CN105316352A

CN105316352A - Carrier for adaptive expression target gene and its preparation method and application

Info

Publication number: CN105316352A
Application number: CN201410366451.7A
Authority: CN
Inventors: 侯小虎; 何成宜; 陈志英
Original assignee: Shenzhen Institute of Advanced Technology of CAS
Current assignee: Shenzhen Institute of Advanced Technology of CAS
Priority date: 2014-07-29
Filing date: 2014-07-29
Publication date: 2016-02-10

Abstract

The invention provides a carrier of an adaptive expression target gene. The carrier comprises a target gene expression box which comprises a liver-specific promoter, a cholesterol regulation element and the target gene. The cholesterol regulation element, the liver-specific promoter and the target gene are operably connected. The invention also provides a preparation method and application of the carrier of the adaptive expression target gene.

Description

Carrier of a kind of modulated expression's goal gene and its preparation method and application

Technical field

The present invention relates to the expression vector for gene therapy, belong to controllable express field, be specifically related to carrier of a kind of modulated expression's goal gene and its preparation method and application.

Background technology

Cholesterol is one of biomembranous important composition composition, plays an important role in many vital processes.But high-caliber cholesterol can cause the serious disease such as atherosclerosis, coronary heart disease in blood, early start pulse atherosclerosis, coronary heart disease is familial hypercholesterolemia (familialhypercholesterolemia, FH) important behaviour (VanCraeyveldE etc., CurrPharmDes, 17 (24): 2575-2591 (2011)).

In blood, the cholesterol of 70% is by low-density lipoprotein (low-densitylipoprotein, and vldl (verylow-densitylipoprotein LDL), VLDL) (Zhang Rui is carried, foreign medical science, geriatrics fascicle, 30 (1): 29-33 (2009)).The metabolism of content to Blood Cholesterol of the LDL in blood has material impact.

Liver cell is the main cell of metabolism LDL, is also uniquely can the cell of excretion of cholesterol.Low density lipoprotein receptor (low-densitylipoproteinreceptor in liver, LDLr) activity plays topmost regulating effect to LDL level in blood plasma, LDLR gene transcribe by Sterol regulatory element binding protein (Sterolregulatoryelementbindingprotein, SREBP) path regulates (Liu Tongfei etc., China's cytobiology journal, 35 (4): 401-409, (2013)).

SREBP is that one belongs to substantially-helix-loop-helix-leucine zipper (basic-helix-loop-helix-leucinezipper, bHLHZ-ip) transcription factor (Yokoyamaetal. of family, Cell75:187-197 (1993)), in mammalian cell, there are 3 members: SREBP-1a, SREBP-lc and SREBP-2.They have different selectivity to different target genes, and wherein that primary activation LDL gene is SREBP-2.

The mechanism of SREBP path is: when the cholesterol of endocytoplasmic reticulum film is in high level, reticulon SREBP cracking activator (SREBPcleavageactivatingprotein, SCAP) sterol sensitive structure territory (sterolsensingdomain, SSD) will in conjunction with cholesterol, SCAP conformation is caused to change, SCAP can in conjunction with the insulin-induced gene (insulininducedgene of reticulon, Insig), form SREBP-2/SCAP/Insig mixture and be retained in endoplasmic reticulum, the synthesis of cholesterol and metabolism is caused to be blocked, low-density lipoprotein in blood plasma can not by metabolism, when the cholesterol of endoplasmic reticulum is in low-level, SCAP dissociates from Insig, SREBP-2/SCAP moves to golgi body from endoplasmic reticulum, core SREBP-2 (SREBP-2n) in nucleus increases, cholesterol regulation element (sterolregulatoryelement in SREBP-2n specific binding LDLR gene promoter, SRE), thus promote transcribing of LDLR gene, and then impel the low-density lipoprotein in blood plasma to carry out metabolism (KassimSH etc. by LDLr path, HumGeneTher, 24 (1): 19-26 (2013), SudhofTC etc., JBiolChem, 262 (22): 10773-10779 (1987), KongWJ etc., JMolMed (Berl), 84 (1): 29-36 (2006), RadhakrishnanA etc., ProcNatlAcadSciUSA, 104 (16): 6511-6518 (2007), SunLP etc., ProcNatlAcadSciUSA, 104 (16): 6519-6526 (2007)).

SREBP, except being regulated by the expression of SREBP path to LDLr, can also be regulated the enzyme in other cholesterol or fatty acid synthesis pathway.Research shows, on transcriptional level, 3-hydroxy-3-methylglutaryl-coenzyme A reductase enzyme (3-hydroxy-3-methylglutarylcoenzymeAreductase, HMGCR) mRNA content is mainly subject to the regulation and control of SREBP.HMGCR is the rate-limiting enzyme of cholesterol biosynthesis, SREBP path and HMGCR path of degrading is two main reverse feedback regulation mechanism (Liu Tongfei etc. of cholesterol metabolic, China's cytobiology journal, 35 (4): 401-409, (2013)).SREBP is also relevant with cholesterol regulation element SRE to the adjustment of HMGCR, SRE is the cis-acting elements of an about 10bp on gene promoter, SRE does not exist only in LDLR gene promoter, some coding cholesterol or fatty acid synthesis pathway in enzyme gene promoter in, also have the existence of SRE in the promotor of such as HMGCR gene and acetyl coenzyme A synthetase gene.SREBP is by transcribing of SRE adjustment LDLR gene or transcribing of other enzyme genes, and then the metabolism of cholesterol regulating or lipid acid (Rawsonetal., Mol.Cell.Biol.4:631-640 (2003)).

At present, medicine for HMGCR degraded path has HMGCR inhibitor (as statins statins), although this pharmacological agent effectively can reduce the cholesterol of in blood plasma 50%, but must take with maintaining treatment effect (VanCraeyveldE etc. all the life, CurrPharmDes, 2011,17 (24): 2575-2591; RahalkarAR etc., MolGenetMetab, 2008,93 (3): 282-294; KassimSH etc., HumGeneTher, 24 (1): 19-26 (2013)).

On the other hand, report is had to be studied for SREBP path from gene level, Chenetal. (2000) adopt AAV carrier, cmv enhancer, actin promoter expression LDLR gene (ChenSJ etc., MolTher, 2 (3): 256-261 (2000)) content of the cholesterol of 40%, is reduced.But this non-liver specificity expression causes other cells and kills and wounds.

Jacobsetal. (2008) adopt AAV carrier Liver specific promoters antitrypsin and apoE enhanser to express LDLR gene (KassimSH etc., HumGeneTher, 24 (1): 19-26 (2013)) content of the cholesterol of 40-60%, is reduced.Although employ tissue-specific promoter, but this phraseology not with cholesterol regulation element SRE, not obvious to the feedback regulation of the content height of cholesterol, the expression of goal gene is comparatively constant, the effect of cholesterol regulating metabolism can not be played, be difficult to the effect reaching long-term treatment.

Therefore, be necessary to provide a kind of liver specifically, the modulability ground carrier of expressing goal gene and its preparation method and application.

Summary of the invention

For solving the problem, first aspect present invention provides a kind of carrier of modulated expression's goal gene, the carrier of described modulated expression's goal gene comprises destination gene expression box, described destination gene expression box comprises: Liver specific promoters, cholesterol regulation element and goal gene, and described cholesterol regulation element, Liver specific promoters are operably connected with goal gene.

Preferably, described goal gene is the encoding gene of protein or the encoding gene of microRNA.

Preferably, described Liver specific promoters is PCK promotor, α-I-antitrypsin promotor, thyroid hormone binding globulin promotor, α-fetoprotein promotor, alcohol dehydrogenase promoter, IGF-II promotor, Factor IX promotor, HBV basal core protein promoter, s2 protein promoter before HBV, throxine-binding globulin promotor, the hybrid promoter of HCR-Ap0CII, HCR-hAAT hybrid promoter, the AAT promotor be combined with the enhancer element of mouse albumin gene, apo E promotor, low-density lipoprotein promotor, pyruvate kinase promoter, lecithin cholesterol acyltransferase promotor, Apolipoprotein H promotor, siderophilin promotor, transthyretin promotor, alpha fibre proteinogen and β _ fibrinogenic promotor, α-I-chymotrypsin inhibitor promotor, α-2-HS glycoprotein promotor, haptoglobin promotor, ceruloplasmin promotor, profibr(in)olysin promotor, complement proteins promotor, Complement C_3 activates the promotor of son, hemopexin promotor and α-I-acid glycoprotein promotor.

Preferably, described cholesterol regulation element is the cis-acting elements in the promotor of gene, and described gene to comprise in cholesterol biosynthesis approach or the encoding gene of enzyme or the encoding gene of receptor protein in fatty acid synthesis pathway.

Preferably, the carrier of described modulated expression's goal gene also comprises at least one in Kozak sequence and liver specificity positioning controling element.

Preferably, the carrier of described modulated expression's goal gene is not containing the skeleton DNA sequence dna of standard plasmid.

Preferably, the carrier of described modulated expression's goal gene is minicircle dna.

Second aspect present invention provides a kind of preparation method of carrier of modulated expression's goal gene, comprising:

By the multiple clone site of Liver specific promoters, cholesterol regulation element and goal gene insertion vector, obtain the carrier of described modulated expression's goal gene, the carrier of described modulated expression's goal gene comprises destination gene expression box, described destination gene expression box comprises: Liver specific promoters, cholesterol regulation element and goal gene, and described cholesterol regulation element, Liver specific promoters are operably connected with goal gene.

Preferably, described carrier is virus vector, prokaryotic plasrnid carrier or eucaryon plasmid carrier.

Preferably, described carrier is minicircle dna empty plasmid, the carrier of described modulated expression's goal gene is minicircle dna matrix grain, described minicircle dna matrix grain obtains not containing the carrier of modulated expression's goal gene of the skeleton DNA sequence dna of standard plasmid after Site-specific recombinase, gained does not comprise destination gene expression box containing the carrier of modulated expression's goal gene of the skeleton DNA sequence dna of standard plasmid, described destination gene expression box comprises: Liver specific promoters, cholesterol regulation element and goal gene, described cholesterol regulation element, Liver specific promoters is operably connected with goal gene.

Preferably, the described carrier not containing modulated expression's goal gene of the skeleton DNA sequence dna of standard plasmid is minicircle dna.

Third aspect present invention provides a kind of application of carrier in the medicine of preparation treatment cholesterol metabolism abnormity disease of modulated expression's goal gene.

Fourth aspect present invention provides a kind of method adopting the vehicle treatment cholesterol metabolism abnormity disease of modulated expression's goal gene, comprises the steps:

S01) carrier of modulated expression's goal gene is prepared;

S02) carrier of described modulated expression's goal gene is operated by following arbitrary step:

A) carrier of described modulated expression's goal gene is used for separately gene therapy;

B) carrier of described modulated expression's goal gene is combined for gene therapy with one or more in chemotherapy, radiotherapy, operation, biotherapy, immunotherapy;

C) adopt the mode of targeting Delivery in body to be directly delivered in patient body by the carrier of described modulated expression's goal gene to treat;

D) first pass through in-vitro transfection technology by the carrier transfection immune effector cell of described modulated expression's goal gene, then described transfection is had in the immune effector cell of the carrier of modulated expression's goal gene defeated time patient body and implement treatment.

Preferably, the carrier of described modulated expression's goal gene is minicircle dna carrier.

Carrier of the modulated expression's goal gene that the invention provides and its preparation method and application has following beneficial effect:

The carrier of modulated expression's goal gene provided by the invention has Liver specific promoters and cholesterol regulation element SRE two kinds of controlling elements simultaneously, due to the vitals that liver is cholesterol metabolic, Liver specific promoters is conducive to the tissue specific expression of the carrier of modulated expression's goal gene, improves the targeting of gene therapy; In addition, the cholesterol regulation element SRE that the present invention adopts can make full use of the natural cholesterol feed-back regulatory mechanism of cell, enhances cell active, suitably to the endogenous capacity that the metabolism of cholesterol regulates.

Accompanying drawing explanation

The plasmid map of the ZY781.bpA empty carrier that Fig. 1 provides for the embodiment of the present invention;

The plasmid map of the parental plasmid ZY781.PLDLR.LDLR.bpA that Fig. 2 provides for the embodiment of the present invention;

The plasmid map of the parental plasmid ZY781.PLDLR.Kozak.LDLR.bpA that Fig. 3 provides for the embodiment of the present invention;

The plasmid map of the parental plasmid ZY781.PLDLR.UTR.LDLR.bpA that Fig. 4 provides for the embodiment of the present invention;

The plasmid map of the parental plasmid ZY781.Enhancer.PLDLR.LDLR.bpA that Fig. 5 provides for the embodiment of the present invention;

The plasmid map of the parental plasmid ZY781.Enhancer.PLDLR.Kozak.LDLR.bpA that Fig. 6 provides for the embodiment of the present invention;

The plasmid map of the parental plasmid ZY781.Enhancer.ApoE.LDLR.bpA that Fig. 7 provides for the embodiment of the present invention;

The plasmid map of the parental plasmid ZY781.Enhancer.ApoE.Kozak.LDLR.bpA that Fig. 8 provides for the embodiment of the present invention;

ZY781.ApoE.bpA (this seminar builds and preserves);

After the transfection HepG 2 cell of the plasmid that Fig. 9 ~ Figure 25 provides for the embodiment of the present invention, the expression of LDLR gene (wherein, Fig. 9 ~ Figure 16 is respectively ZY781.bpA empty carrier, minicircle dna MC.PLDLR.LDLR.bpA, MC.PLDLR.Kozak.LDLR.bpA, MC.PLDLR.UTR.LDLR.bpA, MC.Enhancer.PLDLR.LDLR.bpA, MC.Enhancer.PLDLR.Kozak.LDLR.bpA, after MC.Enhancer.ApoE.LDLR.bpA and MC.Enhancer.ApoE.Kozak.LDLR.bpAA transfection HepG 2 cell, RT-PCR result is carried out to the mRNA of LDLR gene in each group of cell, in Fig. 9 ~ Figure 16, each figure center pillar-1 is control group, post-2 is cholesterol group, post-3 is statin group, " * " represents P<0.05, " * * " represents P<0.01,

Figure 17 comprises figure a ~ figure h, after figure a ~ figure h is respectively ZY781.bpA empty carrier, minicircle dna MC.PLDLR.LDLR.bpA, MC.PLDLR.Kozak.LDLR.bpA, MC.PLDLR.UTR.LDLR.bpA, MC.Enhancer.PLDLR.LDLR.bpA, MC.Enhancer.PLDLR.Kozak.LDLR.bpA, MC.Enhancer.ApoE.LDLR.bpA and MC.Enhancer.ApoE.Kozak.LDLR.bpAA transfection HepG 2 cell, fluorescence microscope is adopted respectively to organize the result of LDLr distribution situation in cell;

Figure 18 ~ Figure 25 is respectively ZY781.bpA empty carrier, minicircle dna MC.PLDLR.LDLR.bpA, MC.PLDLR.Kozak.LDLR.bpA, MC.PLDLR.UTR.LDLR.bpA, MC.Enhancer.PLDLR.LDLR.bpA, MC.Enhancer.PLDLR.Kozak.LDLR.bpA, after MC.Enhancer.ApoE.LDLR.bpA and MC.Enhancer.ApoE.Kozak.LDLR.bpAA transfection HepG 2 cell, the relative value of fluorescence intensity in each group of cell, in Figure 18 ~ Figure 25, each figure center pillar-1 is control group, post-2 is cholesterol group, post-3 is statin group, " * " represents P<0.05, " * * " represents P<0.01, ),

After the transfection HepG 2 cell of the plasmid that Figure 26 ~ Figure 34 provides for the embodiment of the present invention, the expression of LDLR gene (wherein, Figure 26 comprises figure a ~ figure h, figure a ~ figure h is respectively ZY781.bpA empty carrier, minicircle dna MC.PLDLR.LDLR.bpA, MC.PLDLR.Kozak.LDLR.bpA, MC.PLDLR.UTR.LDLR.bpA, MC.Enhancer.PLDLR.LDLR.bpA, MC.Enhancer.PLDLR.Kozak.LDLR.bpA, after MC.Enhancer.ApoE.LDLR.bpA and MC.Enhancer.ApoE.Kozak.LDLR.bpAA transfection HepG 2 cell, fluorescence microscope is adopted respectively to organize the result of cell LDL metabolic condition,

Figure 27 ~ Figure 34 is respectively ZY781.bpA empty carrier, minicircle dna MC.PLDLR.LDLR.bpA, MC.PLDLR.Kozak.LDLR.bpA, MC.PLDLR.UTR.LDLR.bpA, MC.Enhancer.PLDLR.LDLR.bpA, MC.Enhancer.PLDLR.Kozak.LDLR.bpA, after MC.Enhancer.ApoE.LDLR.bpA and MC.Enhancer.ApoE.Kozak.LDLR.bpAA transfection HepG 2 cell, the relative value of each group of cell LDL metabolic capacity, in Figure 27 ~ Figure 34, each figure center pillar-1 is control group, post-2 is cholesterol group, post-3 is statin group, " * " represents P<0.05, " * * " represents P<0.01, ),

The present invention describes in detail

First aspect, the invention provides a kind of carrier of modulated expression's goal gene, the carrier of described modulated expression's goal gene comprises destination gene expression box, described destination gene expression box comprises: Liver specific promoters, cholesterol regulation element and goal gene, and described cholesterol regulation element, Liver specific promoters are operably connected with goal gene.

As used herein, " carrier " refers in genetically engineered research, can insert foreign DNA and the structure that can copy in recipient cell.

As used herein, described " goal gene " refers to all DNA moleculars needing research, as the nucleotide sequence with cell or experimenter's partially or completely allos of being imported into cell.Object fragment can from genomic dna, also can from the DNA of cDNA, synthetic, and current at most be PCR primer (comprising RT-PCR product, multiple PCR products etc.).Goal gene used herein should only not be understood to can the nucleotide sequence of coded polypeptide, also can be understood to endogenous cellular or the nucleotide sequence of the nucleotide sequence portion that is imported into cell or complete complementary, like this, goal gene can produce the RNA molecule (as microRNA, shRNA or siRNA) that can hybridize or suppress endogenous nucleotide sequence to express after transcribing.

As used herein, " DNA " is deoxyribonucleotide, is formed by connecting by 3,5-phosphodiester bond by 4 kinds of main deoxynucleotides (dAMP, dGMP, dCMT and dTMP).

As used herein, the sequence that described " destination gene expression box " is insertion vector multiple clone site, be used at cells goal gene, owing to there is the expression regulation region relative to described goal gene, its goal gene comprised can be transcribed into RNA, some RNA continues to be translated to produce to have required bioactive polypeptide or protein, and some RNA can be processed into microRNA.

As used herein, " RNA " is ribonucleotide, is the polymer be formed by connecting by 3,5-phosphodiester bond by Nucleotide.The base of RNA mainly contains 4 kinds, i.e. VITAMIN B4 (A), guanine (G), cytosine(Cyt) (C), uridylic (U), and wherein, U instead of the T thymus pyrimidine in DNA and becomes the feature base of RNA.

Preferably, described destination gene expression box inserts the multiple clone site of described carrier.

As used herein, " multiple clone site " refers to one section of sequence of synthetic on carrier sequence, containing multiple restriction endonuclease recognized site, can provide multiple insertable position or interleaved plan for foreign DNA.

Further preferably, the encoding gene of described protein includes but not limited to cell receptor, signaling molecule, regulatory factor, antigen or antibody gene.

As used herein, " cell receptor " is preferably on cytolemma or intracellular acceptor, and this receptor energy specific binding target molecules, as the LDLr adopted in one embodiment of the invention.

As used herein, " signaling molecule " is on energy specific binding cytolemma or intracellular receptor, and causes, affects or regulate polypeptide or the protein of corresponding signal path.

As used herein, " regulatory factor " be can transcribe in specific regulatory cells, translate, molecule that metabolism etc. is movable.

As used herein, " antigen " or " antibody " includes but not limited to antibody that is natural, restructuring, antigen or epitope.

As used herein, " antigen " or antibody " include but not limited to people source or mouse source antibody.

As used herein, " antibody " includes but not limited to curative antibody.

As used herein, " antibody " can be single target, two target or many targeting antibodies.

As used herein, " target " refers to antibodies specific ground conjugated antigen, and " two target " refers to that antibody has two sites be combined with antigen-specific, and " many targets " refers to that antibody has the plural site be combined with antigen-specific.

Further preferably, " encoding gene of microRNA " includes but not limited to the gene can transcribing siRNA, shRNA, dsRNA or miRNA.

Preferably, described goal gene is the encoding gene of enzyme or the encoding gene of receptor protein that participate in cholesterol biosynthesis approach.

Preferably, described goal gene is the encoding gene of microRNA, and the encoding gene energy specificity silence of described microRNA participates in the encoding gene of enzyme of cholesterol biosynthesis approach or the expression of the encoding gene of receptor protein.

Preferably, described goal gene is the encoding gene of enzyme or the encoding gene of receptor protein that participate in fatty acid synthesis pathway.

Preferably, described goal gene is the encoding gene of microRNA, and the encoding gene energy specificity silence of described microRNA participates in the encoding gene of enzyme of fatty acid synthesis pathway or the expression of the encoding gene of receptor protein.

Preferably, described goal gene is LDLR gene, 3-hydroxy-3-methylglutaryl-coenzyme A synthetic enzyme (3-hydroxy-3-methylglutarylcoenzymeAsynthase, HMGCS) gene or 3-hydroxy-3-methylglutaryl-coenzyme A reductase enzyme (3-hydroxy-3-methylglutarylcoenzymeAreductase, HMGCR) gene.

Further preferably, the GenBank accession number of described LDLR gene is NM_000527.4.

Further preferably, the GenBank accession number of described HMGCS gene is NM_002130.7.

Further preferably, the GenBank accession number of described HMGCR gene is NM_000859.2.

The above-mentioned nucleotide sequence shown in GenBank accession number NM_000527.4, NM_002130.7 and NM_000859.2 comprises the nucleotide sequence of LDLR gene, HMGCS gene and HMGCR gene respectively.

Preferably, described goal gene includes but not limited to LDLR gene exemplified herein, HMGCS gene or HMGCR gene.

Preferably, the homology >95% (preferably >=98%) that it is the nucleotide sequence of LDLR gene in nucleotide sequence shown in NM_000527.4 that described goal gene comprises with accession number, and aminoacid sequence and the accession number of coding are the identical or substantially identical gene order of the aminoacid sequence of the coding of LDLR gene in nucleotide sequence shown in NM_000527.4.

Preferably, the homology >95% (preferably >=98%) that it is the sequence of HMGCS gene in nucleotide sequence shown in NM_002130.7 that described goal gene comprises with accession number, and aminoacid sequence and the accession number of coding are the identical or substantially identical gene order of the aminoacid sequence of the coding of HMGCS gene in nucleotide sequence shown in NM_002130.7.

Preferably, the homology >95% (preferably >=98%) that it is the sequence of HMGCR gene in nucleotide sequence shown in NM_000859.2 that described goal gene comprises with accession number, and aminoacid sequence and the accession number of coding are the identical or substantially identical gene order of the aminoacid sequence of the coding of HMGCR gene in nucleotide sequence shown in NM_000859.2.

Preferably, described Liver specific promoters behaviour source Liver specific promoters.

As used herein, " promotor " refers to a kind of nucleotide sequence, and it is present in the upstream (5 ' end) of object nucleotide sequence usually, and object nucleotide sequence can be guided to be transcribed into mRNA.Usually, promotor or promoter region provide the recognition site of RNA polymerase and the necessary other factors of correct initiation transcription.

As used herein, " Liver specific promoters " refers to that described promotor can optionally activate in liver cell or come from transcribing in hepatocellular clone.In the present invention, Liver specific promoters is those specific activitys in liver active all high promotors in other any bodily tissue.Usually, the specific activity of Liver specific promoters in liver is high a lot of in other tissue.Such as, the specific activity of this promotor in liver can be as high as few 2 times, at least 3 times, at least 4 times, at least 5 times or at least 10 times in other cell type.The activity of described promotor in liver source cell and reference cell contrasts and instructs the expression in particular organization to stop the ability of the expression in other cell or tissue to measure by it simultaneously.Therefore, Liver specific promoters allows the activity expression of gene in liver and stops the expression of gene in other cell or tissue.

Particularly, in described promotor, part bilingual is:

PCK promotor: pepck promoter); α-I-antitrypsin promotor: AAT promotor; IMA-IGF2BP3-001: IGF-II promotor; Factor IX promotor: FVIII) promotor; HBV basal core protein promoter: BCP promotor; S2 protein promoter before HBV: PreS2 promotor; Throxine-binding globulin promotor: TBG promotor; The hybrid promoter of liver control area-Ap0CII: the hybrid promoter of HCR-Ap0CII; Liver control area-people's antitrypsin promotor: HCR-hAAT hybrid promoter; Lecithin cholesterol acyltransferase promotor: LCAT promotor; Apolipoprotein H promotor: ApoH promotor; Throxine-binding globulin promotor: TBG promotor; Throxine-binding globulin promotor: TBG promotor.

Particularly, in the AAT promotor be combined with the enhancer element of mouse albumin gene, described enhanser is mouse ALB enhanser (Ealb).

Particularly, in described complement proteins promotor, described complement is CIq, CIr, C2, C3, C4, C5, C6, C8, C9, the factor I of complement or factor H.

Preferably, be applicable to Liver specific promoters of the present invention to comprise, but be not restricted to and can find other tissue-specific promoter (NucleicAcidsResearch, J4:D104-D107 (2006)) in tissue-specific promoter database TiProD.

Alternately, the hybrid promoter comprising liver-specific enhancer and Liver specific promoters can be used.The hybrid promoter that such promotor comprises the hybrid promoter of liver control area (HCR)-Ap0CII, HCR-hAAT hybrid promoter, the AAT promotor combined with the enhancer element (Ealb) of mouse albumin gene and apo E promotor, formed by the promotor (Ealb-AATp) of mouse albumin gene enhanser (Ealb) and mouse α-l-antitrypsin (AAT).

As used herein, described PEPCK is preferably Yang, Y.ff., J. etc., GeneMed., pepck promoter described in 5 (5): 417-424 (2003).

As used herein, the promotor of described HBcAg gene is preferably Kramer, M.G., etc., Mol.Ther., the promotor of HBcAg gene described in 7 (3): 375-385 (2003).

As used herein, the promotor of described TBG gene is preferably Wang, the promotor of TBG gene described in L., etal., Proc.Natl.Acad.Sci, 96:3906-3910 (1999).

As used herein, " cholesterol regulation element " cis-acting elements of an about 10bp for gene promoter exists, enter nuclear identification after SREBP albumen is activated and in conjunction with SRE, and promote that the gene promoter with this SRE starts transcribing of downstream gene.

SREBP is relevant with cholesterol regulation element SRE to the adjustment of HMGCR, SRE is the cis-acting elements of an about 10bp on gene promoter, SRE does not exist only in LDLR gene promoter, some coding cholesterol or fatty acid synthesis pathway in enzyme gene promoter in, also have the existence of SRE in the promotor of such as HMGCR gene and acetyl coenzyme A synthetase gene.SREBP is by transcribing of SRE adjustment LDLR gene or transcribing of other enzyme genes, and then the metabolism of cholesterol regulating or lipid acid (Rawsonetal., Mol.Cell.Biol.4:631-640 (2003)).

Preferably, described cholesterol regulation element is the cis-acting elements in the promotor of gene, and described gene comprises the encoding gene of enzyme or the encoding gene of receptor protein in cholesterol biosynthesis approach.

Preferably, described cholesterol regulation element is the cis-acting elements in the promotor of gene, and described gene comprises the encoding gene of enzyme or the encoding gene of receptor protein in fatty acid synthesis pathway.

Further preferably, the encoding gene of enzyme or the encoding gene of receptor protein are LDLR gene, HMGCS gene or HMGCR gene in described cholesterol biosynthesis approach or in fatty acid synthesis pathway.

Still more preferably, the GenBank accession number of described LDLR gene is NM_000527.4.

Still more preferably, the GenBank accession number of described HMGCS gene is NM_002130.7.

Still more preferably, the GenBank accession number of described HMGCR gene is NM_000859.2.

As described herein, the described gene that there is cholesterol regulation element includes but not limited to LDLR gene promoter, HMGCS gene promoter or HMGCR gene promoter.Described cholesterol regulation element is preferably S ü dhofTC etc. at JBiolChem.1987; The cholesterol regulation element reported in 262 (22): 10773-10779.As used herein, described " being operably connected " refers to functional spatial disposition of two or more nucleic acid region or nucleotide sequence.Such as: promoter region is placed in the specific position relative to object nucleotide sequence, what make object nucleotide sequence transcribes the guiding being subject to this promoter region, thus promoter region is " operably connected " on this nucleotide sequence.As herein describedly to be operably connected without the need to being connected continuously between sequence.

Preferably, described cholesterol regulation element, Liver specific promoters are described cholesterol regulation element with the mode that goal gene is operably connected, Liver specific promoters is connected successively, sequentially with goal gene.

Further preferably, described cholesterol regulation element, Liver specific promoters are described cholesterol regulation element with the mode that goal gene is operably connected, Liver specific promoters is connected successively, sequentially, continuously with goal gene.

As used herein, " Kozak sequence " refers to the one section of sequence be present near initiator codon AUG, its translation initial in play an important role.In eukaryotic cell, the initiation site of translation is mostly in the AUG codon site of 5' end.After being combined with the 5' end cap minor structure of mRNA, ribosomal 40S small subunit scans mRNA, until run into first AUG codon.Ribosomal 40S small subunit causes the initial of translation to a great extent to being identified in of nucleotide sequence around AUG, and affects translation efficiency.

The Kozak sequence that different eukaryotic gene expression is taked is different, Kozak sequence preference used herein is behaved, the genetic expression of zooblast time the Kozak sequence taked.

Preferably, described Kozak sequence behave, the genetic expression of zooblast time the Kozak sequence taked.

Further preferably, described Kozak sequence is the one in the nucleotide sequence shown in SEQIDNO:1 ~ 6.

Concrete, the nucleotides sequence shown in described SEQIDNO:1 is classified as GCCNCC aTGg.

Concrete, the nucleotides sequence shown in described SEQIDNO:2 is classified as GCCGCCNCC aTGg.

Concrete, the nucleotides sequence shown in described SEQIDNO:3 is classified as AGNN aTGn.

Concrete, the nucleotides sequence shown in described SEQIDNO:4 is classified as ANN aTGg.

Concrete, the nucleotides sequence shown in described SEQIDNO:5 is classified as ACC aTGg.

Concrete, the nucleotides sequence shown in described SEQIDNO:6 is classified as GACACC aTGg.

As used herein, " aTG" by underscore mark, identify the encoding gene of initiator codon AUG; " N " is in one in A, T, C and G.

Preferably, described ATG can also be ATT or ATA.

Preferably, the nucleotide sequence " GCCNCC shown in SEQIDNO:1 aTGg " in, N is that R, R are preferably A or G.

Preferably, the nucleotide sequence " GCCGCCNCC shown in SEQIDNO:2 aTGg " in, N is that R, R are preferably A or G.

Preferably, the carrier of described modulated expression's goal gene also comprises liver specificity positioning controling element.

As used herein, " liver specificity positioning controling element " (HCR) refers to the nucleotide sequence with DNaseI susceptibility site, this district is found in the downstream of apo E (ApoE) gene, and liver cell control region can strengthen the expression of liver specific genes.

Preferably, described liver specificity positioning controling element is enhanser.

Further preferably, enhanser is the enhanser on LDLR gene promoter.

Preferably, the GenBank accession number of the enhanser on described LDLR gene promoter is U32510.1.

In a preferred embodiment of the invention, the nucleotides sequence of the enhanser of the carrier employing of the modulated expression's goal gene constructed is classified as the 5 ~ 325bp of GenBank accession number for nucleotide sequence shown in U32510.1.

Particularly, the sequence of the enhanser on described LDLR gene promoter obtains by PCR, the method of described PCR is: adopt pLDLR-F provided by the invention (SEQIDNO:11) and LDLR-R (SEQIDNO:12) to be PCR primer, take ZY781.ApoE.bpA as the enhanser obtained after pcr template carries out PCR on described LDLR gene promoter.

LDLR gene promoter does not have liver specificity positioning controling element, liver specificity positioning controling element is placed on before LDLR gene promoter by the present invention, can strengthen the expression of LDLR gene.

As used herein, " skeleton DNA sequence dna " comprises in standard plasmid the DNA sequence dna copying or screen the functions such as the host containing plasmid being responsible for bacterial plasmid, comprises bacterium replication sequence, resistant gene, unmethylated CpG motif etc.

" the skeleton DNA " and " skeleton DNA vector " that adopt herein can exchange.

Preferably, the carrier of described modulated expression's goal gene is not containing the skeleton DNA sequence dna suppressing destination gene expression in parental plasmid.

As used herein, " minicircle dna " refers to not containing the skeleton DNA sequence dna of prokaryotic plasrnid, mainly with the carrier that superhelix exists, can be free on people and mammalian cell chromosomal DNA outward stable lasting expression transcribe or express goal gene.

The minicircle dna that the present invention adopts, owing to eliminating the skeleton DNA sequence dna of bacterial origin, compared with virus vector, plasmid vector, the vivo and vitro genetic expression of minicircle dna decreases the possibility be inflamed with gene silencing, the expression cycle is more of a specified duration, and Gene expression intensities strengthens 10-1000 doubly (ChenZY etc., GeneTher,, 11 (10): 856-864 (2004); Kay etc., NatBiotechnol, 28 (12): 1287-1289 (2010); US Patent No., 897,380B2; MayrhoferP etc., MethodsMolBiol, 542:87-104 (2009)).

Preferably, described minicircle dna comprises any those skilled in the art sequence added as required.

In this paper one embodiment, minicircle dna obtains through Site-specific recombinase in intestinal bacteria by traditional plasmid, minicircle dna lacks the bacterial sequences such as resistant maker gene, replication origin, enhance the security (ChenZY etc. in clinical application, GeneTher, 11 (10): 856-864 (2004); ChenZY etc. .Moleculartherapy, 2008,16 (3): 548-556).

" minicircle dna " and " minicircle dna carrier " that adopt herein can exchange.

Preferably, the carrier of described modulated expression's goal gene is the p2 Ф C31 minicircle dna carrier matrix grain of the multiple clone site gained described destination gene expression box being inserted p2 Ф C31 empty plasmid.

As used herein, " p2 Ф C31 empty plasmid " has attB site and attP site, can recombinate in this attB site and attP site under the effect of Φ C31 recombinase.

Particularly, described empty plasmid p2 Ф C31 construction process is with reference to ChenZY etc., MolecularTherapy, 8 (3), 495-500 (2003), ChenZY etc., HumanGeneTherapy, 16 (1), 126-131 (2005) and US Patent No. 7897380B2.

Further preferably, the carrier of described modulated expression's goal gene is the minicircle dna carrier that described p2 Ф C31 minicircle dna carrier matrix grain obtains through Site-specific recombinase.

Site-specific recombinase mainly contains phiC31 (Φ C31) recombinase system, parA recombinase system, Cre recombinase system (Hu Chunsheng etc., biotechnology communication, 22 (1): 104-109 (2011)).In the method for the minicircle dna carrier that the Site-specific recombinase through minicircle dna carrier matrix grain of the present invention obtains, the described minicircle dna carrier matrix grain with specific recombination site comprises the matrix grain with phiC31 specific recombination site, such as p2 Ф C31 minicircle dna carrier matrix grain; Also comprise and be not limited to have the minicircle dna carrier matrix grain of other specific recombination site, such as there is the minicircle dna carrier matrix grain of parA specific recombination site or Cre specific recombination site.

Preferably, the carrier of described modulated expression's goal gene is the pMC.BESPX minicircle dna matrix grain of the multiple clone site gained described destination gene expression box being inserted pMC.BESPX empty plasmid.

As used herein, " pMC.BESPX empty plasmid " has attB site and attP site, can recombinate in this attB site and attP site under the effect of Φ C31 recombinase.

Particularly, described empty plasmid pMC.BESPX complete genome sequence reference ChenZY etc., NatureBiotechnology, 28, (12), 1289-1291 (2010).

Further preferably, the carrier of described modulated expression's goal gene is the minicircle dna carrier that described pMC.BESPX minicircle dna matrix grain obtains through Site-specific recombinase.

Preferably, the carrier of described modulated expression's goal gene is the ZY781.bpA minicircle dna matrix grain of the multiple clone site gained described destination gene expression box being inserted ZY781.bpA plasmid.

Particularly, described ZY781.bpA is obtained recombinant plasmid in PspOMI and the ScaI site of empty plasmid pMC.BESPX insertion SV40bpA fragment, and the gene accession number of described SV40bpA is NC_001669.1; The complete genome sequence reference ChenZY etc. of described empty plasmid pMC.BESPX, NatureBiotechnology, 28, (12), 1289-1291 (2010).

Further preferably, the carrier of described modulated expression's goal gene is that described ZY781.bpA minicircle dna matrix grain produces minicircle dna carrier through Site-specific recombinase.

As in an embodiment of the invention, with LDLRcDNA, and the LDLR promotor containing SRE is template, with the primer that primer LDLR1 (SEQIDNO:7), LDLR2 (SEQIDNO:8), LDLR3 (SEQIDNO:9), LDLR4 (SEQIDNO:10) are over-lap PCR, the fragment obtained after over-lap PCR is connected on ZY781.bpA by SpeI and SalI double digestion, obtain the carrier of described modulated expression's goal gene, called after ZY781.PLDLR.LDLR.bpA.

In the present invention's preferred embodiment, the nucleotides sequence of described LDLRcDNA is classified as the 188 ~ 2770bp of GenBank accession number for nucleotide sequence shown in NM_000527.4.

In the present invention's preferred embodiment, the nucleotides sequence of described SRE is classified as the 1 ~ 187bp of GenBank accession number for nucleotide sequence shown in NM_000527.4.

In the present invention's preferred embodiment, the nucleotides sequence of described SRE is classified as the 4983 ~ 5169bp of GenBank accession number for nucleotide sequence shown in NG_009060.1.

In the present invention's preferred embodiment, the nucleotides sequence of described LDLR promotor is classified as the 3170 ~ 4982bp of GenBank accession number for nucleotide sequence shown in NG_009060.1.

Preferably, as in an embodiment of the invention, the ZY781.PLDLR.LDLR.bpA minicircle dna matrix grain utilizing the embodiment of the present invention to provide has prepared minicircle dna carrier.Recombinate in attB site on the female plasmid vector of described ZY781.bpA minicircle dna and attP site under the effect of Φ C31 recombinase, make micro-ring matrix grain produce the plasmid backbone DNA circle containing attL site and the minicircle dna containing attR site non-reversiblely.Described skeleton DNA contains the restriction enzyme site of I-Sce1 restriction endonuclease.Described skeleton DNA is linear DNA by I-Sce1 endonuclease digestion, is then degraded by the DNA enzymatic in Host Strains, is conducive to the separating-purifying of minicircle dna.

The carrier of modulated expression's goal gene provided by the invention has Liver specific promoters and cholesterol regulation element SRE two kinds of controlling elements simultaneously, wherein, liver is the vitals of cholesterol metabolic, Liver specific promoters is conducive to the tissue specific expression of the carrier of modulated expression's goal gene, improves the targeting of gene therapy; In addition, the cholesterol regulation element SRE that the present invention adopts can make full use of the natural cholesterol feed-back regulatory mechanism of cell, enhance cell active, suitably to the endogenous capacity that the metabolism of cholesterol regulates, when born of the same parents' inner cholesterol level height, cell is by the regulating effect of feedback regulation approach, reduce synthesis and the metaboilic level of cholesterol, cell reduces the metabolic capacity of LDL, and cells show is the downward of LDLR genetic expression; Otherwise when born of the same parents' inner cholesterol level is low, cells show is the rise of LDLR genetic expression.

The carrier of modulated expression's goal gene provided by the invention preferably also has at least one in liver specificity positioning controling element HCR and Kozak sequence 2 kinds of controlling elements.

The promotor of the carrier of modulated expression's goal gene provided by the invention preferably also has for the one in PLDLR promotor and PApoE promotor.

Those skilled in the art can select suitable SRE, HCR, Kozak and promotor combination according to specific needs, and the present invention is preferably the one in following combination:

1) there are PLDLR promotor, cholesterol regulation element SRE two kinds of controlling elements simultaneously;

2) simultaneously containing PLDLR promotor, cholesterol regulation element SRE, Kozak sequence three kinds of controlling elements;

3) simultaneously containing PLDLR promotor, cholesterol regulation element SRE, liver specificity positioning controling element HCR tri-kinds of controlling elements;

4) there are PLDLR promotor, cholesterol regulation element SRE, liver specificity positioning controling element HCR, Kozak sequence four kinds of controlling elements simultaneously.

Second aspect, the invention provides a kind of preparation method of carrier of modulated expression's goal gene, comprising:

As used herein, " antibody " includes but not limited to curative antibody.

Particularly, in described promotor, part bilingual is:

As described herein, the described gene that there is cholesterol regulation element includes but not limited to LDLR gene promoter, HMGCS gene promoter or HMGCR gene promoter.Described cholesterol regulation element is preferably S ü dhofTC etc. at JBiolChem.1987; The cholesterol regulation element reported in 262 (22): 10773-10779.

Concrete, the nucleotides sequence shown in described SEQIDNO:1 is classified as GCCRCC aUGg.

Concrete, the nucleotides sequence shown in described SEQIDNO:2 is classified as GCCGCCRCC aUGg.

Concrete, the nucleotides sequence shown in described SEQIDNO:3 is classified as AGNN aUGn.

Concrete, the nucleotides sequence shown in described SEQIDNO:4 is classified as ANN aUGg.

Concrete, the nucleotides sequence shown in described SEQIDNO:5 is classified as ACC aUGg.

Concrete, the nucleotides sequence shown in described SEQIDNO:6 is classified as GACACC aUGg.

As used herein, " aUG" by underscore mark, identify initiator codon AUG; " R " is preferably A or G; " N " is in one in A, T, C and G.

As used herein, " liver specificity positioning controling element " refers to the nucleotide sequence with DNaseI susceptibility site, and this district is found in the downstream of apo E (ApoE) gene, and liver cell control region can strengthen the expression of liver specific genes.

Further preferably, enhanser is the enhanser on LDLR gene promoter.

In embodiments of the present invention, the GenBank accession number of described LDLRcDNA is NM_000527.4.

In the embodiment of the present invention, the GenBank accession number of the described LDLR promotor containing SRE is NM_000527.4.

The preparation method of the carrier of modulated expression's goal gene provided by the invention is conventional cloning methods in field, those skilled in the art are according to after suitable SRE, HCR, the Kozak of selection of technical scheme provided by the invention and promotor combination, clone's structure recombinant vectors can be carried out according to the situation of concrete carrier and cloning site, the i.e. carrier of modulated expression's goal gene, such as designs suitable primer, adopts the conventional cloning techniques such as over-lap PCR to clone.

The third aspect, the preparation method that the invention provides a kind of carrier of modulated expression's goal gene or a kind of carrier of modulated expression's goal gene is preparing prevention, treatment or is diagnosing the application in the medicine of cholesterol metabolism abnormity disease.

Preferably, described cholesterol metabolism abnormity disease includes but not limited to early mobilize pulse atherosclerosis, coronary heart disease and FH.

Preferably, the carrier of described modulated expression's goal gene as described in the first aspect of the invention.

Preferably, the preparation method of the carrier of described modulated expression's goal gene as described in respect of the second aspect of the invention.

Fourth aspect, the invention provides a kind of method adopting the vehicle treatment cholesterol metabolism abnormity disease of modulated expression's goal gene, comprising:

S01) carrier of modulated expression's goal gene is prepared;

D) first pass through in-vitro transfection technology by the carrier transfection immune effector cell of described modulated expression's goal gene, then described transfection is had in the immune effector cell of the carrier of modulated expression's goal gene defeated time patient body and implement treatment

Embodiment

The following stated is the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention; can also make some improvements and modifications, these improvements and modifications are also considered as protection scope of the present invention.

Method used in following embodiment is ordinary method if no special instructions, and concrete steps can be see: " Molec μ larCloning:ALaboratoryManual " (Sambrook, J., Russell, DavidW., Molec μ larCloning:ALaboratoryManual, 3 ^rdedition, 2001, NY, ColdSpringHarbor).

The bacterial strain E.coliZYCY10P3S2T that the embodiment of the present invention uses, liver cell HepG2 cell are commercial goods, and the reagent used is commercial goods; The primer and DNA sequence dna synthesize by Shanghai Invitrogen company.

The configuration of the TB substratum that the embodiment of the present invention uses, LB substratum is as follows:

TB substratum: TB gravy powder (Terrificbrothpowder) 47.6g/L; Glycerine 4g/L; PH7.0

LB substratum: tryptone (Tryptone) 10g/L; Yeast powder (Yeastextract) 5g/L; NaCl10g/L; PH7.0

Primer that the embodiment of the present invention adopts is as shown in table 1:

Table 1. embodiment of the present invention the primer

In table 1, the sequence of single underscore mark is the restriction enzyme site of restriction enzyme; Double underline is Kozak sequence; The joint sequence of point type underscore mark over-lap PCR.

In table 1, remarks column SpeI, SalI and SpeI represent the title of the restriction enzyme site contained in corresponding primer.

The over-lap PCR amplification LDLR expression casette that the present invention adopts, the pcr template adopted is the LDLR gene (genebank accession number is NM_000527.4) of tape starting.

Table 1 overlapping PCR primers illustrates:

As shown in table 1, the primer of the over-lap PCR of employing comprises LDLR1-LDLR8, and wherein, the step of over-lap PCR is:

A) primer LDLR1 is adopted, LDLR2, LDLR3 and LDLR4 amplification PLDLR-LDLR gene, step is: LDLR1 (SEQIDNO:7) and LDLR2 (SEQIDNO:8) is first group of primer, LDLR3 (SEQIDNO:9) and LDLR4 (SEQIDNO:10) is second group of primer, during over-lap PCR, first group of primer and second group of primer is first adopted to carry out PCR respectively, obtain first group of DNA fragmentation product and second group of DNA fragmentation product, after products therefrom mixing, be that the 3rd group of primer and mixed product carry out PCR again with LDLR1 (SEQIDNO:7) and LDLR4 (SEQIDNO:10), obtain overlapping PCR products PLDLR-LDLR gene, the overlapping PCR products PLDLR-LDLR gene of gained is for subsequent use, and subsequent cloning steps is originally see inventive embodiments 1 step (1),

Italic in primer LDLR2 (SEQIDNO:8) and primer LDLR3 (SEQIDNO:9) and the sequence of overstriking are the lap sequence of first group of DNA fragmentation product and second group of DNA fragmentation product;

B) primer LDLR1 is adopted, LDLR2, LDLR5 and LDLR4 amplification PLDLR-LDLR gene, step is: LDLR1 (SEQIDNO:7) and LDLR2 (SEQIDNO:8) is first group of primer, LDLR5 (SEQIDNO:11) and LDLR4 (SEQIDNO:10) is second group of primer, during over-lap PCR, first group of primer and second group of primer is first adopted to carry out PCR respectively, obtain first group of DNA fragmentation product and second group of DNA fragmentation product, after products therefrom mixing, be that the 3rd group of primer and mixed product carry out PCR again with LDLR1 (SEQIDNO:7) and LDLR4 (SEQIDNO:10), obtain overlapping PCR products PLDLR.Kozak.LDLR gene, the overlapping PCR products PLDLR.Kozak.LDLR gene of gained is for subsequent use, and subsequent step is originally see inventive embodiments 1 step (1),

Point type underscore identification division in primer LDLR2 (SEQIDNO:8) and primer LDLR5 (SEQIDNO:11) is the joint sequence of over-lap PCR, is the lap sequence of first group of DNA fragmentation product and second group of DNA fragmentation product;

C) primer LDLR1 is adopted, LDLR6, LDLR7 and LDLR4 amplification PLDLR-LDLR gene, step is: LDLR1 (SEQIDNO:7) and LDLR6 (SEQIDNO:12) is first group of primer, LDLR7 (SEQIDNO:13) and LDLR4 (SEQIDNO:10) is second group of primer, during over-lap PCR, first group of primer and second group of primer is first adopted to carry out PCR respectively, obtain first group of DNA fragmentation product and second group of DNA fragmentation product, after products therefrom mixing, be that the 3rd group of primer and mixed product carry out PCR again with LDLR1 (SEQIDNO:7) and LDLR4 (SEQIDNO:10), obtain overlapping PCR products PLDLR.UTR.LDLR gene, the overlapping PCR products PLDLR.UTR.LDLR gene of gained is for subsequent use, and subsequent step is originally see inventive embodiments 1 step (1),

Point type underscore identification division in primer LDLR6 (SEQIDNO:12) and primer LDLR7 (SEQIDNO:13) is the joint sequence of over-lap PCR, i.e. the lap sequence of first group of DNA fragmentation product and second group of DNA fragmentation product.

In table 1, remarks column PLDLR represents that corresponding primer is the primer of LDLR promotor.

In table 1, remarks column UTR represents that corresponding primer is the primer of UTR.

In table 1, remarks column qPCR represents that corresponding primer is the primer for relative quantification PCR (RT-qPCR), comprises LDLR-F (SEQIDNO:17) and LDLR-R (SEQIDNO:18).

Relative quantification PCR (RT-qPCR) internal reference that the embodiment of the present invention adopts is Actin muscle (Actin), and internal reference primer comprises Actin-F (SEQIDNO:19) and Actin-R (SEQIDNO:20).

The relative quantification PCR QiagenQuantiFastSYBRGreenPCRkit test kit that the embodiment of the present invention adopts completes on Rochesystem.Program comprises for 94 DEG C of 5min, 94 DEG C of for20s, 68 DEG C of for20s (40 circulations).

Embodiment one

Build the parental plasmid of preparing minicircle dna, comprise the following steps:

1) with LDLRcDNA (GenBank accession number is for 188 ~ 2770bp of nucleotide sequence shown in NM_000527.4), (nucleotides sequence of LDLR promotor is classified as the 3170 ~ 4982bp of GenBank accession number for nucleotide sequence shown in NG_009060.1 to the LDLR promoter region containing SRE, the nucleotides sequence of SRE is classified as the 4983 ~ 5169bp of GenBank accession number for nucleotide sequence shown in NG_009060.1) be template, respectively with the combination of primers 1 (LDLR1 shown in table 1, LDLR2, LDLR3, LDLR4), combination of primers 2 (LDLR1, LDLR2, LDLR5, LDLR4), combination of primers 3 (LDLR1, LDLR6, LDLR7, LDLR4) for primer carries out over-lap PCR, the concrete steps of over-lap PCR are see " explanation of table 1 overlapping PCR primers " described in the embodiment of the present invention, the object fragment obtained is by after SpeI and SalI double digestion, DNAT4 ligase enzyme is adopted to be connected between SpeI and the SalI site multiple clone site of ZY781.bpA, the recombinant plasmid obtained is called after ZY781.PLDLR.LDLR.bpA after order-checking qualification, ZY781.PLDLR.Kozak.LDLR.bpA, ZY781.PLDLR.UTR.LDLR.bpA.

Wherein, UTR sequence is CAAGTTTGTACAAAAAAGCAGGCT (SEQIDNO:21), and UTR is non-coding region, is used as contrast.

2) be that (pMC.BESPXSpeI and EcoRI inserts HCR and ApoE promotor to template with ZY781.ApoE.bpA, ScaI and PspOMI inserts SV40bpA, described HCR is enhanser, the nucleotides sequence of described enhanser is classified as the 5 ~ 325bp of GenBank accession number for nucleotide sequence shown in U32510.1), the HCR fragment that LDLR-F (SEQIDNO:11) and LDLR-R (SEQIDNO:12) obtains for primer PCR, cut by SpeI enzyme and be connected respectively to ZY781.PLDLR.LDLR.bpA, on ZY781.PLDLR.Kozak.LDLR.bpA, the plasmid obtained is difference called after ZY781.Enhancer.PLDLR.LDLR.bpA after order-checking qualification, ZY781.Enhancer.PLDLR.Kozak.LDLR.bpA.

3) respectively with ZY781.PLDLR.LDLR.bpA, ZY781.PLDLR.Kozak.LDLR.bpA is template, the fragment that LDLR-8 (SEQIDNO:8) and LDLR-4 (SEQIDNO:4) obtains for primer PCR, ZY781.ApoE.bpA is connected to by HindIII and SalI double digestion, the plasmid obtained is difference called after ZY781.Enhancer.ApoE.LDLR.bpA after order-checking qualification, ZY781.Enhancer.ApoE.Kozak.LDLR.bpA.

Result:

A. adopted ZY781.bpA empty carrier plasmid map respectively as shown in Figure 1; The plasmid map of the constructed parental plasmid for the preparation of minicircle dna is respectively as shown in Fig. 2 ~ 8.

B. check order to the constructed parental plasmid for the preparation of minicircle dna, sequencing result shows that the embodiment of the present invention 1 obtains the parental plasmid of minicircle dna.

Embodiment two

Prepare minicircle dna, comprise the following steps:

1) parental plasmid ZY781.PLDLR.LDLR.bpA, ZY781.PLDLR.Kozak.LDLR.bpA, ZY781.PLDLR.UTR.LDLR.bpA, ZY781.Enhancer.PLDLR.LDLR.bpA, ZY781.Enhancer.PLDLR.Kozak.LDLR.bpA, ZY781.Enhancer.ApoE.LDLR.bpA and ZY781.Enhancer.ApoE.Kozak.LDLR.bpA of embodiment 1 being prepared be transformation of E. coli genetically engineered E.coli ZYCY10P3S2T respectively, obtain the E.coliZYCY10P3S2T containing each parental plasmid, be expressed as:

E.coliZYCY10P3S2T(ZY781.PLDLR.LDLR.bpA)、

E.coliZYCY10P3S2T(ZY781.PLDLR.Kozak.LDLR.bpA)、

E.coliZYCY10P3S2T(ZY781.PLDLR.UTR.LDLR.bpA)、

E.coliZYCY10P3S2T(ZY781.Enhancer.PLDLR.LDLR.bpA)、

E.coliZYCY10P3S2T(ZY781.Enhancer.PLDLR.Kozak.LDLR.bpA)、

E.coliZYCY10P3S2T(ZY781.Enhancer.ApoE.LDLR.bpA)、

E.coliZYCY10P3S2T(ZY781.Enhancer.ApoE.Kozak.LDLR.bpA)；

2) E.coliZYCY10P3S2T obtained containing each parental plasmid inoculates respectively:

In 12mL microbial culture pipe, load 5mLTB substratum (kantlex (Kanamycin) final concentration 50 μ g/ml), after inoculation 37 DEG C, 250rpm cultivates 8h.

3) in 2L Erlenmeyer flask, load 400mLTB substratum (Kanamycin final concentration 50 μ g/ml), inoculate by the inoculum size of 1%, then 37 DEG C, 250rpm cultivates 12-16h.

4) induced liquid is prepared: the NaOH (1mol/L) getting the LB substratum of 400mL, 16mL; The pectinose (filtration sterilization, massfraction 20wt%) of 400 μ L.

5) induced liquid that step (4) configures is joined in the nutrient solution in step (3) and induce 5-8h.

6) collected by centrifugation thalline, extracts test kit in a large number with QIAGENPlasmidMegaKit (25) (article No. 12183) plasmid and extracts plasmid.

7) enzyme cuts the standby minicircle dna of system of identification, result display parental plasmid ZY781.PLDLR.LDLR.bpA, ZY781.PLDLR.Kozak.LDLR.bpA, ZY781.PLDLR.UTR.LDLR.bpA, ZY781.Enhancer.PLDLR.LDLR.bpA, ZY781.Enhancer.PLDLR.Kozak.LDLR.bpA, ZY781.Enhancer.ApoE.LDLR.bpA and ZY781.Enhancer.ApoE.Kozak.LDLR.bpA all obtains purer minicircle dna, called after MC.PLDLR.LDLR.bpA respectively, MC.PLDLR.Kozak.LDLR.bpA, MC.PLDLR.UTR.LDLR.bpA, MC.Enhancer.PLDLR.LDLR.bpA, MC.Enhancer.PLDLR.Kozak.LDLR.bpA, MC.Enhancer.ApoE.LDLR.bpA and MC.Enhancer.ApoE.Kozak.LDLR.bpAA.

Embodiment three

Relative quantification PCR (RT-qPCR) detects from mRNA level in-site LDLR expression conditions, comprises the following steps:

A.HepG2 cell cultivates 24h with every hole 3*104 bed board 24 orifice plate.

B.Qiagensuperfect transfection reagent box (article No. 301307), different minicircle dna prepared by embodiment 2 and control plasmid ZY781 transfection HepG 2 cell respectively.

C. often kind of plasmid transfection HepG2 cell arranges three groups, control group (adopting Controlmedium), cholesterol group (adopting Sterolmedium) and statin group (adopting Statinmedium) is respectively according to the difference of cell culture medium, wherein, the cell culture medium of control group is DMEM; The cell culture medium of cholesterol group is in DMEM cell culture medium, add the cholesterol (Sigma that final concentration is 10 μ g/mL, article No. C3045-5G) and final concentration be the 25-HYDROXY CHOLESTEROL (Sigma, article No. H1015-10MG) of 0.5 μ g/mL; The cell culture medium of statin group adds the statins (Sigma, article No. S6196-5MG) that final concentration is 0.75 μ g/mL in DMEM cell culture medium, each group cell cultures 36h (cell culture condition is normal condition).

D. OmegaTotalRNA test kit (article No. R6834-02) is adopted to extract RNA.E. primeScriptRTreagentKitwithgDNAEraser Reverse Transcription box (article No. DRR047A) is adopted to carry out reverse transcription.

F.RTqPCR take Actin as internal reference, and internal reference primer is Actin-F and Actin-R (see table 1).Primer LDLR-F and LDLR-R (see table 1) is specifically for LDLR gene.Relative quantification PCR QiagenQuantiFastSYBRGreenPCRkit test kit (article No. 204054) completes on Rochesystem.Program comprises for 94 DEG C of 5min, 94 DEG C of for20s, 68 DEG C of for20s (40 circulations).

Result:

A. as shown in Fig. 9 ~ 16, Fig. 9 ~ 16 are respectively ZY781.bpA empty carrier, minicircle dna MC.PLDLR.LDLR.bpA, MC.PLDLR.Kozak.LDLR.bpA, MC.PLDLR.UTR.LDLR.bpA, MC.Enhancer.PLDLR.LDLR.bpA, MC.Enhancer.PLDLR.Kozak.LDLR.bpA, after MC.Enhancer.ApoE.LDLR.bpA and MC.Enhancer.ApoE.Kozak.LDLR.bpAA transfection HepG 2 cell, RT-PCR result is carried out to the mRNA of LDLR gene in each group of cell, in Fig. 9 ~ Figure 16, each figure center pillar-1 (in each figure 781, the differences such as PLDLR, as in Fig. 9, post-1 781-1 represents) be control group, post-2 is cholesterol group, for statin group, (P is confidence level to post-3, for repeating the result of 5 parallel laboratory tests, " * " represents P<0.05, " * * " represents P<0.01)

As shown in Figure 9, the RT-PCR result namely after ZY781.bpA empty carrier transfectional cell, in Fig. 9, the LDLR gene expression dose of each group cell is all not high; In addition, in fig .9, although the LDLR gene expression dose of the cholesterol group of Fig. 9 is lower, relative to control group, cholesterol group difference is not obvious; In addition, and relative to control group, the LDLR gene expression dose difference of the statin group of Fig. 9 is also little, illustrates that the feedback regulation of cell to cholesterol levels not containing the carrier of modulated expression's goal gene provided by the invention is not obvious.

And in Figure 10 ~ 11 and Figure 13 ~ 16, each figure control group, cholesterol group and statin group all demonstrate following similar rule, this rule shows as: relative to respective control group, and cholesterol group all demonstrates LDLR down regulation of gene expression in various degree; And relative to respective cholesterol group and control group, the LDLR genetic expression that statin group all demonstrates in various degree is raised; Especially performance is obvious in Figure 10,11,13,14 for described rule; This illustrates in the cell experiment level that provides at the present embodiment 3, minicircle dna MC.PLDLR.LDLR.bpA, MC.PLDLR.Kozak.LDLR.bpA, MC.Enhancer.PLDLR.LDLR.bpA and MC.Enhancer.PLDLR.Kozak.LDLR.bpA provided by the invention, stronger than the regulating power of minicircle dna MC.Enhancer.ApoE.LDLR.bpA and MC.Enhancer.ApoE.Kozak.LDLR.bpAA.Namely in cell experiment level, the carrier of modulated expression's goal gene provided by the invention

There are PLDLR promotor, cholesterol regulation element SRE two kinds of controlling elements simultaneously;

Or simultaneously containing PLDLR promotor, cholesterol regulation element SRE, Kozak sequence three kinds of controlling elements;

Or simultaneously containing PLDLR promotor, cholesterol regulation element SRE, liver specificity positioning controling element HCR tri-kinds of controlling elements

Or when there is PLDLR promotor, cholesterol regulation element SRE, liver specificity positioning controling element HCR, Kozak sequence four kinds of controlling elements simultaneously, the carrier of modulated expression's goal gene provided by the invention is more obvious to the regulating effect of goal gene, and excellent array mode is more selected in 4 kinds of above-mentioned controlling element combinations.

In addition, Figure 12 display: relative to the control group of Figure 12, although cholesterol group demonstrates LDLR down regulation of gene expression, relative to the cholesterol group of Figure 12, the statin group of Figure 12 does not but demonstrate the rise of LDLR genetic expression, this illustrates does not have micro-ring MC.PLDLR.UTR.LDLR.bpA of cholesterol regulation element SRE can not play the effect regulating and express LDLR gene, this illustrates that the carrier of modulated expression's goal gene with cholesterol regulation element SRE is (in the present embodiment from the negative, described goal gene is LDLR gene) regulate the expression of LDLR gene very crucial for cell on one's own initiative according to born of the same parents' inner cholesterol level, the cholesterol regulation element SRE that carrier provided by the invention is transcribed imparts the endogenous capacity of cell regulate and control cholesterol metabolic by cholesterol feedback regulation path, when born of the same parents' inner cholesterol level height (the cholesterol group of the corresponding embodiment of the present invention), cell is by the regulating effect of feedback regulation approach, the synthesis of cholesterol and metaboilic level reduce, cell reduces the metabolic capacity of LDL, namely the downward of LDLR genetic expression is shown as, otherwise, when born of the same parents' inner cholesterol level is low (the statin group of the corresponding embodiment of the present invention), show as the rise of LDLR genetic expression.

Secondly, relative to Fig. 9, Figure 10 ~ 16 all in cell LDLR genetic expression all have rise in various degree, this is due to relative to unloaded (Fig. 9), except cell background is expressed, also has the expression of exogenous carrier, therefore Figure 10 ~ 16 are higher than the expression level of Fig. 9 generally, in addition, effect is started to goal gene stronger, goal gene expression level is generally higher, such as, adopt the ability of PApoE promoter transcription goal gene relative to stronger PLDLR promotor, the control group of Figure 10 ~ 16 is carried out contrast can find: relative to adopting minicircle dna MC.Enhancer.ApoE.LDLR.bpA and MC.Enhancer.ApoE.Kozak.LDLR.bpAA of PApoE promotor than the minicircle dna MC.PLDLR.LDLR.bpA adopting PLDLR promotor, MC.PLDLR.Kozak.LDLR.bpA, MC.Enhancer.PLDLR.LDLR.bpA and MC.Enhancer.PLDLR.Kozak.LDLR.bpA more can raise the expression level of the LDLR gene of cell.

Embodiment four

Detect to the expression amount of LDLR gene and to the metabolic capacity of cell LDL from protein level, comprise the following steps:

A.HepG2 cell cultivates 24h with every hole 0.5*104 bed board 96 orifice plate.

B.Qiagensuperfect transfection reagent box is by different minicircle dna, control plasmid ZY781 transfection HepG 2 cell.

C. transfection HepG 2 cell grows 36h respectively under Controlmedium, Sterolmedium, Statinmedium environment, and described Controlmedium, Sterolmedium, Statinmedium configuration is see embodiment three; In addition, containing comprising people source LDL in often kind of substratum, the institute source LDL that lets others have a look at is connected with DylightTM549 fluorescent probe, can be used to the ability detecting intracellular LDL metabolism.

D. extract substratum out, then each hole adds the LDL-DylightTM549workingsolution of 100ul, and 37 ° are continued incubate3-4h; Described LDL-DylightTM549workingsolution is from LDLuptakeCell-basedassay test kit (Cayman, article No. 10011125-1); The two anti-distribution situations being used for verifying LDLr that the monoclonal antibody (primary antibodie) that this test kit utilizes LDLr special is connected with DylightTM488.

E. LDL-DylightTM549workingsolution is extracted out, change fresh substratum or PBS.Utilize fluorescent microscope, 540nm detects the degree of LDLuptake.

F. extract substratum or PBS out, clean once with TBS.Then add the Cell-basedassayfixativesolution of 100ul/well, incubate10min, fix for cell.3 times are cleaned, each 5min with TBST.

G. 100ul/wellCell-basedassayblockingsolution is added, incubate30min.

H. the primary antibodie after dilution is added, 100ul/well, incubate1h.3 times are cleaned, each 5min with TBST.

I. after lucifuge adds dilution two resist, 100ul/well, incubate1h.3 times are cleaned, each 5min with TBST.

J. fluorescent microscope lucifuge is utilized to detect the distribution situation of 485nmLDLr.

K.ImageJ calculates the relative intensity of LDL metaboilic level.

2, result:

A. the fluoroscopic examination result of step (1-j) as shown in figure 17.Figure 17 comprises figure a ~ figure h, magnification 12.6 times.

After figure a ~ figure h is respectively ZY781.bpA empty carrier, minicircle dna MC.PLDLR.LDLR.bpA, MC.PLDLR.Kozak.LDLR.bpA, MC.PLDLR.UTR.LDLR.bpA, MC.Enhancer.PLDLR.LDLR.bpA, MC.Enhancer.PLDLR.Kozak.LDLR.bpA, MC.Enhancer.ApoE.LDLR.bpA and MC.Enhancer.ApoE.Kozak.LDLR.bpAA transfection HepG 2 cell, adopt fluorescence microscope respectively to organize the result of LDLr distribution situation in cell, namely respectively organize LDLr bis-anti-probe DylightTM488 fluorescence intensity in cell; In figure a ~ figure h, Control, sterol and statin represent control group, cholesterol group and statin group respectively, the HepG2 cell grown under Controlmedium, Sterolmedium, Statinmedium environment in the corresponding step (c) of described control group, cholesterol group and statin group difference.

B. the fluorescence intensity relative value respectively organized of the fluoroscopic examination result of step (1-j) is as shown in Figure 18 ~ Figure 25.Figure 18 ~ Figure 25 is respectively ZY781.bpA empty carrier, minicircle dna MC.PLDLR.LDLR.bpA, MC.PLDLR.Kozak.LDLR.bpA, MC.PLDLR.UTR.LDLR.bpA, MC.Enhancer.PLDLR.LDLR.bpA, MC.Enhancer.PLDLR.Kozak.LDLR.bpA, after MC.Enhancer.ApoE.LDLR.bpA and MC.Enhancer.ApoE.Kozak.LDLR.bpAA transfection HepG 2 cell, the relative value of LDLr bis-anti-probe DylightTM488 fluorescence intensity in each group of cell, the power of fluorescence intensity has reacted the power of cell LDLr expression, the i.e. height of LDLr gene expression dose, in Figure 18 ~ Figure 25, each figure center pillar-1 is control group, and post-2 is cholesterol group, post-3 is statin group, and (P is confidence level, for repeating the result of 5 parallel laboratory tests, " * " represents P<0.05, and " * * " represents P<0.01, ),

C. the fluoroscopic examination result of step (1-k) as shown in figure 26, and the magnification of Figure 26 is 12.6 times.Figure 26 is respectively ZY781.bpA empty carrier, minicircle dna MC.PLDLR.LDLR.bpA, MC.PLDLR.Kozak.LDLR.bpA, MC.PLDLR.UTR.LDLR.bpA, MC.Enhancer.PLDLR.LDLR.bpA, MC.Enhancer.PLDLR.Kozak.LDLR.bpA, after MC.Enhancer.ApoE.LDLR.bpA and MC.Enhancer.ApoE.Kozak.LDLR.bpAA transfection HepG 2 cell, the relative value of the DylightTM549 fluorescent probe fluorescence intensity that LDL connects in each group of cell, in the figure a ~ figure h of Figure 26, Control, sterol and statin represents control group respectively, cholesterol group and statin group, described control group, cholesterol group and statin group be the middle Controlmedium of corresponding step (c) respectively, Sterolmedium, the HepG2 cell grown under Statinmedium environment,

D. the fluorescence intensity relative value respectively organized of the fluoroscopic examination result of step (1-k) is as shown in Figure 27 ~ Figure 34.After Figure 27 ~ Figure 34 is respectively ZY781.bpA empty carrier, minicircle dna MC.PLDLR.LDLR.bpA, MC.PLDLR.Kozak.LDLR.bpA, MC.PLDLR.UTR.LDLR.bpA, MC.Enhancer.PLDLR.LDLR.bpA, MC.Enhancer.PLDLR.Kozak.LDLR.bpA, MC.Enhancer.ApoE.LDLR.bpA and MC.Enhancer.ApoE.Kozak.LDLR.bpAA transfection HepG 2 cell, the DylightTM549 fluorescent probe fluorescence intensity that in each group of cell, LDL connects, the power of fluorescence intensity has reacted the power of cell LDL metabolic capacity.In Figure 27 ~ Figure 34, each figure center pillar-1 is control group, and post-2 is cholesterol group, post-3 is statin group, and (P is confidence level, for repeating the result of 5 parallel laboratory tests, " * " represents P<0.05, and " * * " represents P<0.01; ).

The above results all demonstrates the conclusion similar with embodiment 3, namely relative to ZY781.bpA empty carrier and minicircle dna MC.PLDLR.UTR.LDLR.bpA, minicircle dna MC.PLDLR.LDLR.bpA provided by the invention, MC.PLDLR.Kozak.LDLR.bpA, MC.Enhancer.PLDLR.LDLR.bpA, MC.Enhancer.PLDLR.Kozak.LDLR.bpA, MC.Enhancer.ApoE.LDLR.bpA and MC.Enhancer.ApoE.Kozak.LDLR.bpAA all imparts the ability of cell from main regulation LDLR gene expression dose, when born of the same parents' inner cholesterol level is high, LDLR down regulation of gene expression, LDL metaboilic level reduces, when born of the same parents' inner cholesterol level is low, LDLR genetic expression is raised, and LDL metaboilic level improves, and in the cell experiment level provided at the present embodiment 4, minicircle dna MC.PLDLR.LDLR.bpA, MC.PLDLR.Kozak.LDLR.bpA, MC.Enhancer.PLDLR.LDLR.bpA and MC.Enhancer.PLDLR.Kozak.LDLR.bpA provided by the invention, stronger than the regulating power of minicircle dna MC.Enhancer.ApoE.LDLR.bpA and MC.Enhancer.ApoE.Kozak.LDLR.bpAA, this ability that may start goal gene with PApoE promotor is relative to relevant more by force PLDLR promotor, in cell experiment level, relative to the cell adopting PLDLR promotor, adopt PApoE promotor cell, no matter be control group, cholesterol group or statin group, LDLR gene expression dose is on the whole in relatively high level.

Subordinate list 2 and table 3, the plasmid information that table 2 adopts for the embodiment of the present invention, the information of the Host Strains that table 3 adopts for the embodiment of the present invention:

Table 2

Table 3

The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, all any amendments done within the spirit and principles in the present invention, equivalent replacement and improvement etc., all should be included within protection scope of the present invention.

Claims

1. the carrier of modulated expression's goal gene, it is characterized in that, the carrier of described modulated expression's goal gene comprises destination gene expression box, described destination gene expression box comprises: Liver specific promoters, cholesterol regulation element and goal gene, and described cholesterol regulation element, Liver specific promoters are operably connected with goal gene.

2. the carrier of modulated expression's goal gene as claimed in claim 1, it is characterized in that, described goal gene is the encoding gene of protein or the encoding gene of microRNA.

3. the carrier of modulated expression's goal gene as claimed in claim 1, it is characterized in that, described Liver specific promoters is PCK promotor, α-I-antitrypsin promotor, thyroid hormone binding globulin promotor, α-fetoprotein promotor, alcohol dehydrogenase promoter, IGF-II promotor, Factor IX promotor, HBV basal core protein promoter, s2 protein promoter before HBV, throxine-binding globulin promotor, the hybrid promoter of HCR-Ap0CII, HCR-hAAT hybrid promoter, the AAT promotor be combined with the enhancer element of mouse albumin gene, apo E promotor, low-density lipoprotein promotor, pyruvate kinase promoter, lecithin cholesterol acyltransferase promotor, Apolipoprotein H promotor, siderophilin promotor, transthyretin promotor, alpha fibre proteinogen and β _ fibrinogenic promotor, α-I-chymotrypsin inhibitor promotor, α-2-HS glycoprotein promotor, haptoglobin promotor, ceruloplasmin promotor, profibr(in)olysin promotor, complement proteins promotor, Complement C_3 activates the promotor of son, hemopexin promotor and α-I-acid glycoprotein promotor.

4. the carrier of modulated expression's goal gene as claimed in claim 1, it is characterized in that, described cholesterol regulation element is the cis-acting elements in the promotor of gene, and described gene to comprise in cholesterol biosynthesis approach or the encoding gene of enzyme or the encoding gene of receptor protein in fatty acid synthesis pathway.

5. the carrier of modulated expression's goal gene as claimed in claim 1, it is characterized in that, the carrier of described modulated expression's goal gene also comprises at least one in Kozak sequence and liver specificity positioning controling element.

6. the carrier of modulated expression's goal gene as claimed in claim 1, is characterized in that, the carrier of described modulated expression's goal gene is not containing the skeleton DNA sequence dna of standard plasmid.

7. the carrier of modulated expression's goal gene as claimed in claim 1, it is characterized in that, the carrier of described modulated expression's goal gene is minicircle dna.

8. a preparation method for the carrier of modulated expression's goal gene, is characterized in that, comprising:

9. the preparation method of the carrier of modulated expression's goal gene as claimed in claim 8, it is characterized in that, described carrier is virus vector, prokaryotic plasrnid carrier or eucaryon plasmid carrier.

10. the preparation method of the carrier of modulated expression's goal gene as claimed in claim 8, it is characterized in that, described carrier is minicircle dna empty plasmid, the carrier of described modulated expression's goal gene is minicircle dna matrix grain, described minicircle dna matrix grain obtains not containing the carrier of modulated expression's goal gene of the skeleton DNA sequence dna of standard plasmid after Site-specific recombinase, gained does not comprise destination gene expression box containing the carrier of modulated expression's goal gene of the skeleton DNA sequence dna of standard plasmid, described destination gene expression box comprises: Liver specific promoters, cholesterol regulation element and goal gene, described cholesterol regulation element, Liver specific promoters is operably connected with goal gene.

The preparation method of the carrier of 11. modulated expression's goal gene as claimed in claim 10, is characterized in that, the described carrier not containing modulated expression's goal gene of the skeleton DNA sequence dna of standard plasmid is minicircle dna.

The preparation method of the carrier of 12. modulated expression's goal gene as claimed in claim 8, is characterized in that, described goal gene is the encoding gene of protein or the encoding gene of microRNA.

The preparation method of the carrier of 13. modulated expression's goal gene as claimed in claim 8, it is characterized in that, described Liver specific promoters is PCK promotor, α-I-antitrypsin promotor, thyroid hormone binding globulin promotor, α-fetoprotein promotor, alcohol dehydrogenase promoter, IGF-II promotor, Factor IX promotor, HBV basal core protein promoter, s2 protein promoter before HBV, throxine-binding globulin promotor, the hybrid promoter of HCR-Ap0CII, HCR-hAAT hybrid promoter, the AAT promotor be combined with the enhancer element of mouse albumin gene, apo E promotor, low-density lipoprotein promotor, pyruvate kinase promoter, lecithin cholesterol acyltransferase promotor, Apolipoprotein H promotor, siderophilin promotor, transthyretin promotor, alpha fibre proteinogen and β _ fibrinogenic promotor, α-I-chymotrypsin inhibitor promotor, α-2-HS glycoprotein promotor, haptoglobin promotor, ceruloplasmin promotor, profibr(in)olysin promotor, complement proteins promotor, Complement C_3 activates the promotor of son, hemopexin promotor and α-I-acid glycoprotein promotor.

The preparation method of the carrier of 14. modulated expression's goal gene as claimed in claim 8, it is characterized in that, described cholesterol regulation element is the cis-acting elements in the promotor of gene, and described gene to comprise in cholesterol biosynthesis approach or the encoding gene of enzyme or the encoding gene of receptor protein in fatty acid synthesis pathway.

The preparation method of the carrier of 15. modulated expression's goal gene as claimed in claim 8, is characterized in that, the carrier of described modulated expression's goal gene also comprises at least one in Kozak sequence and liver specificity positioning controling element.

The preparation method of the carrier of 16. modulated expression's goal gene as claimed in claim 8, is characterized in that, the carrier of described modulated expression's goal gene is not containing the skeleton DNA sequence dna of standard plasmid.

The carrier of 17. 1 kinds of modulated expression's goal gene is being prepared prevention, treatment or is being diagnosed the application in the medicine of cholesterol metabolism abnormity disease.

18. 1 kinds of methods adopting the vehicle treatment cholesterol metabolism abnormity disease of modulated expression's goal gene, is characterized in that, comprise the steps:

S01) carrier of modulated expression's goal gene is prepared;