative fluorescence respectively.With the transcriptional activity of definite mouse acat-2 promotor and the section at main transcriptional activity place, find out the transcription factor binding member that plays a major role simultaneously.
Cell Res.1992 is pressed in the cultivation of rat hepatocytes LW-13, and the method described in the 2:139-152 is carried out; Busch is pressed in the cultivation of people's hepatoblastoma cell HepG2, S.J., Barnhart, R.L., Martin, G.A., Flanagan, M.A., and Jackson, R.L., J.Biol.Chem.1990, the described method of 265:22474-22479; Levy is pressed in the cultivation of human colon cancer cell Caco-2, E., Mehran, M., and Seidman, E., FASEB J.1995, the described method of 9:626-635.Contain 10%FBS (wherein Caco-2 is 20%FBS), 10 μ g/ml streptomycin and 10u/ml penicillin in the DMEM substratum, culture condition is all: humidity 95%, 5%CO
2, 37 ℃.
Transfection a few days ago seeds cells into 12 orifice plates, and every porocyte number is 100,000-300, and 000, the cell abundance reaches 50-80% during transfection, and wherein 4 days two kinds of situations are not broken up and are broken up in the transfection of Caco-2 cell.With pGL3-mD50 (being called for short mD50), pGL3-mD51 (mD51), pGL3-mD52 (mD52), pGL3-mD53 (mD53) and negative control plasmid pGL3-Basic (Basic, do not contain promotor) with calcium phosphate method difference transfection LW-13, HepG2 and Caca-2 cell, carry out transient expression research.The transfection parameter is mark plasmid pCH110 and 200 μ l transfection liquid in the every hole 2 μ g sample DNAs, 1ug, 37 ℃ of transfectional cells 12 hours, collecting cell after 44 hours.
Measure plain enzyme (luciferase) vigor of relative fluorescence behind the collecting cell, method is with reference to the product description of Promega company.Judge the transcriptional activity of its upstream promoter according to the plain enzyme activity of relative fluorescence, and the betagalactosidase activity of coexpression is as interior mark, in order to proofread and correct the different transfection efficiency differences that cause of microenvironment (cell density, cell differential cloth, culturing bottle inwall etc.) because of cell cultures.The calculation formula of the plain enzyme activity of relative fluorescence is:
Wherein:
During mensuration with the luciferase gene upstream not the pGL3-Basic of tape starting make negative control, (the beta-galactosidase enzymes vigor O.D. of the beta-galactosidase gene transfection efficiency between the same batch sample
420/ corresponding protein content) fluctuation should be less than 15%, and three parts of parallel sample are made in each experiment.
Experimental result as shown in Figure 3.The result shows: in rat hepatocytes LW-13, people's hepatoblastoma cell HepG2, human colon cancer cell Caco-2, the main transcriptional activity section of mouse ACAT-2 gene promoter all-1030bp～-569bp between.When insert segmental 5 ' end from-1030bp foreshorten to-during 868bp, its transcriptional activity slightly descends; When insert segmental 5 ' end from-1030bp foreshorten to-during 569bp, its transcriptional activity obviously descends.These presentation of results,-1030bp～-569bp, promptly the 98-558 position is the main transcriptional activity section of mouse ACAT-2 gene promoter among the SEQ ID NO:1, explanation simultaneously, in this promotor, a HNF-1 α and two Cdx-2 are main transcription factor binding members.
All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Sequence table
＜110〉Institute Of Biochemistry And Cell Biology, Shanghai Institutes For Biological Sciences, Chinese Academy Of Sciences
＜120〉mouse cholesteryl ester synthetic enzyme-2 gene promoter
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Claims (10)
1. a promotor is characterized in that, it is mouse cholesteryl ester synthetic enzyme-2 promotor, and its nucleotide sequence is selected from down group:
(a) nucleotide sequence shown in the 47-1137 position among the SEQ ID NO:1,
(b) nucleotide sequence shown in the 98-558 position among the SEQ ID NO:1, or
(c) nucleotide sequence shown in the 1-1199 position among the SEQ ID NO:1.
2. promotor as claimed in claim 1 is characterized in that, its nucleotide sequence is the nucleotide sequence shown in the 98-558 position among the SEQ ID NO:1.
3. a carrier is characterized in that, it contains the described promotor of claim 1.
4. carrier as claimed in claim 3 is characterized in that it also contains the foreign gene that described promotor is operably connected.
5. carrier as claimed in claim 4 is characterized in that described foreign gene is a reporter gene.
6. a host cell is characterized in that, it contains the described carrier of claim 3.
7. host cell as claimed in claim 6 is characterized in that described host cell is a mammalian cell.
8. host cell as claimed in claim 7 is characterized in that, described host cell is mouse, rat and people's a cell.
9. the purposes of the described promotor of claim 1 is characterized in that, is used to screen the medicine of treatment cholesterol related diseases.
10. a method of screening the medicine of treatment cholesterol related diseases is characterized in that, comprises step:
(a) under the condition that is fit to growth, cultivate host cell, described host cell contains an expression vector, described expression vector contains described promotor of claim 1 and the reporter gene that links to each other with this promotor operability, wherein in the substratum of first group of host cell, add candidate substances, in the substratum of second group of host cell, do not add candidate substances;
(b) measure the expression amount of reporter gene in first group and the second group of host cell, wherein the expression amount of reporter gene is higher than second group and just represents that this candidate substances is the medicine of treatment cholesterol related diseases in first group of host cell.
Priority Applications (1)
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| CN 02150731 CN1219062C (en) | 2002-11-27 | 2002-11-27 | Mouse cholesterol ester synthetase-2 gene promotor |
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| Application Number | Priority Date | Filing Date | Title |
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| CN 02150731 CN1219062C (en) | 2002-11-27 | 2002-11-27 | Mouse cholesterol ester synthetase-2 gene promotor |
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