CN102816793A

CN102816793A - Single carrier bidirectionally started Tet-on inducible expression system, its construction method and application

Info

Publication number: CN102816793A
Application number: CN2012103005722A
Authority: CN
Inventors: 夏海滨; 陈皓
Original assignee: Shaanxi Normal University
Current assignee: Shaanxi Normal University
Priority date: 2012-08-22
Filing date: 2012-08-22
Publication date: 2012-12-12
Anticipated expiration: 2032-08-22
Also published as: CN102816793B

Abstract

The invention relates to a single carrier bidirectionally started Tet-on inducible expression system, the elements of which are connected as the following sequence: 5-'CMV-TetR-KRAB-SV40polyA-rtTA2S-M2-EB-PminCMV-KB-TRE-Tight-PminCMV-EcoR I-Spe I-SV40polyA-3'. The composite element rtTA2S-M2-EB-PminCMV-KB-TRE on the left side of Tight-PminCMV is connected in the way of 3'-rtTA2S-M2-EB-PminCMV-KB-TRE-5'-5'-Tight-PminCMV-3', and the other elements are all connected in a 5'-3' sequence in the same direction with the system. The enzyme digestion sites EcoR I and Spe I are positioned between the element Tight-PminCMV and the element SV40polyA.

Description

Tet-on abduction delivering system and the construction process and the application of single carrier two-way startup

Technical field

The present invention relates to life science, be specifically related to Tet-on abduction delivering system and the construction process and the application of single carrier two-way startup.

Background technology

Gene therapy is one of treat-ment of getting a good chance of of present oncotherapy field, and in the gene therapy to the regulation and control quickly and accurately of used therapeutic gene to improving curative effect, it is most important to reduce the negative interaction that therapeutic gene brings.Tet-on abduction delivering system is a kind of of tsiklomitsin inducible system, characteristics be add tsiklomitsin or its verivate such as vibra-(Doxycycline, Dox) wait induced drug after, controlled genetic expression is opened.Tet-on abduction delivering system is the abduction delivering system of widespread use, and it is low to have a background, and height is induced multiple, high specific, and inductor does not have advantages such as overt toxicity, is fit to very much the regulation and control to therapeutic gene.Present stage, this system has been applied in the research and treatment of many diseases.But still there is the significantly interference effect of background seepage and modulin overexpression pair cell in existing Tet-on abduction delivering system; The foundation of Tet-o abduction delivering system in the past simultaneously need be regulated and control carrier and two plasmids of reaction carriers, has so reduced the efficient that system sets up.The Tet-on abduction delivering system of single carrier two-way startup can overcome the shortcoming of two pUC pUCs, has low background seepage simultaneously, and is easy to be used for like carriers such as adenovirus, and is therefore most important for oncotherapy.

All be positioned on the carrier through setting up a kind of whole regulator control system and goal gene; And has a Tet-on abduction delivering system of low background seepage and efficient induction multiple; Realization is to range gene, like effective regulating and expressing of reporter gene or Antioncogene TRAIL; And can be applied to as on the virus vector such as adenovirus carrier, make the better vigor of having of its acquisition, for therapy of tumor provides strong instrument.

Summary of the invention

A technical problem to be solved by this invention is to provide a kind of Tet-on abduction delivering system of single carrier two-way startup.

Another technical problem to be solved by this invention is to provide a kind of construction process of Tet-on abduction delivering system of single carrier two-way startup.

To be solved by this invention also have a technical problem to be for the Tet-on abduction delivering system of single carrier two-way startup a kind of new purposes to be provided.

The technical scheme that solves the problems of the technologies described above employing is: the order of connection of the Tet-on abduction delivering system component of single carrier two-way startup is 5-' CMV-TetR-KRAB-SV40polyA-rtTA2S-M2-EB-PminCMV-KB-TRE-Tight-PminCMV-EcoR I-Spe I-SV40polyA-3 '; The mode of connection of the composite component rtTA2S-M2-EB-PminCMV-KB-TRE in Tight-PminCMV left side is 3 '-rtTA2S-M2-EB-PminCMV-KB-TRE-5 '-5 '-Tight-PminCMV-3 ', and all the other elements are all by being linked in sequence with this system's equidirectional 5 '-3 '; Two restriction enzyme site EcoR I and Spe I between elements T ight-PminCMV and element SV40polyA, No.11 in this system's sequence such as the nucleotides sequence tabulation.

The direction of the PminCMV element in TRE element of the present invention left side and the Tight-PminCMV on right side start in the opposite direction, all adjacent with the TRE element.

The goal gene that between the EcoR I of the Tet-on abduction delivering system of the single carrier two-way startup of the present invention and Spe I site, is connected into is reporter gene or oncotherapy gene.

The construction process of the Tet-on abduction delivering system of single carrier two-way startup comprises the steps:

1, makes up the adenovirus E 1 district shuttle vectors that contains Kpn I-Cla I-Not I-Xho I-EcoR I-Spe I restriction endonuclease sites

Use enzyme to cut connection method DNA linker (Kpn I-Cla I-Not I-Xho I-EcoR I-Spe I-Spe I) is connected into adenovirus E 1 district shuttle vectors, obtain needed carrier;

2, amplification makes up the required element of Tet-on abduction delivering system of single carrier two-way startup

Make up the required element of Tet-on abduction delivering system of single carrier two-way startup through PCR amplification, element comprises CMV, TetR-KRAB, SV40polyA, Tight-PminCMV, rtTA2S-M2.

3, make up the intermediate carrier that has composite component TRE-KB-PminCMV-EB-rtTA2S-M2

Cut connection through enzyme; TRE-PminCMV is connected to pShuttle-KSL-SV40polyA-KB; And after using Kpn I enzyme to cut, the T4 archaeal dna polymerase is mended flat terminal, puts down terminal the connection with the T4 dna ligase again; 304 Kpn I site is disappeared, obtain the TRE-KB-PminCMV element; Again rtTA2S-M2 is connected to the 3 ' end of TRE-KB-PminCMV; With quadrat method the EcoR I site between TRE-KB-PminCMV and rtTA2S-M2 is disappeared, obtain having the intermediate carrier pShuttle-TRE-KB-PminCMV-EB-rtTA2S-M2-SV40polyA of composite component TRE-KB-PminCMV-EB-rtTA2S-M2.

4, make up the Tet-on abduction delivering system of single carrier two-way startup

Use the ligase enzyme connection method; With element 5 '-CMV-3 '; 5 '-TetR-KRAB-3 ' successively is connected on the pShuttle-KSL-SV40polyA; 5 '-SV40polyA-3 ' and 3 '-rtTA2S-M2-TRE-PminCMV-5 ' is connected to 5 '-SV40polyA-3 '-3 '-rtTA2S-M2-TRE-PminCMV-5 '; And be connected to the 3 ' end of elements T etR-KRAB together, again 5 '-Tight-PminCMV-3 is connected to the 5 ' end of 3 '-rtTA2S-M2-TRE-PminCMV-5 ', the Tet-on abduction delivering system of single carrier two-way startup.

The purposes of the Tet-on abduction delivering system of single carrier two-way startup in preparation treatment colorectal carcinoma medicine.

Effective constituent prepares the Tet-on abduction delivering system of carrying single carrier two-way startup in the E1 district and the adenovirus carrier of regulating and expressing goal gene TRAIL, and preparation medicine for treating tumor thing uses with the form of conventional injection.Described medicinal conventional formulation contains as the preparation E1 district of activeconstituents and carries the Tet-on abduction delivering system of single carrier two-way startup and the adenovirus carrier of regulating and expressing goal gene TRAIL, and this activeconstituents mixes like the organic or inorganic liquid excipient that is suitable for intravenous administration with pharmaceutically acceptable carrier in preparation.

The purposes of the Tet-on abduction delivering system of single carrier two-way startup in preparation treatment colorectal carcinoma medicine; Preparation E1 carries the Tet-on abduction delivering system of single carrier two-way startup and the adenovirus carrier of regulating and expressing goal gene TRAIL in the district; Preparation treatment colorectal carcinoma medicine uses with injection liquid, and it is following to prepare draw raw material and proportioning thereof of this medicine 1000:

E1 carries in the district Tet-on abduction delivering system of single carrier two-way startup and the adenovirus carrier 2 * 10 of regulating and expressing goal gene TRAIL ¹⁵Vp

Water for injection adds to 2000mL

The preparation method is undertaken by the ordinary method of pharmaceutics injection.Every 2mL, every mL contain activeconstituents E1 district and carry the Tet-on abduction delivering system of single carrier two-way startup and the adenovirus carrier 1 * 10 of regulating and expressing goal gene TRAIL ¹²Vp.

1 injection every day during use, by the knurl volume calculation, the knurl body<3cm ³, consumption is 2 * 10 ¹²Promptly 1 in vp virus; 3cm ³<the knurl body<5cm ³, consumption is 3 * 10 ¹²Promptly 1.5 in vp virus; Knurl Ti>5cm ³, consumption is 4 * 10 ¹²Promptly 2 in vp virus, each multiple spot intratumor injection.Treat continuous 5 days of the medication course of treatment.During the medication simultaneously by the oral vibra-of conventional antibiotic dosage.

The present invention has improved the structure of Tet-on abduction delivering system; The expression of whole controlling elements and goal gene all has been structured on the carrier; Overcome the inefficiencies of 2 plasmid Tet-on systems; Regulation and control to genetic expression simultaneously have exactness, and can be applied to like virus vector such as adenovirus carriers, make the treatment of gene safer and effective.

Description of drawings

Fig. 1 is the structure flow process of carrier pShuttle-KSL-SV40polyA-KB.

Fig. 2 is the structure flow process that has the intermediate carrier of composite component TRE-KB-PminCMV-EB-rtTA2S-M2.

Fig. 3 is the Tet-on abduction delivering system constructing flow process and the structure of single carrier two-way startup.

Fig. 4 is the structure of carrier pE1-Bi-Tet-on.

Fig. 5 is the structure of carrier pE1-r-Tet-on.

The Tet-on abduction delivering system of Fig. 6 list carrier two-way startup is to the regulating and controlling effect of eGFP genetic expression.

The Tet-on abduction delivering system of Fig. 7 list carrier two-way startup is to the regulating and controlling effect of Flu genetic expression.

The temporal induction curve of the Tet-on abduction delivering system of Fig. 8 list carrier two-way startup.

The Tet-on abduction delivering system drug dose of Fig. 9 list carrier two-way startup relies on curve.

The going of the Tet-on abduction delivering system of Figure 10 list carrier two-way startup closed closed curve behind the medicine.

The Tet-on abduction delivering system of Figure 11 list carrier two-way startup is to the Western Blot detected result of the regulating and controlling effect of FAM76B-Flag genetic expression.

Figure 12 is that the adenovirus carrier of the E1 district Tet-on abduction delivering system and the regulating and expressing trail dna that have single carrier two-way startup is in external growth-inhibiting effect to human colon cancer cell SW480.

Embodiment

To further explain of the present invention, but the invention is not restricted to these embodiment below in conjunction with accompanying drawing and embodiment.

Embodiment 1

The Tet-on abduction delivering system of single carrier two-way startup of present embodiment, the order of connection of its element are 5-' CMV-TetR-KRAB-SV40polyA-rtTA2S-M2-EB-PminCMV-KB-TRE-Tigh t-PminCMV-EcoR I-Spe I-SV40polyA-3 '.

The mode of connection of the composite component rtTA2S-M2-EB-PminCMV-KB-TRE in Tight-PminCMV left side is 3 '-rtTA2S-M2-EB-PminCMV-KB-TRE-5 '-5 '-Tight-PminCMV-3 ', and all the other elements are all by being linked in sequence with this system's equidirectional 5 '-3 '; Two restriction enzyme site EcoR I and Spe I are between elements T ight-PminCMV and element SV40polyA.No.11 in this system's sequence such as the nucleotides sequence tabulation.

Its construction process step is following:

The two-way polyadenylic acid signal SV40polyA sequence of restriction enzyme site Xba I and Bgl II is contained at synthetic two ends in the biological Shanghai of student on commission worker ltd; Be connected on the carrier pUC19-SV40polyA, be connected through SV40polyA fragment that obtains after the DNA agarose electrophoresis purifying and recovering and the adenovirus E 1 district shuttle vectors pShuttle that cuts through same enzyme (purchase is from addgene company) through behind Xba I and the Bgl II double digestion.The T4 ligase enzyme connects, and condition of contact is: 2 μ l enzymes are cut the purifying fragment, 5 μ l, 2 * T4 ligase enzyme damping fluid; 0.5 μ l pShuttle carrier, 0.5 μ l T4 ligase enzyme, 2 μ l tri-distilled waters; 25 ℃ connect 1 hour, with the DH5 α cell that connects the product transformed competence colibacillus.And coat in the LB flat board of the kantlex that contains 30 μ g/ml.Picking colony is inoculated in the LB nutrient solution of kantlex of 30ug/ml, after 14-16 hour, extracts DNA through the alkaline bleach liquor cleavage method, identifies the acquisition positive colony through Xba I and Bgl II double digestion.The carrier called after pShuttle-SV40polyA that obtains sees Fig. 1.After this carrier being cut through EcoR I enzyme, it is flat to use the T4DNA polysaccharase that sticky end is mended again, and reaction system is: 20 μ l enzymes are cut product; 3 μ l EcoR I enzyme cutting buffering liquids, 1 μ l 10mM dNTP, 1 μ l T4DNA polysaccharase; 25 μ l tri-distilled waters, 37 ℃ were reacted 1 hour.Mend flat terminal product, put down terminal the connection, condition of contact is: 1 μ l mends flat end products; 5 μ l, 2 * T4 ligase enzyme damping fluid, 0.5 μ l T4 ligase enzyme, 3.5 μ l tri-distilled waters; 25 ℃ connect 1 hour; Make the EcoR I be positioned on last 2668 of the former pShuttle disappear, the carrier of acquisition is called pShuttle-SV40polyA-EB, sees Fig. 1.With carrier pShuttle-SV40polyA-EB behind Kpn I and Xba I double digestion; Be connected with DNAlinker (Kpn I-Cla I-Not I-Xho I-EcoR I-Spe I-Spe I); The positive colony that obtains is called pShuttle-KSL-SV40polyA, sees Fig. 1.Carrier pShuttle-KSL-SV40polyA is cut through Kpn I enzyme; After the T4DNA polysaccharase is equalled the sticky end benefit; After the T4 ligase enzyme connects; Kpn I site on the carrier pShuttle-KSL-SV40polyA is disappeared, and the positive colony of acquisition is called pShuttle-KSL-SV40polyA-KB, sees Fig. 1.

The peGFPN1 carrier is available from CLONTECH company; PeGFPN1 is the polymerase chain reaction template, through the sequence of polymerase chain reaction acquisition increasing human cytomegalic inclusion disease virus promotor CMV, and in 5 ' end introducing Kpn I site; 3 ' end is introduced Cla I site, and P1-P2 is a pair of primer P1: GGTACCAATAGTTATTAATAGTAA, P2: ATCGATGATCTGACGGTTCACTAA.The PCR amplification condition is: 94 ℃, 2 minutes, and 94 ℃, 50 seconds, 55 ℃, 60 seconds, 72 ℃, 60 seconds, 30 circulations.Overlapping polymerase chain reaction product is to reclaim fragment after 1.0% the agarose electrophoresis through mass concentration.The polymerase chain reaction fragment is connected with pGEMT easy carrier.Condition of contact is: 2 μ l enzymes are cut the purifying fragment; 5 μ l, 2 * T4 ligase enzyme damping fluid, 0.5 μ l pGEMT easy carrier, 0.5 μ l T4 ligase enzyme; 2 μ l tri-distilled waters; 25 ℃ connect 1 hour, with the DH5 α cell that connects the product transformed competence colibacillus, and coat in the LB flat board of the penbritin that contains 100 μ g/ml.Picking colony is inoculated in the LB nutrient solution of the penbritin that contains 100 μ g/ml, after 14～16 hours, extracts DNA through the alkaline bleach liquor cleavage method, through the enzyme evaluation positive colony of cutting and check order.Institute is obtained positive colony called after pGEMT/CMV.

Carrier pLVCT-tTR-KRAB buys from addgene company; With pLVCT-tTR-KRAB is the polymerase chain reaction template; Obtain tsiklomitsin Transcriptional Silencing vitamin T etR-KRAB sequence through the polymerase chain reaction; And in 5 ' end introducing Cla I site, it is a pair of primer P3:A that 3 ' end is introduced Not I site P3-P4 ATCGATATGGCTAGATTAGATAAA, P4:T GCGGCCGCTTAAACTGATGATTTGAT.The PCR amplification condition is: 94 ℃, 2 minutes, and 94 ℃, 50 seconds, 55 ℃, 60 seconds, 72 ℃, 70 seconds, 30 circulations.Product reclaims the back and is connected with pGEMT easy carrier as previously mentioned, and the positive colony of acquisition is called pGEMT/TetR-KRAB.

With the pUC19-SV40polyA described in the step 1 is template, and same PCR amplification obtains the SV40polyA element, and introduces Not I site at 5 ' end, and 3 ' end is introduced Xba I site, and P5-P6 is a pair of amplimer, P5: GCGGCCGCCACAGCGGGGAGATCCAG, P6:C TCTAGACTCATGGCTGCGCCCCGA, the PCR amplification condition is: 94 ℃, 2 minutes, 94 ℃, 50 seconds, 55 ℃, 60 seconds, 72 ℃, 30 seconds, 30 circulations.Product reclaims the back and is connected with pGEMT easy carrier as previously mentioned, and the positive colony of acquisition is called pGEMT/SV40polyA.

Use synthetic primer P7-P8 to be template and primer, the minimum human cytomegalic inclusion disease virus promotor of the rigorous type of PCR amplification Tight-PminCMV sequence, and in 5 ' end introducing Sal I site, 3 ' end is introduced EcoR I site, P7:G GTCGACTAGGCGTGTACGGTGGGAGGCCTATATAAGCAGAGCTCGT, P8:G GAATTCGCGATCTGACGGTTCACTAAACGAGCTCTGCTTATATAGG.Synthase Kettenreaktion amplification condition is: 94 ℃, 2 minutes, and 94 ℃, 50 seconds, 55 ℃, 60 seconds; 72 ℃, 20 seconds; 30 circulations, product are reclaimed the back through agarose electrophoresis and are connected with pGEMT easy carrier, and the positive colony of acquisition is called pGEMT/Tight-PminCMV.

Carrier pTet-on advanced; Available from CLONTECH company; With pTet-on advanced is the polymerase chain reaction template, obtains trans tsiklomitsin activating transcription factor rtTA2S-M2 sequence through the polymerase chain reaction, and introduces EcoR I site at 5 ' end; 3 ' end is introduced Spe I site, and P9-P10 is a pair of amplimer P9:G GAATTCATGTCTAGACTGGACAAG, P10:G ACTAGTTTACCCGGGGAGCATGTC.Synthase Kettenreaktion amplification condition is: 94 ℃, 2 minutes, and 94 ℃, 50 seconds, 55 ℃, 60 seconds, 72 ℃, 60 seconds, 30 circulations.Product reclaims the back and is connected with pGEMT easy carrier as previously mentioned, and the positive colony of acquisition is called pGEMT/rtTA2S-M2.

Carrier pTRE2pur carrier is available from CLONTECH company; Behind Xho I and EcoR I double digestion, be to reclaim the minimum human cytomegalic inclusion disease virus promotor TRE-PminCMV fragment that has tsiklomitsin response element TRE after 1.0% the agarose electrophoresis through reaction product through massfraction, be connected with the carrier pShuttle-KSL-SV40polyA-KB (described in the step 1) that cuts through same enzyme; Condition of contact is: the TRE-PminCMV fragment that 2 μ l enzymes are cut purifying; 5 μ l, 2 * T4 ligase enzyme damping fluid, 0.5 μ l pShuttle-KSL-SV40polyA-KB carrier, 0.5 μ l T4 ligase enzyme; 2 μ l tri-distilled waters; 25 ℃ connect 1 hour, connect product and are transformed into competent DH5 α cell, the picking clone; Identify that through Xho I and EcoR I double digestion the positive colony that the back is obtained is called pShuttle-TRE-PminCMV-SV40polyA, sees Fig. 2.PShuttle-TRE-PminCMV-SV40polyA is cut through Kpn I enzyme; The T4DNA polysaccharase is mended sticky end flat; After the T4DNA ligase enzyme connects; Kpn I site in the TRE-PminCMV fragment is disappeared, and conversion and enzyme are cut and are identified that the positive colony that the back obtains is called pShuttle-TRE-KB-PminCMV-SV40polyA, sees Fig. 2.With carrier pGEMT/rtTA2S-M2 behind EcoR I and Spe I double digestion; Be to reclaim after 1.0% the agarose electrophoresis to obtain fragment rtTA2S-M2 through massfraction; It is connected with the carrier pShuttle-TRE-KB-PminCMV-SV40polyA that cuts through same enzyme; The positive colony that obtains is called pShuttle-TRE-KB-PminCMV-rtTA2S-M2-SV40polyA, sees Fig. 2.This carrier is cut through EcoR I enzyme again; Mend flat end, after the connection, the EcoR I site between the TRE-KB-PminCMV-rtTA2S-M2 is disappeared; The positive colony that obtains is called pShuttle-TRE-KB-PminCMV-EB-rtTA2S-M2-SV40polyA, sees Fig. 2.This carrier is the intermediate carrier that needs in the building process, comprises composite component TRE-KB-PminCMV-EB-rtTA2S-M2 on it.

With carrier pGEMT/CMV process Kpn I and Cla I double digestion; And be to reclaim the CMV fragment that obtains after 1.0% the agarose electrophoresis to be connected with the carrier pShuttle-KSL-SV40polyA that cuts through same enzyme through massfraction, transform and enzyme is cut the positive colony of identifying acquisition and is called pShuttle-CMV-SV40polyA.With carrier pGEMT/TetR-KRAB through Cla I and Not I double digestion and through TetR-KRAB fragment that recovery after 1.0% the agarose electrophoresis obtains with cut through same enzyme after carrier pShuttle-CMV-SV40polyA be connected; Conversion and enzyme are cut evaluation, and the positive colony of acquisition is called pShuttle-CMV-TetR-KRAB-SV40polyA.Carrier pGEMT/SV40polyA is through Not I and Xba I double digestion; And be to reclaim after 1.0% the agarose electrophoresis to obtain the SV40polyA fragment through massfraction; The intermediate carrier pShuttle-TRE-KB-PminCMV-EB-rtTA2S-M2-SV40polyA that obtains in the step 2 is through Spe I and Xho I double digestion; And be to reclaim after 1.0% the agarose electrophoresis to obtain composition element fragment 5 '-TRE-KB-PminCMV-EB-rtTA2S-M2-3 ' through massfraction, carrier S huttle-CMV-TetR-KRAB-SV40polyA is behind Not I and Xho I double digestion, and the while is connected with fragment TRE-KB-PminCMV-EB-rtTA2S-M2 with fragment SV40polyA; Condition of contact is: 2 μ l fragment SV40polyA; 2 μ l fragment TRE-KB-PminCMV-EB-rtTA2S-M2,5 μ l, 2 * T4 ligase enzyme damping fluid, 0.5 μ l pShuttle-CMV-TetR-KRAB-SV40polyA carrier; 0.5 μ l T4 ligase enzyme, 25 ℃ connect 1 hour.Through transforming; Enzyme is cut and is identified that the positive colony that the back obtains is called pShuttle-CMV-TetR-KRAB-SV40polyA-rtTA2S-M2-EB-PminCMV-KB-TRE-SV40polyA; The mode of connection of composite component 5 ' in this carrier-TRE-KB-PminCMV-EB-rtTA2S-M2-3 ' is that its 3 ' end links to each other with the 3 ' end of CMV-TetR-KRAB-SV40polyA, and 5 ' end is positioned at Xho I site on the carrier.Carrier pGEMT/Tight-PminCMV is behind Sal I and EcoR I double digestion; After 2.0% agarose electrophoresis, reclaim and obtain fragment Tight-PminCMV; With its be connected with carrier pShuttle-CMV-TetR-KRAB-SV40polyA-rtTA2S-M2-EB-PminCMV-KB-TRE-SV40polyA after EcoR I enzyme is cut through the Xho I; Conversion and enzyme are cut evaluation; The positive colony that obtains is pShuttle-CMV-TetR-KRAB-SV40polyA-rtTA2S-M2-EB-PminCMV-KB-TRE-Tight-PminCMV-SV40polyA; And with its called after pE1-K-Bi-Tet-on; See Fig. 3,5-' the CMV-TetR-KRAB-SV40polyA-rtTA2S-M2-EB-PminCMV-KB-TRE-Tigh t-PminCMV-EcoR I-Spe I-SV40polyA-3 ' structure on this carrier is the Tet-on abduction delivering system of single carrier two-way startup, can insert goal gene or DNA linker between the EcoR I on the carrier-Spe I site.The Tet-on abduction delivering system of constructed single carrier two-way startup has the two-way startup structure in the present embodiment; The Tight-PminCMV on TRE right side starts the expression of goal gene; The PminCMV in TRE left side then expresses activating transcription factor rtTA2S-M2, and activating transcription factor rtTA2S-M2 can combine the TRE sequence, activates the genetic expression of TRE both sides simultaneously; So rtTA2S-M2 has formed self-activated structure under self promoter regulation.And this structure is when induced drug does not exist, the expression of activating transcription factor self seldom, decapacitation reduces outside the background seepage when goal gene is non-induces, and also can reduce the interference of rtTA2S-M2 pair cell.And this system has also introduced Transcriptional Silencing vitamin T etR-KRAB, and the product albumen of this gene can combine the TRE sequence; Suppress the genetic expression of TRE both sides, reduce the background seepage, and after adding the induced drug vibra-; TetR-KRAB can break away from the TRE sequence, remove transcripting suppressioning action, and meanwhile incitant rtTA2S-M2 can change the structure phase under drug effect; And be attached on the TRE sequence, activate self and the expression of goal gene.And the TRE sequence is positioned at the system middle part in this system architecture; So eliminated that TetR-KRAB has to the transcripting suppressioning action about TRE upstream and downstream 2KB; So system applies is to as on the virus vector such as adenovirus the time; Can not have influence on the viral genome gene transcription, thereby reduce influence the adenovirus packing.

Made up carrier pShuttle-SV40polyA-rtTA2S-M2-EB-PminCMV-KB-TRE-Tight-Pmi nCMV-SV40polyA simultaneously; And called after pE1-Bi-Tet-on; See Fig. 4, its construction process and carrier construction pE1-K-Bi-Tet-on are similar, just are not connected into these two elements of CMV and TetR-KRAB; Thereafter SV40polyA, TRE-KB-PminCMV-EB-rtTA2S-M2 and Tight-PminCMV all are connected among the carrier pShuttle-KSL-SV40polyA by above-mentioned restriction enzyme site.In addition, made up pE1-CMV-rtTA2S-M2-SV40polyA-TRE-Tight-PminCMV as contrast, other is called pE1-r-Tet-on; See Fig. 5; Its constructional feature is non-two-way startup, and rtTA2S-M2 is started by the CMV promotor, and goal gene is started by TRE-PminCMV.

Embodiment 2

The Tet-on abduction delivering system of single carrier two-way startup of the expression eGFP gene of present embodiment; The order of connection of element is 5-' CMV-TetR-KRAB-SV40polyA-rtTA2S-M2-EB-PminCMV-KB-TRE-Tigh t-PminCMV-EcoR I-eGFP-Spe I-SV40polyA-3 ', has inserted reporter gene eGFP in two restriction enzyme site EcoR I and the Spe I.

Its construction process step is following:

This embodiment 1-4 step and embodiment 1 are identical.

5, the Tet-on abduction delivering system of single carrier two-way startup of construction expression eGFP gene

With carrier pLVCT-tTR-KRAB is template, PCR amplification eGFP gene, and in 5 ' end introducing EcoR I site, 3 ' end is introduced Spe I site.P11-P12 is a pair of amplimer, P11:C GAATTCATGGTGAGCAAGGGCGAG, P12:C ACTAGTTTACTTGTACAGCTCGTC.Product reclaims the back and is connected with pGEMT easy carrier, and the positive colony of acquisition is called pGEMT/eGFP.After using EcoR I and Spe I double digestion; Reclaim purifying and obtain fragment; Be connected with the carrier pE1-K-Bi-Tet-on that cuts through same enzyme; The positive colony that obtains is called pE1-K-Bi-Tet-on-eGFP, and 5-' the CMV-TetR-KRAB-SV40polyA-rtTA2S-M2-EB-PminCMV-KB-TRE-Tigh t-PminCMV-EcoR I-eGFP-Spe I-SV40polyA-3 ' structure on this carrier is the Tet-on abduction delivering system of single carrier two-way startup of expressing the eGFP gene.Be as contrast in addition; The eGFP fragment is connected in the carrier pcDNA3.1 of EcoR I and Xba I double digestion; The carrier that obtains is called pcDNA3.1/eGFP; Be connected into the carrier that obtains among the pE1-r-Tet-on and be called pE1-r-Tet-on-eGFP, be connected among the pE1-Bi-Tet-on, the carrier of acquisition is called pE1-Bi-Tet-on-eGFP.

6, test express the eGFP gene single carrier two-way startup Tet-on abduction delivering system induce performance

PE1-K-Bi-Tet-on-eGFP, pE1-Bi-Tet-on-eGFP, pE1-r-Tet-on-eGFP, pcDNA3.1/eGFP, pcDNA3.1 carrier that present embodiment is made up are transfected into the HEK293 cell.Changing the DMEM substratum that contains 10% NBCS after 4 hours continues to cultivate.Every kind of plasmid transfection two dish cells, and in wherein adding vibra-in the dish cell, making its final concentration is 2 μ g/ml, observes the expression intensity and the background seepage situation of each carrier after 48 hours.The use inverted fluorescence microscope is taken a picture, observe respectively organize carrier induce with non-induction state under the expression of eGFP, experimental result is seen Fig. 6; Visible by Fig. 6; 5 are empty carrier pcDNA3.1 group, and this organizes negative control group, no matter whether adds the equal redgreen fluorescent signal of inductor vibra-; 4 are the pcDNA3.1/eGFP group, and this organizes positive control group, and no matter whether adding vibra-all has the green fluorescence signal; 3 is pE1-r-Tet-on-eGFP, and the Tet-on system of this unilateral initiative structure is visible a little green fluorescence signal when not adding powerful mycin, and there is the background seepage in prompting; 2 are the pE1-Bi-Tet-on-eGFP group, and this group has the two-way startup structure, and the background seepage obviously is less than the pE1-r-Tet-on-eGFP group; 1 is the pE1-K-Bi-Tet-on-eGFP group; It is the Tet-on abduction delivering set of systems of single carrier two-way startup; This group have only added vibra-after, the green fluorescence signal just appears, explain that native system has low background seepage; And add its green fluorescence strength of signal of vibra-and positive control basically identical, what native system was described induces intensity enough.

Embodiment 3

Expression Lampyridea luciferase genes (the firefly luciferase of present embodiment; The Tet-on abduction delivering system of single carrier two-way startup Flu); The order of connection of element is 5-' CMV-TetR-KRAB-SV40polyA-rtTA2S-M2-EB-PminCMV-KB-TRE-Tigh t-PminCMV-EcoR I-Flu-Spe I-SV40polyA-3 ', has inserted reporter gene Flu in two restriction enzyme site EcoR I and the Spe I.

Its construction process step is following:

This embodiment 1-4 step and embodiment 1 are identical.

5, the Tet-on abduction delivering system of single carrier two-way startup of construction expression Flu gene

Carrier Luciferase-pcDNA3 is template available from addgene company with carrier Luciferase-pcDNA3, PCR amplification Flu gene, and in 5 ' end introducing EcoR I site, 3 ' end is introduced Spe I site.P13-P14 is a pair of amplimer, P13:C GAATTCATGGAAGACGCCAAAAAC, P14:C ACTAGTTTACACGGCGATCTTTCC.Product reclaims the back and is connected with pGEMT easy carrier as previously mentioned, and the positive colony of acquisition is called pGEMT/Flu.After using EcoR I and Spe I double digestion, reclaim purifying and obtain fragment, be connected with the carrier pE1-K-Bi-Tet-on that cuts through same enzyme, the positive colony of acquisition is called pE1-K-Bi-Tet-on-Flu.5-' CMV-TetR-KRAB-SV40polyA-rtTA2S-M2-EB-PminCMV-KB-TRE-Tigh t-PminCMV-EcoR I-Flu-Spe I-SV40polyA-3 ' structure on this carrier is the Tet-on abduction delivering system of single carrier two-way startup of expressing the Flu gene.Be as contrast in addition, the Flu fragment connected in the carrier pcDNA3.1 of EcoR I and Xba I double digestion that the carrier of acquisition is called pcDNA3.1/Flu.

6, test express the Flu gene single carrier two-way startup Tet-on abduction delivering system induce performance

Inoculation HEK293 cell treats that to 24 orifice plates cell density reaches at 70% o'clock, uses Lipofectamine ^TM2000 lipofectamine box transfectional cells; The pE1-K-Bi-Tet-on-Flu of mole number such as get respectively, negative control pcDNA3.1 and positive control pcDNA3.1/Flu plasmid and sea pansy luciferase confidential reference items plasmid pRL-CMV (available from Promega company) (0.4 μ g) cotransfection are gone into the HEK293 cell, two groups of cells of each plasmid transfection; Every group of 3 repetitions; The substratum that transfection more renewed after 4 hours, and in wherein adding vibra-in one group of cell, making its final concentration is 2 μ g/ml.Collecting cell after 48 hours, the method lysing cell that uses two luciferase assay test kit Dual-Luciferase Reporter Assay System (available from Promega company) to provide, the column criterion of going forward side by side luciferase vitality test.Measuring method is following:

(1) cell culture medium in 24 orifice plates is sopped up, every hole adds the 1 * lysis Buffer 200mL that provides in the test kit, and cracking is 10 minutes under the shaking table 100rpm.

(2) collect every empty split product and go in the Eppendorf pipe, centrifugal 10 minutes of 4 ℃ of following 16000g collect supernatant and go in the Eppendorf pipe of new precooling.

(3) use white 96 orifice plates, every hole adds lysate 20 μ L, and every hole adds Photinus pyralis LUC substrate 50 μ L, carries out the luciferase vitality test first time.

(4) every hole adds 1 * Stop&Glo reaction solution, stops Lampyridea luciferase vigor, and sea pansy luciferase reaction substrate is provided, and carries out the luciferase vitality test second time.

(5) draw the plain enzyme activity of standard fluorescence=Photinus pyralis LUC energy value (relative light unit, RLU)/sea pansy luciferase energy value (RLU).

Through contrasting the plain enzyme activity value of the standard fluorescence of 3 kinds of carriers before and after inducing, come quantitative response carry single carrier two-way startup Tet-on abduction delivering system carrier pE1-K-Bi-Tet-on induce intensity, background seepage degree; Induce multiple, the result sees Fig. 7, and 6 are the pE1-K-Bi-Tet-on-Flu group among the figure; This group shows significantly induces difference, and the high multiple (137 times) of inducing, and the background seepage is very little; When not inducing, expression amount is near the negative control level, 7 positive contrasts; 8 negative control groups, these two groups are not all had and significantly induce difference.Explain that the pE1-K-Bi-Tet-on carrier has good inducibility.

7, the Tet-on abduction delivering system time of measuring single carrier two-way startup of expressing the Flu gene is induced curve

Inoculation CHO-K1 cell treats that to 24 orifice plates cell density reaches at 70% o'clock, uses Lipofectamine ^TM2000 lipofectamine boxes divide transfectional cell, and experiment is divided into 12 groups, and 3 every group parallel appearance are gone into cell with carrier pE1-K-Bi-Tet-on-Flu and pRL-CMV cotransfection, change the DMEM that contains 10% NBCS after 4 hours and continue to cultivate; In every porocyte, add vibra-; Making its final concentration is 2 μ g/ml; Collected the plain enzyme activity value of one group of raji cell assay Raji standard fluorescence in per 4 hours; Numerical value is depicted as the graphic representation about the time, thereby obtains expressing the temporal induction curve of Tet-on abduction delivering system of single carrier two-way startup of Flu gene, see Fig. 8.The result can find out that the intensity of inducing of system reaches maximum after 40 hours, proves this system induction rapid speed.

8, measure the drug dose dependence curve of the Tet-on abduction delivering system of single carrier two-way startup of expressing the Flu gene

Inoculation CHO-K1 cell treats that to 24 orifice plates cell density reaches at 70% o'clock, uses Lipofectamine ^TM2000 lipofectamine boxes divide transfectional cell, and experiment is divided into 6 groups every group two parallel appearance, and transfection plasmid pE1-K-Bi-Tet-on-Flu and pRL-CMV go into the HEK293 cell, change the DMEM that contains 10% NBCS after 4 hours and continue to cultivate; Every group adds vibra-respectively, makes final concentration be respectively 10ng/ml, 100ng/ml; 1000ng/ml, 2000ng/ml, 4000ng/ml; 8000ng/ml, collecting cell after 48 hours, bioassay standard luciferase energy value; And obtain expressing the drug dose curve of Tet-on abduction delivering system of single carrier two-way startup of Flu gene, see Fig. 9.Can confirm that by figure this system has maximum to induce intensity under the vibra-concentration of 2 μ g/ml.

9, close closed curve after inductor is removed by the Tet-on abduction delivering system of single carrier two-way startup of mensuration expression Flu gene

Inoculation CHO-K1 cell treats that to 24 orifice plates cell density reaches at 70% o'clock, uses Lipofectamine ^TM2000 lipofectamine boxes divide transfectional cell, and experiment is divided into 6 groups every group three parallel appearance, and all transfection pE1-K-Bi-Tet-on-Flu and pRL-CMV go into the HEK293 cell, change the DMEM that contains 10% NBCS after 4 hours and continue to cultivate; Every group all adds vibra-; Making final concentration is 2000ng/ml, changes the DMEM that contains 10% NBCS after 72 hours and continues to cultivate, and no longer add vibra-; Collect one group of sample every day; Bioassay standard luciferase energy value, thus obtain expressing the Flu gene single carrier two-way startup Tet-on abduction delivering system go close closed curve behind the medicine, the result sees Figure 10.Can see among the figure remove induced drug after, the abduction delivering system after 24 hours expression amount just drop to below 10% of high expression level amount, prove that the Tet-on abduction delivering system of single carrier two-way startup has the characteristic of quick closedown.

Embodiment 4

The Tet-on abduction delivering system of single carrier two-way startup of the expressed fusion protein FAM76B-Flag of present embodiment; The order of connection of element is 5-' CMV-TetR-KRAB-SV40polyA-rtTA2S-M2-EB-PminCMV-KB-TRE-Tigh t-PminCMV-EcoR I-Xho I-FAM76B-Flag-SV40polyA-3 ', has successively inserted DNA linker (EcoR I-Xho I-Spe I) and reporter gene FAM76B-Flag in two restriction enzyme site EcoR I and the Spe I.

Its construction process step is following:

This embodiment 1-4 step and embodiment 1 are identical.

5, the Tet-on abduction delivering system of single carrier two-way startup of construction expression fusion protein F AM76B-Flag

With human cDNA library is template, PCR amplification FAM76B-Flag antigen-4 fusion protein gene, and introduce through Xho I site at 5 ' end, 3 ' end is introduced Xba I site.Primer P15-P16 is a pair of amplimer, P15: CTCGAGATGGCGGCCTCGGCCCTG, P16: TCTAGATTATTTGTCATCGTCATCTTTGTAATCAGGAGATGTTAGTATGCT.Gene is connected to the positive colony that obtains on the pGEMT easy carrier and is called pGEMT/FAM76B-Flag, and purifying and recovering obtains fragment FAM76B-Flag behind Xho I and Xba I double digestion.Article two, synthetic dna single chain, L1:AATTCCTCGAGA, L2:CTAGTCTCGAGG, two strands are diluted to 20 μ M concentration, respectively get 20 μ l and mix, and annealing at room temperature two hours then forms DNA linker (EcoR I-Xho I-Spe I).Carrier pE1-K-Bi-Tet-on is connected with DNA linker (EcoR I-Xho I-Spe I) behind EcoR I and Spe I double digestion, and the positive colony of acquisition is called pE1-K-Bi-Tet-on-ESL.Carrier pE1-K-Bi-Tet-on-ESL obtains carrier through Xho I and Spe I double digestion; Be connected the conversion back with fragment FAM76B-Flag and obtain positive colony; Called after pE1-K-Bi-Tet-on-FAM76B-Flag, the structure of 5-' the CMV-TetR-KRAB-SV40polyA-rtTA2S-M2-EB-PminCMV-KB-TRE-Tigh t-PminCMV-EcoR I-Xho I-FAM76B-Flag-SV40polyA-3 ' on this carrier is the Tet-on abduction delivering system of single carrier two-way startup of expressing the FAM76B-Flag gene.Fragment FAM76B-Flag is connected in the carrier pcDNA3.1 of Xho I and Xba I double digestion, and the carrier of acquisition is called pcDNA3.1/FAM76B-Flag.

6, the background seepage situation of the Tet-on abduction delivering system of single carrier two-way startup of mensuration expressed fusion protein FAM76B-Flag

Cultivate the HEK293 cell in 4 60mm flat boards; Treat that the cell fraction of coverage reaches at 50% o'clock, use calcium phosphate method respectively the plasmid pE1-K-Bi-Tet-on-FAM76B-Flag, positive control plasmid pcDNA3.1/FAM76B-Flag and the negative control pcDNA3.1 that in embodiment 3 steps 5, obtain of transfection go into the HEK293 cell; Every group of plasmid transfection 2 dishes.Put in 37 ℃ of cell culture incubators, change the DMEM that contains 10% NBCS after 4 hours and continue to cultivate; And in every group wherein a dish add vibra-in the cell, making its final concentration is 2 μ g/ml.Harvested cell after 48 hours, cracking is also gathered in the crops albumen, afterwards 6 kinds of albumen is respectively got appearance on the sample of an amount of volume, carries out the SDS-PAGE electrophoresis.Semidrying from gel electrotransfer albumen appearance to pvdf membrane.One anti-is mouse anti Flag tag monoclonal antibody; Two anti-(ECL luminous detection result sees Figure 11 for Horseradish Peroxidase, HRP) the anti-mouse IgG of labelled goat for the dilution horseradish peroxidase; 9,10 negative contrast pcDNA3.1 groups among the figure; 11,12 for carrying the carrier pE1-K-Bi-Tet-on-FAM76B-Flag of the Tet-on abduction delivering system group of single carrier two-way startup, and 13,14 positive contrast pcDNA3.1/FAM76B-Flag organize, and the result can find out; Can inducible expression carrier pE1-K-Bi-Tet-on-FAM76B-Flag when adding vibra-can detect the expression of FAM76B-Flag; Intensity is consistent with positive controls, detects the expression less than FAM76B-Flag when not adding vibra-, and is consistent with negative control.Western Blot detects less than the background seepage, and fruit has proved that this system has exactness when non-inducing.

Embodiment 5

The Tet-on abduction delivering system of single carrier two-way startup of the expression TRAIL of present embodiment; The order of connection of element is 5-' CMV-TetR-KRAB-SV40polyA-rtTA2S-M2-EB-PminCMV-KB-TRE-Tigh t-PminCMV-EcoR I-Xho I-TRAIL-Spe I-SV40polyA-3 '; Successively insert DNAlinker (EcoR I-Xho I-Spe I) and therapeutic gene TRAIL between two restriction enzyme site EcoR I and the Spe I; And the Tet-on abduction delivering system that will express single carrier two-way startup of TRAIL recombinates on the adenovirus skeleton carrier, obtains to have the Tet-on abduction delivering system of single carrier two-way startup and the recombinant adenovirus of regulating and expressing therapeutic gene TRAIL.

Step 1-4 is with embodiment 1.

5, the Tet-on abduction delivering system of single carrier two-way startup of construction expression therapeutic gene TRAIL

With human cDNA library is template, through design primer-oligomerization PCR amplification people Trail gene extracellular fragment, i.e. and aminoacid sequence 114-281, P17-P18 is a pair of amplimer, P17:A CTCGAGATGGTGAGAGAAAGAGGTCCTCAG, P18:A TCTAGATTAGCCAACTAAAAAGGCC.Product is after 1% agarose gel electrophoresis purifying and recovering; With this fragment subclone in pGEMT easy carrier, through enzyme cut and check order identify after, with positive colony through Xho I and Xba I enzyme cut trail dna fragment that the digestion back also obtains after the purifying and recovering and the carrier pE1-K-Bi-Tet-on-ESL that cuts through Xho I and Spe I enzyme (embodiment 4; Obtain in the step 5) connect; Condition of contact is: 4 μ l enzymes are cut the purifying fragment, 5 μ l, 2 * T4 ligase enzyme damping fluid, and 0.5 μ l enzyme is cut carrier; 0.5 μ l T4 ligase enzyme, 25 ° of C connect 1 hour.Cut through conversion, extraction DNA, enzyme subsequently and identify that obtaining positive colony is exactly to carry the Tet-on abduction delivering system of single carrier two-way startup and the adenovirus E 1 district shuttle vectors of abduction delivering TRAIL, called after pE1-K-Bi-Tet-on-Trail.(embodiment 1 with the carrier pShuttle-CMV-SV40polyA that cuts through Xho I and Spe I enzyme with the trail dna fragment with method; Obtain in the step 4) connect; The positive colony that obtains is called pE1-CMV-TRAIL, and this carrier is the adenovirus E 1 district shuttle vectors of non-abduction delivering trail dna.The eGFP gene fragment of in addition EcoR I and Xba I enzyme being cut acquisition be connected through the carrier pShuttle-CMV-SV40polyA of EcoR I with Spe I double digestion; The positive colony that obtains is called pE1-CMV-eGFP, and this carrier is for expressing the adenovirus E 1 district shuttle vectors of eGFP.

6, make up the E1 district and carry the Tet-on abduction delivering system of single carrier two-way startup and the adenovirus carrier of regulating and expressing goal gene TRAIL

Adopt calcium phosphate method the linearizing Tet-on abduction delivering system regulation that carries single carrier two-way startup of Pac I to be expressed the E1 district shuttle vectors pE1-K-Bi-Tet-on-Trail of goal gene TRAIL and through the linearizing adenovirus skeleton of Pac I pAdEasy-1 (available from addgene company) 3:1 cotransfection HEK 293 clones in molar ratio.After 7～10 days; It is thus clear that tangible cytopathy (cytopathic effect; CPE), pass through centrifugal results virus stock solution used then, after 2～3 virus amplification of taking turns; Further the virus stock solution used that is obtained is infected the cell cultures plate of 10 150cm, be used for follow-up being further purified thereby amplification obtains the virus stock solution used of q.s.The cell of the virus that infects through multigelation 3 times (returning switching in 37 ° of C water-baths and the alcohol dry ice) after centrifugal 15 minutes of 3500rpm; The Tet-on abduction delivering system of single carrier two-way startup and the adenovirus carrier of regulating and expressing goal gene TRAIL, called after Ad-K-Bi-Tet-on-Trail carry in E1 district through twice cesium chloride density gradient ultracentrifugation acquisition purifying.With method pE1-CMV-TRAIL and pE1-CMV-eGFP are called Ad-CMV-TRAIL and Ad-CMV-eGFP with the virus that pAdEasy-1 reorganization packing obtains respectively.With micro-spectrophotometer (Nanodrop), test sample article 260nm light absorption value is again according to formula: virus titer (vp/mL)=A260nm * 1.1 * 10 ¹²24 orifice plates are cultivated HEK293 to every porocyte number about 2 * 10 ⁴Individual, use massfraction viral liquid to be diluted to 7 concentration (10 as the 2%DMEM substratum ^-3, 10 ^-4, 10 ^-5, 10 ^-6, 10 ^-7, 10 ^-8, 10 ^-9), each concentration repeats 3, and every hole adds viral dilution liquid 400 μ L.Other stays the hole that does not add viral liquid as negative control.Under 37 ℃, incubator was cultivated 5-7 days, every day observation of cell, the number of CPE appears in every hole that counts.In viral liquid dilution gradient is 1 * 10 ^-9Under the condition, calculate viral vigor (IU/mL)=CPE number * 2.5 * 10 ⁹Calculate the titre/vigor of virus.In once testing together, titre/energy value of virus of A d-K-Bi-Tet-on-Trail is 76vp/IU, and titre/energy value of virus of A d-CMV-TRAIL is 220vp/IU.Can find out that the Tet-on abduction delivering system on virus vector can effectively regulate and control trail dna to be expressed, reduce in the wrapping process the viral vigor that the cytotoxicity because of trail dna causes and descend.

Embodiment 6

Water for injection adds to 2000mL

In order to verify that the present invention is applied to adenovirus carrier; Beneficial effect as treatment colorectal carcinoma medicine; The contriver adopts that E1 carries in the district Tet-on abduction delivering system of single carrier two-way startup and the adenovirus carrier Ad-K-Bi-Tet-on-Trail (following Ad-K-Bi-Tet-on-Trail) of regulating and expressing goal gene TRAIL in the embodiment of the invention 5; Carry out the growth-inhibiting experiment of external colon cancer cell SW480, the experiment situation is following:

The human colon cancer cell SW480 that takes the logarithm vegetative period is inoculated in 96 orifice plates after 0.1% trysinization, every porocyte number is 2 * 10 ⁴, add 5 * 10 respectively in every hole ⁶The adenovirus Ad-CMV-eGFP of vp (negative control), adenovirus Ad-CMV-TRAIL (positive control), adenovirus Ad-K-Bi-Tet-on-Trail, and PBS (blank).In these 4 groups, every group all is divided into two groups, wherein in the group; Add the vibra-that final concentration is 2 μ g/ml; Use mtt assay to measure 570nm place light absorption value after 72 hours, the growth-inhibiting effect of adenovirus to human colon cancer cell SW480 respectively organized in reflection, and experimental result is seen Figure 12.15 are the PBS group among Figure 12, and 16 are the Ad-CMV-eGFP group, and 17 are the Ad-CMV-TRAIL group; 18 are the Ad-K-Bi-Tet-on-Trail group; The result shows that Ad-K-Bi-Tet-on-Trail is that the Tet-on abduction delivering system of single carrier two-way startup and the adenovirus carrier of regulating and expressing goal gene TRAIL carry in the E1 district, under the situation that does not add vibra-, with blank and negative control group basically identical; There is not cell growth inhibition; And behind the adding vibra-, show the obvious growth restraining effect, approaching with positive controls.The Tet-on abduction delivering system that this list carrier two-way startup is described can be successfully applied to adenovirus carrier, and can regulate and control the expression of Antioncogene TRAIL.

Nucleotide or aminoacid sequence table

< 110>Shaanxi Normal University

< 120>the Tet-on abduction delivering system of single carrier two-way startup and construction process and application

<160>13

<210>1

<211>420

<212>DNA

< 213>synthetic

<220>

<221>PolyA_signal

<400>1

tctagacaca?gcggggagat?ccagacatga?taagatacat?tgatgagttt?ggacaaacca 60

caactagaat?gcagtgaaaa?aaatgcttta?tttgtgaaat?ttgtgatgct?attgctttat 120

ttgtaaccat?tataagctgc?aataaacaag?ttaacaacaa?caattgcatt?cattttatgt 180

ttcaggttca?gggggaggtg?tgggaggttt?tttaaagcaa?gtaaaacctc?tacaaatgtg 240

gtatggctga?ttatgatccg?gctgcctcgc?gcgtttcggt?gatgacggtg?aaaacctctg 300

acacatgcag?ctcccggaga?cggtcacagc?ttgtctgtaa?gcggatgccg?ggagcagaca 360

agcccgtcag?ggcgcgtcag?cgggtgttgg?cgggtgtcgg?ggcgcagcca?tgagagatct 420

<210>2

<211>44

<212>DNA

< 213>synthetic

<220>

<221>misc_recomb

<400>2

ggtaccatcg?atgcggccgc?ctcgaggaat?tcactagtac?tagt 44

<210>3

<211>603

<212>DNA

< 213>human cytomegalic inclusion disease virus

<220>

<221>Promoter

<400>3

ggtaccaata?gttattaata?gtaatcaatt?acggggtcat?tagttcatag?cccatatatg 60

gagttccgcg?ttacataact?tacggtaaat?ggcccgcctg?gctgaccgcc?caacgacccc 120

cgcccattga?cgtcaataat?gacgtatgtt?cccatagtaa?cgccaatagg?gactttccat 180

tgacgtcaat?gggtggagta?tttacggtaa?actgcccact?tggcagtaca?tcaagtgtat 240

catatgccaa?gtacgccccc?tattgacgtc?aatgacggta?aatggcccgc?ctggcattat 300

gcccagtaca?tgaccttatg?ggactttcct?acttggcagt?acatctacgt?attagtcatc 360

gctattacca?tggtgatgcg?gttttggcag?tacatcaatg?ggcgtggata?gcggtttgac 420

tcacggggat?ttccaagtct?ccaccccatt?gacgtcaatg?ggagtttgtt?ttggcaccaa 480

aatcaacggg?actttccaaa?atgtcgtaac?aactccgccc?cattgacgca?aatgggcggt 540

aggcgtgtac?ggtgggaggt?ctatataagc?agagctggtt?tagtgaaccg?tcagatcatc 600

gat 603

<210>4

<211>1022

<212>DNA

< 213>synthetic

<220>

<221>CDS

<400>4

atcgatatgg?ctagattaga?taaaagtaaa?gtgattaaca?gcgcattaga?gctgcttaat 60

gaggtcggaa?tcgaaggttt?aacaacccgt?aaactcgccc?agaagctagg?tgtagagcag 120

cctacattgt?attggcatgt?aaaaaataag?cgggctttgc?tcgacgcctt?agccattgag 180

atgttagata?ggcaccatac?tcacttttgc?cctttagaag?gggaaagctg?gcaagatttt 240

ttacgtaata?acgctaaaag?ttttagatgt?gctttactaa?gtcatcgcga?tggagcaaaa 300

gtacatttag?gtacacggcc?tacagaaaaa?cagtatgaaa?ctctcgaaaa?tcaattagcc 360

tttttatgcc?aacaaggttt?ttcactagag?aatgcattat?atgcactcag?cgctgtgggg 420

cattttactt?taggttgcgt?attggaagat?caagagcatc?aagtcgctaa?agaagaaagg 480

gaaacaccta?ctactgatag?tatgccgcca?ttattacgac?aagctatcga?attatttgat 540

caccaaggtg?cagagccagc?cttcttattc?ggccttgaat?tgatcatatg?cggattagaa 600

aaacaactta?aatgtgaaag?tgggtcgcca?aaaaagaaga?gaaaggtcga?cggcggtggt 660

gctttgtctc?ctcagcactc?tgctgtcact?caaggaagta?tcatcaagaa?caaggagggc 720

atggatgcta?agtcactaac?tgcctggtcc?cggacactgg?tgaccttcaa?ggatgtattt 780

gtggacttca?ccagggagga?gtggaagctg?ctggacactg?ctcagcagat?cgtgtacaga 840

aatgtgatgc?tggagaacta?taagaacctg?gtttccttgg?gttatcagct?tactaagcca 900

gatgtgatcc?tccggttgga?gaagggagaa?gagccctggc?tggtggagag?agaaattcac 960

caagagaccc?atcctgattc?agagactgca?tttgaaatca?aatcatcagt?ttaagcggcc 1020

gc 1022

<210>5

<211>72

<212>DNA

< 213>synthetic

<220>

<221>Promoter

<400>5

gtcgactagg?cgtgtacggt?gggaggccta?tataagcaga?gctcgtttag?tgaaccgtca 60

gatcgcgaat?tc 72

<210>6

<211>759

<212>DNA

< 213>synthetic

<220>

<221>CDS

<400>6

gaattcatgt?ctagactgga?caagagcaaa?gtcataaacg?gcgctctgga?attactcaat 60

ggagtcggta?tcgaaggcct?gacgacaagg?aaactcgctc?aaaagctggg?agttgagcag 120

cctaccctgt?actggcacgt?gaagaacaag?cgggccctgc?tcgatgccct?gccaatcgag 180

atgctggaca?ggcatcatac?ccacttctgc?cccctggaag?gcgagtcatg?gcaagacttt 240

ctgcggaaca?acgccaagtc?attccgctgt?gctctcctct?cacatcgcga?cggggctaaa 300

gtgcatctcg?gcacccgccc?aacagagaaa?cagtacgaaa?ccctggaaaa?tcagctcgcg 360

ttcctgtgtc?agcaaggctt?ctccctggag?aacgcactgt?acgctctgtc?cgccgtgggc 420

cactttacac?tgggctgcgt?attggaggaa?caggagcatc?aagtagcaaa?agaggaaaga 480

gagacaccta?ccaccgattc?tatgccccca?cttctgagac?aagcaattga?gctgttcgac 540

cggcagggag?ccgaacctgc?cttccttttc?ggcctggaac?taatcatatg?tggcctggag 600

aaacagctaa?agtgcgaaag?cggcgggccg?gccgacgccc?ttgacgattt?tgacttagac 660

atgctcccag?ccgatgccct?tgacgacttt?gaccttgata?tgctgcctgc?tgacgctctt 720

gacgattttg?accttgacat?gctccccggg?taaactagt 759

<210>7

<211>377

<212>DNA

< 213>synthetic

<220>

<221>Promoter

<400>7

ctcgagttta?ccactcccta?tcagtgatag?agaaaagtga?aagtcgagtt?taccactccc 60

tatcagtgat?agagaaaagt?gaaagtcgag?tttaccactc?cctatcagtg?atagagaaaa 120

gtgaaagtcg?agtttaccac?tccctatcag?tgatagagaa?aagtgaaagt?cgagtttacc 180

actccctatc?agtgatagag?aaaagtgaaa?gtcgagttta?ccactcccta?tcagtgatag 240

agaaaagtga?aagtcgagtt?taccactccc?tatcagtgat?agagaaaagt?gaaagtcgag 300

ctcggtaccc?taggcgtgta?cggtgggagg?cctatataag?cagagctcgt?ttagtgaacc 360

gtcagatcgc?ggaattc 377

<210>8

<211>372

<212>DNA

< 213>synthetic

<220>

<221>mutation

<400>8

ctcgagttta?ccactcccta?tcagtgatag?agaaaagtga?aagtcgagtt?taccactccc 60

tatcagtgat?agagaaaagt?gaaagtcgag?tttaccactc?cctatcagtg?atagagaaaa 120

gtgaaagtcg?agtttaccac?tccctatcag?tgatagagaa?aagtgaaagt?cgagtttacc 180

actccctatc?agtgatagag?aaaagtgaaa?gtcgagttta?ccactcccta?tcagtgatag 240

agaaaagtga?aagtcgagtt?taccactccc?tatcagtgat?agagaaaagt?gaaagtcgag 300

ctcgctaggc?gtgtacggtg?ggaggcctat?ataagcagag?ctcgtttagt?gaaccgtcag 360

atcgcggaat?tc 372

<210>9

<211>1125

<212>DNA

< 213>synthetic

<220>

<221>misc_feature

<400>9

ctcgagttta?ccactcccta?tcagtgatag?agaaaagtga?aagtcgagtt?taccactccc 60

tatcagtgat?agagaaaagt?gaaagtcgag?tttaccactc?cctatcagtg?atagagaaaa 120

gtgaaagtcg?agtttaccac?tccctatcag?tgatagagaa?aagtgaaagt?cgagtttacc 180

actccctatc?agtgatagag?aaaagtgaaa?gtcgagttta?ccactcccta?tcagtgatag 240

agaaaagtga?aagtcgagtt?taccactccc?tatcagtgat?agagaaaagt?gaaagtcgag 300

ctcgctaggc?gtgtacggtg?ggaggcctat?ataagcagag?ctcgtttagt?gaaccgtcag 360

atcgcggaat?tcatgtctag?actggacaag?agcaaagtca?taaacggcgc?tctggaatta 420

ctcaatggag?tcggtatcga?aggcctgacg?acaaggaaac?tcgctcaaaa?gctgggagtt 480

gagcagccta?ccctgtactg?gcacgtgaag?aacaagcggg?ccctgctcga?tgccctgcca 540

atcgagatgc?tggacaggca?tcatacccac?ttctgccccc?tggaaggcga?gtcatggcaa 600

gactttctgc?ggaacaacgc?caagtcattc?cgctgtgctc?tcctctcaca?tcgcgacggg 660

gctaaagtgc?atctcggcac?ccgcccaaca?gagaaacagt?acgaaaccct?ggaaaatcag 720

ctcgcgttcc?tgtgtcagca?aggcttctcc?ctggagaacg?cactgtacgc?tctgtccgcc 780

gtgggccact?ttacactggg?ctgcgtattg?gaggaacagg?agcatcaagt?agcaaaagag 840

gaaagagaga?cacctaccac?cgattctatg?cccccacttc?tgagacaagc?aattgagctg 900

ttcgaccggc?agggagccga?acctgccttc?cttttcggcc?tggaactaat?catatgtggc 960

ctggagaaac?agctaaagtg?cgaaagcggc?gggccggccg?acgcccttga?cgattttgac 1020

ttagacatgc?tcccagccga?tgcccttgac?gactttgacc?ttgatatgct?gcctgctgac 1080

gctcttgacg?attttgacct?tgacatgctc?cccgggtaaa?ctagt 1125

<210>10

<211>1129

<212>DNA

< 213>synthetic

<220>

<221>mutation

<400>10

ctcgagttta?ccactcccta?tcagtgatag?agaaaagtga?aagtcgagtt?taccactccc 60

tatcagtgat?agagaaaagt?gaaagtcgag?tttaccactc?cctatcagtg?atagagaaaa 120

gtgaaagtcg?agtttaccac?tccctatcag?tgatagagaa?aagtgaaagt?cgagtttacc 180

actccctatc?agtgatagag?aaaagtgaaa?gtcgagttta?ccactcccta?tcagtgatag 240

agaaaagtga?aagtcgagtt?taccactccc?tatcagtgat?agagaaaagt?gaaagtcgag 300

ctcgctaggc?gtgtacggtg?ggaggcctat?ataagcagag?ctcgtttagt?gaaccgtcag 360

atcgcggaat?taattcatgt?ctagactgga?caagagcaaa?gtcataaacg?gcgctctgga 420

attactcaat?ggagtcggta?tcgaaggcct?gacgacaagg?aaactcgctc?aaaagctggg 480

agttgagcag?cctaccctgt?actggcacgt?gaagaacaag?cgggccctgc?tcgatgccct 540

gccaatcgag?atgctggaca?ggcatcatac?ccacttctgc?cccctggaag?gcgagtcatg 600

gcaagacttt?ctgcggaaca?acgccaagtc?attccgctgt?gctctcctct?cacatcgcga 660

cggggctaaa?gtgcatctcg?gcacccgccc?aacagagaaa?cagtacgaaa?ccctggaaaa 720

tcagctcgcg?ttcctgtgtc?agcaaggctt?ctccctggag?aacgcactgt?acgctctgtc 780

cgccgtgggc?cactttacac?tgggctgcgt?attggaggaa?caggagcatc?aagtagcaaa 840

agaggaaaga?gagacaccta?ccaccgattc?tatgccccca?cttctgagac?aagcaattga 900

gctgttcgac?cggcagggag?ccgaacctgc?cttccttttc?ggcctggaac?taatcatatg 960

tggcctggag?aaacagctaa?agtgcgaaag?cggcgggccg?gccgacgccc?ttgacgattt 1020

tgacttagac?atgctcccag?ccgatgccct?tgacgacttt?gaccttgata?tgctgcctgc 1080

tgacgctctt?gacgattttg?accttgacat?gctccccggg?taaactagt 1129

<210>11

<211>3648

<212>DNA

< 213>synthetic

<220>

<221>misc_feature

<400>11

ggtaccaata?gttattaata?gtaatcaatt?acggggtcat?tagttcatag?cccatatatg 60

gagttccgcg?ttacataact?tacggtaaat?ggcccgcctg?gctgaccgcc?caacgacccc 120

cgcccattga?cgtcaataat?gacgtatgtt?cccatagtaa?cgccaatagg?gactttccat 180

tgacgtcaat?gggtggagta?tttacggtaa?actgcccact?tggcagtaca?tcaagtgtat 240

catatgccaa?gtacgccccc?tattgacgtc?aatgacggta?aatggcccgc?ctggcattat 300

gcccagtaca?tgaccttatg?ggactttcct?acttggcagt?acatctacgt?attagtcatc 360

gctattacca?tggtgatgcg?gttttggcag?tacatcaatg?ggcgtggata?gcggtttgac 420

tcacggggat?ttccaagtct?ccaccccatt?gacgtcaatg?ggagtttgtt?ttggcaccaa 480

aatcaacggg?actttccaaa?atgtcgtaac?aactccgccc?cattgacgca?aatgggcggt 540

aggcgtgtac?ggtgggaggt?ctatataagc?agagctggtt?tagtgaaccg?tcagatcatc 600

gatatggcta?gattagataa?aagtaaagtg?attaacagcg?cattagagct?gcttaatgag 660

gtcggaatcg?aaggtttaac?aacccgtaaa?ctcgcccaga?agctaggtgt?agagcagcct 720

acattgtatt?ggcatgtaaa?aaataagcgg?gctttgctcg?acgccttagc?cattgagatg 780

ttagataggc?accatactca?cttttgccct?ttagaagggg?aaagctggca?agatttttta 840

cgtaataacg?ctaaaagttt?tagatgtgct?ttactaagtc?atcgcgatgg?agcaaaagta 900

catttaggta?cacggcctac?agaaaaacag?tatgaaactc?tcgaaaatca?attagccttt 960

ttatgccaac?aaggtttttc?actagagaat?gcattatatg?cactcagcgc?tgtggggcat 1020

tttactttag?gttgcgtatt?ggaagatcaa?gagcatcaag?tcgctaaaga?agaaagggaa 1080

acacctacta?ctgatagtat?gccgccatta?ttacgacaag?ctatcgaatt?atttgatcac 1140

caaggtgcag?agccagcctt?cttattcggc?cttgaattga?tcatatgcgg?attagaaaaa 1200

caacttaaat?gtgaaagtgg?gtcgccaaaa?aagaagagaa?aggtcgacgg?cggtggtgct 1260

ttgtctcctc?agcactctgc?tgtcactcaa?ggaagtatca?tcaagaacaa?ggagggcatg 1320

gatgctaagt?cactaactgc?ctggtcccgg?acactggtga?ccttcaagga?tgtatttgtg 1380

gacttcacca?gggaggagtg?gaagctgctg?gacactgctc?agcagatcgt?gtacagaaat 1440

gtgatgctgg?agaactataa?gaacctggtt?tccttgggtt?atcagcttac?taagccagat 1500

gtgatcctcc?ggttggagaa?gggagaagag?ccctggctgg?tggagagaga?aattcaccaa 1560

gagacccatc?ctgattcaga?gactgcattt?gaaatcaaat?catcagttta?agcggccgcc 1620

acagcgggga?gatccagaca?tgataagata?cattgatgag?tttggacaaa?ccacaactag 1680

aatgcagtga?aaaaaatgct?ttatttgtga?aatttgtgat?gctattgctt?tatttgtaac 1740

cattataagc?tgcaataaac?aagttaacaa?caacaattgc?attcatttta?tgtttcaggt 1800

tcagggggag?gtgtgggagg?ttttttaaag?caagtaaaac?ctctacaaat?gtggtatggc 1860

tgattatgat?ccggctgcct?cgcgcgtttc?ggtgatgacg?gtgaaaacct?ctgacacatg 1920

cagctcccgg?agacggtcac?agcttgtctg?taagcggatg?ccgggagcag?acaagcccgt 1980

cagggcgcgt?cagcgggtgt?tggcgggtgt?cggggcgcag?ccatgagtct?agtttacccg 2040

gggagcatgt?caaggtcaaa?atcgtcaaga?gcgtcagcag?gcagcatatc?aaggtcaaag 2100

tcgtcaaggg?catcggctgg?gagcatgtct?aagtcaaaat?cgtcaagggc?gtcggccggc 2160

ccgccgcttt?cgcactttag?ctgtttctcc?aggccacata?tgattagttc?caggccgaaa 2220

aggaaggcag?gttcggctcc?ctgccggtcg?aacagctcaa?ttgcttgtct?cagaagtggg 2280

ggcatagaat?cggtggtagg?tgtctctctt?tcctcttttg?ctacttgatg?ctcctgttcc 2340

tccaatacgc?agcccagtgt?aaagtggccc?acggcggaca?gagcgtacag?tgcgttctcc 2400

agggagaagc?cttgctgaca?caggaacgcg?agctgatttt?ccagggtttc?gtactgtttc 2460

tctgttgggc?gggtgccgag?atgcacttta?gccccgtcgc?gatgtgagag?gagagcacag 2520

cggaatgact?tggcgttgtt?ccgcagaaag?tcttgccatg?actcgccttc?cagggggcag 2580

aagtgggtat?gatgcctgtc?cagcatctcg?attggcaggg?catcgagcag?ggcccgcttg 2640

ttcttcacgt?gccagtacag?ggtaggctgc?tcaactccca?gcttttgagc?gagtttcctt 2700

gtcgtcaggc?cttcgatacc?gactccattg?agtaattcca?gagcgccgtt?tatgactttg 2760

ctcttgtcca?gtctagacat?gaattaattc?cgcgatctga?cggttcacta?aacgagctct 2820

gcttatatag?gcctcccacc?gtacacgcct?agcgagctcg?actttcactt?ttctctatca 2880

ctgataggga?gtggtaaact?cgactttcac?ttttctctat?cactgatagg?gagtggtaaa 2940

ctcgactttc?acttttctct?atcactgata?gggagtggta?aactcgactt?tcacttttct 3000

ctatcactga?tagggagtgg?taaactcgac?tttcactttt?ctctatcact?gatagggagt 3060

ggtaaactcg?actttcactt?ttctctatca?ctgataggga?gtggtaaact?cgactttcac 3120

ttttctctat?cactgatagg?gagtggtaaa?ctcgactagg?cgtgtacggt?gggaggccta 3180

tataagcaga?gctcgtttag?tgaaccgtca?gatcgcgaat?tcactagtac?tagacacagc 3240

ggggagatcc?agacatgata?agatacattg?atgagtttgg?acaaaccaca?actagaatgc 3300

agtgaaaaaa?atgctttatt?tgtgaaattt?gtgatgctat?tgctttattt?gtaaccatta 3360

taagctgcaa?taaacaagtt?aacaacaaca?attgcattca?ttttatgttt?caggttcagg 3420

gggaggtgtg?ggaggttttt?taaagcaagt?aaaacctcta?caaatgtggt?atggctgatt 3480

atgatccggc?tgcctcgcgc?gtttcggtga?tgacggtgaa?aacctctgac?acatgcagct 3540

cccggagacg?gtcacagctt?gtctgtaagc?ggatgccggg?agcagacaag?cccgtcaggg 3600

cgcgtcagcg?ggtgttggcg?ggtgtcgggg?cgcagccatg?agagatct 3648

<210>12

<211>18

<212>DNA

< 213>synthetic

<220>

<221>misc_recomb

<400>12

gaattcctcg?agactagt 18

<210>13

<211>522

<212>DNA

< 213>people

<220>

<221>CDS

<400>13

ctcgagatgg?tgagagaaag?aggtcctcag?agagtagcag?ctcacataac?tgggaccaga 60

ggaagaagca?acacattgtc?ttctccaaac?tccaagaatg?aaaaggctct?gggccgcaaa 120

ataaactcct?gggaatcatc?aaggagtggg?cattcattcc?tgagcaactt?gcacttgagg 180

aatggtgaac?tggtcatcca?tgaaaaaggg?ttttactaca?tctattccca?aacatacttt 240

cgatttcagg?aggaaataaa?agaaaacaca?aagaacgaca?aacaaatggt?ccaatatatt 300

tacaaataca?caagttatcc?tgaccctata?ttgttgatga?aaagtgctag?aaatagttgt 360

tggtctaaag?atgcggaata?tggactctat?tccatctatc?aagggggaat?atttgagctt 420

aaggaaaatg?acagaatttt?tgtttctgta?acaaatgagc?acttgataga?catggaccat 480

gaagccagtt?ttttcggggc?ctttttagtt?ggctaatcta?ga 522?。

Claims

1. the Tet-on abduction delivering system of a single carrier two-way startup; It is characterized in that: the order of connection of its element is 5-' CMV-TetR-KRAB-SV40polyA-rtTA2S-M2-EB-PminCMV-KB-TRE-Tigh t-PminCMV-EcoR I-Spe I-SV40polyA-3 '; The mode of connection of the composite component rtTA2S-M2-EB-PminCMV-KB-TRE in Tight-PminCMV left side is 3 '-rtTA2S-M2-EB-PminCMV-KB-TRE-5 '-5 '-Tight-PminCMV-3 ', and all the other elements are all by being linked in sequence with this system's equidirectional 5 '-3 '; Two restriction enzyme site EcoR I and Spe I are between elements T ight-PminCMV and element SV40polyA, and this system's sequence is following:

ggtaccaata?gttattaata?gtaatcaatt?acggggtcat?tagttcatag?cccatatatg 60

gagttccgcg?ttacataact?tacggtaaat?ggcccgcctg?gctgaccgcc?caacgacccc 120

cgcccattga?cgtcaataat?gacgtatgtt?cccatagtaa?cgccaatagg?gactttccat 180

tgacgtcaat?gggtggagta?tttacggtaa?actgcccact?tggcagtaca?tcaagtgtat 240

catatgccaa?gtacgccccc?tattgacgtc?aatgacggta?aatggcccgc?ctggcattat 300

gcccagtaca?tgaccttatg?ggactttcct?acttggcagt?acatctacgt?attagtcatc 360

gctattacca?tggtgatgcg?gttttggcag?tacatcaatg?ggcgtggata?gcggtttgac 420

tcacggggat?ttccaagtct?ccaccccatt?gacgtcaatg?ggagtttgtt?ttggcaccaa 480

aatcaacggg?actttccaaa?atgtcgtaac?aactccgccc?cattgacgca?aatgggcggt 540

aggcgtgtac?ggtgggaggt?ctatataagc?agagctggtt?tagtgaaccg?tcagatcatc 600

gatatggcta?gattagataa?aagtaaagtg?attaacagcg?cattagagct?gcttaatgag 660

gtcggaatcg?aaggtttaac?aacccgtaaa?ctcgcccaga?agctaggtgt?agagcagcct 720

acattgtatt?ggcatgtaaa?aaataagcgg?gctttgctcg?acgccttagc?cattgagatg 780

ttagataggc?accatactca?cttttgccct?ttagaagggg?aaagctggca?agatttttta 840

cgtaataacg?ctaaaagttt?tagatgtgct?ttactaagtc?atcgcgatgg?agcaaaagta 900

catttaggta?cacggcctac?agaaaaacag?tatgaaactc?tcgaaaatca?attagccttt 960

ttatgccaac?aaggtttttc?actagagaat?gcattatatg?cactcagcgc?tgtggggcat 1020

tttactttag?gttgcgtatt?ggaagatcaa?gagcatcaag?tcgctaaaga?agaaagggaa 1080

acacctacta?ctgatagtat?gccgccatta?ttacgacaag?ctatcgaatt?atttgatcac 1140

caaggtgcag?agccagcctt?cttattcggc?cttgaattga?tcatatgcgg?attagaaaaa 1200

caacttaaat?gtgaaagtgg?gtcgccaaaa?aagaagagaa?aggtcgacgg?cggtggtgct 1260

ttgtctcctc?agcactctgc?tgtcactcaa?ggaagtatca?tcaagaacaa?ggagggcatg 1320

gatgctaagt?cactaactgc?ctggtcccgg?acactggtga?ccttcaagga?tgtatttgtg 1380

gacttcacca?gggaggagtg?gaagctgctg?gacactgctc?agcagatcgt?gtacagaaat 1440

gtgatgctgg?agaactataa?gaacctggtt?tccttgggtt?atcagcttac?taagccagat 1500

gtgatcctcc?ggttggagaa?gggagaagag?ccctggctgg?tggagagaga?aattcaccaa 1560

gagacccatc?ctgattcaga?gactgcattt?gaaatcaaat?catcagttta?agcggccgcc 1620

acagcgggga?gatccagaca?tgataagata?cattgatgag?tttggacaaa?ccacaactag 1680

aatgcagtga?aaaaaatgct?ttatttgtga?aatttgtgat?gctattgctt?tatttgtaac 1740

cattataagc?tgcaataaac?aagttaacaa?caacaattgc?attcatttta?tgtttcaggt 1800

tcagggggag?gtgtgggagg?ttttttaaag?caagtaaaac?ctctacaaat?gtggtatggc 1860

tgattatgat?ccggctgcct?cgcgcgtttc?ggtgatgacg?gtgaaaacct?ctgacacatg 1920

cagctcccgg?agacggtcac?agcttgtctg?taagcggatg?ccgggagcag?acaagcccgt 1980

cagggcgcgt?cagcgggtgt?tggcgggtgt?cggggcgcag?ccatgagtct?agtttacccg 2040

gggagcatgt?caaggtcaaa?atcgtcaaga?gcgtcagcag?gcagcatatc?aaggtcaaag 2100

tcgtcaaggg?catcggctgg?gagcatgtct?aagtcaaaat?cgtcaagggc?gtcggccggc 2160

ccgccgcttt?cgcactttag?ctgtttctcc?aggccacata?tgattagttc?caggccgaaa 2220

aggaaggcag?gttcggctcc?ctgccggtcg?aacagctcaa?ttgcttgtct?cagaagtggg 2280

ggcatagaat?cggtggtagg?tgtctctctt?tcctcttttg?ctacttgatg?ctcctgttcc 2340

tccaatacgc?agcccagtgt?aaagtggccc?acggcggaca?gagcgtacag?tgcgttctcc 2400

agggagaagc?cttgctgaca?caggaacgcg?agctgatttt?ccagggtttc?gtactgtttc 2460

tctgttgggc?gggtgccgag?atgcacttta?gccccgtcgc?gatgtgagag?gagagcacag 2520

cggaatgact?tggcgttgtt?ccgcagaaag?tcttgccatg?actcgccttc?cagggggcag 2580

aagtgggtat?gatgcctgtc?cagcatctcg?attggcaggg?catcgagcag?ggcccgcttg 2640

ttcttcacgt?gccagtacag?ggtaggctgc?tcaactccca?gcttttgagc?gagtttcctt 2700

gtcgtcaggc?cttcgatacc?gactccattg?agtaattcca?gagcgccgtt?tatgactttg 2760

ctcttgtcca?gtctagacat?gaattaattc?cgcgatctga?cggttcacta?aacgagctct 2820

gcttatatag?gcctcccacc?gtacacgcct?agcgagctcg?actttcactt?ttctctatca 2880

ctgataggga?gtggtaaact?cgactttcac?ttttctctat?cactgatagg?gagtggtaaa 2940

ctcgactttc?acttttctct?atcactgata?gggagtggta?aactcgactt?tcacttttct 3000

ctatcactga?tagggagtgg?taaactcgac?tttcactttt?ctctatcact?gatagggagt 3060

ggtaaactcg?actttcactt?ttctctatca?ctgataggga?gtggtaaact?cgactttcac 3120

ttttctctat?cactgatagg?gagtggtaaa?ctcgactagg?cgtgtacggt?gggaggccta 3180

tataagcaga?gctcgtttag?tgaaccgtca?gatcgcgaat?tcactagtac?tagacacagc 3240

ggggagatcc?agacatgata?agatacattg?atgagtttgg?acaaaccaca?actagaatgc 3300

agtgaaaaaa?atgctttatt?tgtgaaattt?gtgatgctat?tgctttattt?gtaaccatta 3360

taagctgcaa?taaacaagtt?aacaacaaca?attgcattca?ttttatgttt?caggttcagg 3420

gggaggtgtg?ggaggttttt?taaagcaagt?aaaacctcta?caaatgtggt?atggctgatt 3480

atgatccggc?tgcctcgcgc?gtttcggtga?tgacggtgaa?aacctctgac?acatgcagct 3540

cccggagacg?gtcacagctt?gtctgtaagc?ggatgccggg?agcagacaag?cccgtcaggg 3600

cgcgtcagcg?ggtgttggcg?ggtgtcgggg?cgcagccatg?agagatct 3648。

2. according to the Tet-on abduction delivering system of the described single carrier two-way startup of claim 1, it is characterized in that: the direction of the PminCMV element in described TRE element left side and the Tight-PminCMV on right side start in the opposite direction, all adjacent with the TRE element.

3. according to the Tet-on abduction delivering system of the described single carrier two-way startup of claim 1, it is characterized in that: the goal gene that between EcoR I and Spe I site, is connected into is reporter gene or oncotherapy gene.

4. the construction process of the Tet-on abduction delivering system of a single carrier two-way startup is characterized in that it comprises the steps:

(1) makes up the adenovirus E 1 district shuttle vectors that contains Kpn I-Cla I-Not I-Xho I-EcoR I-Spe I restriction endonuclease sites

(2) amplification makes up the required element of Tet-on abduction delivering system of single carrier two-way startup

Make up the required element of Tet-on abduction delivering system of single carrier two-way startup through PCR amplification, element comprises CMV, TetR-KRAB, SV40polyA, Tight-PminCMV, rtTA2S-M2;

(3) make up the intermediate carrier that has composite component TRE-KB-PminCMV-EB-rtTA2S-M2

Cut connection through enzyme; TRE-PminCMV is connected to pShuttle-KSL-SV40polyA-KB; And after using Kpn I enzyme to cut, the T4DNA polysaccharase is mended flat terminal, puts down terminal the connection with the T4DNA ligase enzyme again; 304 Kpn I site is disappeared, obtain the TRE-KB-PminCMV element; Again rtTA2S-M2 is connected to the 3 ' end of TRE-KB-PminCMV; With quadrat method the EcoR I site between TRE-KB-PminCMV and rtTA2S-M2 is disappeared, obtain having the intermediate carrier pShuttle-TRE-KB-PminCMV-EB-rtTA2S-M2-SV40polyA of composite component TRE-KB-PminCMV-EB-rtTA2S-M2;

(4) the Tet-on abduction delivering system of the single carrier two-way startup of structure

5. the purposes in the colorectal carcinoma medicine is treated in preparation by the Tet-on abduction delivering system of the single carrier two-way startup of claim 1.

6. treat the purposes in the colorectal carcinoma medicine according to the Tet-on abduction delivering system of the described single carrier two-way startup of claim 5 in preparation; It is characterized in that: the Tet-on abduction delivering system of single carrier two-way startup uses in preparation treatment colorectal carcinoma medicine; Effective constituent prepares the Tet-on abduction delivering system of carrying single carrier two-way startup in the E1 district and the adenovirus carrier of regulating and expressing goal gene TRAIL, and preparation medicine for treating tumor thing uses with the form of conventional injection liquid.