CN101748123A

CN101748123A - Manufacturing method of adjustable liver damage animal model and special DNA fragment thereof

Info

Publication number: CN101748123A
Application number: CN200810227638A
Authority: CN
Inventors: 邓宏魁; 宋希军; 多曙光; 郭玉珊
Original assignee: Peking University
Current assignee: Peking University
Priority date: 2008-11-27
Filing date: 2008-11-27
Publication date: 2010-06-23
Anticipated expiration: 2028-11-27
Also published as: CN101748123B

Abstract

The invention discloses a manufacturing method of an adjustable liver damage animal model and a special DNA fragment thereof; a DNA fragment I is formed by sequentially connecting liver super promoters and antisense tetracycline activator coding genes of a Tet-on gene expression system from upstream to downstream; an DNI fragment II is formed by sequentially connecting tetracycline reaction factors, promoters and prourokinase activator coding genes of the Tet-off/Tet-on gene expression system from upstream to downstream; a recombination expression vector carrying the DNI fragment II is used for converting mammalian cells, the cells are cultivated to obtain recombinant adenovirus. The DNA fragment I is led in the animal to obtain a transgenosis first-construction animal; the transgenosis first-construction animal and scid/bg animal are back-crossed to obtain the animal carrying the DNA fragment I with the scid/bg background; the recombinant adenovirus is used for injecting the animal carrying the DNA fragment I with the scid/bg background, so as to obtain the adjustable liver damage animal model; in the model, the prourokinase activator expression is activated by doxycycline, the expression intensity is changed along with the change of dosage, the specificity is very high.

Description

The making method of adjustable liver damage animal model and special DNA fragment thereof

Technical field

The present invention relates to a kind of making method and special DNA fragment thereof of adjustable liver damage animal model.

Background technology

Hepatitis B virus (HBV) and hepatitis C virus (HCV) serious threat human beings'health, the whole world have 3.6 hundred million hepatitis B patients and 1.7 hundred million the third hepatopaths approximately, and 1,500,000 deaths are arranged every year approximately.In 10 years of past, making significant headway aspect these diseases of treatment, but still do not setting up effective healing means, and prior treatment method develops better antiviral and remains the task of top priority usually with side effect.

At present, new antiviral mainly at cell levels to testing and estimating, be used for clinical before, must on animal model, test its activity and toxicity.The medicine of anti-HBV and HCV can only carry out studying in the body with chimpanzee, because the reason of economic and moral aspect, the chimpanzee test has certain limitation, so attempting to set up the humanized mouse model of liver, people are used for HCV and HBV medicine and developing vaccines, obtain some progress, as the liver of the source liver cell reconstruction Alb-uPA transgenic mice of choosing, such mouse can be infected by HBV and HCV.

In order to study intravital blood coagulation hemolytic system, people such as Janice L.Hechei have made up the transgenic mice of albumin promoter driving uPA, overexpression uPA in the liver cell of this transgenic mice, the uPA in the blood plasma rises, and causes internal hemorrhage easily and cause death in newborn mice.Have two lower strains of uPA expression level to be survived, but only about half of transgenic mouse birth back is dead among the offspring.The expression of the intravital uPA of transgenic mice should be stable, but the activity of the detected uPA in the blood of the mouse of two strains descends in time gradually, and the blood coagulation ability is also recovered gradually, and after about 1-2 month, mouse blood coagulation ability is recovered fully.The liver of the transgenic mice in one age in week is complete white; During age in week, the red area of some 1mm-1cm that are dispersed in has appearred in the liver to 3-5; 6-8 week can be observed the red area that is linked to be sheet.Two strains all are this situations, illustrate with genetically modified integration site irrelevant.These phenomenons show that the tissue of white is original, and red spot occurred afterwards.Some cells of further investigation discovery have been left out foreign gene by homologous recombination, finally can rebuild whole liver by increasing, and this explanation liver cell has powerful multiplication capacity, rebuilds mouse liver for the external source liver cell foundation is provided.The Alb-uPA mouse is backcrossed on the immunodeficient mouse scid/bg background, the mouse liver of being transplanted to the Alb-uPA heterozygosis from the liver cell of the isolating virus-free infection of hepatectomy patient and isozygotying, finder source liver cell can reach the reconstruction of higher degree in the mouse body that transgenosis is isozygotied, and can be infected by HBV and HCV, and in the mouse liver of transgenosis heterozygosis, almost can't see the liver cell in people source.

Another important models that is used to transplant research be Fah-/-mouse, tyrosinemia can take place in the mouse of Fah defective, tyrosinemia is a kind of heredity amino acid metabolism disease, lack the FAA lytic enzyme in patient's body, tyrosine is in vivo during metabolism, owing to FAA can not accumulate in hydrolysis, thereby cause the damage of liver cell.NTBC is a 4-hydroxyphenylpyruvate dioxygenase inhibitor, can stop the accumulation of FAA, therefore takes the symptom that NTBC can improve tyrosinemia effectively.This mouse can the normal growth breeding by feeding NTBC.Because the FAH defective can cause hepatocellular death, so can be used as the model of hepatocyte transplantation on this model theory.Report 1000 normal mouse liver cells be transplanted to the FAH mouse, just can rebuild Fah-/-liver of mouse.But in very long following period of time, all do not use Fah-/-model carries out the humanized report of liver.

Though people source liver cell can be good at rebuilding the Alb-uPA mouse, but this mouse has several shortcomings to hinder its application: 1) breeding difficulty: after the mouse birth of transgenosis heterozygosis, there is half to cause death approximately, the homozygote mouse is all dead in one month, the mouse that can only obtain to isozygoty by the mating of heterozygosis mouse, according to genetic development, it is homozygote that the offspring has 1/4 mouse approximately, it is lethal to remove new life, is difficult to obtain the mouse of isozygotying.2) the transplanting window is little; Homozygous mouse can be dead about one month, so must rebuild the liver of mouse as early as possible, transplant time after birth 7-14 days usually.3) operation is inconvenient, and 7-14 days mouse is not easy to operation technique.

In the intestinal bacteria E.coli, TetR (Tet repressor) is a negative regulation tetracyclin resistance operon.TetR is attached to the expression of blocking gene on the Tet operon sequence when not having tsiklomitsin.When tsiklomitsin was arranged, TetR discharged from operon sequence, and gene begins to express Tet-off system that Here it is.Different with the Tet-Off system, the Tet-On system uses the trans Tet that has two sudden changes to suppress sub-rTetRs (reverse Tetrepressors), merge the land of rTetRs and the active region of VP16, and what senior tet-on system used is minimum VP16 active region.The land of rTetRs combines with the tet operon sequence when doxycycline (Doxycycline tetracycline derivant) or tsiklomitsin are arranged, and starts the expression of goal gene.The expression that the senior tet-On system that obtains by the optimization to the tet-on system has improved goal gene, toxicity reduces, the proteic clone of easier acquisition stably express.Senior tet-On system has following advantage mutually: 1) very tight regulation and control: because the rtTA and the TRE-Tight that optimize, the background expression level of goal gene is low.2) have no side effect: because do not have corresponding sequence, the tetO sequence that acts on TRE-Tight that the land of rtTA-Adavanced is very special in mammalian cell in the eukaryotic gene group.3) efficiently fast inducing action: Dox can be in 30 minutes the expression of goal gene be improved 1000 times, and other inducible system speed is slow, can not induce fully.4) absolute expression levels height: the high expression level of Tet system even be higher than the direct startup of CMV promotor has the Tet-Off transient expression of reporting in the HeLa cell stronger 35 times than CMV.5) induced drug: Tc and Dox price are relatively cheap, nontoxic, and result's repeatability is high.Based on above advantage, we use senior tet-on system to set up model.

Adenovirus carrier is a kind of carrier that is used for therapeutic transgene of development in recent years, and it is low that it has toxicity, the high and unconformable characteristics of efficient.Discover, import the intravital adenovirus of mouse, main infected liver cell by blood.Therefore can use adenovirus carrier that foreign gene is imported mouse liver.

Summary of the invention

The making method and the special DNA fragment thereof that the purpose of this invention is to provide a kind of adjustable liver damage animal model.

Dna fragmentation I provided by the invention comprises to the downstream successively from the upstream: the antisense tetracycline of liver specific promoter, Tet-on gene expression system activates sub-encoding gene.

Described liver specific promoter can adopt multiple promotor, as the mouse albumin promoter.

Described dna fragmentation I also comprises the enhanser that is positioned at mouse albumin promoter upstream, and described enhanser and described mouse albumin promoter form following 1) or 2) or 3) or 4) fusion dna:

1) in the sequence table sequence 3 from the dna molecular shown in the deoxyribonucleotide of 5 ' terminal 29-2364 position;

2) its nucleotide sequence is the dna molecular shown in the sequence 3 in the sequence table;

3) under stringent condition with 1) or 2) dna sequence dna hybridization that limits and dna molecular with identical function;

4) with sequence table in the dna sequence dna that limits of sequence 3 have 90% above homology, and have the dna molecular of identical function.

It is following 5 that described antisense tetracycline activates son) or 6) protein:

5) protein of forming by the aminoacid sequence shown in the sequence in the sequence table 2;

6) with the aminoacid sequence of sequence 2 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and have antisense tetracycline activate subfunction by sequence 2 deutero-protein.

Described dna fragmentation I also comprise the position be positioned at the polyA that antisense tetracycline activates sub-encoding gene downstream, it is following 7 that the antisense tetracycline of described connection polyA activates sub-encoding gene) or 8) or 9) dna molecular:

7) its nucleotide sequence is the dna molecular shown in the sequence 1 in the sequence table;

8) under stringent condition with 7) the dna sequence dna hybridization that limits and the described antisense tetracycline of encoding activate the dna molecular of son;

9) with sequence table in the dna sequence dna that limits of sequence 1 have 90% above homology, and coding has the protein DNA molecule of antisense tetracycline activation subfunction;

The recombinant expression vector that contains described dna fragmentation I also belongs to protection scope of the present invention.

The present invention also protects by the tsiklomitsin response factor (TRE) of Tet-off/Tet-on gene expression system, promotor and uPA activator encoding gene by the order in upstream to the downstream dna fragmentation II that is connected in sequence.

Described uPA activator is following 10) or 11) protein:

10) protein of forming by the aminoacid sequence shown in the sequence in the sequence table 5;

11) with the aminoacid sequence of sequence 5 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and have uPA activator function by sequence 5 deutero-protein.

The encoding gene of described uPA activator is following 12) or 13) or 14) or 15) dna molecular:

12) in the sequence table sequence 4 from the dna molecular shown in the deoxyribonucleotide of 5 ' terminal 44-1346 position;

13) its nucleotide sequence is the dna molecular shown in the sequence 4 in the sequence table;

14) under stringent condition with 12) or 13) the dna sequence dna hybridization that limits and the dna molecular of the described uPA activator of encoding;

15) dna sequence dna with sequence 4 qualifications has 90% above homology, and the protein DNA molecule of encoding and having uPA activator function.

Described promotor is following 16) or 17) or 18) dna molecular:

16) its nucleotide sequence is the dna molecular shown in the sequence 6 in the sequence table;

17) under stringent condition with 16) dna sequence dna hybridization that limits and dna molecular with identical function;

18) with sequence table in the dna sequence dna that limits of sequence 6 have 90% above homology, and have the dna molecular of identical function.

Described tsiklomitsin response factor (TRE) is following 19) or 20) or 21) dna molecular:

19) its nucleotide sequence is the dna molecular shown in the sequence 7 in the sequence table;

20) under stringent condition with 19) dna sequence dna hybridization that limits and dna molecular with identical function;

21) with sequence table in the dna sequence dna that limits of sequence 7 have 90% above homology, and have the dna molecular of identical function.

The recombinant expression vector that contains described dna fragmentation II also belongs to protection scope of the present invention.

Described recombinant expression vector can import adenovirus expression carrier with described dna fragmentation II and obtain.

Described recombinant expression vector specifically can be Adeno-TRE-uPA, and described Adeno-TRE-uPA imports the Adeno-X carrier with described dna fragmentation II and obtains.

The recombinant adenovirus that the described recombinant expression vector of cultivation obtains in mammalian cell also belongs to protection scope of the present invention.

Described mammalian cell specifically can be the HEK293 cell.

Above-mentioned stringent condition can be at 6 * SSC, in the solution of 0.5%SDS, 65 ℃ of hybridization down, uses 2 * SSC then, and 0.1%SDS and 1 * SSC, 0.1%SDS respectively wash film once.

The present invention also protects a kind of making method of liver damage animal model, may further comprise the steps:

1) described dna fragmentation I is imported in the animal, obtain the transgenic founder animal;

2) described transgenic founder animal and scid/bg animal are backcrossed, obtain the animal that carries dna fragmentation I of scid/bg background;

3) with the animal that carries dna fragmentation I of described recombinant adenovirus injection scid/bg background, obtain adjustable liver injury animal.

Described animal specifically can be mouse.

After common backcross for 3 times, can obtain the mouse of the background of immune deficiency.In the actually operating, can backcross 3-5 time.

Use the liver injury model mouse that method of the present invention obtains, overcome the shortcoming of Alb-uPA mouse, expression of gene is activated by doxycycline (derivative of tsiklomitsin), and expression intensity changes with the dosage variation of Dox, and maximum expression intensity and CMV promotor directly start quite.This systemic characteristic is very high, does not almost have nonspecific effect.

Following embodiment is convenient to understand better the present invention, but does not limit the present invention.

Description of drawings

Fig. 1 is the electrophorogram of pcr amplification uPA gene.

Fig. 2 cuts evaluation figure for the enzyme of pGEMT-uPA plasmid.

Fig. 3 cuts evaluation figure for the enzyme of PTRE-uPA.

Fig. 4 cuts evaluation figure for the enzyme of Adeno-TRE-uPA

Fig. 5 is the segmental electrophoresis result of Alb-rtTA.

Fig. 6 is the PCR qualification result of transgenic mice.

Fig. 7 is that the RT-PCR of transgenic mice identifies.

Fig. 8 is the immunohistochemical analysis result of model mice.

Fig. 9 is the RT-PCR analytical results of model mice.

Figure 10 is the HE coloration result of model mice.

Figure 11 is the evaluation that liver injury mouse model external source liver cell of the present invention is rebuild.

Embodiment

Experimental technique among the following embodiment if no special instructions, is ordinary method.

The structure of embodiment 1, pAlb-rtTA-polyA plasmid

One, the structure of Psk-rtTA-polyA plasmid

1, cuts pTet-On-Advanced carrier (Clontech company with restriction enzyme EcorI and 37 ℃ of enzymes of HindIII, Catalog No.:630930) spends the night, after enzyme is cut end, enzyme is cut product and is carried out 1% agarose gel electrophoresis, reclaims the rtTA-polyA fragment that test kit reclaims about 1.2kb with ancient cooking vessel state glue.The dna fragmentation that reclaims is dissolved in the 10ul ultrapure water.The dna fragmentation that reclaims is checked order, see the sequence 1 in the sequence table, the aminoacid sequence of rtTA genes encoding is seen the sequence 2 in the sequence table.

2, cut pBluscript carrier (Fermentas company) with restriction enzyme EcorI and 37 ℃ of enzymes of HindIII and spend the night, after enzyme was cut end, enzyme was cut product and is carried out 1% agarose gel electrophoresis, reclaimed the pSK carrier framework that test kit reclaims about 3kb with ancient cooking vessel state glue.The dna fragmentation that reclaims is dissolved in the 10ul ultrapure water.

3, the pSK carrier framework that rtTA-polyA fragment that step 1 is obtained and step 2 obtain is hatched 2h for 16 ℃, connects.

4, (day root biochemical technology company limited CB104-03), puts on ice and slowly thaws, and adds the connection product of 10ul step 3, mixing to get 1 pipe (100ul) TOP10 competent cell; Continue to place 30min in the ice-water bath; Heat shock 90sec is left standstill in 42 ℃ of water-baths; Be transferred to rapidly and cool off 1-2min in the ice-water bath; Add 600ul and do not contain antibiotic LB substratum, pat mixing gently, put 37 ℃ of shaking table incubation 45min.(Amp 125U/ml), respectively is coated with 100ul bacterium liquid to get the LB solid medium flat board of two ammonia benzyl resistances; After room temperature left standstill and treats that bacterium liquid absorbs, 37 ℃ of incubators were inverted and are cultivated 10-15hr.

5, from the agar plate that transforms, choose single colony inoculation to the LB solid medium of ammonia benzyl resistance (Amp, 125U/ml) in 37 ℃ of cultivations.

Be extracted in the plasmid of the bacterium colony of growing on the substratum, carry out enzyme with restriction enzyme HindIII and HEcorI and cut, carry out agarose gel electrophoresis then.The band that shows about 1.2kb and about 3kb on the gel.Enzyme is cut the male plasmid check order, the result shows and has obtained the purpose plasmid, with its called after Psk-rtTA-polyA.

Two, the structure of pAlb-rtTA-polyA plasmid

1, the acquisition of Alb promotor/enhanser

To be arranged in the enhanser fragment of the 1.99kb between mouse albumin gene 5 ' the upstream 10.4-8.5kb and the promoter fragment of 0.33kb and be fused into Alb promotor/enhanser that length is 2.3kb at pAlb-hGH, concrete experimental technique is referring to document (Pinkert CA et al.An ALB enhancer located 10 kb upstreamfunctions along with its promoter to direct efficient, liverspecificexpression in transgenic mice.Genes Dev 1987; 1:268-276.) (Peking University).Alb promotor/the enhancer sequence that obtains is seen the sequence 3 in the sequence table.

2, cut pAlb-Hgh 3-4 hour with restriction enzyme BamH I enzyme; Add the T4 archaeal dna polymerase then, smooth end; Add restriction enzyme Sac I then and carry out enzyme and cut, obtain Alb promotor/enhanser fragment of 2.3kb.

3, cut Psk-rtTA-polyA 3-4 hour with restriction enzyme EcorI enzyme; Add the T4 archaeal dna polymerase then, smooth end; Add restriction enzyme Sac I then and carry out enzyme and cut, obtain the carrier segments of 4.2kb.

4, the Alb promotor/enhanser fragment of step 2 preparation and the carrier segments mixing of step 3 preparation are hatched, connect.

5, with connect product transform the TOP10 competent cell (day root biochemical technology company limited, CB104-03).(Amp 125U/ml), is coated with 100ul bacterium liquid to get the LB solid medium flat board of ammonia benzyl resistance; After room temperature left standstill and treats that bacterium liquid absorbs, 37 ℃ of incubators were inverted and are cultivated 10-15hr.

6, from the agar plate that transforms, choose single colony inoculation to the LB solid medium of ammonia benzyl resistance (Amp, 125U/ml) in 37 ℃ of cultivations.

Be extracted in the plasmid of the bacterium colony of growing on the substratum, carry out enzyme with restriction enzyme SacI and KpnI and cut, carry out agarose gel electrophoresis then.Show the band of about 3.5kb and about 3kb on the gel, show to have obtained the purpose plasmid, its called after pAlb-rtTA-polyA.

Embodiment 2, making TRE-PminCMV-uPA adenovirus

One, amplification uPA activator (uPA) gene

The a pair of primer of uPA mRNA sequence (NM_008873) design according to mouse among the Gene bank is as follows:

Upstream primer (29bp): 5 '-GGC GCTA ATGAAAGTCTGGCTGGCGAG-3 ';

Downstream primer (29bp): 5 '-TCT GGTACCGAGAGGACGGTCAGCATGGG-3 '.

In the above primer, what underscore marked is restriction enzyme site, and tiltedly runic is expressed for the Kozak sequence is used for strengthening.Upstream primer is positioned at the initial ATG of uPA mRNA, contains the NheI restriction enzyme site.Downstream primer is positioned at 76bp place, terminator codon downstream, contains Kpn I restriction enzyme site.

1, extracting CD-1 mouse (available from dimension tonneau China) kidney RNA and reverse transcription and become cDNA, is template with cDNA, adopts above-mentioned primer, uses the Pyrobest DNA Polymerase of TaKaRa to carry out the PCR reaction.

2, the PCR product is after gel electrophoresis, use DNA fast purifying/recovery test kit (Beijing ancient cooking vessel state biotech firm, cat.no A014-1) and reclaim, ultraviolet spectrophotometer is measured A260/280 OD, estimate the concentration and the purity of PCR product, the DNA of recovery be stored in-20 ℃ standby.The electrophorogram of PCR product is seen Fig. 1.

3, the PCR product of Hui Shouing through external add A reaction after, be connected into the pGEM-T carrier (available from Promega, catalog number: A3600).

1. add the A reaction

In 200ul PCR pipe, add following solution (cumulative volume 10ul):

PCR product 7.2ul

10×PCR?buffer 1ul

dNTP(2.5mM) 0.8ul

Taq archaeal dna polymerase (Takara article No. DRR001A) 0.2ul

Reaction is 30 minutes under 70 ℃ of conditions.

2) the PCR product cloning is gone into the pGEM-T carrier

1. ligation adds following solution to final volume 10ul in the 1.5ml centrifuge tube:

2×T4DNA?Ligase?buffer 5ul；

pGEM-T(50g/ul) 0.5ul；

Add the PCR product 3.5ul behind the A;

T4 dna ligase (Promega M1801) 1ul;

After the incubated at room 1 hour, be used for transformation experiment.

2. transform

Getting 5ul connection product joins in the 100ul Top10 competence, ice bath 30 minutes, hatched 90 seconds for 42 ℃, ice bath is after 2 minutes again, it is transferred in the 0.5ml LB nutrient solution, cultivated 45 minutes under 37 ℃, 100rpm condition, draw 50ul then and evenly coat on the 10cm LB flat board of handling through IPTG and X-gal, cultivated 18 hours under 37 ℃ of conditions.

3. recombinate the selecting and identifying of positive bacterium colony

Select 8～10 single bacterium colonies of finely disseminated white and go into respectively in the 5ml liquid LB substratum, cultivated 12 hours under 37 ℃, 250rpm condition, when treating that substratum becomes muddiness, divide and get 1ul bacterium liquid and be used for PCR and identify reorganization, preserve under 4 ℃ of conditions of all the other bacterium liquid.

The Wizard Plus SV Minipreps DNA Purification System test kit of using Promega company extracts the plasmid that PCR identifies positive strain, identifies with restriction enzyme M1uI and NheI.The positive plasmid of plasmid that 3kb and two DNA bands of 1.3kb occur.The results are shown in Figure 2.Among Fig. 2, M:marker; 1: the plasmid of positive strain; 2:pGEM-T carrier (negative control).With positive plasmid called after pGEMT-uPA.

Send Beijing three biological limited liability company of rich polygala roots to check order the pGEMT-uPA plasmid, sequencing result shows and has obtained correct uPA.

Two, preparation Adeno-TRE-uPA adenovirus

System for use in carrying is the Adeno-X Tet-on expression system of Clontech company, article No. 631050.

1, makes up pTRE-uPA

With NheI and KpnI the uPA fragment is scaled off from the pGEMT-uPA plasmid, be connected on the skeleton of the pTRE-Shuttle2 carrier (Clontech, Adeno-X Tet-on expression system, article No. 631050) that digested with NheI and KpnI.Conversion reaction is the same.The bacterium colony of that resistance of picking card is cultivated, and enzyme is cut the screening positive clone (see figure 3), is pTRE-uPA.

2, make up Adeno-TRE-uPA

With restriction enzyme PI-Sce I and I-Ceu I the TRE-PminCMV-uPA fragment of 3kb is scaled off from pTRE-uPA, be cloned into adenovirus carrier Adeno-X (Adeno-X Tet-on expressionsystem, article No. 631050) in, obtains Adeno-TRE-uPA.Connect before the product conversion, connect product with swaI digestion and eliminate background.The bacterium colony of picking Amp resistance is cultivated, the PCR screening positive clone, and positive colony is cut the affirmation (see figure 4) with enzyme.

3, packing is produced the Adeno-TRE-uPA adenovirus

1) transfection is 12-24 hour before, inoculation (1-2) * 10 in the 6cm culture dish ⁶HEK293 cell (Adeno-XTet-on expression system, article No. 631050) is in order to raise the efficiency transfection when cell 50%-70%.

2) cell is at 37 ℃, 5%CO ₂Condition is cultivated.

3) each 6cm culture dish is with the Adeno-TRE-uPA of calcium phosphate or liposome transfection 10ug Pac I digestion.

4) after one day, detect the cytopathy reaction every day.

5) after the week, transitional cell is to 15ml centrifuge tube (making sure to keep in mind not use trysinization), and the cell that pastes closely can scrape off transfer gently.

6) room temperature 1500g * 5min centrifugal enrichment cell.

7) supernatant discarded, resuspended being deposited among the aseptic PBS of 500ul.

8) three cracking cells of multigelation: in dry ice/ethanol bath, freeze, molten in 37 ℃ of water-baths, but do not reach 37 ℃, each back vortex concussion cell that melts.

9) after the freeze thawing three times, frozen in-20 ℃, or be used for for the 10th step.

10) add the 250ul cell lysate in the culture dish of a 6cm, directly lysate is added in the substratum, a Zhou Yihou should see the cytopathy reaction.

11) if the cell more than 50% comes off from culture dish, the cell lysate of-70 ℃ of frozen fronts if do not see the cytopathy reaction, illustrates that virus titer is too low.

4, amplification recombinant adenovirus

1) infect preceding 24 hours inoculation HEK 293 cells to the T75 culturing bottle, cell infects when growing to the 50-70% of culturing bottle.

2) cell is at 37 ℃, 5%CO ₂Condition is cultivated.

3) second day, change liquid with the substratum that contains adenovirus, the virus of the corresponding 1-5pfu of each cell.

4) 37 ℃, 5%CO ₂Condition was cultivated 90 minutes.

5) change the fresh growth medium of 10ml.

6) 37 ℃, 5%CO ₂Condition was cultivated 3-4 days.

7) detect the cytopathy reaction, when 50% cell detachment, the transitional cell suspension centrifuge tube of falling 15ml makes sure to keep in mind not use trysinization, and the cell that pastes closely can scrape off transfer gently.

8) freeze-thaw method results virus.

9) (Clontech Cat.No.631028) measures virus titer with Adeno-X Rapid Titer Kit.

The preparation of embodiment 3, liver injury mouse

One, the preparation of Alb-rtTA mouse

(1) the segmental enzyme of Alb-rtTA cut, recovery and purifying

1, cutting the pAlb-rtTA-polyA plasmid with restriction enzyme SacI and 37 ℃ of enzymes of KpnI spends the night.

2, enzyme is cut product and is carried out 1% agarose gel electrophoresis.Electrophoresis result is seen Fig. 5.Downcut the band of 3.5kb size, reclaim test kit (ancient cooking vessel state) with DNA glue and reclaim DNA.

3, be further purified dna fragmentation with Promega Wizard DNA clear-up kit (Cat:A 7280).

4, the DNA behind TE (pH 7.5 for 7mM Tris-HCl, 0.15mM EDTA) the dissolving purifying.

(2) preparation is for the DNA sample of procaryotic injection

Measure the OD value of DNA sample, DNA is diluted to 2-5ng/ul with TE; 12000g * 10min (4 ℃) is centrifugal; The careful sucking-off of last 1/3 part solution with after centrifugal is sub-packed in some centrifuge tubes, and is frozen in-20 ℃ of refrigerators.

(3) the protokaryon microinjection is made the Alb-rtTA transgenic mice

To use the protokaryon microinjection by the pAlb-rtTA linearizing dna fragmentation that as above the step purifying is good and make the pAlb-rtTA transgenic mice, concrete experimental procedure is as follows:

1, choose female ICR mouse in 4～5 ages in week (available from dimension tonneau China), the vaginal tract sealing, as donor, about afternoon 3:00, every mouse peritoneal injection 10IU pregnant mare serum (PMSG);

2, after 47～48 hours, every mouse peritoneal is injected people's trophoblast hormone HCG of 10 IU, and mates with normal public mouse; The only of the right age female mouse of peek (more than 2 monthly ages) is chosen the female mouse of oestrusing of vaginal tract flush as acceptor in addition, mates with the public mouse of ligation;

3, observe donor, acceptor before the next morning 9:00, have smart bolt person to take out standby.The acceptor cage is taken out and is performed quarantine measures;

4, about 10:30, disconnected neck is put to death donor, and whole uterine tube is taken out in operation, puts into and contains 0.05% Unidasa M2 liquid, and microscopically is torn ampulla of uterine tube with tweezers, and zygote is promptly together flowed out in company with granulosa cell;

5, examine the zygote that is placed in the Unidasa M2 liquid, when the granulosa cell around the zygote breaks away from,, put into M2 liquid and wash, be placed at last in the M16 liquid and put into 5%CO the zygote sucking-off ₂, 37 ℃ of incubators are cultivated;

6, examine under a microscope, it is full to select cell, and zona pellucida is clear, and the apparent zygote of male pronucleus is stand-by;

7, install and to hold ovum pin and entry needle, make its end be parallel to Stage microscope, splash into a M2 liquid, cover paraffin oil, move into zygote to be injected in the central authorities of depression slide.The bubble of DNA in entry needle should be walked by formerly whole bullets.

8, under high power lens, entry needle is touched ovum holding tube, DNA is slowly flowed out and control its flow; Pressure-vaccum zygote makes it be in the optimum position repeatedly, and entry needle is thrust the male pronucleus of zygote, promptly withdraws from until seeing that protokaryon expands.The zygote of injecting He do not inject is placed apart up and down, be unlikely to mix and stir, after injection finishes, put into 5%CO ₂, 37 ℃ of incubators are cultivated.

9, with acceptor anesthesia, injection measurement is 1% vetanarcol 0.01ml/g, abdominal injection.Operation is taken out ovary and is connected uterine tube, fixes with fatty tweezer, finds the uterine tube opening at microscopically.Draw injection after cultivate the zygote that survives, the absorption method is to inhale one section long M2 earlier, inhales a bubble, draws zygote then, closely arranges as far as possible, inhales one section liquid again, inhales a bubble, inhales one section liquid again, three bubbles of totally four sections liquid.Remove long that section liquid, remaining liquid is roughly about 1cm, about bubble 0.2cm.To transplant the mouth of pipe and insert the uterine tube mouth, gently the liquid in the grafts will be blown into, and see that ampulla of uterine tube expands and clearly see three bubbles, promptly transplant successfully.Ovary is put back to the abdominal cavity together with uterine tube, suture muscles and skin.

10, acceptor was weighed once every a week, when increasing than weighing for the first time for the second time, can tentatively judge pregnancy.The newborn mouse childbirth in 19～21 days of operation back is transgenosis (Founder) mouse of establishing.

(4) evaluation of male mouse

1, PCR identifies

Positive mouse tail genomic dna is carried out pcr analysis, identify whether it is the transgenic positive mouse.

Adopt Alb-rtTA to stride gene primer and carry out the PCR detection.Upstream primer is based on Alb promoter sequence (Alb/p), and downstream primer (primer II) is based on the rtTA gene order, and target fragment is 700bp.Primer sequence is as follows:

Upstream primer (primer I): 5 '-tgg ctg aaa gtt act gtg gga-3 ';

Downstream primer (primer II): 5 '-aga tgg tgc gct cct gga cgt-3 '.

In 32 positive mouse, have 4 PCR and be accredited as the positive, be transgenic mice.The PCR qualification result of 4 transgenic mices is seen Fig. 6.Among Fig. 6, PC: plasmid pAlb-rtTA-polyA (positive control); 1-5,2-9,2-10, a 4-6:4 transgenic mice; NC:ICR mouse (negative control).

2, RT-PCR identifies

The procaryotic injection transgenic method, the purpose fragment is a random integration, the situation of transgene silencing and express spectra mistake takes place sometimes, for the rtTA that confirms to obtain transgenic mice whether express and express spectra whether correct, the method that adopts RT-PCR tissue to the heart, liver, lung, kidney and the pancreas of mouse on the mRNA level detects.

4 transgenic mices are all expressed rtTA, and express spectra is correct, and are strong special at the liver expression goal gene, except liver, have only kidney that faint expression is arranged.Wherein the qualification result of a transgenic mice is seen Fig. 7.

Two, backcross and obtain the Alb-rtTA transgenic mice of immune deficiency background

In order to accept the external source liver cell, need the mouse background of this model conversion, and have only the mouse of immune deficiency could be by the adenovirus repeated infection to immune deficiency.Scid/bg is a kind of immunodeficient mouse, no sophisticated T cell and B cell in the body, NK cell disappearance.This mouse can be accepted foreign cell and tissue, thus we with the Alb-rtTA mouse through repeatedly backcrossing, be converted to the Scid/bg background.

(1) the Alb-rtTA transgenic mice under the conventional environment is transferred to the SPF environment by the c-section purification

1, selects the Alb-rtTA transgenic mice of the mating purification object of cutting open the belly not in 6～8week age.Post-coitum is observed cloudy bolt after second day continuous a couple of days, and writes down cloudy bolt and the date occurs, and as becoming pregnant the date, 19 ± 1d is as the expected date of childbirth for gestation, i.e. the c-section date.

2, to the pregnant mouse of the expected date of childbirth, after disconnected cervical vertebra is put to death, soak 2～3s with 2% Peracetic Acid immediately, promptly on operating table, fix, cut open the belly, clamp nearly anus virgin uterine neck with aseptic mosquito forceps, cut off the uterus with aseptic scissors from anus one side of mosquito forceps again, after taking out whole uterus, put 2% Peracetic Acid liquid and soak 1～2s, in the aqueduct liquid (thimerosal) of operation shield retaining, the uterus is transferred in the aseptic operation shield retaining rapidly, cut the uterus, take out the young baby, with sterile gauze little young whole body is wiped clean, scarlet to little young whole body again, till freely breathing.

3, the young baby alive of c-section gained, (tie up the aseptic ICR mouse that tonneau China Experimental Animal Center is raised, mating shifts to an earlier date in 3～4d) boxes than the Alb-rtTA mouse of cutting open the belly, and every nurse is for about 8 of breasts to put into the nurse mouse, and produce after little son fully contacts with nurse, put to death nurse and produce little son.

4, be transferred to aseptic shield retaining from the operation shield retaining and raise, 21d is from breast.After every microorganism detection is qualified, is transferred in the SPF barrier system and raises.

5, changed the Alb-rtTA mouse of raising in the SPF barrier system over to, per season censorship is (5～10) once, make every microorganism detection.

(2) the Alb-rtTA transgenic mice of acquisition scid/bg background

The transgenic mice and the scid/bg mouse (available from dimension tonneau China) that purify are backcrossed under the SPF environment, and mouse and scid/bg mouse that the back method by PCR of at every turn backcrossing detects transgenic positive are backcrossed next time.After backcrossing for 3 times, the scid gene test of mouse is found that transgenic mice has carried pure and mild scid site, IgG is detected find that content far below normal mouse, illustrates that mouse has obtained the background of immune deficiency.In order further to reduce the immunizing power of mouse, mouse is continued to backcross.The Alb-rtTA mouse of the scid/bg background that obtains after backcrossing for 5 times carries out the test of step 3.

Three, the acquisition of liver injury model mouse

The Alb-rtTA transgenic mice of 10 immune deficiency backgrounds is divided into two groups, 5 every group (first group and second group).10 ICR mouse are divided into two groups, 5 every group (the 3rd group and the 4th group).First group and the 3rd group of mouse in the following whole test stage with the Dox aqueous solution (1mg/ml) as tap water.Second group and the 4th group of mouse in the following whole experimental phase with ortho-water as tap water.Each is organized mouse and injects adenovirus Adeno-TRE-uPA 5 * 10 respectively ⁹/ only, dissect mouse after three days and get liver organization, carry out RT-PCR and immunohistochemical analysis.

For the injection adenovirus, DOX inductive Alb-rtTA transgenic mice was opened the abdominal cavity discovery in 1 day behind virus injection, and the mouse liver outward appearance is pale, and this hepar damnification feature with the Alb-uPA mouse of report is consistent.The pcr analysis result shows that respectively organizing mouse is all infected by adenovirus uPA.Immunohistochemical analysis the results are shown in Figure 8.Among Fig. 8, A) wild-type mice; B) Dox inductive wild-type mice; C) Alb-rtTA transgenic positive mouse; D) Dox inductive Alb-rtTA transgenic mice.Have only DOX inductive Alb-rtTA mouse can detect the expression of uPA, the liver cell of statistical result showed more than 80% all expressed uPA.And in its excess-three kind mouse, all do not detect uPA.The RT-PCR analytical results is seen Fig. 9.Among Fig. 9, WT) wild-type mice; WT+DOX) Dox inductive wild-type mice; Alb-rtTA) Alb-rtTA transgenic positive mouse; Alb-rtTA+Dox) Dox inductive Alb-rtTA transgenic mice.The result of RT-PCR conforms to the immunohistochemical methods result, promptly has only two elements to exist simultaneously and just can start expression with uPA under the DOX inductive situation.The HE coloration result is seen Figure 10.Among Figure 10, A) DOX inductive wild-type mice; B) DOX inductive Alb-rtTA transgenic mice.The wild-type mice cellular form is normal, and the damage characteristic of cavity sample appears in the stem cell of Alb-rtTA transgenic mice.

Four, the hepatocellular reconstruction of external source

1, the acquisition of external source liver cell

With tribromoethyl alcohol with GFP transgenic mice (available from dimension tonneau China) anesthesia after, open the abdominal cavity, the separation gate vein, the scalp acupuncture that will have flexible pipe thrusts portal vein, bulldog clamp is fixed.Flexible pipe connects syringe, pours into PBE50ml and PBC 50ml successively.Cut liver then, WB washes liver.Tear the coating of liver with tweezers, sieve filtration cell with cell.800rpm * 1min is centrifugal, carefully sops up supernatant, uses the WBD re-suspended cell, and is centrifugal; Use DF12 (GIBICO) washed twice again, trypan blue dyeing detects the liver cell survival rate.

Perfusion damping fluid (PB) prescription (PH7.4): NaCl 27g, KCl 1.26g, NaHCO ₃6.3g, Glucose2.4g, Hepes 14.34g, H ₂O 3000ml.

The prescription of PBE (PH7.4): EDTA 0.37g, PB 500ml.

The prescription of PBC (PH7.4): Collagenase 4.2g, CaCl ₂-2H ₂O 0.275g, PB 500ml.

The prescription of WB (PH7.4): NaCl 17.5g, KCl 1.15g, CaCl ₂-2H ₂O 0.325g, Hepes 5.95g, BSA 2.5g, H ₂O 2500ml.

The prescription of WBD (PH7.4): MgCl ₂-6H ₂O 0.15g, MgSO ₄-7H ₂O 0.15g, DNase 0.15g, WB 1500ml.

2, the hepatocellular reconstruction of external source

The whole test stage as tap water, is respectively injected adenovirus Adeno-TRE-uPA 5 * 10 with the Alb-rtTA transgenic mice of 10 immune deficiency backgrounds with the Dox aqueous solution (1mg/ml) ⁹/ only, obtain the liver injury mouse model.

At second day (testing the 4th day) liver transplantation cell of injection adenovirus, every mouse transplants 5 * 10 ⁵Individual.Transplant per 7 days every the injected in mice 5 * 10 in back ⁹Adenovirus promotes the propagation of foreign cell.The per injection adenovirus detects the effect of transplanting after 7 days.

Hepatocellular implantation method is as follows: with tribromoethyl alcohol model mice is anaesthetized, side position is on experiment table.Spleen position, left side skin is picked hair, cut off skin and muscle layer, the about 1cm of openings of sizes.Isolate spleen with tweezers, from the spleen end liver cell is injected spleen slowly with syringe.After having annotated, have an acupuncture treatment the position, hole in case hemorrhage with toe-in.Then spleen is put back to original position, successively suture muscles layer and skin layer.Mouse after the transplanting is put on the insulation tire and is incubated to reviving.

The results are shown in Figure 11.Among Figure 11, P1-P5 promotes once to five times for transplanting back adenovirus Adeno-TRE-uPA.For the injection adenovirus, and DOX inductive Alb-rtTA transgenic mice, can observe liver 4 days the time behind virus injection has point-like regeneration, and the regeneration zone of joint knot shape appears in liver in the time of 7 days, and whole liver recovers fully after 10 days.The GFP liver cell is rebuild the mouse liver of uPA damage.After promoting for the first time, can detect the cell mass of 4-5 cell in the liver of mouse; After promoting for the second time, these cell masses are further bred; Promote for three times the back cell mass to reach more than 100 cell, the reconstruction degree reaches 20%; Promote for four times the back cell mass to begin to link in flakes, the reconstruction rate reaches 50%; Continue to promote that with adenovirus uPA foreign cell reconstruction rate is near 80%.

Sequence table

＜110〉Peking University

＜120〉making method of adjustable liver damage animal model and special DNA fragment thereof

<130>CGGNARY81956

<160>7

<210>1

<211>1228

<212>DNA

＜213〉artificial sequence

<220>

<223>

<400>1

gaattcacca?tgtctagact?ggacaagagc?aaagtcataa?acggcgctct?ggaattactc 60

aatggagtcg?gtatcgaagg?cctgacgaca?aggaaactcg?ctcaaaagct?gggagttgag 120

cagcctaccc?tgtactggca?cgtgaagaac?aagcgggccc?tgctcgatgc?cctgccaatc 180

gagatgctgg?acaggcatca?tacccacttc?tgccccctgg?aaggcgagtc?atggcaagac 240

tttctgcgga?acaacgccaa?gtcattccgc?tgtgctctcc?tctcacatcg?cgacggggct 300

aaagtgcatc?tcggcacccg?cccaacagag?aaacagtacg?aaaccctgga?aaatcagctc 360

gcgttcctgt?gtcagcaagg?cttctccctg?gagaacgcac?tgtacgctct?gtccgccgtg 420

ggccacttta?cactgggctg?cgtattggag?gaacaggagc?atcaagtagc?aaaagaggaa 480

agagagacac?ctaccaccga?ttctatgccc?ccacttctga?gacaagcaat?tgagctgttc 540

gaccggcagg?gagccgaacc?tgccttcctt?ttcggcctgg?aactaatcat?atgtggcctg 600

gagaaacagc?taaagtgcga?aagcggcggg?ccggccgacg?cccttgacga?ttttgactta 660

gacatgctcc?cagccgatgc?ccttgacgac?tttgaccttg?atatgctgcc?tgctgacgct 720

cttgacgatt?ttgaccttga?catgctcccc?gggtaactaa?gtaaggatcc?agacatgata 780

agatacattg?atgagtttgg?acaaaccaca?actagaatgc?agtgaaaaaa?atgctttatt 840

tgtgaaattt?gtgatgctat?tgctttattt?gtaaccatta?taagctgcaa?taaacaagtt 900

aacaacaaca?attgcattca?ttttatgttt?caggttcagg?gggaggtgtg?ggaggttttt 960

taaagcaagt?aaaacctcta?caaatgtggt?atggctgatt?atgatcctgc?aagcctcgtc 1020

gtctggccgg?accacgctat?ctgtgcaagg?tccccggacg?cgcgctccat?gagcagagcg 1080

cccgccgccg?aggcaagact?cgggcggcgc?cctgcccgtc?ccaccaggtc?aacaggcggt 1140

aaccggcctc?ttcatcggga?atgcgcgcga?ccttcagcat?cgccggcatg?tcccctggcg 1200

gacgggaagt?atcagctcga?ccaagctt 1228

<210>2

<211>248

<212>PRT

＜213〉artificial sequence

<220>

<223>

<400>2

Met?Ser?Arg?Leu?Asp?Lys?Ser?Lys?Val?Ile?Asn?Gly?Ala?Leu?Glu?Leu

1 5 10 15

Leu?Asn?Gly?Val?Gly?Ile?Glu?Gly?Leu?Thr?Thr?Arg?Lys?Leu?Ala?Gln

20 25 30

Lys?Leu?Gly?Val?Glu?Gln?Pro?Thr?Leu?Tyr?Trp?His?Val?Lys?Asn?Lys

35 40 45

Arg?Ala?Leu?Leu?Asp?Ala?Leu?Pro?Ile?Glu?Met?Leu?Asp?Arg?His?His

50 55 60

Thr?His?Phe?Cys?Pro?Leu?Glu?Gly?Glu?Ser?Trp?Gln?Asp?Phe?Leu?Arg

65 70 75 80

Asn?Asn?Ala?Lys?Ser?Phe?Arg?Cys?Ala?Leu?Leu?Ser?His?Arg?Asp?Gly

85 90 95

Ala?Lys?Val?His?Leu?Gly?Thr?Arg?Pro?Thr?Glu?Lys?Gln?Tyr?Glu?Thr

100 105 110

Leu?Glu?Asn?Gln?Leu?Ala?Phe?Leu?Cys?Gln?Gln?Gly?Phe?Ser?Leu?Glu

115 120 125

Asn?Ala?Leu?Tyr?Ala?Leu?Ser?Ala?Val?Gly?His?Phe?Thr?Leu?Gly?Cys

130 135 140

Val?Leu?Glu?Glu?Gln?Glu?His?Gln?Val?Ala?Lys?Glu?Glu?Arg?Glu?Thr

145 150 155 160

Pro?Thr?Thr?Asp?Ser?Met?Pro?Pro?Leu?Leu?Arg?Gln?Ala?Ile?Glu?Leu

165 170 175

Phe?Asp?Arg?Gln?Gly?Ala?Glu?Pro?Ala?Phe?Leu?Phe?Gly?Leu?Glu?Leu

180 185 190

Ile?Ile?Cys?Gly?Leu?Glu?Lys?Gln?Leu?Lys?Cys?Glu?Ser?Gly?Gly?Pro

195 200 205

Ala?Asp?Ala?Leu?Asp?Asp?Phe?Asp?Leu?Asp?Met?Leu?Pro?Ala?Asp?Ala

210 215 220

Leu?Asp?Asp?Phe?Asp?Leu?Asp?Met?Leu?Pro?Ala?Asp?Ala?Leu?Asp?Asp

225 230 235 240

Phe?Asp?Leu?Asp?Met?Leu?Pro?Gly

245

<210>3

<211>2372

<212>DNA

＜213〉artificial sequence

<220>

<223>

<400>3

ctggagctcc?accgcggtgg?cggccgctct?agcttcctta?gcatgacgtt?ccactttttt 60

ctaaggtgga?gcttacttct?ttgatttgat?cttttgtgaa?acttttggaa?attacccatc 120

ttcctaagct?tctgcttctc?tcagttttct?gcttgctcat?tccacttttc?cagctgaccc 180

tgccccctac?caacattgct?ccacaagcac?aaattcatcc?agagaaaata?aattctaagt 240

tttatagttg?tttggatcgc?ataggtagct?aaagaggtgg?caacccacac?atccttaggc 300

atgagcttga?ttttttttga?tttagaacct?tcccctctct?gttcctagac?tacactacac 360

attctgcaag?catagcacag?agcaatgttc?tactttaatt?actttcattt?tcttgtatcc 420

tcacagccta?gaaaataacc?tgcgttacag?catccactca?gtatcccttg?agcatgaggt 480

gacactactt?aacataggga?cgagatggta?ctttgtgtct?cctgctctgt?cagcagggca 540

ctgtacttgc?tgataccagg?gaatgtttgt?tcttaaatac?catcattccg?gacgtgtttg 600

ccttggccag?ttttccatgt?acatgcagaa?agaagtttgg?actgatcaat?acagtcctct 660

gcctttaaag?caataggaaa?aggccaactt?gtctacgttt?agtatgtggc?tgtagaaagg 720

gtatagatat?aaaaattaaa?actaatgaaa?tggcagtctt?acacattttt?ggcagcttat 780

ttaaagtctt?ggtgttaagt?acgctggagc?tgtcacagct?accaatcagg?catgtctggg 840

aatgagtaca?cggggaccat?aagttactga?cattcgtttc?ccattccatt?tgaatacaca 900

cttttgtcat?ggtattgctt?gctgaaattg?ttttgcaaaa?aaaacccctt?caaattcata 960

tatattattt?taataaatga?attttaattt?atctcaatgt?tataaaaaag?tcaattttaa 1020

taattaggta?cttatatacc?caataatatc?taacaatcat?ttttaaacat?ttgtttattg 1080

agcttattat?ggatgaatct?atctctatat?actctatata?ctctaaaaaa?gaagaaagac 1140

catagacaat?catctatttg?atatgtgtaa?agtttacatg?tgagtagaca?tcagatgctc 1200

catttctcac?tgtaatacca?tttatagtta?cttgcaaaac?taactggaat?tctaggactt 1260

aaatatttta?agttttagct?gggtgactgg?ttggaaaatt?ttaggtaagt?actgaaacca 1320

agagattata?aaacaataaa?ttctaaagtt?ttagaagtga?tcataatcaa?atattaccct 1380

ctaatgaaaa?tattccaaag?ttgagctaca?gaaatttcaa?cataagataa?ttttagctgt 1440

aacaatgtaa?tttgttgtct?attttctttt?gagatacagt?tttttctgtc?tagctttggc 1500

tgtcctggac?cttgctctgt?agaccaggtt?ggtcttgaac?tcagagatct?gcttgcctct 1560

gccttgcaag?tgctaggatt?aaaagcatgt?gccaccactg?cctggctaca?atctatgttt 1620

tataagagat?tataaagctc?tggctttgtg?acattaatct?ttcagataat?aagtcttttg 1680

gattgtgtct?ggagaacata?cagactgtga?gcagatgttc?agaggtatat?ttgcttaggg 1740

gtgaattcaa?tctgcagcaa?taattatgag?cagaattact?gacacttcca?ttttatacat 1800

tctacttgct?gatctatgaa?acatagataa?gcatgcaggc?attcatcata?gttttcttta 1860

tctggaaaaa?cattaaatat?gaaagaagca?ctttattaat?acagtttaga?tgtgttttgc 1920

catcttttaa?tttcttaaga?aatactaagc?tgatgcagag?tgaagagtgt?gtgaaaagca 1980

gtggtgcagc?ttggcttgaa?ctcgttctcc?agcttgggat?cgacctgcag?gcatgcttcc 2040

atgccaaggc?ccacactgaa?atgctcaaat?gggagacaaa?gagattaagc?tcttatgtaa 2100

aatttgctgt?tttacataac?tttaatgaat?ggacaaagtc?ttgtgcatgg?gggtgggggt 2160

ggggttagag?gggaacagct?ccagatggca?aacatacgca?agggatttag?tcaaacaact 2220

ttttggcaaa?gatggtatga?ttttgtaatg?gggtaggaac?caatgaaatg?cgaggtaagt 2280

atggttaatg?atctacagtt?attggttaaa?gaagtatatt?agagcgagtc?tttctgcaca 2340

cagatcacct?ttcctatcaa?ccccgggatc?aa 2372

<210>4

<211>1414

<212>DNA

＜213〉Mus mouse kind (Mus musculus)

<400>4

gctgcagtca?ccgaactgct?gtctagagcc?cagcggcact?accatgaaag?tctggctggc 60

gagcctgttc?ctctgcgcct?tggtggtgaa?aaactctgaa?ggtggcagtg?tacttggagc 120

tcctgatgaa?tcaaactgtg?gctgtcagaa?cggaggtgta?tgcgtgtcct?acaagtactt 180

ctccagaatt?cgccgatgca?gctgcccaag?gaaattccag?ggggagcact?gtgagataga 240

tgcatcaaaa?acctgctatc?atggaaatgg?tgactcttac?cgaggaaagg?ccaacactga 300

taccaaaggt?cggccctgcc?tggcctggaa?tgcgcctgct?gtccttcaga?aaccctacaa 360

tgcccacaga?cctgatgcta?ttagcctagg?cctggggaaa?cacaattact?gcaggaaccc 420

tgacaaccag?aagcgaccct?ggtgctatgt?gcagattggc?ctaaggcagt?ttgtccaaga 480

atgcatggtg?catgactgct?ctcttagcaa?aaagccttct?tcgtctgtag?accaacaagg 540

cttccagtgt?ggccagaagg?ctctaaggcc?ccgctttaag?attgttgggg?gagaattcac 600

tgaggtggag?aaccagccct?ggttcgcagc?catctaccag?aagaacaagg?gaggaagtcc 660

tccctccttt?aaatgtggtg?ggagtctcat?cagtccttgc?tgggtggcca?gtgccgcaca 720

ctgcttcatt?caactcccaa?agaaggaaaa?ctacgttgtc?tacctgggtc?agtcgaagga 780

gagctcctat?aatcctggag?agatgaagtt?tgaggtggag?cagctcatct?tgcacgaata 840

ctacagggaa?gacagcctgg?cctaccataa?tgatattgcc?ttgctgaaga?tacgtaccag 900

cacgggccaa?tgtgcacagc?catccaggtc?catacagacc?atctgcctgc?ccccaaggtt 960

tactgatgct?ccgtttggtt?cagactgtga?gatcactggc?tttggaaaag?agtctgaaag 1020

tgactatctc?tatccaaaga?acctgaaaat?gtccgtcgta?aagcttgttt?ctcatgaaca 1080

gtgtatgcag?ccccactact?atggctctga?aattaattat?aaaatgctgt?gtgctgcgga 1140

cccagagtgg?aaaacagatt?cctgcaaggg?cgattctgga?ggaccgctta?tctgtaacat 1200

cgaaggccgc?ccaactctga?gtgggattgt?gagctggggc?cgaggatgtg?cagagaaaaa 1260

caagcccggt?gtctacacga?gggtctcaca?cttcctggac?tggattcaat?cccacattgg 1320

agaagagaaa?ggtctggcct?tctgatggcc?ctcaggtagc?tgagggaaga?aacagatggg 1380

tcacttgttc?ccatgctgac?cgtcctctct?gcaa 1414

<210>5

<211>433

<212>PRT

＜213〉Mus mouse kind (Mus musculus)

<400>5

Met?Lys?Val?Trp?Leu?Ala?Ser?Leu?Phe?Leu?Cys?Ala?Leu?Val?Val?Lys

1 5 10 15

Asn?Ser?Glu?Gly?Gly?Ser?Val?Leu?Gly?Ala?Pro?Asp?Glu?Ser?Asn?Cys

20 25 30

Gly?Cys?Gln?Asn?Gly?Gly?Val?Cys?Val?Ser?Tyr?Lys?Tyr?Phe?Ser?Arg

35 40 45

Ile?Arg?Arg?Cys?Ser?Cys?Pro?Arg?Lys?Phe?Gln?Gly?Glu?His?Cys?Glu

50 55 60

Ile?Asp?Ala?Ser?Lys?Thr?Cys?Tyr?His?Gly?Asn?Gly?Asp?Ser?Tyr?Arg

65 70 75 80

Gly?Lys?Ala?Asn?Thr?Asp?Thr?Lys?Gly?Arg?Pro?Cys?Leu?Ala?Trp?Asn

85 90 95

Ala?Pro?Ala?Val?Leu?Gln?Lys?Pro?Tyr?Asn?Ala?His?Arg?Pro?Asp?Ala

100 105 110

Ile?Ser?Leu?Gly?Leu?Gly?Lys?His?Asn?Tyr?Cys?Arg?Asn?Pro?Asp?Asn

115 120 125

Gln?Lys?Arg?Pro?Trp?Cys?Tyr?Val?Gln?Ile?Gly?Leu?Arg?Gln?Phe?Val

130 135 140

Gln?Glu?Cys?Met?Val?His?Asp?Cys?Ser?Leu?Ser?Lys?Lys?Pro?Ser?Ser

145 150 155 160

Ser?Val?Asp?Gln?Gln?Gly?Phe?Gln?Cys?Gly?Gln?Lys?Ala?Leu?Arg?Pro

165 170 175

Arg?Phe?Lys?Ile?Val?Gly?Gly?Glu?Phe?Thr?Glu?Val?Glu?Asn?Gln?Pro

180 185 190

Trp?Phe?Ala?Ala?Ile?Tyr?Gln?Lys?Asn?Lys?Gly?Gly?Ser?Pro?Pro?Ser

195 200 205

Phe?Lys?Cys?Gly?Gly?Ser?Leu?Ile?Ser?Pro?Cys?Trp?Val?Ala?Ser?Ala

210 215 220

Ala?His?Cys?Phe?Ile?Gln?Leu?Pro?Lys?Lys?Glu?Asn?Tyr?Val?Val?Tyr

225 230 235 240

Leu?Gly?Gln?Ser?Lys?Glu?Ser?Ser?Tyr?Asn?Pro?Gly?Glu?Met?Lys?Phe

245 250 255

Glu?Val?Glu?Gln?Leu?Ile?Leu?His?Glu?Tyr?Tyr?Arg?Glu?Asp?Ser?Leu

260 265 270

Ala?Tyr?His?Asn?Asp?Ile?Ala?Leu?Leu?Lys?Ile?Arg?Thr?Ser?Thr?Gly

275 280 285

Gln?Cys?Ala?Gln?Pro?Ser?Arg?Ser?Ile?Gln?Thr?Ile?Cys?Leu?Pro?Pro

290 295 300

Arg?Phe?Thr?Asp?Ala?Pro?Phe?Gly?Ser?Asp?Cys?Glu?Ile?Thr?Gly?Phe

305 310 315 320

Gly?Lys?Glu?Ser?Glu?Ser?Asp?Tyr?Leu?Tyr?Pro?Lys?Asn?Leu?Lys?Met

325 330 335

Ser?Val?Val?Lys?Leu?Val?Ser?His?Glu?Gln?Cys?Met?Gln?Pro?His?Tyr

340 345 350

Tyr?Gly?Ser?Glu?Ile?Asn?Tyr?Lys?Met?Leu?Cys?Ala?Ala?Asp?Pro?Glu

355 360 365

Trp?Lys?Thr?Asp?Ser?Cys?Lys?Gly?Asp?Ser?Gly?Gly?Pro?Leu?Ile?Cys

370 375 380

Asn?Ile?Glu?Gly?Arg?Pro?Thr?Leu?Ser?Gly?Ile?Val?Ser?Trp?Gly?Arg

385 390 395 400

Gly?Cys?Ala?Glu?Lys?Asn?Lys?Pro?Gly?Val?Tyr?Thr?Arg?Val?Ser?His

405 410 415

Phe?Leu?Asp?Trp?Ile?Gln?Ser?His?Ile?Gly?Glu?Glu?Lys?Gly?Leu?Ala

420 425 430

Phe

<210>6

<211>129

<212>DNA

＜213〉artificial sequence

<220>

<223>

<400>6

taggcgtgta?cggtgggagg?cctatataag?cagagctcgt?ttagtgaacc?gtcagatcgc 60

ctggagacgc?catccacgct?gttttgacct?ccatagaaga?caccgggacc?gatccagcct 120

ccgcggccc 129

<210>7

<211>312

<212>DNA

＜213〉artificial sequence

<220>

<223>

<400>7

tttaccactc?cctatcagtg?atagagaaaa?gtgaaagtcg?agtttaccac?tccctatcag 60

tgatagagaa?aagtgaaagt?cgagtttacc?actccctatc?agtgatagag?aaaagtgaaa 120

gtcgagttta?ccactcccta?tcagtgatag?agaaaagtga?aagtcgagtt?taccactccc 180

tatcagtgat?agagaaaagt?gaaagtcgag?tttaccactc?cctatcagtg?atagagaaaa 240

gtgaaagtcg?agtttaccac?tccctatcag?tgatagagaa?aagtgaaagt?cgagctcggt 300

acccgggtcg?ag 312

Claims

1.DNA fragment I comprises to the downstream successively from the upstream: the antisense tetracycline of liver specific promoter, Tet-on gene expression system activates sub-encoding gene.

2. dna fragmentation I as claimed in claim 1 is characterized in that: described liver specific promoter is the mouse albumin promoter.

3. dna fragmentation I as claimed in claim 2, it is characterized in that: described dna fragmentation I also comprises the enhanser that is positioned at mouse albumin promoter upstream, described enhanser and mouse albumin promoter form fusion dna, and described fusion dna is following 1) or 2) or 3) or 4) DNA:

4. as arbitrary described dna fragmentation I in the claim 1 to 3, it is characterized in that: it is following 5 that described antisense tetracycline activates son) or 6) protein:

5. dna fragmentation I as claimed in claim 4 is characterized in that: described antisense tetracycline activates sub-encoding gene downstream and connects polyA, and it is following 7 that the antisense tetracycline of described connection polyA activates sub-encoding gene) or 8) or 9) dna molecular:

9) with sequence table in the dna sequence dna that limits of sequence 1 have 90% above homology, and coding has the protein DNA molecule of antisense tetracycline activation subfunction.

6. the recombinant expression vector that contains arbitrary described dna fragmentation I in the claim 1 to 5.

By tsiklomitsin response factor, promotor and the uPA activator encoding gene of Tet-off/Tet-on gene expression system by the order in upstream to the downstream dna fragmentation II that is connected in sequence.

8. dna fragmentation II as claimed in claim 7 is characterized in that: described uPA activator is following 10) or 11) protein:

9. dna fragmentation II as claimed in claim 8 is characterized in that: the encoding gene of described uPA activator is following 12) or 13) or 14) or 15) dna molecular:

10. as arbitrary described dna fragmentation II among the claim 7-9, it is characterized in that: described promotor is following 16) or 17) or 18) dna molecular:

11. as arbitrary described dna fragmentation II among the claim 7-10, it is characterized in that: described tsiklomitsin response factor is following 19) or 20) or 21) dna molecular:

12. contain the recombinant expression vector of arbitrary described dna fragmentation II in the claim 7 to 11.

13. recombinant expression vector as claimed in claim 12 is characterized in that: described recombinant expression vector imports adenovirus expression carrier with arbitrary described dna fragmentation II in the claim 7 to 11 and obtains.

14. recombinant expression vector as claimed in claim 13 is characterized in that: described recombinant expression vector is Adeno-TRE-uPA;

Described Adeno-TRE-uPA imports the Adeno-X carrier with arbitrary described dna fragmentation II in the claim 7 to 11 and obtains.

15. in mammalian cell, cultivate the recombinant adenovirus that arbitrary described recombinant expression vector obtains in the claim 12 to 14.

16. recombinant adenovirus as claimed in claim 15 is characterized in that: described mammalian cell is the HEK293 cell.

17. the making method of a liver damage animal model may further comprise the steps:

1) arbitrary described dna fragmentation I among the claim 1-5 is imported in the animal, obtain the transgenic founder animal;

2) described transgenic founder animal and scid/bg animal are backcrossed, obtain the animal of carrying dna fragmentation I of scid/bg background;

3) with the animal of carrying dna fragmentation I of claim 15 or 16 described recombinant adenovirus injection scid/bg backgrounds, obtain adjustable liver injury animal.

18. method as claimed in claim 17 is characterized in that: described animal is a mouse.