CN106676135A

CN106676135A - Alb-uPA-teton lentiviral vector and preparation method and application thereof

Info

Publication number: CN106676135A
Application number: CN201710051562.2A
Authority: CN
Inventors: 白家驷; 毛青
Original assignee: First Affiliated Hospital of TMMU
Current assignee: First Affiliated Hospital of TMMU
Priority date: 2017-01-23
Filing date: 2017-01-23
Publication date: 2017-05-17

Abstract

The invention relates to an Alb-uPA-teton lentiviral vector and a preparation method and application thereof. The Alb-uPA-teton lentiviral vector contains an albumin promoter PAlb and a urokinase type plasminogen activator gene uPA which are reversely connected, wherein the albumin promoter PAlb is used for regulating the expression of a tetracycline trans-activator rtTA, and the urokinase type plasminogen activator gene uPA is regulated by a PTight promoter to express. The Alb-uPA-teton lentiviral vector has the advantages that the ability of establishing a system is realized, and the expression of uPA in an NOD mouse is detected.

Description

Alb-uPA-teton slow virus carriers and its preparation method and application

Technical field

The invention belongs to gene function field, and in particular to Alb-uPA-teton slow virus carriers, the carrier is further related to Preparation method and application.

Background technology

In addition to people and primate, there is no natural toy infection host in people's hepatitis viruss, this serious obstruction Research to people's hepatites virus infections mechanism and antiviral drugs.At present only chimpanzee is to study people's hepatites virus infections most Good experiment in vivo model, but it is subject to many restrictions such as ethics and funds using shielded rare primate.Therefore It is a difficult problem urgently to be resolved hurrily to set up small animal model cheap and easy to get.The mice that can carry human liver tissue that developed recently gets up moves Thing model is that hepatites virus infections mechanism and antiviral drugs research provide good platform.It has been investigated that urokinase type is fine Molten protease (urokinaseplasminogen activator, uPA) mouse liver injury model, is the reason for carrying human liver tissue Think mouse model.But the urokinase type fibrinolytic protein enzyme transgenic mice obtained by transgenic method, filial generation is newborn Mortality rate is high, screening difficulty is big, the structure of the chimeric liver mouse model of people is hindered significantly, is promoted and is used.

The Chinese patent of Publication No. CN102628063A discloses PAlb-uPA slow virus carriers, and the carrier is thin in mice Born of the same parents' level can overexpression urokinase type plasminogen activator uPA, but through a large amount of animal procaryotic injection experimental verifications, The filial generation (founder0) for obtaining transgenic mice by the carrier fails to filter out the transgenic mice for carrying purpose fragment, that is, exist The biological effectiveness of genes of interest can not be expressed in mice body.Producing reason may be as follows：(1) the carrying genes of interest for building Slow viruss plasmid, there occurs routed solution Jing after in-situ injection is transferred in mice body to germ cell, it is impossible to complete or weigh completely Newly it is inserted randomly in mouse genome, causes purpose fragment to be beyond expression；(2) slow viruss of the carrying genes of interest for building Plasmid, the injection of Jing mouse fertilized eggs cell in-situ, random fashion is incorporated in mouse genome, and purpose fragment puts in order generation Change, it is impossible to which the biological efficiency of purpose fragment is expressed in the requirement according to original design；(3) may put in order and not send out Raw to change, the simply arrangement between purpose fragment is affected by genome space structure, disturbs the expression of purpose fragment.

Therefore, it is badly in need of being improved existing pAlb-uPA slow virus carriers, is obtained in that filial generation carries purpose fragment Transgenic mice, so as to prepare urokinase type fibrinolytic protein enzyme mouse liver injury model, there is provided carry human liver tissue receptor Mice.

The content of the invention

In view of this, an object of the present invention be provide it is a kind of with independently build be ability Alb-uPA-TetOn Slow virus carrier；The second object of the present invention is the preparation method for providing Alb-uPA-TetOn slow virus carriers；The present invention's The three of purpose are the application for providing Alb-uPA-TetOn slow virus carriers in prepare transgenosis mice；The purpose of the present invention Four be application that offer is prepared in urokinase type fibrinolytic protein enzyme mouse liver injury model.

To reach above-mentioned purpose, the present invention provides following technical scheme：

Alb-uPA-TetOn slow virus carriers, the Alb-uPA-TetOn slow virus carriers contain the white egg of Opposite direction connection White promoter PAlb and urokinase type plasminogen activator gene uPA, the albumin promoter PAlb regulation and control tetracycline is anti- Formula activates sub- rtTA expression, and the urokinase type plasminogen activator gene uPA is expressed by PTight promoter regulations；

The nucleotide sequence of the albumin promoter PAlb is as shown in SEQ ID NO.34；The urokinase-type plasminogen The nucleotide sequence of activator gene uPA is as shown in SEQ ID NO.33；The nucleoside of the tetracycline transactivator rtTA Acid sequence is as shown in SEQ ID NO.31；The nucleotide sequence of the PTight promoteres is as shown in SEQ ID NO.32.

In the present invention, the albumin promoter PAlb and PTight promoter of the Alb-uPA-TetOn slow virus carriers 5 ' end be connected with insulator.

In the present invention, the terminator for terminating urokinase type plasminogen activator gene uPA expression is rabbit beta Globulin PolyA, the terminator for terminating tetracycline transactivator rtTA expression is bovine growth hormone gene polyA.

Most preferably, the nucleotide sequence of the Alb-uPA-TetOn slow virus carriers is as shown in SEQ ID NO.26.

2nd, the preparation method of the Alb-uPA-TetOn slow virus carriers, comprises the steps：

(1) will clone's urokinase type fibrinolytic protein enzyme gene uPA, rabbit beta Globulin PolyA and bovine growth hormone gene PolyA is connected on the pTRE Tight plasmids of Jing EcoR I and the double digestions of Sal I by SLIC technologies, obtains 1969-pTRE- UPA-PA transition vectors；

(2) albumin promoter Alb and tetracycline transactivator rtTA are connected to into Jing restriction endonucleases by SLIC technologies On the 1969-pTRE-uPA-PA excessive vectors of AscI enzyme action, 1969-PTRE-uPA-rTTA-Alb excessive vectors are obtained；

(3) 1969-PTRE-uPA-rTTA-Alb excessive vectors are reclaimed Jing after Xhol and Sacll enzyme action 5549bp fragments, Then recovery product is connected with the insulator with Xhol and Sacll enzyme action sticky ends, obtains final product Alb-uPA-TetOn slow viruss Carrier.

Preferably, the cloning process of urokinase type fibrinolytic protein enzyme gene uPA is as follows：It is with PAlb-uPA slow virus carriers Template, sequence shown in SEQ ID NO.1 and SEQ ID NO.2 carries out amplification acquisition for primer；

Rabbit beta Globulin PolyA cloning process is as follows：With PAlb-uPA slow virus carriers as template, SEQ ID NO.3 and Sequence shown in SEQ ID NO.4 carries out amplification acquisition for primer；

Bovine growth hormone gene polyA cloning process is as follows：With PAlb-uPA slow virus carriers as template, SEQ ID Sequence shown in NO.5 and SEQ ID NO.6 carries out amplification acquisition for primer；

Albumin promoter Alb cloning process is as follows：With PAlb-uPA slow virus carriers as template, SEQ ID NO.9 and Sequence shown in SEQ ID NO.10 carries out amplification acquisition for primer；

Tetracycline transactivator rtTA cloning process is as follows：With PAlb-uPA slow virus carriers as template, SEQ ID Sequence shown in NO.7 and SEQ ID NO.8 carries out amplification acquisition for primer.

3rd, application of the Alb-uPA-TetOn slow virus carriers in prepare transgenosis mice.

4th, the Alb-uPA-TetOn slow virus carriers are in urokinase type fibrinolytic protein enzyme mouse liver injury model is prepared Application.

The beneficial effects of the present invention is：The invention discloses Alb-uPA-TetOn slow virus carriers, the carrier by It is improved on existing PAlb-uPA slow virus carriers, purpose fragment pAlb and uPA is carried out into Opposite direction connection, and in whole purpose The two ends of fragment add respectively insulator, and to block because of influencing each other that radom insertion site difference causes, the carrier has only Vertical building is ability, and slow viruss plasmid energy normal expression uPA after Alb-uPA-TetOn slow virus carrier in-situ injection is lived biology Property, can be consequently used for preparing urokinase type fibrinolytic protein enzyme mouse liver injury model；To people's hepatites virus infections mechanism and anti- Virus drugs research is significant.

Description of the drawings

In order that the purpose of the present invention, technical scheme and beneficial effect are clearer, the present invention provides drawings described below and carries out Explanation：

Fig. 1 is that (1,2,3,4 are respectively select four to 1969-pTRE-uPA-PA carrier digestion verification results in figure Positive colony；DL represents markerDL2000；T represents marker T14).

Fig. 2 be 1969-PTRE-uPA-rTTA-Alb carriers PCR detection figure (DL represents Marker DL2000,1-1 in figure, 1-2,1-3,1-4,1-6,1-7,1-8,2-1,2-2,2-3,2-4,2-5,2-6,3-1,3-2,3-3,3-4,3-7,3-8,4-2, 4-3,4-4,4-7 and 4-8 represent respectively different positive monoclonals, purpose fragment 530bp).

Fig. 3 carries out respectively enzyme action result (1- for 1969-PTRE-uPA-rTTA-Alb carriers with EcoRl, ApaLl and Sacl 4,1-8 represent different strains plasmid, and DL represents markerDL2000；T represents marker T14).

Fig. 4 represents 1969-Alb-uPA-teton-final plasmid maps.

Fig. 5 be 1969-Alb-uPA-teton-final plasmid PCR testing results (DL represents Marker DL2000 in figure, 1-1,1-5,1-6,2-2,2-4,2-5,3-1,3-2,3-3,3-4,3-6,3-7,4-2,4-3,4-6 represent different positive Dan Ke It is grand).

Fig. 6 is that (DL is represented 1969-Alb-uPA-teton-final plasmid ApaLl, Sphl and EcoRl enzyme action result markerDL2000；T represents marker T14, and 3-1,3-2,3-3 and 3-4 represent different strains plasmid).

Fig. 7 is that (15,17 represent positive transgenic Mus to preliminary experiment transgenic mice PCR testing results, and P is expressed as positive right According to B6 is negative control, and N is blank；Figure below is β-actin internal references).

Fig. 8 is preliminary experiment transgenic mice PCR testing results (21,25,27,28,30,38,39,47,49,50,54 and 67 Positive transgenic Mus are represented, P is expressed as positive control, and B6 is negative control, and N is blank；Figure below is β-actin internal references).

Specific embodiment

Below in conjunction with accompanying drawing, the preferred embodiments of the present invention are described in detail.

Embodiment 1, plasmid construction

1st, PrimerSTAR PCR amplifications associated clip

With PAlb-uPA slow virus carriers as template, sequence shown in table 1 enters performing PCR amplification clone's urokinase type for primer pair Fibrinolytic protein enzyme gene uPA, albumin promoter Alb (pAlb), tetracycline transactivator (rtTA), rabbit beta Globulin PolyA (Rabbit's beta globin PolyA and bovine growth hormone gene polyA (bGHPA), wherein albumin promoter PAlb Nucleotide sequence is as shown in SEQ ID NO.34；The nucleotide sequence such as SEQ of urokinase type plasminogen activator gene uPA Shown in ID NO.33；The nucleotide sequence of tetracycline transactivator rtTA is as shown in SEQ ID NO.31；PTight promoteres Nucleotide sequence as shown in SEQ ID NO.32；As shown in table 2, PCR reaction conditions are as shown in table 3 for PCR reaction systems.

The primer that table 1.PCR reactions are used

Table 2.PCR reaction systems

Reacted constituent	Single reaction addition (μ l)
		5×Tap buffer	10
DNTP Mix, 2.5mM each	4
		Forward primer	1
Downstream primer	1
		Module DNA (50ng/ μ L)	1
PrimeSTAR archaeal dna polymerases	0.5
		The water of nuclease free	32.5
Cumulative volume	50

Table 3.PCR cycling conditions

Amplified production is carried out into electrophoresis, purpose fragment is reclaimed, it is standby.

(2) 1969-pTRE-uPA-PA transition vectors are built

PTRE Tight plasmids are carried out into double digestion using EcoR I and Sal I, then by uPA, Rabbit beta of clone Globin PolyA, bGHPA are connected on pTRE Tight plasmids by SLIC technologies, and linked system is as shown in table 4.

Table 4.TRE-uPA-PA linked systems

Reaction composition	Single reaction addition (μ l)
		PTRE carriers	7.3
UPA	7.1
		Rabbit-globin-PA	1
SLIC	3.6
		bGHPA	1
Total scale of construction	20

Connection product is converted to DH5 α competent cells, and is coated on the LB fine jades containing ampicillin (AMP) resistance On fat flat board, culture 16 hours is inverted in 37 DEG C of constant incubators, then picking positive colony extracts plasmid after amplification culture Carry out carrying out enzyme action identification with ApaLI, Sac and EcoRI, electrophoresis result is as shown in Figure 1.Digestion verification is correctly named as 1969-PTRE-uPA-PA。

(3) 1969-PTRE-uPA-rTTA-Alb transition vectors are built

The 1969-PTRE-uPA-PA carriers of structure are carried out into 37 DEG C of enzyme action with ASCI restriction endonucleases overnight, then using SLIC By on rTTA and pAlb to 1969-PTRE-uPA-PA carriers, the carrier of successful connection is named as technology：1969-PTRE-uPA- RTTA-Alb, linked system is shown in Table 5.By the 1969-PTRE-uPA-rTTA-Alb carriers conversion DH5 α impressions of successful connection State cell, and be coated on the LB agar plates containing ampicillin (AMP) resistance, training is inverted in 37 DEG C of constant incubators Support 16 hours, picking monoclonal.

Table 5PTRE-uPA-PA linked systems

Reaction composition	Single reaction addition (μ l)
		1969-PTRE-uPA-PA	8
RTTA	2
		ALB-Promoter	2
SLIC	8
		Cumulative volume	20

The monoclonal of picking is enlarged into culture, then enters performing PCR detection using following primer：

1969Alb-TF1:5’-ggatgctgtaacgcaggtta-3’(SEQ ID NO.11)；

1969PTRE-TR1:5’-tcactgcattctagttgtgg-3’(SEQ ID NO.12)；

PCR reaction conditions are as follows：95 DEG C of denaturations 3min；95 DEG C of degeneration 30sec, 58 DEG C of annealing 30sec, 72 DEG C of extensions 1min/kb, circulates 30 times；Extend 5min after 72 DEG C, amplified production is entered into row agarose gel electrophoresis, as a result as shown in Figure 2.

Then the clone of PCR tests positives is randomly choosed, then with EcoRl, ApaLl and Sacl enzyme action is carried out respectively, Digestion products enter row agarose gel electrophoresis, as a result as shown in Figure 3.

(4) carrier of 1969-Alb-uPA-teton-final mesh is built

Using restricted enzyme Xhol and Sall double digestion 1969-PTRE-uPA-rTTA-Alb, 37 DEG C of water-baths overnight, 5549bp and the bands of 2705bp two are obtained, 5549bp fragments are reclaimed；Simultaneously with identical Xhol and Sacll double digestions band Xhol and HS4insulator (the GenBank of Sacll restriction enzyme sites：U78775.2).After the 5549bp fragments for reclaiming and enzyme action HS4insulator is attached, and the correct rear carrier of connection is named as into 1969-Alb-uPA-teton-final, and its structure is such as Shown in Fig. 4.The 1969-Alb-uPA-teton-final carriers of successful connection are converted into DH5 α competent cells, and is coated on and is contained Have on the LB agar plates of ampicillin (AMP) resistance, culture 16 hours, picking Dan Ke are inverted in 37 DEG C of constant incubators It is grand.

1969Alb-TF1:5’-ggatgctgtaacgcaggtta-3’(SEQ ID NO.13)；

Insulator-tF1:5’-tccaggacggagtcagtgaggcg-3’(SEQ ID NO.14)；

PCR reaction conditions are as follows：95 DEG C of denaturations 3min；95 DEG C of degeneration 30sec, 58 DEG C of annealing 30sec, 72 DEG C of extensions 1min/kb；Extend 5min after 72 DEG C, amplified production is entered into row agarose gel electrophoresis, as a result as shown in Figure 5.

Then the clone of PCR tests positives is randomly choosed, then with ApaLl, Sphl and EcoRl enzyme action is carried out respectively, Digestion products enter row agarose gel electrophoresis, as a result as shown in Figure 6.As a result show, purpose fragment is successfully connected in carrier, success Obtain 1969-Alb-uPA-teton-final carriers.

Need to carry out sequencing identification after the completion of carrier construction, sequencing is as shown in table 6 using primer.

Relevant primer used in the sequencing of table 6.

It is used for next step Jing after sequence verification is correct, wherein 1969-Alb-uPA-teton-final carriers sequencing shows core Nucleotide sequence as shown in SEQ ID NO.26, referred to as Alb-uPA-TetOn slow virus carriers.The carrier albumin promoter PAlb and urokinase type plasminogen activator gene uPA Opposite direction connections, wherein albumin promoter PAlb regulation and control tetracyclines are anti- Formula activates sub- rtTA expression and is terminated expressing by bovine growth hormone gene polyA, urokinase type plasminogen activator gene uPA Expressed by PTight promoter regulations and terminated by rabbit beta Globulin PolyA to express, swash in tetracycline transactivator rtTA and urine The expression of enzyme type plasminogen activating agent gene uPA；Albumin promoter PAlb upstreams are connected with albumin enhancer, albumin 5 ' ends of promoter PAlb and PTight promoteres are also associated with insulator, and at pAlb and uPA backward chainings two are connected with respectively It is available for PCR detections identification fragment PA-1 (HA tag) and PA-2 (Histag), insulator inner side to increase double E1/E2 signs enzymes, have The expression of uPA is detected beneficial to southern bolt.

Detection of expression in embodiment 2, animal body

1st, Alb-uPA-teton transgenic mices are cultivated

From non-obese diabetes (NOD/LtJ) mice as experiment porch, the Mus fertility is stronger, sets up in the Mus Panimmunity defect Mus are available for later stage hybridization to select on platform, are that the depth cultivation of transgenic mice lays the foundation.It is specifically chosen NOD/ShiLtJNju mices (Nanjing University's animal pattern institute) are used as cultivation Mus.

2nd, procaryotic injection is implemented

Procaryotic injection is divided into two steps：

(1) preliminary experiment:1969-Alb-uPA-teton-final plasmids are carried out into enzyme action, electrophoresis with I-Ceu I restriction endonucleases Separate, reclaim purpose fragment sequence (2239-10325), carry out DNA purification, then procaryotic injection, 17 sons are produced in filial generation Mus.After wean, take mousetail and enter performing PCR amplification and sequencing identification murine genes type, obtain 1 positive mice.

(2) formal experiment：Procaryotic injection is carried out using purifying DNA fragment by Pre-testing procedure, filial generation is produced 59 filial mices, deposited It is living 58.After wean, take mousetail and enter performing PCR amplification and sequencing identification murine genes type, (head builds to obtain 13 positive mices Mus founder0).

3、Alb-uPA-teton：Transgenic mice (head builds Mus founder0 generations) is screened

After procaryotic injection in preliminary experiment, producing 17 newborn generation mices carries out cutting tail (5g/ is only), and collect blood is carried out MRNA extractions, PCR amplifications, electrophoresis and sequencing identification, as shown in table 7, amplification condition is as follows for detection primer：95 DEG C of denaturations 5min；95 DEG C of degeneration 30s, 60 DEG C of annealing 30s, 72 DEG C of extension 45s, circulate 35 times；Extend 2min, last 16 DEG C of guarantors after 72 DEG C Deposit.

The transgenic mice PCR detection primers of table 7.

Amplified production enters row agarose gel electrophoresis, as a result as shown in Figure 7.As seen from the figure, 17 ZISHU of preliminary experiment In, two pcr amplified fragments of only No. 15 uPA-seqR1 product 778bp and Alb-gtR1 product 540bp are the positive.

Formal experiment transgenic mice is detected in the same manner, as a result as shown in Figure 8.As a result show, formal experiment In, there are uPA-seqR1 product 778bp and Alb-gtR1 product two pcr amplified fragments of 540bp to be positive in 58 ZISHU Some 15#, 21#, 25#, 27#, 28#, 30#, 32#, 38#, 39#, 47#, 49#, 50#, 54# and No. 67#.P represents positive control, B6 is negative control, and N is blank；Internal reference is β-actin.Therefore 21#, 25#, 27#, 28#, 30#, 32#, 38#, 39# are chosen, 47#, 49#, 50#, 54#, 67# are positive Mus.

The above results show that the 1969-Alb-uPA-teton-final carriers that the present invention builds are obtained in that carrying purpose The transgenic mice of gene, possesses and independently builds the ability for being.

The Chinese patent for being suitable for Publication No. CN102628063A in the same manner discloses PAlb-uPA slow viruss Carrier can not obtain the transgenic mice for carrying genes of interest, can only obtain expression urokinase-type plasminogen in cellular level The mouse liver cell of activator uPA.

Finally illustrate, preferred embodiment above is only unrestricted to illustrate technical scheme, although logical Cross above preferred embodiment to be described in detail the present invention, it is to be understood by those skilled in the art that can be In form and in details various changes are made to it, without departing from claims of the present invention limited range.

<110>The First Affiliated Hospital of Third Military Medical University of PLA；

<120>Alb-uPA-teton slow virus carriers and its preparation method and application

<160> 34

<210> 1

<211> 36

<212> DNA

<213>Artificial sequence

<220>

<223>UPA forward primer

<400> 1

caccgggacc gatccagcct atgaaagtct ggcgag 36

<210> 2

<211> 20

<212> DNA

<213>Artificial sequence

<220>

<223>UPA downstream primers

<400> 2

tcagaaggcc agacctttct 20

<210> 3

<211> 40

<212> DNA

<213>Artificial sequence

<220>

<223>Rabbit beta Globulin PolyA forward primer

<400> 3

agaaaggtct ggccttctga gatctttttc cctctgccaa 40

<210> 4

<211> 20

<212> DNA

<213>Artificial sequence

<220>

<223>Rabbit beta Globulin PolyA downstream primers

<400> 4

agtagtcagg agaggaggaa 20

<210> 5

<211> 40

<212> DNA

<213>Artificial sequence

<220>

<223>BGHPA forward primer

<400> 5

ttcctcctct cctgactact taagggttcc gcaagctcta 40

<210> 6

<211> 54

<212> DNA

<213>Artificial sequence

<220>

<223>BGHPA downstream primers

<400> 6

gatccccggg taccgagctc ccgcggggcg cgccctagag ctcgctgatc agcc 54

<210> 7

<211> 40

<212> DNA

<213>Artificial sequence

<220>

<223>RTTA forward primer

<400> 7

ggctgatcag cgagctctag ttacccgggg agcatgtcaa 40

<210> 8

<211> 20

<212> DNA

<213>Artificial sequence

<220>

<223>RTTA downstream primers

<400> 8

atgtctagac tggacaagag 20

<210> 9

<211> 40

<212> DNA

<213>Artificial sequence

<220>

<223>Alb forward primer

<400> 9

ctcttgtcca gtctagacat tttgccagag gctagtgggg 40

<210> 10

<211> 46

<212> DNA

<213>Artificial sequence

<220>

<223>Alb downstream primers

<400> 10

gatccccggg taccgagctc ccgcggtagc ttccttagca tgacgt 46

<210> 11

<211> 20

<212> DNA

<213>Artificial sequence

<220>

<223>Primer 1969Alb-TF1

<400> 11

ggatgctgta acgcaggtta 20

<210> 12

<211> 20

<212> DNA

<213>Artificial sequence

<220>

<223>Primer 1969PTRE-TR1

<400> 12

tcactgcatt ctagttgtgg 20

<210> 13

<211> 20

<212> DNA

<213>Artificial sequence

<220>

<223>Primer 1969Alb-TF1

<400> 13

ggatgctgta acgcaggtta 20

<210> 14

<211> 23

<212> DNA

<213>Artificial sequence

<220>

<223>Primer I nsulator-tF1

<400> 14

tccaggacgg agtcagtgag gcg 23

<210> 15

<211> 20

<212> DNA

<213>Artificial sequence

<220>

<223>Primer 1969PTRE-TF1

<400> 15

gagctcgttt agtgaaccgt 20

<210> 16

<211> 20

<212> DNA

<213>Artificial sequence

<220>

<223>Primer 1969uPA-seqF1

<400> 16

aattcactga ggtggagaac 20

<210> 17

<211> 40

<212> DNA

<213>Artificial sequence

<220>

<223>Primer 1969-Rabit-PA-F

<400> 17

agaaaggtct ggccttctga gatctttttc cctctgccaa 40

<210> 18

<211> 40

<212> DNA

<213>Artificial sequence

<220>

<223>Primer 1969-Rabit-PA-F

<400> 18

ggctgatcag cgagctctag ttacccgggg agcatgtcaa 40

<210> 19

<211> 40

<212> DNA

<213>Artificial sequence

<220>

<223>Primer 1969-Rabit-PA-F

<400> 19

agaaaggtct ggccttctga gatctttttc cctctgccaa 40

<210> 20

<211> 20

<212> DNA

<213>Artificial sequence

<220>

<223>Primer 1969-Alb-seqF1

<400> 20

agcgagtttc cttgtcgtca 20

<210> 21

<211> 20

<212> DNA

<213>Artificial sequence

<220>

<223>Primer 1969-Alb-seqF2

<400> 21

cctctgaaca tctgctcaca 20

<210> 22

<211> 20

<212> DNA

<213>Artificial sequence

<220>

<223>Primer 1969-Alb-seqF3

<400> 22

tcagcaagca ataccatgac 20

<210> 23

<211> 20

<212> DNA

<213>Artificial sequence

<220>

<223>Primer 1969PTRE-TR1#

<400> 23

tcactgcatt ctagttgtgg 20

<210> 24

<211> 20

<212> DNA

<213>Artificial sequence

<220>

<223>Primer 1969Alb-uPA-seqR1

<400> 24

ggtcaaaaca gcgtggatgg 20

<210> 25

<211> 20

<212> DNA

<213>Artificial sequence

<220>

<223>Primer 1969Alb-uPA-seqR1

<400> 25

gtagtgtagt ctaggaacag 20

<210> 26

<211> 10761

<212> DNA

<213>Artificial sequence

<220>

<223>1969-Alb-uPA-teton-final carrier sequences

<400> 26

gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt 60

cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt 120

tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat 180

aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt 240

ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg 300

ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga 360

tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc 420

tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac 480

actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg 540

gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca 600

acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg 660

gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg 720

acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg 780

gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag 840

ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg 900

gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct 960

cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac 1020

agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact 1080

catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga 1140

tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt 1200

cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct 1260

gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc 1320

taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc 1380

ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc 1440

tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg 1500

ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt 1560

cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg 1620

agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg 1680

gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt 1740

atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag 1800

gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt 1860

gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta 1920

ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt 1980

cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc 2040

cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca 2100

acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc 2160

cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg 2220

accatgatta cgaattcttc gctaccttag gaccgttata gttacgtcac tgactccgtc 2280

ctggagttgg atgagagata atggccttac gttgtgccag gggagggtcg ggctggattt 2340

agcaagattt accttctcca aagagcggtg ctgcagtggc acagctgccc acggaggtgg 2400

gggggtcacc gtccctggag gtgatgaaga actgtgggga tgtggcactg agggacatgg 2460

ccagtgggca cggtgggtgg gttggggttg gtcttgggga tcttggaggg cttttccagc 2520

cttcatgatt tgacgattgt atgaacatct acatggcaat tctccagctg cctgtcccag 2580

tcctactgac ccagctgtat ctctccaggc aagctcttcc accccttctg cttgcatcca 2640

gacaccatca aacatgcagg ctcagacaca gggaccagca gtgtctgtgg cctttttgtg 2700

ctcctctcca tgctgggttt taacttgctc tttgtccttc tatcctatct tcttatcctt 2760

aaggctgttc tgaacgctgt gacttggaga gtgtcccaga gccctcaaca cctgcatgtc 2820

ccacgtccat gctgtcctgc acttccttat ccccaagatc tgcctctccg tgatgcactg 2880

aattggcaaa catgtgtcac cccagaccaa caatgtcaca gcaaactccc ccttgatagg 2940

acaaggggga atggctttac actgagacag gggaggtttg ggttggatat gaggaggcag 3000

tttttccccc agagggtggt gacgcactga acaggttgcc caaggaggct gtggatgccc 3060

catccctgca ggcattcaag gccaggctgg atgtggctct gggcagcctg ggctgctggt 3120

tgatgaccct gcacatagca gggggttgga tctggatgag cactgtgctc ctttgcaacc 3180

caggccgttc tatgattctg tcattctaaa tctctctttc agcctaaagc tttttccccg 3240

tatcccccca ggtgtctgca ggctcaaaga gcagcgagaa gcgttcagag gaaagcgatc 3300

ccgtgccacc ttccccgtgc ccgggctgtc cccgcacgct gccggctcgg ggatgcgggg 3360

ggagcgccgg accggagcgg agccccgggc ggctcgctgc tgccccctag cgggggaggg 3420

acgtaattac atccctgggg gctttggggg ggggctgtcc ctctagcgag atcgatgcgg 3480

ccgcactagt taattaagaa ttcgagctcg gtacccgggg atcctctaga gatatcgtcg 3540

acctgcaggc atgctcgagt ttaccactcc ctatcagtga tagagaaaag tgaaagtcga 3600

gtttaccact ccctatcagt gatagagaaa agtgaaagtc gagtttacca ctccctatca 3660

gtgatagaga aaagtgaaag tcgagtttac cactccctat cagtgataga gaaaagtgaa 3720

agtcgagttt accactccct atcagtgata gagaaaagtg aaagtcgagt ttaccactcc 3780

ctatcagtga tagagaaaag tgaaagtcga gtttaccact ccctatcagt gatagagaaa 3840

agtgaaagtc gagctcggta cccgggtcga gtaggcgtgt acggtgggag gcctatataa 3900

gcagagctcg tttagtgaac cgtcagatcg cctggagacg ccatccacgc tgttttgacc 3960

tccatagaag acaccgggac cgatccagcc tatgaaagtc tggctggcga gcctgttcct 4020

ctgcgccttg gtggtgaaaa actctgaagg tggcagtgta cttggagctc ctgatgaatc 4080

aaactgtggc tgtcagaacg gaggtgtatg cgtgtcctac aagtacttct ccagaattcg 4140

ccgatgcagc tgcccaagga aattccaggg ggagcactgt gagatagatg catcaaaaac 4200

ctgctatcat ggaaatggtg actcttaccg aggaaaggcc aacactgata ccaaaggtcg 4260

gccctgcctg gcctggaatg cgcctgctgt ccttcagaaa ccctacaatg cccacagacc 4320

tgatgctatt agcctaggcc tggggaaaca caattactgc aggaaccctg acaaccagaa 4380

gcgaccctgg tgctatgtgc agattggcct aaggcagttt gtccaagaat gcatggtgca 4440

tgactgctct cttagcaaaa agccttcttc gtctgtagac caacaaggct tccagtgtgg 4500

ccagaaggct ctaaggcccc gctttaagat tgttggggga gaattcactg aggtggagaa 4560

ccagccctgg ttcgcagcca tctaccagaa gaacaaggga ggaagtcctc cctcctttaa 4620

atgtggtggg agtctcatca gtccttgctg ggtggccagt gccgcacact gcttcattca 4680

actcccaaag aaggaaaact acgttgtcta cctgggtcag tcgaaggaga gctcctataa 4740

tcctggagag atgaagtttg aggtggagca gctcatcttg cacgaatact acagggaaga 4800

cagcctggcc taccataatg atattgcctt gctgaagata cgtaccagca cgggccaatg 4860

tgcacagcca tccaggtcca tacagaccat ctgcctgccc ccaaggttta ctgatgctcc 4920

gtttggttca gactgtgaga tcactggctt tggaaaagag tctgaaagtg actatctcta 4980

tccaaagaac ctgaaaatgt ccgtcgtaaa gcttgtttct catgaacagt gtatgcagcc 5040

ccactactat ggctctgaaa ttaattataa aatgctgtgt gctgcggacc cagagtggaa 5100

aacagattcc tgcaagggcg attctggagg accgcttatc tgtaacatcg aaggccgccc 5160

aactctgagt gggattgtga gctggggccg aggatgtgca gagaaaaaca agcccggtgt 5220

ctacacgagg gtctcacact tcctggactg gattcaatcc cacattggag aagagaaagg 5280

tctggccttc tgagatcttt ttccctctgc caaaaattat ggggacatca tgaagcccct 5340

tgagcatctg acttctggct aataaaggaa atttattttc attgcaatag tgtgttggaa 5400

ttttttgtgt ctctcactcg gaaggacata tgggagggca aatcatttaa aacatcagaa 5460

tgagtatttg gtttagagtt tggcaacata tgcccatatg ctggctgcca tgaacaaagg 5520

ttggctataa agaggtcatc agtatatgaa acagccccct gctgtccatt ccttattcca 5580

tagaaaagcc ttgacttgag gttagatttt ttttatattt tgttttgtgt tatttttttc 5640

tttaacatcc ctaaaatttt ccttacatgt tttactagcc agatttttcc tcctctcctg 5700

actacttaag ggttccgcaa gctctagtcg agccccagct ggttctttcc gcctcagaag 5760

ccatagagcc caccgcatcc ccagcatgcc tgctattgtc ttcccaatcc tcccccttgc 5820

tgtcctgccc caccccaccc cccagaatag aatgacacct actcagacaa tgcgatgcaa 5880

tttcctcatt ttattaggaa aggacagtgg gagtggcacc ttccagggtc aaggaaggca 5940

cgggggaggg gcaaacaaca gatggctggc aactagaagg cacagtcgag gctgatcagc 6000

gagctctagt tacccgggga gcatgtcaag gtcaaaatcg tcaagagcgt cagcaggcag 6060

catatcaagg tcaaagtcgt caagggcatc ggctgggagc atgtctaagt caaaatcgtc 6120

aagggcgtcg gccggcccgc cgctttcgca ctttagctgt ttctccaggc cacatatgat 6180

tagttccagg ccgaaaagga aggcaggttc ggctccctgc cggtcgaaca gctcaattgc 6240

ttgtctcaga agtgggggca tagaatcggt ggtaggtgtc tctctttcct cttttgctac 6300

ttgatgctcc tgttcctcca atacgcagcc cagtgtaaag tggcccacgg cggacagagc 6360

gtacagtgcg ttctccaggg agaagccttg ctgacacagg aacgcgagct gattttccag 6420

ggtttcgtac tgtttctctg ttgggcgggt gccgagatgc actttagccc cgtcgcgatg 6480

tgagaggaga gcacagcgga atgacttggc gttgttccgc agaaagtctt gccatgactc 6540

gccttccagg gggcagaagt gggtatgatg cctgtccagc atctcgattg gcagggcatc 6600

gagcagggcc cgcttgttct tcacgtgcca gtacagggta ggctgctcaa ctcccagctt 6660

ttgagcgagt ttccttgtcg tcaggccttc gataccgact ccattgagta attccagagc 6720

gccgtttatg actttgctct tgtccagtct agacattttg ccagaggcta gtggggttga 6780

taggaaaggt gatctgtgtg cagaaagact cgctctaata tacttcttta accaataact 6840

gtagatcatt aaccatactt acctcgcatt tcattggttc ctaccccatt acaaaatcat 6900

accatctttg ccaaaaagtt gtttgactaa atcccttgcg tatgtttgcc atctggagct 6960

gttcccctct aaccccaccc ccacccccat gcacaagact ttgtccattc attaaagtta 7020

tgtaaaacag caaattttac ataagagctt aatctctttg tctcccattt gagcatttca 7080

gtgtgggcct tggcatggaa gcatgcctgc aggtcgatcc caagctggag aacgagttca 7140

agccaagctg caccactgct tttcacacac tcttcactct gcatcagctt agtatttctt 7200

aagaaattaa aagatggcaa aacacatcta aactgtatta ataaagtgct tctttcatat 7260

ttaatgtttt tccagataaa gaaaactatg atgaatgcct gcatgcttat ctatgtttca 7320

tagatcagca agtagaatgt ataaaatgga agtgtcagta attctgctca taattattgc 7380

tgcagattga attcacccct aagcaaatat acctctgaac atctgctcac agtctgtatg 7440

ttctccagac acaatccaaa agacttatta tctgaaagat taatgtcaca aagccagagc 7500

tttataatct cttataaaac atagattgta gccaggcagt ggtggcacat gcttttaatc 7560

ctagcacttg caaggcagag gcaagcagat ctctgagttc aagaccaacc tggtctacag 7620

agcaaggtcc aggacagcca aagctagaca gaaaaaactg tatctcaaaa gaaaatagac 7680

aacaaattac attgttacag ctaaaattat cttatgttga aatttctgta gctcaacttt 7740

ggaatatttt cattagaggg taatatttga ttatgatcac ttctaaaact ttagaattta 7800

ttgttttata atctcttggt ttcagtactt acctaaaatt ttccaaccag tcacccagct 7860

aaaacttaaa atatttaagt cctagaattc cagttagttt tgcaagtaac tataaatggt 7920

attacagtga gaaatggagc atctgatgtc tactcacatg taaactttac acatatcaaa 7980

tagatgattg tctatggtct ttcttctttt ttagagtata tagagtatat agagatagat 8040

tcatccataa taagctcaat aaacaaatgt ttaaaaatga ttgttagata ttattgggta 8100

tataagtacc taattattaa aattgacttt tttataacat tgagataaat taaaattcat 8160

ttattaaaat aatatatatg aatttgaagg ggtttttttt gcaaaacaat ttcagcaagc 8220

aataccatga caaaagtgtg tattcaaatg gaatgggaaa cgaatgtcag taacttatgg 8280

tccccgtgta ctcattccca gacatgcctg attggtagct gtgacagctc cagcgtactt 8340

aacaccaaga ctttaaataa gctgccaaaa atgtgtaaga ctgccatttc attagtttta 8400

atttttatat ctataccctt tctacagcca catactaaac gtagacaagt tggccttttc 8460

ctattgcttt aaaggcagag gactgtattg atcagtccaa acttctttct gcatgtacat 8520

ggaaaactgg ccaaggcaaa cacgtccgga atgatggtat ttaagaacaa acattccctg 8580

gtatcagcaa gtacagtgcc ctgctgacag agcaggagac acaaagtacc atctcgtccc 8640

tatgttaagt agtgtcacct catgctcaag ggatactgag tggatgctgt aacgcaggtt 8700

attttctagg ctgtgaggat acaagaaaat gaaagtaatt aaagtagaac attgctctgt 8760

gctatgcttg cagaatgtgt agtgtagtct aggaacagag aggggaaggt tctaaatcaa 8820

aaaaaatcaa gctcatgcct aaggatgtgt gggttgccac ctctttagct acctatgcga 8880

tccaaacaac tataaaactt agaatttatt ttctctggat gaatttgtgc ttgtggagca 8940

atgttggtag ggggcagggt cagctggaaa agtggaatga gcaagcagaa aactgagaga 9000

agcagaagct taggaagatg ggtaatttcc aaaagtttca caaaagatca aatcaaagaa 9060

gtaagctcca ccttagaaaa aagtggaacg tcatgctaag gaagctaccg cggcgcgcct 9120

cactgactcc gtcctggagt tggatgagag ataatggcct tacgttgtgc caggggaggg 9180

tcgggctgga tttagcaaga tttaccttct ccaaagagcg gtgctgcagt ggcacagctg 9240

cccacggagg tgggggggtc accgtccctg gaggtgatga agaactgtgg ggatgtggca 9300

ctgagggaca tggccagtgg gcacggtggg tgggttgggg ttggtcttgg ggatcttgga 9360

gggcttttcc agccttcatg atttgacgat tgtatgaaca tctacatggc aattctccag 9420

ctgcctgtcc cagtcctact gacccagctg tatctctcca ggcaagctct tccacccctt 9480

ctgcttgcat ccagacacca tcaaacatgc aggctcagac acagggacca gcagtgtctg 9540

tggccttttt gtgctcctct ccatgctggg ttttaacttg ctctttgtcc ttctatccta 9600

tcttcttatc cttaaggctg ttctgaacgc tgtgacttgg agagtgtccc agagccctca 9660

acacctgcat gtcccacgtc catgctgtcc tgcacttcct tatccccaag atctgcctct 9720

ccgtgatgca ctgaattggc aaacatgtgt caccccagac caacaatgtc acagcaaact 9780

cccccttgat aggacaaggg ggaatggctt tacactgaga caggggaggt ttgggttgga 9840

tatgaggagg cagtttttcc cccagagggt ggtgacgcac tgaacaggtt gcccaaggag 9900

gctgtggatg ccccatccct gcaggcattc aaggccaggc tggatgtggc tctgggcagc 9960

ctgggctgct ggttgatgac cctgcacata gcagggggtt ggatctggat gagcactgtg10020

ctcctttgca acccaggccg ttctatgatt ctgtcattct aaatctctct ttcagcctaa10080

agctttttcc ccgtatcccc ccaggtgtct gcaggctcaa agagcagcga gaagcgttca10140

gaggaaagcg atcccgtgcc accttccccg tgcccgggct gtccccgcac gctgccggct10200

cggggatgcg gggggagcgc cggaccggag cggagccccg ggcggctcgc tgctgccccc10260

tagcggggga gggacgtaat tacatccctg ggggctttgg gggggggctg tccctctagc10320

gagttcgcta ccttaggacc gttatagtta cgcatgcaag cttggcactg gccgtcgttt10380

tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc10440

cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt10500

tgcgcagcct gaatggcgaa tggcgcctga tgcggtattt tctccttacg catctgtgcg10560

gtatttcaca ccgcatatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa10620

gccagccccg acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg10680

catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac10740

cgtcatcacc gaaacgcgcg a 10761

<210> 27

<211> 20

<212> DNA

<213>Artificial sequence

<220>

<223>Primer 1969PTRE-TF1

<400> 27

gagctcgttt agtgaaccgt 20

<210> 28

<211> 20

<212> DNA

<213>Artificial sequence

<220>

<223>Primer 1969uPA-seqR1

<400> 28

ttgaatgaag cagtgtgcgg 20

<210> 29

<211> 20

<212> DNA

<213>Artificial sequence

<220>

<223>Primer 1969rTTA-gtF1

<400> 29

ggctgctcaa ctcccagctt 20

<210> 30

<211> 20

<212> DNA

<213>Artificial sequence

<220>

<223>Primer 1969Alb-gtR1

<400> 30

agagtgaaga gtgtgtgaaa 20

<210> 31

<211> 747

<212> DNA

<213>Artificial sequence

<220>

<223>RtTA coding region sequences

<400> 31

atgtctagac tggacaagag caaagtcata aacggcgctc tggaattact caatggagtc 60

ggtatcgaag gcctgacgac aaggaaactc gctcaaaagc tgggagttga gcagcctacc 120

ctgtactggc acgtgaagaa caagcgggcc ctgctcgatg ccctgccaat cgagatgctg 180

gacaggcatc atacccactt ctgccccctg gaaggcgagt catggcaaga ctttctgcgg 240

aacaacgcca agtcattccg ctgtgctctc ctctcacatc gcgacggggc taaagtgcat 300

ctcggcaccc gcccaacaga gaaacagtac gaaaccctgg aaaatcagct cgcgttcctg 360

tgtcagcaag gcttctccct ggagaacgca ctgtacgctc tgtccgccgt gggccacttt 420

acactgggct gcgtattgga ggaacaggag catcaagtag caaaagagga aagagagaca 480

cctaccaccg attctatgcc cccacttctg agacaagcaa ttgagctgtt cgaccggcag 540

ggagccgaac ctgccttcct tttcggcctg gaactaatca tatgtggcct ggagaaacag 600

ctaaagtgcg aaagcggcgg gccggccgac gcccttgacg attttgactt agacatgctc 660

ccagccgatg cccttgacga ctttgacctt gatatgctgc ctgctgacgc tcttgacgat 720

tttgaccttg acatgctccc cgggtaa 747

<210> 32

<211> 319

<212> DNA

<213>Artificial sequence

<220>

<223>PTight promoter coding region sequences

<400> 32

tttactccct atcagtgata gagaacgtat gtcgagttta ctccctatca gtgatagaga 60

acgatgtcga gtttactccc tatcagtgat agagaacgta tgtcgagttt actccctatc 120

agtgatagag aacgtatgtc gagtttactc cctatcagtg atagagaacg tatgtcgagt 180

ttatccctat cagtgataga gaacgtatgt cgagtttact ccctatcagt gatagagaac 240

gtatgtcgag gtaggcgtgt acggtgggag gcctatataa gcagagctcg tttagtgaac 300

cgtcagatcg cctggagaa 319

<210> 33

<211> 1302

<212> DNA

<213>Artificial sequence

<220>

<223>Urokinase type plasminogen activator gene uPA coding region sequences

<400> 33

atgaaagtct ggctggcgag cctgttcctc tgcgccttgg tggtgaaaaa ctctgaaggt 60

ggcagtgtac ttggagctcc tgatgaatca aactgtggct gtcagaacgg aggtgtatgc 120

gtgtcctaca agtacttctc cagaattcgc cgatgcagct gcccaaggaa attccagggg 180

gagcactgtg agatagatgc atcaaaaacc tgctatcatg gaaatggtga ctcttaccga 240

ggaaaggcca acactgatac caaaggtcgg ccctgcctgg cctggaatgc gcctgctgtc 300

cttcagaaac cctacaatgc ccacagacct gatgctatta gcctaggcct ggggaaacac 360

aattactgca ggaaccctga caaccagaag cgaccctggt gctatgtgca gattggccta 420

aggcagtttg tccaagaatg catggtgcat gactgctctc ttagcaaaaa gccttcttcg 480

tctgtagacc aacaaggctt ccagtgtggc cagaaggctc taaggccccg ctttaagatt 540

gttgggggag aattcactga ggtggagaac cagccctggt tcgcagccat ctaccagaag 600

aacaagggag gaagtcctcc ctcctttaaa tgtggtggga gtctcatcag tccttgctgg 660

gtggccagtg ccgcacactg cttcattcaa ctcccaaaga aggaaaacta cgttgtctac 720

ctgggtcagt cgaaggagag ctcctataat cctggagaga tgaagtttga ggtggagcag 780

ctcatcttgc acgaatacta cagggaagac agcctggcct accataatga tattgccttg 840

ctgaagatac gtaccagcac gggccaatgt gcacagccat ccaggtccat acagaccatc 900

tgcctgcccc caaggtttac tgatgctccg tttggttcag actgtgagat cactggcttt 960

ggaaaagagt ctgaaagtga ctatctctat ccaaagaacc tgaaaatgtc cgtcgtaaag 1020

cttgtttctc atgaacagtg tatgcagccc cactactatg gctctgaaat taattataaa 1080

atgctgtgtg ctgcggaccc agagtggaaa acagattcct gcaagggcga ttctggagga 1140

ccgcttatct gtaacatcga aggccgccca actctgagtg ggattgtgag ctggggccga 1200

ggatgtgcag agaaaaacaa gcccggtgtc tacacgaggg tctcacactt cctggactgg 1260

attcaatccc acattggaga agagaaaggt ctggccttct ga 1302

<210> 34

<211> 2339

<212> DNA

<213>Artificial sequence

<220>

<223>Albumin promoter pAlb coding region sequences

<400> 34

tctagcttcc ttagcatgac gttccacttt tttctaaggt ggagcttact tctttgattt 60

gatcttttgt gaaacttttg gaaattaccc atcttcctaa gcttctgctt ctctcagttt 120

tctgcttgct cattccactt ttccagctga ccctgccccc taccaacatt gctccacaag 180

cacaaattca tccagagaaa ataaattcta agttttatag ttgtttggat cgcataggta 240

gctaaagagg tggcaaccca cacatcctta ggcatgagct tgattttttt tgatttagaa 300

ccttcccctc tctgttccta gactacacta cacattctgc aagcatagca cagagcaatg 360

ttctacttta attactttca ttttcttgta tcctcacagc ctagaaaata acctgcgtta 420

cagcatccac tcagtatccc ttgagcatga ggtgacacta cttaacatag ggacgagatg 480

gtactttgtg tctcctgctc tgtcagcagg gcactgtact tgctgatacc agggaatgtt 540

tgttcttaaa taccatcatt ccggacgtgt ttgccttggc cagttttcca tgtacatgca 600

gaaagaagtt tggactgatc aatacagtcc tctgccttta aagcaatagg aaaaggccaa 660

cttgtctacg tttagtatgt ggctgtagaa agggtataga tataaaaatt aaaactaatg 720

aaatggcagt cttacacatt tttggcagct tatttaaagt cttggtgtta agtacgctgg 780

agctgtcaca gctaccaatc aggcatgtct gggaatgagt acacggggac cataagttac 840

tgacattcgt ttcccattcc atttgaatac acacttttgt catggtattg cttgctgaaa 900

ttgttttgca aaaaaaaccc cttcaaattc atatatatta ttttaataaa tgaattttaa 960

tttatctcaa tgttataaaa aagtcaattt taataattag gtacttatat acccaataat 1020

atctaacaat catttttaaa catttgttta ttgagcttat tatggatgaa tctatctcta 1080

tatactctat atactctaaa aaagaagaaa gaccatagac aatcatctat ttgatatgtg 1140

taaagtttac atgtgagtag acatcagatg ctccatttct cactgtaata ccatttatag 1200

ttacttgcaa aactaactgg aattctagga cttaaatatt ttaagtttta gctgggtgac 1260

tggttggaaa attttaggta agtactgaaa ccaagagatt ataaaacaat aaattctaaa 1320

gttttagaag tgatcataat caaatattac cctctaatga aaatattcca aagttgagct 1380

acagaaattt caacataaga taattttagc tgtaacaatg taatttgttg tctattttct 1440

tttgagatac agttttttct gtctagcttt ggctgtcctg gaccttgctc tgtagaccag 1500

gttggtcttg aactcagaga tctgcttgcc tctgccttgc aagtgctagg attaaaagca 1560

tgtgccacca ctgcctggct acaatctatg ttttataaga gattataaag ctctggcttt 1620

gtgacattaa tctttcagat aataagtctt ttggattgtg tctggagaac atacagactg 1680

tgagcagatg ttcagaggta tatttgctta ggggtgaatt caatctgcag caataattat 1740

gagcagaatt actgacactt ccattttata cattctactt gctgatctat gaaacataga 1800

taagcatgca ggcattcatc atagttttct ttatctggaa aaacattaaa tatgaaagaa 1860

gcactttatt aatacagttt agatgtgttt tgccatcttt taatttctta agaaatacta 1920

agctgatgca gagtgaagag tgtgtgaaaa gcagtggtgc agcttggctt gaactcgttc 1980

tccagcttgg gatcgacctg caggcatgct tccatgccaa ggcccacact gaaatgctca 2040

aatgggagac aaagagatta agctcttatg taaaatttgc tgttttacat aactttaatg 2100

aatggacaaa gtcttgtgca tgggggtggg ggtggggtta gaggggaaca gctccagatg 2160

gcaaacatac gcaagggatt tagtcaaaca actttttggc aaagatggta tgattttgta 2220

atggggtagg aaccaatgaa atgcgaggta agtatggtta atgatctaca gttattggtt 2280

aaagaagtat attagagcga gtctttctgc acacagatca cctttcctat caaccccgg 2339

Claims

1.Alb-uPA-TetOn slow virus carriers, it is characterised in that：The Alb-uPA-TetOn slow virus carriers contain reversely The albumin promoter PAlb and urokinase type plasminogen activator gene uPA of connection, the albumin promoter PAlb is adjusted Control tetracycline transactivator rtTA expression, the urokinase type plasminogen activator gene uPA is adjusted by PTight promoteres Control expression；

The nucleotide sequence of the albumin promoter PAlb is as shown in SEQ ID NO.34；The urokinase type plasminogen swashs The nucleotide sequence of agent gene uPA living is as shown in SEQ ID NO.33；The nucleotides sequence of the tetracycline transactivator rtTA Row are as shown in SEQ ID NO.31；The nucleotide sequence of the PTight promoteres is as shown in SEQ ID NO.32.

2. Alb-uPA-TetOn slow virus carriers according to claim 1, it is characterised in that：The Alb-uPA-TetOn is slow 5 ends of the albumin promoter PAlb and PTight promoter of viral vector are connected with insulator.

3. Alb-uPA-TetOn slow virus carriers according to claim 1, it is characterised in that：Terminate urokinase-type plasminogen The terminator of activator gene uPA expression is rabbit beta Globulin PolyA, terminates the end of tetracycline transactivator rtTA expression Only son is bovine growth hormone gene polyA.

4. Alb-uPA-TetOn slow virus carriers according to claim 1, it is characterised in that：The albumin promoter ' end is also associated with Mouse albumin enhancer for the 5 of PAlb.

5. Alb-uPA-TetOn slow virus carriers according to claim 2, it is characterised in that：The Alb-uPA-TetOn is slow The nucleotide sequence of viral vector is as shown in SEQ ID NO.26.

6. the preparation method of Alb-uPA-TetOn slow virus carriers described in any one of Claims 1 to 5, it is characterised in that include Following steps：

(1) clone's urokinase type fibrinolytic protein enzyme gene uPA, rabbit beta Globulin PolyA and bovine growth hormone gene polyA are led to Cross SLIC technologies to be connected on the pTRE Tight plasmids of Jing EcoR I and the double digestions of Sal I, obtain 1969-pTRE-uPA-PA Transition vector；

(2) albumin promoter Alb and tetracycline transactivator rtTA is connected to into Jing restriction endonuclease AscI by SLIC technologies On the 1969-pTRE-uPA-PA excessive vectors of enzyme action, 1969-PTRE-uPA-rTTA-Alb excessive vectors are obtained；

(4) 1969-PTRE-uPA-rTTA-Alb excessive vectors are reclaimed into 5549bp fragments Jing after Xhol and Sacll enzyme action, then Recovery product is connected with the insulator with Xhol and Sacll enzyme action sticky ends, Alb-uPA-TetOn slow viruss load is obtained final product Body.

7. the preparation method of Alb-uPA-TetOn slow virus carriers according to claim 5, it is characterised in that：Urokinase type The cloning process of fibrinolytic protein enzyme gene uPA is as follows：With PAlb-uPA slow virus carriers as template, SEQ ID NO.1 and SEQ Sequence carries out amplification acquisition for primer shown in ID NO.2；

Rabbit beta Globulin PolyA cloning process is as follows：With PAlb-uPA slow virus carriers as template, SEQ ID NO.3 and SEQ ID Sequence shown in NO.4 carries out amplification acquisition for primer；

Bovine growth hormone gene polyA cloning process is as follows：With PAlb-uPA slow virus carriers as template, SEQ ID NO.5 and Sequence shown in SEQ ID NO.6 carries out amplification acquisition for primer；

Albumin promoter Alb cloning process is as follows：With PAlb-uPA slow virus carriers as template, SEQ ID NO.9 and SEQ Sequence carries out amplification acquisition for primer shown in ID NO.10；

Tetracycline transactivator rtTA cloning process is as follows：With PAlb-uPA slow virus carriers as template, SEQ ID NO.7 and Sequence shown in SEQ ID NO.8 carries out amplification acquisition for primer.

8. application of the Alb-uPA-TetOn slow virus carriers in prepare transgenosis mice described in any one of Claims 1 to 5.

9. Alb-uPA-TetOn slow virus carriers described in any one of Claims 1 to 5 are to prepare urokinase type fibrinolytic protein enzyme little Application in Hepar Mus damage model.