CN110229849A - Spatiotemporal database type uPA gene expression non-virus carrier and its preparation method and application - Google Patents

Abstract

The present invention provides a kind of spatiotemporal database type uPA gene expression non-virus carriers, the spatiotemporal database type uPA gene expression non-virus carrier includes spatiotemporal database uPA expression casette, the spatiotemporal database uPA expression casette includes: Liver specific promoters, uPA mutated gene and unstable structure domain, and the uPA mutated gene is the uPA gene for inserting endoplasmic reticulum retention signal gene.The regulator control system of the spatiotemporal database type uPA gene expression non-virus carrier is tighter, simpler；UPA gene can be controlled to be expressed in specific time particular space, make other organs from the risk of toxicity.

Description

Spatiotemporal database type uPA gene expression non-virus carrier and its preparation method and application

Technical field

The present invention relates to genetic engineering field more particularly to a kind of spatiotemporal database type uPA gene expression non-virus carrier and Preparation method and application.

Background technique

Hepatitis B (HBV) infection has stringent species specificity, and it is long only to infect a small number of spirits such as people, gorilla and tree shrew Class animal's liver.Common experimental animal model such as mouse, rat etc. does not infect HBV.Due to lacking reliable and stable HBV sense Animal model is contaminated, the research of HBV infection/pathogenic mechanism and viral resistant strategies is constrained.Establish humanization chimera liver mouse Model is effective way the bottleneck that overcoming.Liver has very strong regeneration potential.The base of humanization chimera hepatic model Present principles are: by expressing hepatotoxicity wind agitation gene in host's liver, human-induced host hepatocytes are dead, stimulate the source of people of transplanting Hepatocyte growth realizes the substitution of source of people liver cell, to form the chimera liver of humanization.In fact, humanization is chimeric The key core of body liver is that the suitable hepatotoxicity wind agitation expression vector of design.According to the difference of hepatotoxicity wind agitation gene, at present Main development 3 kinds of people-mouse Chimera hepatic model in the world.The first is Alb (albumin, albumin)-uPA (urokinase plasminogen activator, plasma urokinase-type plasminogen activator)-SCID (severe combined Immune deficiency, severe combined immunodeficiency) transgenic mice, abbreviation uPA transgenic mice.Second is FRG small Tri- base of mouse, i.e. Fah (fumaryl acetoacetate hydrolase, fumaroyl acetoacetate hydrolase)/Rag2/Il2rg Because knocking out mouse.The third is AFC8 mouse, i.e. FKBP (FK506 binding domain, FK506 binding domain)-Caspase 8 (caspase -8) fusion protein transgenic mice.In three, there is uPA transgenic mice both remaining to be difficult to the height reached Horizontal source of people liver cell substitution rate (up to 90% or more).

UPA, i.e. urokinase type plasminogen activator, belong to serine protein hydrolase family, and uPA is catalyzed plasminogen It is converted into fibrinolysin, starts fibrin and extracellular matrix protein dissolves cascade reaction.In general, uPA mainly physiology, The processes such as differentiation, migration, tissue reconstruction, extracellular matrix degradation, the tumor-infiltrated and transfer of cell are participated under pathological conditions.It is existing In the building for having uPA transgenic mice, due to not having controlled overexpression uPA that can cause serious bleeding and organ damage, make , breeding difficulty high at the newborn mice death rate.UPA can cause hepatocyte death and promote liver regeneration, in its toxic side effect Under the premise of can be effectively controlled, uPA gene is still considered as being optimal for constructing the hepatotoxicity wind agitation of chimera hepatic model Gene.

Currently, the improved method for uPA gene regulation expression system is the Tet-on tetracycline based on AAV viral vectors Switch the uPA expression system of regulation.Tet-on-uPA includes two parts: constitutive expression or the rtTA of liver specificity expression (reverse tetracycline-controlled trans-activator, tetracycline control trans- transcription activating because Son) and tetracycline response element (tetracycline-response element, TRE) control under uPA expression vector (TRE-uPA).Tet-on-uPA can effectively control uPA in the expression of feature time, better than initial Alb- by tetracycline uPA。

Tet-on regulator control system consists of two parts, more complicated.When therefore, for constructing Tet transgenic animals first Two transgenic animals strains of rtTA and TRE-uPA need to be constructed, is hybridized, can not settle at one go, whole process is sufficiently complex simultaneously And it takes time and effort very much.Although uPA is secretory protein, it is thin in liver in addition, uPA expression is in time by Tetracycline regulation Secretion subsequently into the circulatory system, and then is transported to the other internal organs of whole body to extracellular after expressing in born of the same parents, spatial distribution still not by Control, still there is the risk for causing other organ toxicities.Meanwhile the AAV viral vectors that Tet-on-uPA is used in the prior art, system It is standby complicated, inconvenient for use.

Summary of the invention

The purpose of the present invention is to provide a kind of spatiotemporal database type uPA gene expression non-virus carriers, it is intended to solve existing The expression system of uPA gene can not regulate and control the problem of its induction time and space simultaneously in technology.

For achieving the above object, The technical solution adopted by the invention is as follows:

A kind of spatiotemporal database type uPA gene expression non-virus carrier, the non-viral load of the spatiotemporal database type uPA gene expression Body includes spatiotemporal database uPA expression casette, and the spatiotemporal database uPA expression casette includes: Liver specific promoters, uPA Mutated gene and unstable structure domain, the uPA mutated gene are the uPA gene for inserting endoplasmic reticulum retention signal gene；

And a kind of preparation method of spatiotemporal database type uPA gene expression non-virus carrier, include the following steps:

Synthesize the DNA fragmentation of Liver specific promoters, uPA mutated gene and unstable structure domain；

By the multiple cloning sites of above-mentioned DNA fragmentation insertion carrier, it is non-viral to obtain the spatiotemporal database type uPA gene expression Carrier, the spatiotemporal database type uPA gene expression non-virus carrier include spatiotemporal database uPA expression casette, air-conditioning when described Controlling uPA expression casette includes: Liver specific promoters, uPA mutated gene and unstable structure domain；Wherein, the uPA mutation Gene is the uPA gene for inserting endoplasmic reticulum retention signal gene.

And a kind of spatiotemporal database type uPA gene expression non-virus carrier is in preparing humanization chimera hepatic model Application.

Compared with prior art, a kind of spatiotemporal database type uPA gene expression non-virus carrier of the present invention, when described Empty regulation type uPA gene expression non-virus carrier includes spatiotemporal database uPA expression casette, the spatiotemporal database uPA gene table It include: Liver specific promoters, uPA mutated gene and unstable structure domain up to box.The spatiotemporal database type uPA gene expression is non- In viral vectors, containing Liver specific promoters, enables and control downstream gene when uPA gene expression and carried out in liver cell It is specific expressed；UPA mutated gene is inserted, and the uPA mutated gene is the uPA for inserting endoplasmic reticulum retention signal gene Gene, so as to the distribution of strict control uPA gene, the position for regulating and controlling its distribution is only limitted in liver cell, and uPA is made to be mutated base The distribution space position of cause has obtained tight control；A unstable structure domain is also inserted, when not adding trimethoprim or first When oxygen benzyl piperidine derivatives, the unstable structure domain and the fusion protein of uPA mutated gene are unstable, can be fast in liver cell Speed is degraded；Spatiotemporal database uPA expression casette of the present invention is carried out when using trimethoprim or trimethoprim derivative Inducing expression can be such that the fusion protein of unstable structure domain and uPA mutated gene stablizes, and be accumulated in liver cell, make uPA Gene can be regulated and controled by stablizing, unstable by control addition inducer trimethoprim or the time of trimethoprim derivative, control The fusion protein of constant domain and uPA mutated gene stablizes the time of expression, makes it be induced to express in specific time, and table It is strictly limited in liver cell, will not be secreted to extracellular up to rear uPA mutant protein, will not more enter the circulatory system and influence complete The other internal organs of body, the regulator control system of the spatiotemporal database type uPA gene expression non-virus carrier are tighter, simpler；It can Control uPA gene is expressed in specific time particular space, makes other organs from the risk of toxicity.

A kind of preparation method of spatiotemporal database type uPA gene expression non-virus carrier, the carrier are non-viral load Body, operate with it is easier, meanwhile, the preparation method handy and safe is easy to operate, easy to use, and success rate is high, is applicable in Property is extensive.

A kind of spatiotemporal database type uPA gene expression non-virus carrier is preparing humanization chimera hepatic model In application process, preparation method handy and safe operates with easier, success rate height, and applicability is extensive.

Detailed description of the invention

Fig. 1 is pMC.BESXP map provided in an embodiment of the present invention.

Fig. 2 is that the spatiotemporal database type uPA gene is prepared in the seamless cloning process of utilization provided in an embodiment of the present invention Express the flow chart of non-virus carrier.

Fig. 3 is spatiotemporal database type uPA gene expression minicircle dna matrix grain (pMC.ApoE- provided in an embodiment of the present invention MuPA-DD map).

It includes Liver specific promoters, uPA mutated gene and unstable structure domain that Fig. 4, which is provided in an embodiment of the present invention, Spatiotemporal database uPA expression casette.

Fig. 5 be it is provided in an embodiment of the present invention include Liver specific promoters, uPA mutated gene and unstable structure domain, The spatiotemporal database uPA expression casette of polyA element.

Fig. 6 is the non-viral minicircle dna (ApoE-muPA- of spatiotemporal database type uPA gene expression provided in an embodiment of the present invention DD MC) map.

Fig. 7 is DD-GFP expression vector schematic diagram and GFP-DD expression vector schematic diagram provided in an embodiment of the present invention.

Fig. 8 is the expression regulation result of DD-GFP expression vector and GFP-DD expression vector provided in an embodiment of the present invention.

Fig. 9 is the relationship of the induced concentration of trimethoprim provided in an embodiment of the present invention and the expression quantity of destination protein.

Figure 10 is Western Blot detection ApoE-muPA-DD carrier provided in an embodiment of the present invention in liver cell The result figure of expression.

Figure 11 be ApoE-muPA-DD transfected hepatocytes provided in an embodiment of the present invention system after TMP is induced in fibrin Cracking result on former agar plate.

Specific embodiment

To keep the purpose, technical solution and technical effect of the embodiment of the present invention clearer, the present invention will be implemented below Technical solution in example is clearly and completely described, it is clear that and described embodiments are some of the embodiments of the present invention, and The embodiment being not all of.In conjunction with the embodiment in the present invention, those of ordinary skill in the art are not making creative work Under the premise of every other embodiment obtained, shall fall within the protection scope of the present invention.

In the description of the present invention, it is to be understood that, term " first ", " second " are used for description purposes only, and cannot It is interpreted as indication or suggestion relative importance or implicitly indicates the quantity of indicated technical characteristic.Define as a result, " the One ", the feature of " second " can explicitly or implicitly include one or more of the features.In the description of the present invention, The meaning of " plurality " is two or more, unless otherwise specifically defined.

In the present invention, described " structural domain " refers to the region in large biological molecule with specific structure and standalone feature, Refer in particular to such region in protein.Structural domain is a kind of layer of structure between second level and tertiary structure, is albumen The fundamental structural unit and protein function unit of matter tertiary structure.In multi-domain proteins, different structural domains is normal Often it is associated from different functions.

In the present invention, described " unstable structure domain " refers to Destabilizing Domain, the unstable knot Structure domain is a kind of small-sized full source of people protein structure domain, it is ensured that the stability of fusion payload albumen, can hold very much It changes places and is added in cell or gene therapy product, correlation function is opened or closed by conformational change, be by the U.S. Obsidian Therapeutic and Celgene company cooperates the unstable structure domain (Destabilizing of joint development Domain, DD) technology, which can be used for controlling the expression of two kinds of immune-regulating factors IL12 and CD40L.

In the present invention, described " uPA " i.e. urokinase type plasminogen activator, belongs to serine protein hydrolase man Race, uPA can be catalyzed plasminogen and be converted into fibrinolysin, start fibrin and extracellular matrix protein dissolves cascade reaction.

In the present invention, described " endoplasmic reticulum retention signal " refers to the one of the structure and function protein carboxyl terminal of endoplasmic reticulum A tetrapeptide array: Lys-Asp-Glu-Leu-COO-, i.e. KDEL signal sequence.This section of sequence has accordingly on the film of golgiosome Receptor, once will be combined into golgiosome by the receptor on golgiosome, formed reflux vesicle transported back endoplasmic reticulum, institute The sequence is known as endoplasmic reticulum retention signal.

In the present invention, described " promoter " refers to a kind of nucleic acid sequence, is typically found in the upper of purpose nucleic acid sequence Trip (5 '-end), can activate RNA polymerase, be allowed to that the specificity of transcription initiation is accurately combined and had with template DNA.

In the present invention, described " Liver specific promoters " refer to that the promoter can be activated selectively in stem cell Or the transcription in the cell line of stem cell.In the present invention, Liver specific promoters refer to those usually in liver The activity promoter all higher than the activity in other any bodily tissues.In general, activity of the Liver specific promoters in liver It is more much higher than in its hetero-organization.Therefore, Liver specific promoters allow gene in activity expression of the liver in that and prevent base Because of the expression in other cell or tissues.The purpose of " Liver specific promoters " described in insertion is the purpose in order to control downstream Gene carries out specifically expressing in liver cell.

In the present invention, described " carrier " refers in genetic engineering research, exogenous DNA can be inserted and can be in recipient cell The structure replicated in born of the same parents.

In the present invention, described " multiple cloning sites " refer to the artificial synthesized DNA fragmentation contained on carrier, thereon It is the insertion site of exogenous DNA containing multiple single restriction enzyme sites.

Present example provides a kind of spatiotemporal database type uPA gene expression non-virus carrier, the spatiotemporal database type uPA base Because expression non-virus carrier includes spatiotemporal database uPA expression casette, the spatiotemporal database uPA expression casette includes: liver spy Specific Promoters, uPA mutated gene and unstable structure domain, the uPA mutated gene are to insert endoplasmic reticulum retention signal base The uPA gene of cause.

Specifically, the spatiotemporal database type uPA gene expression non-virus carrier includes spatiotemporal database uPA expression casette, Wherein, described " spatiotemporal database uPA expression casette " is the base sequence for being inserted into vector multiple cloning site, it is therefore an objective to for thin UPA gene is expressed in born of the same parents.Specifically, the spatiotemporal database uPA expression casette includes: Liver specific promoters, uPA Mutated gene and unstable structure domain.

" the spatiotemporal database uPA expression casette " inserts a unstable structure domain, the unstable structure domain with The fusion protein of uPA mutated gene is unstable, and in the case where specific small molecule ligand is not present, fusion protein is in liver cell It is interior to be degraded rapidly；And by utilizing specific small molecule trimethoprim or trimethoprim derivative to the spatiotemporal database UPA expression casette carries out inducing expression, the fusion protein of unstable structure domain and uPA mutated gene can be made to stablize, and product Tire out in liver cell, uPA gene is enable to be stablized, it is derivative by control addition inducer trimethoprim or trimethoprim The time of object, the fusion protein that can control unstable structure domain and uPA mutated gene stablize the time expressed, make it specific Be induced to express in time, it is ensured that uPA gene expression non-virus carrier can the expression in time to uPA gene adjust Control.

Preferably, the unstable structure domain is connected with liver specific promoter, uPA mutated gene, of the invention preferred In embodiment, the unstable structure domain may connect to the C-terminal or N-terminal of uPA mutated gene, and unstable structure domain and uPA are mutated Gene connects to form fusion protein, the case where specific small molecule ligand trimethoprim or trimethoprim derivative is not present Under, it is unstable to be formed by fusion protein, can be degraded quickly in the cell；If specific small molecule ligand methoxy benzyl is added Pyridine or trimethoprim derivative carry out inducing expression, unstable structure domain connect with uPA mutated gene the fusion protein to be formed compared with Stablize, can be accumulated in the cell.In the specific embodiment of the invention, the unstable structure domain fusion is in the uPA The C-terminal of mutated gene can strictly limit its destination protein and be stranded in liver cell, will not be transported to extracellular, will not more enter The circulatory system and influence the other internal organs of whole body.

In the specific embodiment of the invention, the following (Sequence in such as sequence table of unstable structure domain base sequence No.1):

5’-ATGATCTCTCTGATTGCCGCTCTGGCCGTGGACTACGTGATCGGGATGGAAAACGCTATGCCATG GAATCTGCCCGCCGATCTGGCTTGGTTCAAGAGGAACACCCTGAACAAGCCAGTGATCATGGGCAGACACACTTGG GAGTCCATTGGCCGGCCCCTGCCTGGACGCAAGAACATCATTCTGAGCTCCCAGCCCTCTACCGACGACAGGGTGA CATGGGTGAAAAGTGTGGACGAAGCCATTGCCGCTTGCGGAGATGTGCCCGAGATCATGGTCATCGGCGGAGGGAG AGTGATCGAGCAGTTCCTGCCTAAGGCCCAGAAACTGTACCTGACTCACATTGACGCTGAGGTGGAAGGGGACACC CATTTTCCTGATTATGAGCCAGACGATTGGGAAAGCGTGTTCTCCGAGTTTCACGACGCCGATGCTCAGAATTCTC ATAGTTATTGCTTTGAGATCCTGGAAAGGAGA-3’。

Specifically, the spatiotemporal database uPA expression casette further includes uPA mutated gene, wherein the uPA is mutated base Because inserting the uPA gene of endoplasmic reticulum retention signal gene.

Preferably, the endoplasmic reticulum retention signal gene can make an addition to the C-terminal of uPA gene or at least one end of N-terminal, described Endoplasmic reticulum retention signal gene can be selected from making an addition to the C-terminal of uPA gene, make an addition to the N-terminal of uPA gene or making an addition to uPA gene C-terminal and N-terminal any mode.Specifically, being inserted into endoplasmic reticulum retention signal gene in uPA gene, can strictly control The distribution of uPA gene processed, the position for regulating and controlling its distribution are only limitted in liver cell, obtain the distribution space position of uPA mutated gene Tight control is arrived.

In the specific embodiment of the invention, the endoplasmic reticulum retention signal gene includes C-terminal endoplasmic reticulum retention signal gene And N-terminal endoplasmic reticulum retention signal gene；

Specifically, C-terminal endoplasmic reticulum retention signal amino acid sequence is following (Sequence No.2 in such as sequence table), containing can Promote auxiliary amino acid sequence of the KDEL signal in conjunction with the kdel receptor being located on golgiosome for " EEDTSE " and " KDEL " signal sequence (underscore):

EEDTSEKDEL。

The C-terminal endoplasmic reticulum retention signal base sequence is following (Sequence No.3 in such as sequence table):

5’-GAAGAAGATACCTCTGAAAAAGATGAGCTC-3’。

Specifically, the N-terminal retention signal amino acid sequence is following (Sequence No.4 in such as sequence table): wherein RR (Arg-Arg) retention signal is " MHRRRSRSCREDQKP " (1-15 amino acids)；Lip33 spacer peptide is " VIDDQRDLISNNEQLPMLGRRPGAPESKCSR " (16-46 amino acids)；Cross-film sequence is " GALYTGFSILVTLLLAGQATTAYFL " (47-71 amino acids):

MHRRRSRSCREDQKPVIDDQRDLISNNEQLPMLGRRPGAPESKCSRGALYTGFSILVTLLLAGQATTA YFL。

The N-terminal retention signal base sequence is following (Sequence No.5 in such as sequence table): 5 '-ATGCACAGGAGG AGAAGCAGGAGCTGTCGGGAAGATCAGAAGCCAGTCATCGATGATCAGCGCGACCTTATCTCCAACAATGAGCAAC TGCCCATGCTGGGCCGGCGCCCTGGGGCCCCGGAGAGCAAGTGCAGCCGCGGAGCCCTGTACACAGGCTTTTCCAT CCTGGTGACTCTGCTCCTCGCTGGCCAGGCCACCACCGCCTACTTCCTG-3’。

In the specific embodiment of the invention, the uPA mutated gene is that C-terminal is added to C-terminal endoplasmic reticulum retention signal gene And N-terminal is added to the uPA gene of N-terminal endoplasmic reticulum retention signal gene, the following (Sequence in such as sequence table of base sequence No.6):

5’-ATGCACAGGAGGAGAAGCAGGAGCTGTCGGGAAGATCAGAAGCCAGTCATCGATGATCAGCGCGA CCTTATCTCCAACAATGAGCAACTGCCCATGCTGGGCCGGCGCCCTGGGGCCCCGGAGAGCAAGTGCAGCCGCGGA GCCCTGTACACAGGCTTTTCCATCCTGGTGACTCTGCTCCTCGCTGGCCAGGCCACCACCGCCTACTTCCTGTACC AGCAGCAGGTTCCATCGAACTGTGACTGTCTAAATGGAGGAACATGTGTGTCCAACAAGTACTTCTCCAACATTCA CTGGTGCAACTGCCCAAAGAAATTCGGAGGGCAGCACTGTGAAATAGATAAGTCAAAAACCTGCTATGAGGGGAAT GGTCACTTTTACCGAGGAAAGGCCAGCACTGACACCATGGGCCGGCCCTGCCTGCCCTGGAACTCTGCCACTGTCC TTCAGCAAACGTACCATGCCCACAGATCTGATGCTCTTCAGCTGGGCCTGGGGAAACATAATTACTGCAGGAACCC AGACAACCGGAGGCGACCCTGGTGCTATGTGCAGGTGGGCCTAAAGCCGCTTGTCCAAGAGTGCATGGTGCATGAC TGCGCAGATGGAAAAAAGCCCTCCTCTCCTCCAGAAGAATTAAAATTTCAGTGTGGCCAAAAGACTCTGAGGCCCC GCTTTAAGATTATTGGGGGAGAATTCACCACCATCGAGAACCAGCCCTGGTTTGCGGCCATCTACAGGAGGCACCG GGGGGGCTCTGTCACCTACGTGTGTGGAGGCAGCCTCATCAGCCCTTGCTGGGTGATCAGCGCCACACACTGCTTC ATTGATTACCCAAAGAAGGAGGACTACATCGTCTACCTGGGTCGCTCAAGGCTTAACTCCAACACGCAAGGGGAGA TGAAGTTTGAGGTGGAAAACCTCATCCTACACAAGGACTACAGCGCTGACACGCTTGCTCACCACAATGACATTGC CTTGCTGAAGATCCGTTCCAAGGAGGGCAGGTGTGCGCAGCCATCCCGGACTATACAGACCATCTGCCTGCCCTCG ATGTATAACGATCCCCAGTTTGGCACAAGCTGTGAGATCACTGGCTTTGGAAAAGAGAATTCTACCGACTATCTCT ATCCGGAGCAGCTGAAAATGACTGTTGTGAAGCTGATTTCCCACCGGGAGTGTCAGCAGCCCCACTACTACGGCTC TGAAGTCACCACCAAAATGCTGTGTGCTGCTGACCCACAGTGGAAAACAGATTCCTGCCAGGGAGACTCAGGGGGA CCCCTCGTCTGTTCCCTCCAAGGCCGCATGACTTTGACTGGAATTGTGAGCTGGGGCCGTGGATGTGCCCTGAAGG ACAAGCCAGGCGTCTACACGAGAGTCTCACACTTCTTACCCTGGATCCGCAGTCACACCAAGGAAGAGAATGGCCT GGCCCTCGAAGAAGATACCTCTGAAAAAGATGAGCTC-3’。

Specifically, the spatiotemporal database uPA expression casette includes Liver specific promoters.

Preferably, the Liver specific promoters are selected from apo E promoter, albumin promoter, phosphoenolpyruvate Pyruvate carboxykinase promoter, α-I- antitrypsin promoter, thyroid hormone binding globulin promoter, α-fetoprotein Before promoter, alcohol dehydrogenase promoter, IGF-II promoter, Factor IX promoter, HBV basal core protein promoter, HBV S2 protein promoter, throxine-binding globulin promoter, the hybrid promoter of HCR-Ap0CII, HCR-hAAT heterozygosis open Mover, the AAT promoter in conjunction with the enhancer element of mouse albumin gene, low-density lipoprotein promoter, pyruvic acid swash Enzyme promoters, lecithin cholesterol acyltransferase promoter, Apolipoprotein H promoter, siderophillin promoter, first shape Parathyrine transporter promoter, the promoter of alpha fibre proteinogen and beta fibers proteinogen, α-I- anti-chymotrypsin promoter, α- 2-HS glycoprotein promoter, ceruloplasmin promoter, plasminogen promoter, is mended at haptoglobin promoter Times of body protein promoter, the promoter of Complement C_3 activation, hemopexin promoter and α-I- acidoglycoprotein promoter It anticipates one kind.

In the specific embodiment of the invention, the Liver specific promoters select apo E promoter, select and carry rouge egg White E promoter is primarily to the target gene in control downstream carries out specifically expressing in liver cell.The apo E starting The base sequence of son is following (Sequence No.7 in such as sequence table):

5’-TAGGCTCAGAGGCACACAGGAGTTTCTGGGCTCACCCTGCCCCCTTCCAACCCCTCAGTTCCCAT CCTCCAGCAGCTGTTTGTGTGCTGCCTCTGAAGTCCACACTGAACAAACTTCAGCCTACTCATGTCCCTAAAATGG GCAAACATTGCAAGCAGCAAACAGCAAACACACAGCCCTCCCTGCCTGCTGACCTTGGAGCTGGGGCAGAGGTCAG AGACCTCTCTGGGCCCATGCCACCTCCAACATCCACTCGACCCCTTGGAATTTCGGTGGAGAGGAGCAGAGGTTGT CCTGGCGTGGTTTAGGTAGTGTGAGAGGGGTACCCGGGGATCTTGCTACCAGTGGAACAGCCACTAAGGATTCTGC AGTGAGAGCAGAGGGCCAGCTAAGTGGTACTCTCCCAGAGACTGTCTGACTCACGCCACCCCCTCCACCTTGGACA CAGGACGCTGTGGTTTCTGAGCCAGGTACAATGACTCCTTTCGGTAAGTGCAGTGGAAGCTGTACACTGCCCAGGC AAAGCGTCCGGGCAGCGTAGGCGGGCGACTCAGATCCCAGCCAGTGGACTTAGCCCCTGTTTGCTCCTCCGATAAC TGGGGTGACCTTGGTTAATATTCACCAGCAGCCTCCCCCGTTGCCCCTCTGGATCCACTGCTTAAATACGGACGAG GACAGGGCCCTGTCTCCTCAGCTTCAGGCACCACCACTGACCTGGGACAGTGAATGATCCCCCTGATCTGCGGCCT CGACGGTATCGAT-3’。

Preferably, the spatiotemporal database uPA expression casette further includes polyA element, and it is mainly anti-that polyA element is added Only newly synthesized mRNA is degraded, so having very important meaning to the stability of mRNA.The poly A is Niu Sheng Long hormone poly A (bovine growth hormone poly A, abbreviation bpA), signal sequence are following (in such as sequence table Sequence No.8):

5’-CTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAA GGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTC TGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGG CTCTATGG-3’。

In a preferred embodiment of the invention, according to apo E promoter, uPA mutated gene and unstable structure domain Sequence successively synthesize full length DNA segment (ApoE-muPA-DD).The full length DNA segment of obtained " ApoE-muPA-DD " Including apo E promoter, selecting the promoter is hybrid promoter, can control the target gene in downstream strongly in liver Specifically expressing is carried out in cell；UPA mutated gene has been sequentially connected it, wherein the uPA mutated gene is stagnant to insert endoplasmic reticulum The uPA gene of signal gene is stayed, the endoplasmic reticulum retention signal gene makes an addition to the C-terminal and N-terminal of uPA gene, in uPA gene C-terminal and N-terminal are added to endoplasmic reticulum retention signal gene, can enhance the stability of uPA gene distribution, being capable of strict control The distribution of uPA gene, the position for regulating and controlling its distribution are only limitted in liver cell, obtain the distribution space position of uPA mutated gene Tight control；Furthermore it is sequentially connected unstable structure domain, the unstable structure domain is connected to the C of uPA mutated gene End, can strictly limit destination protein in the specific time that specific small molecule is induced, only be distributed in liver cell, no Can be transported to it is extracellular, more will not enter the circulatory system and influence the other internal organs of whole body.The full length DNA of " ApoE-muPA-DD " Segment base sequence is as follows: (Sequence No.9 in such as sequence table):

5’-TAGGCTCAGAGGCACACAGGAGTTTCTGGGCTCACCCTGCCCCCTTCCAACCCCTCAGTTCCCAT CCTCCAGCAGCTGTTTGTGTGCTGCCTCTGAAGTCCACACTGAACAAACTTCAGCCTACTCATGTCCCTAAAATGG GCAAACATTGCAAGCAGCAAACAGCAAACACACAGCCCTCCCTGCCTGCTGACCTTGGAGCTGGGGCAGAGGTCAG AGACCTCTCTGGGCCCATGCCACCTCCAACATCCACTCGACCCCTTGGAATTTCGGTGGAGAGGAGCAGAGGTTGT CCTGGCGTGGTTTAGGTAGTGTGAGAGGGGTACCCGGGGATCTTGCTACCAGTGGAACAGCCACTAAGGATTCTGC AGTGAGAGCAGAGGGCCAGCTAAGTGGTACTCTCCCAGAGACTGTCTGACTCACGCCACCCCCTCCACCTTGGACA CAGGACGCTGTGGTTTCTGAGCCAGGTACAATGACTCCTTTCGGTAAGTGCAGTGGAAGCTGTACACTGCCCAGGC AAAGCGTCCGGGCAGCGTAGGCGGGCGACTCAGATCCCAGCCAGTGGACTTAGCCCCTGTTTGCTCCTCCGATAAC TGGGGTGACCTTGGTTAATATTCACCAGCAGCCTCCCCCGTTGCCCCTCTGGATCCACTGCTTAAATACGGACGAG GACAGGGCCCTGTCTCCTCAGCTTCAGGCACCACCACTGACCTGGGACAGTGAATGATCCCCCTGATCTGCGGCCT CGACGGTATCGATAAGCTTGGCATTCCGGTACTGTTGGTAAAGCCACCATGCACAGGAGGAGAAGCAGGAGCTGTC GGGAAGATCAGAAGCCAGTCATCGATGATCAGCGCGACCTTATCTCCAACAATGAGCAACTGCCCATGCTGGGCCG GCGCCCTGGGGCCCCGGAGAGCAAGTGCAGCCGCGGAGCCCTGTACACAGGCTTTTCCATCCTGGTGACTCTGCTC CTCGCTGGCCAGGCCACCACCGCCTACTTCCTGTACCAGCAGCAGGTTCCATCGAACTGTGACTGTCTAAATGGAG GAACATGTGTGTCCAACAAGTACTTCTCCAACATTCACTGGTGCAACTGCCCAAAGAAATTCGGAGGGCAGCACTG TGAAATAGATAAGTCAAAAACCTGCTATGAGGGGAATGGTCACTTTTACCGAGGAAAGGCCAGCACTGACACCATG GGCCGGCCCTGCCTGCCCTGGAACTCTGCCACTGTCCTTCAGCAAACGTACCATGCCCACAGATCTGATGCTCTTC AGCTGGGCCTGGGGAAACATAATTACTGCAGGAACCCAGACAACCGGAGGCGACCCTGGTGCTATGTGCAGGTGGG CCTAAAGCCGCTTGTCCAAGAGTGCATGGTGCATGACTGCGCAGATGGAAAAAAGCCCTCCTCTCCTCCAGAAGAA TTAAAATTTCAGTGTGGCCAAAAGACTCTGAGGCCCCGCTTTAAGATTATTGGGGGAGAATTCACCACCATCGAGA ACCAGCCCTGGTTTGCGGCCATCTACAGGAGGCACCGGGGGGGCTCTGTCACCTACGTGTGTGGAGGCAGCCTCAT CAGCCCTTGCTGGGTGATCAGCGCCACACACTGCTTCATTGATTACCCAAAGAAGGAGGACTACATCGTCTACCTG GGTCGCTCAAGGCTTAACTCCAACACGCAAGGGGAGATGAAGTTTGAGGTGGAAAACCTCATCCTACACAAGGACT ACAGCGCTGACACGCTTGCTCACCACAATGACATTGCCTTGCTGAAGATCCGTTCCAAGGAGGGCAGGTGTGCGCA GCCATCCCGGACTATACAGACCATCTGCCTGCCCTCGATGTATAACGATCCCCAGTTTGGCACAAGCTGTGAGATC ACTGGCTTTGGAAAAGAGAATTCTACCGACTATCTCTATCCGGAGCAGCTGAAAATGACTGTTGTGAAGCTGATTT CCCACCGGGAGTGTCAGCAGCCCCACTACTACGGCTCTGAAGTCACCACCAAAATGCTGTGTGCTGCTGACCCACA GTGGAAAACAGATTCCTGCCAGGGAGACTCAGGGGGACCCCTCGTCTGTTCCCTCCAAGGCCGCATGACTTTGACT GGAATTGTGAGCTGGGGCCGTGGATGTGCCCTGAAGGACAAGCCAGGCGTCTACACGAGAGTCTCACACTTCTTAC CCTGGATCCGCAGTCACACCAAGGAAGAGAATGGCCTGGCCCTCGAAGAAGATACCTCTGAAAAAGATGAGCTCCA CCGGTCGATGATCTCTCTGATTGCCGCTCTGGCCGTGGACTACGTGATCGGGATGGAAAACGCTATGCCATGGAAT CTGCCCGCCGATCTGGCTTGGTTCAAGAGGAACACCCTGAACAAGCCAGTGATCATGGGCAGACACACTTGGGAGT CCATTGGCCGGCCCCTGCCTGGACGCAAGAACATCATTCTGAGCTCCCAGCCCTCTACCGACGACAGGGTGACATG GGTGAAAAGTGTGGACGAAGCCATTGCCGCTTGCGGAGATGTGCCCGAGATCATGGTCATCGGCGGAGGGAGAGTG ATCGAGCAGTTCCTGCCTAAGGCCCAGAAACTGTACCTGACTCACATTGACGCTGAGGTGGAAGGGGACACCCATT TTCCTGATTATGAGCCAGACGATTGGGAAAGCGTGTTCTCCGAGTTTCACGACGCCGATGCTCAGAATTCTCATAG TTATTGCTTTGAGATCCTGGAAAGGAGATAA-3’。

In a preferred embodiment of the invention, according to apo E promoter, uPA mutated gene and unstable structure domain, It is as shown in Figure 5 that the sequence of poly A element successively synthesizes full length DNA segment (ApoE-muPA-DD-polyA).

The spatiotemporal database type uPA gene expression non-virus carrier provided in an embodiment of the present invention, the spatiotemporal database Type uPA gene expression non-virus carrier includes spatiotemporal database uPA expression casette, the spatiotemporal database uPA expression casette packet It includes: Liver specific promoters, uPA mutated gene and unstable structure domain.The non-viral load of the spatiotemporal database type uPA gene expression In body, containing Liver specific promoters, enables and control downstream gene when uPA gene expression and carry out specificity in liver cell Expression；UPA mutated gene is inserted, and the uPA mutated gene is the uPA gene for inserting endoplasmic reticulum retention signal gene, So as to the distribution of strict control uPA gene, the position for regulating and controlling its distribution is only limitted in liver cell, makes uPA mutated gene Distribution space position has obtained tight control；A unstable structure domain is also inserted, when not adding trimethoprim or methoxy benzyl When piperidine derivatives, the unstable structure domain and the fusion protein of uPA mutated gene are unstable, being capable of quilt rapidly in liver cell Degradation；Spatiotemporal database uPA expression casette of the present invention is induced when using trimethoprim or trimethoprim derivative Expression can be such that the fusion protein of unstable structure domain and uPA mutated gene stablizes, and be accumulated in liver cell, make uPA gene It can be regulated and controled by stablizing, by control addition inducer trimethoprim or the time of trimethoprim derivative, control unstable knot The fusion protein of structure domain and uPA mutated gene stablizes the time of expression, makes it be induced to express in specific time, inducing expression It is strictly limited afterwards to be distributed in liver cell, will not be transported to extracellular, will not more enter the circulatory system and to influence whole body other dirty Device, the regulator control system of the spatiotemporal database type uPA gene expression non-virus carrier are tighter, simpler；UPA base can be controlled Because being expressed in specific time particular space, make other organs from the risk of toxicity.

Correspondingly, the embodiment of the invention also provides a kind of preparations of spatiotemporal database type uPA gene expression non-virus carrier Method includes the following steps:

S01. the DNA fragmentation of Liver specific promoters, uPA mutated gene and unstable structure domain is synthesized；

S02. by the multiple cloning sites of above-mentioned DNA fragmentation insertion carrier, it is non-to obtain the spatiotemporal database type uPA gene expression Viral vectors, the spatiotemporal database type uPA gene expression non-virus carrier includes spatiotemporal database uPA expression casette, when described Air-conditioning control uPA expression casette includes: Liver specific promoters, uPA mutated gene and unstable structure domain；Wherein, the uPA Mutated gene is the uPA gene for inserting endoplasmic reticulum retention signal gene.

Specifically, synthesizing Liver specific promoters, uPA mutated gene and unstable structure domain in above-mentioned steps S01 DNA fragmentation.Preferably, according to the sequence successively synthetic DNA of apo E promoter, uPA mutated gene and unstable structure domain Segment (ApoE-muPA-DD)；The full length DNA segment of obtained " ApoE-muPA-DD " includes apo E promoter, Selecting the promoter is hybrid promoter, and the target gene that can control downstream strongly carries out specifically expressing in liver cell； UPA mutated gene has been sequentially connected it, wherein the uPA mutated gene is the uPA base for inserting endoplasmic reticulum retention signal gene Cause, the endoplasmic reticulum retention signal gene make an addition to the C-terminal and N-terminal of uPA gene, are added in the C-terminal and N-terminal of uPA gene Endoplasmic reticulum retention signal gene can enhance the stability of uPA gene distribution, be capable of the distribution of strict control uPA gene, regulation Its position being distributed is only limitted in liver cell, and the distribution space position of uPA mutated gene is made to have obtained tight control；It is sequentially connected Unstable structure domain, the unstable structure domain are connected to the C-terminal of uPA mutated gene, can strictly limit destination protein and exist It in the specific time that specific small molecule is induced, is only distributed in liver cell, will not be distributed to extracellular, will not more enter and follow Loop system and influence the other internal organs of whole body.In the full length DNA segment base sequence of " ApoE-muPA-DD " such as sequence table Sequence No.9。

Specifically, by the multiple cloning sites of above-mentioned DNA fragmentation insertion carrier, obtaining the space-time in above-mentioned steps S02 Regulation type uPA gene expression non-virus carrier, the spatiotemporal database type uPA gene expression non-virus carrier includes spatiotemporal database UPA expression casette, the spatiotemporal database uPA expression casette include: Liver specific promoters, uPA mutated gene and shakiness Constant domain；Wherein, the uPA mutated gene is the uPA gene for inserting endoplasmic reticulum retention signal gene.

Preferably, the carrier is selected from any one of prokaryotic plasrnid carrier, eucaryon plasmid carrier.Of the invention specific real It applies in example, the preferred pMC.BESXP of carrier.The profile information of carrier pMC.BESXP is shown in Figure of description 1, the carrier overall length 4082bp, the recombination site containing attB and attP.

In a preferred embodiment of the invention, suitable genetic engineering bacterium is selected to carry out carrier conversion according to selected carrier.Institute Stating genetic engineering bacterium can be selected from a variety of coli strains, such as ZYCY10P3S2T, DH5 α, TOP10, JM109 any. In the specific embodiment of the invention, the preferred ZYCY10P3S2T of genetic engineering bacterium carry out using.

Preferably, selection pMC.BESXP carrier is used together with ZYCY10P3S2T engineering bacteria, is expressed in ZYCY10P3S2T Φ C31 recombination enzyme effect under, pMC.BESXP carrier spontaneous can recombinate, and form two small ring-shaped DNA molecules: one is micro- AttR site of the circular DNA (minicircle, MC) containing only destination gene expression frame and 36bp), another is plasmid backbone DNA group At small ring (replication origin containing Plasmid DNA, antibiotics resistance gene and I-SceI*32 restriction enzyme site etc.).Wherein, I- SceI*32 refers to the I-SceI restriction enzyme site of 32 tandem sequence repeats, so that by the plasmid backbone DNA small ring formed and parental plasmid (pMC.BESXP) it can be identified and be dropped by the I-SceI restriction endonuclease of engineering bacteria oneself expression in engineering bacteria ZYCY10P3S2T thallus Solution.Finally, a kind of ring-shaped DNA molecule of minicircle dna is only left in engineering bacteria ZYCY10P3S2T thallus.Only work as PMC.BESXP carrier combination ZYCY10P3S2T engineering bacteria uses, and could generate minicircle dna.Other carriers and engineering bacteria ZYCY10P3S2T or carrier pMC.BESXP and other coli strains (commonly such as DH5 α, TOP10, JM109 etc.) Minicircle dna cannot be generated.And minicircle dna molecule is formed by due to the position attR containing only destination gene expression frame and 36bp Point, there is an advantage in that: firstly, MC molecule is small, it is half of ordinary plasmids more than；Therefore it is easier thin into host Born of the same parents, and then its internal transfection efficiency is higher.Secondly as eliminating gene silencing effect caused by plasmid backbone DNA ingredient (gene silencing effect), MC can be expressed steadily in the long term in vivo；In addition, MC is free of resistance antibiotics resistance gene, The worry of resistant gene diffusion is avoided, therefore there is very high safety.Moreover, because MC is free of the plasmid bone of bacterial origin Frame DNA ingredient, therefore avoid immune response of the body to bacterial origin DNA ingredient.

Preferably, it is described by above-mentioned DNA fragmentation insertion carrier multiple cloning sites the step of in, can be selected it is seamless clone or Any method of specific digestion connection, mainly will be in the multiple cloning sites of DNA fragmentation insertion carrier.

By taking above-mentioned " ApoE-muPA-DD " segment as an example, above-mentioned DNA fragmentation is inserted by carrier using the method for seamless clone The multiple cloning sites of pMC.BESXP include the following steps (such as attached drawing 2):

S201. carrier pMC.BESXP is subjected to double digestion linearisation through restriction enzyme SpeI, SalI, obtained linear Change carrier；

S202. using the DNA fragmentation of above-mentioned synthesis as template, overlapping primers priFOR and priREV is used to carry out PCR Amplification obtains PCR product；

S203. the linearized vector and the PCR product are mixed, In-Fusion recombinase is added and merge instead It answers, obtains the spatiotemporal database type uPA gene expression minicircle dna matrix grain (pMC.ApoE-muPA-DD).

Specifically, the system of the double digestion linearisation is as follows in above-mentioned steps S201:

The condition of the double digestion linearisation is 37 DEG C, 4h.

In above-mentioned steps S202, the specific base sequence of the overlapping primers priFOR and priREV is as follows:

PriFOR (Sequence No.11 in sequence table):

5'-CCCGGGCGCGACTAGTTAGGCTCAGAGGCACACAGG-3'；

PriREV (Sequence No.10 in sequence table)

5'-GCCCCCATGGGTCGACTTATCTCCTTTCCAGGATCTCAAAGCAATAACT-3'；

The system of the PCR amplification is as follows:

1 μ l of KOD-Plus high-fidelity DNA polymerase (TOYOBO).

The pcr amplification reaction condition is as follows:

25 circulations.

In above-mentioned steps S203, the linearized vector and the PCR product are mixed, In-Fusion recombination is added Enzyme carries out fusion reaction,

The fusion reaction system is as follows:

Reaction condition is as follows:

50 DEG C of reaction 15min.

The spatiotemporal database type uPA gene expression minicircle dna matrix grain (pMC.ApoE-muPA- is obtained by above-mentioned reaction DD), map such as Fig. 3 of the spatiotemporal database type uPA gene expression minicircle dna matrix grain (pMC.ApoE-muPA-DD), it is described The size of minicircle dna matrix grain is 7kb.The spatiotemporal database type uPA gene expression minicircle dna matrix grain includes spatiotemporal database UPA expression casette (such as Fig. 4), the spatiotemporal database uPA expression casette include: Liver specific promoters, uPA mutated gene With unstable structure domain；Wherein, the uPA mutated gene is the uPA gene for inserting endoplasmic reticulum retention signal gene.

The preparation method of spatiotemporal database type uPA gene expression minicircle dna matrix grain provided in an embodiment of the present invention, the carrier For non-virus carrier, operate with it is easier, meanwhile, the preparation method handy and safe is easy to operate, easy to use, at Power is high, and applicability is extensive.

Correspondingly, the embodiment of the invention also provides a kind of spatiotemporal database type uPA gene expression non-virus carrier or one kind The application of the preparation method of spatiotemporal database type uPA gene expression non-virus carrier.

Preferably, suitable genetic engineering bacterium is selected to carry out carrier conversion according to selected carrier.The genetic engineering bacterium can Selected from a variety of coli strains, such as ZYCY10P3S2T, DH5 α, TOP10, JM109 any.Of the invention specific real Apply in example, the preferred ZYCY10P3S2T of genetic engineering bacterium carry out using.The spatiotemporal database type uPA gene expression minicircle dna The step of converting that matrix grain (pMC.ApoE-muPA-DD) is transformed into genetic engineering bacterium ZYCY10P3S2T is as follows:

G01. genetic engineering bacterium ZYCY10P3S2T is thawed on ice to liquid；

G02. addition 1-2 μ L (≤50ng) described spatiotemporal database type uPA gene expression non-virus carrier thaws extremely to described In the genetic engineering bacterium ZYCY10P3S2T of liquid, it is uniformly mixed so as to obtain the first mixture, is placed in 30-40 minutes on ice；

G03. first mixture is placed in heat shock 80-90s in 40-42 DEG C of water-bath, is immediately placed in 1-2min on ice, then Be added the not antibiotic LB liquid medium of 180-200 μ L, mix, be placed in 37 DEG C, 180-200rpm culture 40-60min obtain To the second mixture；

G04. 5-10 μ the second mixture of L is taken to be coated on the LB solid plate culture medium of antibiotic containing kanamycin, 37 DEG C culture 12-14h, wherein final concentration of the 0.1% of kanamycins antibiotic.

The spatiotemporal database type uPA gene expression non-virus carrier is transformed into genetic engineering bacterium ZYCY10P3S2T, is passed through 37 DEG C of culture 12-14h cultivate to obtain recon, then carry out monoclonal sequence verification to the recon.

The positive clone molecule that above-mentioned screening obtains is seeded to the TB culture medium containing kanamycins (final concentration 0.1%), 37 DEG C shaking table culture 12-16h, adds the induced medium containing arabinose, (32 DEG C of shaking table cultures under the induction of arabinose 8h), it is finally carried out with plasmid DNA purification kit (QIAGEN EndoFree Plasmid Mega Kit, Qiagen, Germany) It isolates and purifies, obtains the non-viral minicircle dna of spatiotemporal database type uPA gene expression (ApoE-muPA-DD MC), the minicircle dna Map such as Fig. 5, the size of the minicircle dna (ApoE-muPA-DD MC) is 3kb.

It is described in the specific embodiment of the invention

Specifically, utilizing the non-viral minicircle dna (ApoE- of spatiotemporal database type uPA gene expression for screening and obtaining MuPA-DD MC) transfected hepatocytes and inducing expression is carried out, specific steps are as follows:

It D01. is 1x10 according to every hole inoculum density in 6 orifice plates⁶A cell inoculation cell；

D02. at 37 DEG C, 5%CO₂Under conditions of, it is obtained within culture medium culture 24 hours with DMEM (containing 10% fetal calf serum) Cell to be transfected；

D03. the ApoE-muPA-DD MC plasmid being transferred to the cell to be transfected using transfection reagent must recombinate carefully Born of the same parents are incubated for 24 hours；

D04. trimethoprim is added in the recombinant cell culture base, until final concentration of 10 μM of trimethoprim, then into Row induction 24 hours；UPA gene can be detected.

A kind of spatiotemporal database type uPA gene expression non-virus carrier provided in an embodiment of the present invention is to prepare humanization chimeric In the application process of body hepatic model, preparation method handy and safe operates with easier, success rate height, and applicability is extensive.

The above-mentioned spatiotemporal database type uPA gene expression non-virus carrier of the present invention is further illustrated with specific embodiment below And preparation method thereof.

Carrier pMC.BESXP used in the embodiment of the present invention, bacterial strain E.coli ZYCY10P3S2T are commercial goods, Used reagent is commercial goods；The primer and DNA sequence dna are synthesized by Shanghai Invitrogen company.

The configuration of LB culture medium used in the embodiment of the present invention is as follows:

LB culture medium: tryptone (Tryptone) 10g/L；Yeast powder (Yeastextract) 5g/L；NaCl10g/ L；pH7.0

The used primer of the embodiment of the present invention is as shown in table 1:

1 primer sequence table of table

Embodiment 1

Determine the DNA fragmentation of synthesis Liver specific promoters, uPA mutated gene and unstable structure domain, including following step It is rapid:

(1) it determines the position in unstable structure domain, is instruction group with fluorophor GFP, constructs GFP-DD expression vector, Compare influence of the unstable structure domain of different location to transgenic regulation.Specific step is as follows:

1. DD structural domain is placed in GFP fluorescence indicator N-terminal (DD-GFP) or C-terminal (GFP-DD) respectively, respectively structure Build DD-GFP expression vector and GFP-DD expression vector (Fig. 6)；

2. transfecting 293T cell respectively using above-mentioned DD-GFP expression vector and GFP-DD expression vector, and methoxy benzyl is added Pyridine inducer is induced, and is observed in fluorescence microscope.

Interpretation of result: being analyzed by Fig. 7, and when 10 μM of TMP are induced, DD-GFP and GFP-DD expressions of both amount is suitable； When without TMP, GFP-DD signal is very faint, and DD-GFP still has relatively large number of protein residue；It was found that DD is placed in C-terminal to GFP Expression regulation it is tighter.

Further, the trimethoprim induction GFP-DD transfection of various concentration (0 μM, 0.01 μM, 1 μM, 10 μM) is utilized 293T cell, obtained result such as Fig. 8, when trimethoprim induced concentration is higher, then destination protein amount is higher, destination protein amount with TMP concentration is positively correlated.

(2) according to the sequence successively synthetic DNA segment of apo E promoter, uPA mutated gene and unstable structure domain (ApoE-muPA-DD)；Wherein, the uPA mutated gene is the uPA gene for inserting endoplasmic reticulum retention signal gene, described interior Matter net retention signal gene makes an addition to the C-terminal and N-terminal of uPA gene.Sequence in synthesized DNA base sequence such as sequence table No.9。

Embodiment 2

Above-mentioned DNA fragmentation is inserted into carrier pMC.BESXP by seamless clone, the specific steps are as follows:

(1) linearized vector is prepared, carrier pMC.BESXP is subjected to double digestion line through restriction enzyme SpeI, SalI Property, obtain linearized vector；

Specifically, the system of the double digestion linearisation is as follows:

The condition of the double digestion linearisation is 37 DEG C, 4h.

(2) over-lap PCR is carried out.Using the DNA fragmentation of above-mentioned synthesis as template, using overlapping primers priFOR and PriREV carries out PCR amplification and obtains PCR product；

Specifically, the system of the PCR amplification is as follows:

1 μ l of KOD-Plus high-fidelity DNA polymerase (TOYOBO)

The pcr amplification reaction condition is as follows:

25 circulations

(3) seamless clone's connection is carried out.The linearized vector and the PCR product are mixed, In-Fusion weight is added Group enzyme carries out PCR reaction, obtains the spatiotemporal database type uPA gene expression non-virus carrier (pMC.ApoE-muPA-DD)；

Specifically, the linearized vector and the PCR product are mixed, In-Fusion recombinase is added and is merged Reaction,

The fusion reaction system is as follows:

Reaction condition is as follows:

50 DEG C, 15min

The spatiotemporal database type uPA gene expression non-virus carrier (pMC.ApoE-muPA- is obtained by above-mentioned reaction DD).The spatiotemporal database type uPA gene expression non-virus carrier includes spatiotemporal database uPA expression casette, the spatiotemporal database UPA expression casette includes: Liver specific promoters, uPA mutated gene and unstable structure domain；Wherein, the uPA is mutated base Because inserting the uPA gene of endoplasmic reticulum retention signal gene.

Embodiment 3

Above-mentioned spatiotemporal database type uPA gene expression non-virus carrier is converted to genetic engineering bacterium ZYCY10P3S2T and is prepared At micro-loop, specific step of converting is as follows:

(1) genetic engineering bacterium ZYCY10P3S2T is thawed on ice to liquid；

(2) addition 1 μ L (≤50ng) the spatiotemporal database type uPA gene expression non-virus carrier of stating thaws to described to liquid In the genetic engineering bacterium ZYCY10P3S2T of state, it is uniformly mixed so as to obtain the first mixture, is placed in 30 minutes on ice；

(3) first mixture is placed in heat shock 90s in 42 DEG C of water-baths, is immediately placed in 2min on ice, add 200 μ L Not antibiotic LB liquid medium, mix, be placed in 37 DEG C, 180-200rpm culture 40min obtain the second mixture；

(4) 10 the second mixtures of μ L is taken to be coated on the LB solid plate culture medium of antibiotic containing kanamycin, 37 DEG C Cultivate 12h, wherein final concentration of the 0.1% of kanamycins antibiotic.

The spatiotemporal database type uPA gene expression non-virus carrier is transformed into genetic engineering bacterium ZYCY10P3S2T, is passed through 37 DEG C of culture 12-14h cultivate to obtain recon, then carry out positive-selecting to the recon, carry out monoclonal sequence verification.

The positive clone molecule that above-mentioned screening obtains is isolated and purified with plasmid DNA purification kit, air-conditioning when obtaining The non-viral minicircle dna of control type uPA gene expression (ApoE-muPA-DD MC).

Embodiment 4

Utilize the non-viral minicircle dna of spatiotemporal database type uPA gene expression (ApoE-muPA-DD MC) for screening and obtaining Transfected hepatocytes simultaneously carry out inducing expression.

Specific step is as follows:

It (1) is 1x10 according to every hole inoculum density in 6 orifice plates⁶A cell inoculation cell；

(2) at 37 DEG C, under conditions of 5%CO2, with DMEM (contain 10% fetal calf serum) obtain within culture medium culture 24 hours to Transfect cell；

(3) using transfection reagent Lipofectamine 2000 by the ApoE-muPA-DD MC plasmid be transferred to it is described to Transfection cell obtains recombinant cell, is incubated for 24 hours；

(4) trimethoprim is added in the recombinant cell culture base, until final concentration of 10 μM of trimethoprim, then carry out Induction 24 hours；UPA gene can be detected.

Using expression of the Western Blot detection ApoE-uPA-DD carrier in liver cell, specific detecting step is as follows:

(1) prepare protein sample: collecting the cell that ApoE-uPA-DD carrier is expressed and use cell pyrolysis liquid (RIPA lysate, the green skies) cracking is further processed to obtain protein sample；

(2) electrophoresis: after appropriate sample-loading buffer is added in the protein sample, boiling water is heated 3-5 minutes, and albumen is allowed to become Property, after cooling, sample-adding to PAGE gel well, 80-100V electrophoresis 1 hour

(3) transferring film: using in wet type membrane-transferring device (Bio-Rad, the U.S.), 300mA transferring film 1 hour, by albumen from SDS- PAGE gel is transferred to pvdf membrane

(4) it closes: after pvdf membrane cleaning, the closing of Western Blot confining liquid is added；

(5) primary antibody is incubated for: being added diluted uPA antibody (Abcam, Britain), is incubated at room temperature 1 hour

(6) secondary antibody is incubated for: the secondary antibody of diluted horseradish peroxidase (HRP) label is added, is incubated at room temperature 1 hour

(7) it develops the color: utilizing in ECL chemical illuminating reagent (Cell Signaling, the U.S.), detect albumen.

Testing result such as Fig. 9, as shown in Figure 9, Control (blank control) and ApoE-uPA group (positive control) are not sent out Changing；ApoE-uPA-DD carrier transfected hepatocytes system is after 24 hours, then plus TMP induction (10 μM), intracellular uPA albumen Amount at any time accumulation gradually increase, when ApoE-uPA-DD be added to TMP induction 17 or 24 it is small when after (Lane 4), Western Blot detects apparent destination protein band；And induced when ApoE-uPA-DD is not added to TMP, it is equal at each time point Only very faint band, display uPA protein level is very low, illustrates not add after inserting unstable structure domain DD Trimethoprim is induced, and the fusion protein of uPA and DD can be degraded rapidly；And when being added to trimethoprim and being induced, The fusion protein of uPA and DD will do it expression, while the increase of the induction time with trimethoprim, and what is be distributed is intracellular UPA protein content accumulate gradually increase at any time.

UPA protein active is monitored using fibrinogen plate assay, specific steps are as follows:

(1) agar that 1% is prepared with PBS, after heating and melting, is cooled to 40-50 DEG C, toward being added in the agar of 15mL thawing Fibrinogen 5mg and fibrin ferment 60U

(2) culture dish is poured into, plate (thickness 5mm or so) is paved

(3) it after solidifying, is spiled on agar with the glass tube that diameter is 4mm

(4) it is added uPA protein sample (cell lysate) to be measured or uPA standard items (Sigma, the U.S.) in aperture, 37 DEG C It is incubated for 4 hours.It can determine whether uPA activity according to the size of cracking circle.

As shown in Figure 10, ApoE-muPA-DD transfected hepatocytes system, after TMP is induced, in fibrinogen agar plate Upper addition cell lysate, can produce obvious cracking circle, and Tu10Zhong, A are positive control (uPA albumen, Sigma)；B is blank pair According to；C is cell lysate；The result shows that muPA maintains urokinase activity.

The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention Made any modifications, equivalent replacements, and improvements etc., should all be included in the protection scope of the present invention within mind and principle.

SEQUENCE LISTING

<110>advanced institute

<120>a kind of spatiotemporal database type uPA gene expression non-virus carrier and preparation method thereof

<130> 2019.5.23

<160> 11

<170> PatentIn version 3.3

<210> 1

<211> 477

<212> DNA

<213>artificial synthesized

<400> 1

atgatctctc tgattgccgc tctggccgtg gactacgtga tcgggatgga aaacgctatg 60

ccatggaatc tgcccgccga tctggcttgg ttcaagagga acaccctgaa caagccagtg 120

atcatgggca gacacacttg ggagtccatt ggccggcccc tgcctggacg caagaacatc 180

attctgagct cccagccctc taccgacgac agggtgacat gggtgaaaag tgtggacgaa 240

gccattgccg cttgcggaga tgtgcccgag atcatggtca tcggcggagg gagagtgatc 300

gagcagttcc tgcctaaggc ccagaaactg tacctgactc acattgacgc tgaggtggaa 360

ggggacaccc attttcctga ttatgagcca gacgattggg aaagcgtgtt ctccgagttt 420

cacgacgccg atgctcagaa ttctcatagt tattgctttg agatcctgga aaggaga 477

<210> 2

<211> 10

<212> PRT

<213>artificial synthesized

<400> 2

Glu Glu Asp Thr Ser Glu Lys Asp Glu Leu

1 5 10

<210> 3

<211> 30

<212> DNA

<213>artificial synthesized

<400> 3

gaagaagata cctctgaaaa agatgagctc 30

<210> 4

<211> 71

<212> PRT

<213>artificial synthesized

<400> 4

Met His Arg Arg Arg Ser Arg Ser Cys Arg Glu Asp Gln Lys Pro Val

1 5 10 15

Ile Asp Asp Gln Arg Asp Leu Ile Ser Asn Asn Glu Gln Leu Pro Met

20 25 30

Leu Gly Arg Arg Pro Gly Ala Pro Glu Ser Lys Cys Ser Arg Gly Ala

35 40 45

Leu Tyr Thr Gly Phe Ser Ile Leu Val Thr Leu Leu Leu Ala Gly Gln

50 55 60

Ala Thr Thr Ala Tyr Phe Leu

65 70

<210> 5

<211> 213

<212> DNA

<213>artificial synthesized

<400> 5

atgcacagga ggagaagcag gagctgtcgg gaagatcaga agccagtcat cgatgatcag 60

cgcgacctta tctccaacaa tgagcaactg cccatgctgg gccggcgccc tggggccccg 120

gagagcaagt gcagccgcgg agccctgtac acaggctttt ccatcctggt gactctgctc 180

ctcgctggcc aggccaccac cgcctacttc ctg 213

<210> 6

<211> 1470

<212> DNA

<213>artificial synthesized

<400> 6

atgcacagga ggagaagcag gagctgtcgg gaagatcaga agccagtcat cgatgatcag 60

cgcgacctta tctccaacaa tgagcaactg cccatgctgg gccggcgccc tggggccccg 120

gagagcaagt gcagccgcgg agccctgtac acaggctttt ccatcctggt gactctgctc 180

ctcgctggcc aggccaccac cgcctacttc ctgtaccagc agcaggttcc atcgaactgt 240

gactgtctaa atggaggaac atgtgtgtcc aacaagtact tctccaacat tcactggtgc 300

aactgcccaa agaaattcgg agggcagcac tgtgaaatag ataagtcaaa aacctgctat 360

gaggggaatg gtcactttta ccgaggaaag gccagcactg acaccatggg ccggccctgc 420

ctgccctgga actctgccac tgtccttcag caaacgtacc atgcccacag atctgatgct 480

cttcagctgg gcctggggaa acataattac tgcaggaacc cagacaaccg gaggcgaccc 540

tggtgctatg tgcaggtggg cctaaagccg cttgtccaag agtgcatggt gcatgactgc 600

gcagatggaa aaaagccctc ctctcctcca gaagaattaa aatttcagtg tggccaaaag 660

actctgaggc cccgctttaa gattattggg ggagaattca ccaccatcga gaaccagccc 720

tggtttgcgg ccatctacag gaggcaccgg gggggctctg tcacctacgt gtgtggaggc 780

agcctcatca gcccttgctg ggtgatcagc gccacacact gcttcattga ttacccaaag 840

aaggaggact acatcgtcta cctgggtcgc tcaaggctta actccaacac gcaaggggag 900

atgaagtttg aggtggaaaa cctcatccta cacaaggact acagcgctga cacgcttgct 960

caccacaatg acattgcctt gctgaagatc cgttccaagg agggcaggtg tgcgcagcca 1020

tcccggacta tacagaccat ctgcctgccc tcgatgtata acgatcccca gtttggcaca 1080

agctgtgaga tcactggctt tggaaaagag aattctaccg actatctcta tccggagcag 1140

ctgaaaatga ctgttgtgaa gctgatttcc caccgggagt gtcagcagcc ccactactac 1200

ggctctgaag tcaccaccaa aatgctgtgt gctgctgacc cacagtggaa aacagattcc 1260

tgccagggag actcaggggg acccctcgtc tgttccctcc aaggccgcat gactttgact 1320

ggaattgtga gctggggccg tggatgtgcc ctgaaggaca agccaggcgt ctacacgaga 1380

gtctcacact tcttaccctg gatccgcagt cacaccaagg aagagaatgg cctggccctc 1440

gaagaagata cctctgaaaa agatgagctc 1470

<210> 7

<211> 762

<212> DNA

<213>artificial synthesized

<400> 7

taggctcaga ggcacacagg agtttctggg ctcaccctgc ccccttccaa cccctcagtt 60

cccatcctcc agcagctgtt tgtgtgctgc ctctgaagtc cacactgaac aaacttcagc 120

ctactcatgt ccctaaaatg ggcaaacatt gcaagcagca aacagcaaac acacagccct 180

ccctgcctgc tgaccttgga gctggggcag aggtcagaga cctctctggg cccatgccac 240

ctccaacatc cactcgaccc cttggaattt cggtggagag gagcagaggt tgtcctggcg 300

tggtttaggt agtgtgagag gggtacccgg ggatcttgct accagtggaa cagccactaa 360

ggattctgca gtgagagcag agggccagct aagtggtact ctcccagaga ctgtctgact 420

cacgccaccc cctccacctt ggacacagga cgctgtggtt tctgagccag gtacaatgac 480

tcctttcggt aagtgcagtg gaagctgtac actgcccagg caaagcgtcc gggcagcgta 540

ggcgggcgac tcagatccca gccagtggac ttagcccctg tttgctcctc cgataactgg 600

ggtgaccttg gttaatattc accagcagcc tcccccgttg cccctctgga tccactgctt 660

aaatacggac gaggacaggg ccctgtctcc tcagcttcag gcaccaccac tgacctggga 720

cagtgaatga tccccctgat ctgcggcctc gacggtatcg at 762

<210> 8

<211> 225

<212> DNA

<213>artificial synthesized

<400> 8

ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc 60

tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc 120

tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt 180

gggaagacaa tagcaggcat gctggggatg cggtgggctc tatgg 225

<210> 9

<211> 2756

<212> DNA

<213>artificial synthesized

<400> 9

taggctcaga ggcacacagg agtttctggg ctcaccctgc ccccttccaa cccctcagtt 60

cccatcctcc agcagctgtt tgtgtgctgc ctctgaagtc cacactgaac aaacttcagc 120

ctactcatgt ccctaaaatg ggcaaacatt gcaagcagca aacagcaaac acacagccct 180

ccctgcctgc tgaccttgga gctggggcag aggtcagaga cctctctggg cccatgccac 240

ctccaacatc cactcgaccc cttggaattt cggtggagag gagcagaggt tgtcctggcg 300

tggtttaggt agtgtgagag gggtacccgg ggatcttgct accagtggaa cagccactaa 360

ggattctgca gtgagagcag agggccagct aagtggtact ctcccagaga ctgtctgact 420

cacgccaccc cctccacctt ggacacagga cgctgtggtt tctgagccag gtacaatgac 480

tcctttcggt aagtgcagtg gaagctgtac actgcccagg caaagcgtcc gggcagcgta 540

ggcgggcgac tcagatccca gccagtggac ttagcccctg tttgctcctc cgataactgg 600

ggtgaccttg gttaatattc accagcagcc tcccccgttg cccctctgga tccactgctt 660

aaatacggac gaggacaggg ccctgtctcc tcagcttcag gcaccaccac tgacctggga 720

cagtgaatga tccccctgat ctgcggcctc gacggtatcg ataagcttgg cattccggta 780

ctgttggtaa agccaccatg cacaggagga gaagcaggag ctgtcgggaa gatcagaagc 840

cagtcatcga tgatcagcgc gaccttatct ccaacaatga gcaactgccc atgctgggcc 900

ggcgccctgg ggccccggag agcaagtgca gccgcggagc cctgtacaca ggcttttcca 960

tcctggtgac tctgctcctc gctggccagg ccaccaccgc ctacttcctg taccagcagc 1020

aggttccatc gaactgtgac tgtctaaatg gaggaacatg tgtgtccaac aagtacttct 1080

ccaacattca ctggtgcaac tgcccaaaga aattcggagg gcagcactgt gaaatagata 1140

agtcaaaaac ctgctatgag gggaatggtc acttttaccg aggaaaggcc agcactgaca 1200

ccatgggccg gccctgcctg ccctggaact ctgccactgt ccttcagcaa acgtaccatg 1260

cccacagatc tgatgctctt cagctgggcc tggggaaaca taattactgc aggaacccag 1320

acaaccggag gcgaccctgg tgctatgtgc aggtgggcct aaagccgctt gtccaagagt 1380

gcatggtgca tgactgcgca gatggaaaaa agccctcctc tcctccagaa gaattaaaat 1440

ttcagtgtgg ccaaaagact ctgaggcccc gctttaagat tattggggga gaattcacca 1500

ccatcgagaa ccagccctgg tttgcggcca tctacaggag gcaccggggg ggctctgtca 1560

cctacgtgtg tggaggcagc ctcatcagcc cttgctgggt gatcagcgcc acacactgct 1620

tcattgatta cccaaagaag gaggactaca tcgtctacct gggtcgctca aggcttaact 1680

ccaacacgca aggggagatg aagtttgagg tggaaaacct catcctacac aaggactaca 1740

gcgctgacac gcttgctcac cacaatgaca ttgccttgct gaagatccgt tccaaggagg 1800

gcaggtgtgc gcagccatcc cggactatac agaccatctg cctgccctcg atgtataacg 1860

atccccagtt tggcacaagc tgtgagatca ctggctttgg aaaagagaat tctaccgact 1920

atctctatcc ggagcagctg aaaatgactg ttgtgaagct gatttcccac cgggagtgtc 1980

agcagcccca ctactacggc tctgaagtca ccaccaaaat gctgtgtgct gctgacccac 2040

agtggaaaac agattcctgc cagggagact cagggggacc cctcgtctgt tccctccaag 2100

gccgcatgac tttgactgga attgtgagct ggggccgtgg atgtgccctg aaggacaagc 2160

caggcgtcta cacgagagtc tcacacttct taccctggat ccgcagtcac accaaggaag 2220

agaatggcct ggccctcgaa gaagatacct ctgaaaaaga tgagctccac cggtcgatga 2280

tctctctgat tgccgctctg gccgtggact acgtgatcgg gatggaaaac gctatgccat 2340

ggaatctgcc cgccgatctg gcttggttca agaggaacac cctgaacaag ccagtgatca 2400

tgggcagaca cacttgggag tccattggcc ggcccctgcc tggacgcaag aacatcattc 2460

tgagctccca gccctctacc gacgacaggg tgacatgggt gaaaagtgtg gacgaagcca 2520

ttgccgcttg cggagatgtg cccgagatca tggtcatcgg cggagggaga gtgatcgagc 2580

agttcctgcc taaggcccag aaactgtacc tgactcacat tgacgctgag gtggaagggg 2640

acacccattt tcctgattat gagccagacg attgggaaag cgtgttctcc gagtttcacg 2700

acgccgatgc tcagaattct catagttatt gctttgagat cctggaaagg agataa 2756

<210> 10

<211> 36

<212> DNA

<213>artificial synthesized

<400> 10

cccgggcgcg actagttagg ctcagaggca cacagg 36

<210> 11

<211> 49

<212> DNA

<213>artificial synthesized

<400> 11

gcccccatgg gtcgacttat ctcctttcca ggatctcaaa gcaataact 49

Claims (10)

1. a kind of spatiotemporal database type uPA gene expression non-virus carrier, which is characterized in that the spatiotemporal database type uPA gene table It include spatiotemporal database uPA expression casette up to non-virus carrier, the spatiotemporal database uPA expression casette includes: liver specificity Promoter, uPA mutated gene and unstable structure domain, the uPA mutated gene are to insert endoplasmic reticulum retention signal gene UPA gene.

2. spatiotemporal database type uPA gene expression non-virus carrier according to claim 1, which is characterized in that the shakiness Constant domain base sequence is as follows:

5’-ATGATCTCTCTGATTGCCGCTCTGGCCGTGGACTACGTGATCGGGATGGAAAACGCTATGCCATGGAAT CTGCCCGCCGATCTGGCTTGGTTCAAGAGGAACACCCTGAACAAGCCAGTGATCATGGGCAGACACACTTGGGAGT CCATTGGCCGGCCCCTGCCTGGACGCAAGAACATCATTCTGAGCTCCCAGCCCTCTACCGACGACAGGGTGACATG GGTGAAAAGTGTGGACGAAGCCATTGCCGCTTGCGGAGATGTGCCCGAGATCATGGTCATCGGCGGAGGGAGAGTG ATCGAGCAGTTCCTGCCTAAGGCCCAGAAACTGTACCTGACTCACATTGACGCTGAGGTGGAAGGGGACACCCATT TTCCTGATTATGAGCCAGACGATTGGGAAAGCGTGTTCTCCGAGTTTCACGACGCCGATGCTCAGAATTCTCATAG TTATTGCTTTGAGATCCTGGAAAGGAGA-3’。

3. spatiotemporal database type uPA gene expression non-virus carrier according to claim 1, which is characterized in that the endoplasm Net retention signal gene includes C-terminal endoplasmic reticulum retention signal gene and N-terminal endoplasmic reticulum retention signal gene；

Wherein, C-terminal endoplasmic reticulum retention signal gene order is as follows:

5'-GAAGAAGATACCTCTGAAAAAGATGAGCTC-3'；

N-terminal endoplasmic reticulum retention signal gene order is as follows:

5’-ATGCACAGGAGGAGAAGCAGGAGCTGTCGGGAAGATCAGAAGCCAGTCATCGATGATCAGCGCGACCTT ATCTCCAACAATGAGCAACTGCCCATGCTGGGCCGGCGCCCTGGGGCCCCGGAGAGCAAGTGCAGCCGCGGAGCCC TGTACACAGGCTTTTCCATCCTGGTGACTCTGCTCCTCGCTGGCCAGGCCACCACCGCCTACTTCCTG-3’。

4. spatiotemporal database type uPA gene expression non-virus carrier according to claim 1, which is characterized in that the liver is special Specific Promoters are selected from apo E promoter, albumin promoter, phosphoenolpyruvate carboxykinase promoter, α-I- Antitrypsin promoter, thyroid hormone binding globulin promoter, α-fetoprotein promoter, alcohol dehydrogenase promoter, S2 protein promoter, thyroxine-before IGF-II promoter, Factor IX promoter, HBV basal core protein promoter, HBV Haptoglobin promoter, the hybrid promoter of HCR-Ap0CII, HCR-hAAT hybrid promoter, with mouse albumin gene AAT promoter, the low-density lipoprotein promoter, pyruvate kinase promoter, lecithin-cholesterol acyl of enhancer element combination Based transferase promoter, Apolipoprotein H promoter, siderophillin promoter, transthyretin promoter, alpha fibre The promoter of proteinogen and beta fibers proteinogen, α-I- anti-chymotrypsin promoter, α -2-HS glycoprotein promoter, haptoglobin Promoter, ceruloplasmin promoter, plasminogen promoter, complement protein promoter, Complement C_3 activation Promoter, hemopexin promoter and α-I- acidoglycoprotein promoter any one.

5. spatiotemporal database type uPA gene expression non-virus carrier according to claim 1 to 4, which is characterized in that institute At least one end of the C-terminal of uPA gene, N-terminal can be made an addition to by stating endoplasmic reticulum retention signal gene.

6. spatiotemporal database type uPA gene expression non-virus carrier according to claim 1 to 4, which is characterized in that institute Stating unstable structure domain may connect to the C-terminal or N-terminal of uPA mutated gene.

7. a kind of preparation method of spatiotemporal database type uPA gene expression non-virus carrier, includes the following steps:

Synthesize the DNA fragmentation of Liver specific promoters, uPA mutated gene and unstable structure domain；

By the multiple cloning sites of above-mentioned DNA fragmentation insertion carrier, the non-viral load of the spatiotemporal database type uPA gene expression is obtained Body, the spatiotemporal database type uPA gene expression non-virus carrier include spatiotemporal database uPA expression casette, the spatiotemporal database UPA expression casette includes: Liver specific promoters, uPA mutated gene and unstable structure domain；Wherein, the uPA is mutated base Because inserting the uPA gene of endoplasmic reticulum retention signal gene.

8. the preparation method of spatiotemporal database type uPA gene expression non-virus carrier according to claim 7, feature exist In,

According to the sequence successively synthetic DNA segment of apo E promoter, uPA mutated gene and unstable structure domain；

By the multiple cloning sites of above-mentioned DNA fragmentation insertion carrier pMC.BESXP, the spatiotemporal database type uPA gene expression is obtained Non-virus carrier, the spatiotemporal database type uPA gene expression non-virus carrier includes spatiotemporal database uPA expression casette, described Spatiotemporal database uPA expression casette successively includes: apo E promoter, uPA mutated gene and unstable structure domain；Wherein, The uPA mutated gene is the uPA gene for inserting endoplasmic reticulum retention signal gene, the endoplasmic reticulum retention signal gene addition In the C-terminal and N-terminal of uPA gene.

9. the preparation method of spatiotemporal database type uPA gene expression non-virus carrier according to claim 8, feature exist In,

The genetic engineering bacterium of conversion for the spatiotemporal database type uPA gene expression non-virus carrier is ZYCY10P3S2T.

10. a kind of spatiotemporal database type uPA gene expression non-virus carrier is preparing the application in humanization chimera hepatic model.