CN102352369A - Carrier T-VISA for expressing target genes at high efficiency and high specificity in tumor cells - Google Patents
Carrier T-VISA for expressing target genes at high efficiency and high specificity in tumor cells Download PDFInfo
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- CN102352369A CN102352369A CN201110300441XA CN201110300441A CN102352369A CN 102352369 A CN102352369 A CN 102352369A CN 201110300441X A CN201110300441X A CN 201110300441XA CN 201110300441 A CN201110300441 A CN 201110300441A CN 102352369 A CN102352369 A CN 102352369A
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Abstract
The invention discloses a carrier for expressing target genes at high efficiency and high specificity in tumor cells, which is an annular carrier with the sequence shown as SEQ ID NO. 1. The carrier T-VISA can express the target genes at high efficiency and high specificity in the tumor cells, the expression effect is the same as that of a cytomegalovirus (CMV) promoter, and the carrier T-VISA has the characteristic that the expression in normal tissue cells is very low. Therefore, the carrier T-VISA provided by the invention has wide application values that: 1, the carrier T-VISA can be used for driving any target genes to express at high efficiency and high specificity in the tumor cells, including gene reporting such as luciferase and green fluorescent protein (GFP) and gene treatment genes such as p53 (cell apoptosis protein genes), IL-2 (immune regulation protein genes) and the like; and 2, the carrier T-VISA can be used for the study and the application of tumor biology, tumor signal conduction, tumor gene regulation and control and gene treatment after being combined with corresponding target genes.
Description
Technical field:
The invention belongs to biomedicine field, be specifically related to a kind of in tumour cell the carrier T-VISA of efficient high specifically expressing target gene.
Background technology:
In tumour cell, efficient high specifically expressing target gene is the important method of research oncobiology, tumor signal conduction, oncogene regulation and control and gene therapy.The tumor-specific promoters that adopts is expressed target gene at present, and its activity is low, and efficient is poor, is difficult to oncobiology, signal conduction and gene regulating are furtherd investigate, and particularly causes genetic treatment of tumor in clinical, to use.Developing history surplus the gene therapy existing 20 year, people hope that it can become the effective alternative medicine outside the excision chemicotherapy etc.The most cytomegalovirus promoter (CMV) that adopts of therapy of tumor is a highest active at present promotor at present; But this promotor does not have specificity; Also high expression level goal gene in normal cell; Though the clinical trial product of being developed has certain curative effect; But toxic side effect is big, can't obtain the approval of SFDA (Chinese food and drug administration) and U.S. FDA (FDA) and is used for clinical.Someone uses the tomour specific promotor, has significantly reduced toxic side effect, but because active low, it is very limited that its curative effect also becomes.Therefore, a kind of carrier that high specific efficiently expresses target gene or therapeutic gene in tumour cell of exploitation is very important.
Summary of the invention:
The purpose of this invention is to provide a kind of in tumour cell the carrier T-VISA of efficient high specifically expressing target gene.
Of the present invention in tumour cell the carrier T-VISA of efficient high specifically expressing target gene be a circular vectors, its base sequence is shown in SEQ ID NO.1.
The present invention inserts reporter gene luciferase Luciferase gene and constitutes the T-VISA-Luc plasmid in the T-VISA carrier, and activity and the specificity of carrier T-VISA are estimated.
With T-VISA-Luc plasmid transfection ovarian cancer cell line and normal cell system, the result shows that the Luciferase gene expresses in ovarian cancer cell line, but in normal ovarian clone, expresses very low.The result shows that can be in the ovarian cancer cell efficient high specific of T-VISA carrier expresses target gene.
With T-VISA-Luc plasmid transfection breast cancer cell, three cloudy breast cancer cells, breast carcinoma stem cell system and normal cell system; The result is presented at ten breast cancer cell lines and three breast carcinoma stem cell cordings have very high reactivity, but in normal cell system, expresses very low.In breast cancer cell line, breast carcinoma stem cell system and normal cell system, the activity of T-VISA is respectively 1.8 (1.7-4.3) of CMV, 2.6 (2.0-3.2) and 0.006 times.The result shows the T-VISA carrier at breast cancer cell, can express target gene by efficient high specific in three cloudy breast cancer cells and the breast carcinoma stem cell system.
Further detect activity and the specificity of T-VISA carrier in the mouse interior tumor cell.In-situ inoculating ovary cell line Hey8 in the Balb/c nude mice, after 2 weeks, the liposome through tail vein injection 50 μ g comprise the T-VISA-Luc plasmid uses the living imaging appearance to dynamic observe target gene Luciferase.The result shows that CMV-Luc causes Luciferase at mouse lung tissue, tumor tissues and whole body normal tissue expression, but T-VISA-Luc high expression level in the mouse tumor tissue is very low at the whole body normal tissue expression.The result show T-VISA-Luc in the mouse body in the ovarian cancer cell efficient high specific express target gene Luciferase.
In sum, T-VISA carrier of the present invention can be in tumour cell efficient high specifically expressing target gene, expression effect is suitable with the CMV promotor, but it also has the extremely low advantage of expressing in normal tissue cell.
Therefore T-VISA carrier of the present invention is with a wide range of applications: 1, can utilize the T-VISA carrier to drive any target gene efficient high specific in tumour cell and express; Comprise reporter gene such as Luciferase (luciferase); GFP (green fluorescent protein) and therapeutic gene such as p53 (apoptosis protein gene), IL-2 (immunoregulation protein gene) etc.2, the T-VISA carrier can with carry out oncobiology after corresponding target genes combines, the research and the application of tumor signal conduction, oncogene regulation and control and gene therapy.
Description of drawings:
Fig. 1 is a plasmid map; Wherein Figure 1A is the T-VISA-Luc plasmid map that inserts reporter gene Luciferase; Figure 1B is a CMV-Luc plasmid structural representation; Wherein CMV is meant cytomegalovirus promoter; Be that present activity does not have specificity promoter the most by force; Fig. 1 C is a T-VISA-Luc plasmid structural representation, is the luciferase expression plasmid that is made up of hTERT (human telomerase promotor) and VISA (integration amplification system);
Fig. 2 is the design sketch of T-VISA-Luc efficient high specifically expressing target gene Luciferase in ovarian cancer cell;
Fig. 3 be T-VISA-Luc at breast cancer cell, the design sketch of efficient high specifically expressing target gene Luciferase in three cloudy breast cancer cells and the breast carcinoma stem cell system;
Fig. 4 be T-VISA-Luc in the mouse body in the ovarian cancer cell efficient high characteristic express the figure of target gene Luciferase.
Embodiment:
Following examples are to further specify of the present invention, rather than limitation of the present invention.
Embodiment 1:
One, T-VISA carrier:
Clone telomerase promoter hTERT, length is 418bp, its sequence has tumor cell specific shown in SEQ ID NO.2, but its activity in tumour cell less than 1% of CMV promoter activity, therefore can't widespread use.This promotor is inserted the basic plasmid promoter sequence of TSTA part; Form T-TSTA; Again WPRE is inserted 3 '-UTR of target gene among the T-TSTA; Can strengthen the stability action of mRNA; Make up hTERT-VP16-GAL4-WPRE (VP16-GAL4-WPRE Integrated Systemic Amplifier, VISA, conformability system amplifier) regulator control system thus; Abbreviate T-VISA (conformability system amplifier) carrier as, its sequence is shown in SEQ ID NO.1.
In the T-VISA carrier, hTERT promotor control Gal4-VP2 (VP16 of two copies has the intensive transcripting activating characteristic) antigen-4 fusion protein gene; The Gal4-VP2 fusion rotein combines with Gal4 response element G5E4T, starts transcribing in a large number of target gene or goal gene.
This T-VISA carrier mainly comprises hTERT promotor (its sequence is shown in SEQ ID N0.2), WPRE (its sequence is shown in SEQ ID NO.3), G5E4T (its sequence is shown in SEQ ID NO.4) and Gal4-VP2 gene (its sequence is shown in SEQ ID NO.5).
The T-VISA carrier of present embodiment can downcut through enzyme and state the T-VISA-Luc plasmid, utilizes enzyme Nhel/BgIII that the excision of luciferase Luciferase gene is obtained.
Two, the structure of T-VISA-Luc plasmid
Clone's construction step of pGL3.T-VISA-Luc (being the T-VISA-Luc plasmid) plasmid
(1) pCRII-TOPO-hTERT plasmid clone
1, ordinary method is extracted the DNA of mammary cancer MCF-7 cell (buying from U.S. ATCC company).
2, with this DNA as template, the PCR primer of design amplification hTERT, the hTERT PCR upper reaches (Forward) and downstream (Reverse) primer:
Forward：5′-ata?tct?aga?ggc?ccc?tcc?ctc?ggg?tta?ccc?cac?agc-3′(XbaI)
Reverse：5′-ata?gat?ctt?atg?cgg?ccg?ccc?acg?tgc?gca?gca?gga?cgc?agc?gc-3′。
3, be template with mammary cancer MCF-7 cell DNA, hTERT PCR primer (Forward:5 '-ata tct aga ggc ccc tcc ctc ggg tta ccc cac agc-3 '; Reverse:5 '-ata gat ctt atg cgg ccg ccc acg tgc gcagca gga cgc agc gc-3 '), Pfu DNA polymerase, dNTP and PCR reaction solution; Amplification obtains the hTERT promotor, and (416to+1) product, its sequence is shown in SEQ ID NO.2.Its PCR reaction system: 10 * PCR buffer5 μ l, each 1 μ l of the upper reaches (Forward) and downstream (Reverse) primer, the MCF-7 cell DNA is template 100ng DNA, 25mM MgCl
23 μ l, 10mM dNTPs 1 μ l, Pfu-Taq (1U/ μ l), last moisturizing to 50 μ l.The PCR reaction conditions: 94 ℃ of thermally denature 5min, 94 ℃ of sex change 1min, 55 ℃ of annealing 1min, 72 ℃ are extended 2min, carry out 30 circulations altogether; Last 72 ℃ are extended 10min.
4, the PCR product reclaims the test kit recovery with the PCR DNA of Qiangen company (U.S.), reclaims about 418bp size segment.Method is with reference to its specification sheets.
5, with the PCR product 2 μ l that reclaim, pCRII-TOPO (Invitrogen, Carlsbad, CA) 1 μ l, DNA ligase buffer1 μ l, last moisturizing to 10 μ l, 4 ℃ of ligation 10min.
6,2 μ l are connected product and 50 μ l Ecoli DH
5After competent cell mixes, ice bath 30min.The 42 ℃ of moving reaction of water-bath heat shock 45sec place 3min then on ice.Add the LB substratum 500 μ l of preheating, jolt 30min at 37 ℃ of following 250rpm.
7, get 200 μ l LB substratum bacterium liquid, on the coating ampicillinLB culture plate, cultivated 16 hours down at 37 ℃.The picking positive monoclonal extracts plasmid.
8, digested plasmid is identified, carries out sequencing analysis, and confirmation hTERT promotor (its sequence is shown in SEQ ID NO.2) has been inserted among the cloning vector pCRII-TOPO called after pCRII-TOPO-hTERT.
(2) pGL3-hTERT-Luc plasmid clone
1, cut the pCRII-TOPO-hTERT plasmid with the KpnI/Xho1 enzyme, (Promega company, Madison WI), contain luciferase Luciferase gene in this plasmid to cut the pGL-3-basic plasmid with the KpnI/Xho1 enzyme.Enzyme is cut system: 10 * BufferA2 μ l, KpnI/Xho11 μ l, plasmid 5 μ g, moisturizing to 20 μ l, 37 ℃ of water-baths 1 hour.
2, enzyme is cut product electrophoresis in 1 * TAE Agarose gel, and 120v electrophoresis 30 minutes reclaims hTERT (418bp size) segment and pGL-3-Basis (4792bp size) segment.
3, enzyme is cut product and is reclaimed the test kit recovery with Qiangen company (U.S.) pcr dna, and method is with reference to its specification sheets.
4, the hTERT product 6 μ l of Hui Shouing, the pGL-3-basic product 2 μ l of recovery, DNA ligase 1 μ l, buffer 1 μ l, last moisturizing to 10 μ l, 4 ℃ of ligations 4 hours.
5,2 μ l are connected product and 50 μ lE.coliDH
5After competent cell mixes, ice bath 30min.The 42 ℃ of moving reaction of water-bath heat shock 45sec place 3min then on ice.Add the LB substratum 500 μ l of preheating, jolt 30min at 37 ℃ of following 250rpm.
6, get 200 μ lLB substratum bacterium liquid, on the coating AmpicillinLB culture plate, cultivated 16 hours down at 37 ℃.The picking positive monoclonal extracts plasmid.
7, digested plasmid is identified, carries out sequencing analysis, and confirmation hTERT promotor has been inserted among the carrier pGL-3-basic called after pGL3-hTERT-Luc plasmid.
(3) pGL3-Luc-WPRE plasmid clone
1, (Promega company, Madison WI), are used DNA polymerase flush end then to cut the pGEM-3Z-WPRE plasmid with the Asp718/SalI enzyme; With the SmalI enzyme cut the pGL-3-basic plasmid (Promega company, Madison, WI).Enzyme is cut system: 10X Buffer A2 μ l, Asp7181 μ l/SalI 1 μ l, plasmid 5 μ g, moisturizing to 20 μ l, 37 ℃ of water-baths 1 hour.
2, enzyme is cut product electrophoresis in 1XTAE Agarose gel, and 120v electrophoresis 30 minutes reclaims WPRE (590bp size) segment and pG-L3-Basis (about 4800bp size) segment.
3, enzyme is cut product (U.S. pcr dna is reclaimed test kit and reclaims, and method is with reference to its specification sheets with Qiangen company.
4, the WPRE product 6 μ l of Hui Shouing, the pGL-3-basic product 2 μ l of recovery, DNA ligase 1 μ l, buffer 1 μ l, last moisturizing to 10 μ l, 4 ℃ of ligations 4 hours.
5, after 2 μ l connect product and 50 μ lE.coliDH5 competent cells mix, ice bath 30min.The 42 ℃ of moving reaction of water-bath heat shock 45sec place 3min then on ice.Add the LB substratum 500 μ l of preheating, jolt 30min at 37 ℃ of following 250rpm.
6, get 200 μ lLB substratum bacterium liquid, on the coating AmpicillinLB culture plate, cultivated 16 hours down at 37 ℃.The picking positive monoclonal extracts plasmid.
7, digested plasmid is identified, carries out sequencing analysis, confirms that WPRE has inserted among the pGL-3-basic called after pGL3-Luc-WPRE plasmid.
(4) pGL3-hTERT-TSTA-Luc plasmid clone
1, cuts pCRII-TOPO-hTERT plasmid (on seeing) with the HindIII/NotI enzyme, use DNA polymerase flush end then; (Invitrogen, Carlsbad CA), use DNA polymerase flush end then to cut the pGL3-TSTA-Luc plasmid with the MSCI/NheI enzyme.Enzyme is cut system: 10X Buffer A2 μ l, HindIII/NotI or MSCI/NheI1 μ l, plasmid 5 μ g, moisturizing to 20 μ l, 37 ℃ of water-baths 1 hour.
2, enzyme is cut product electrophoresis in 1XTAE Agarose gel, and 120v electrophoresis 30 minutes reclaims hTERT (418bp size) segment and GL3-TSTA-Luc (7300bp size) segment.
3, enzyme is cut product and is reclaimed the test kit recovery with Qiangen company (U.S.) pcr dna, and method is with reference to its specification sheets.
4, the hTERT product 6 μ l of Hui Shouing, the pGL33-TSTA-Luc product 2 μ l of recovery, DNA ligase 1 μ l, buffer 1 μ l, last moisturizing to 10 μ l, 4 ℃ of ligations 4 hours.
5, after 2 μ l connect product and 50 μ l E.coli DH5 competent cells mix, ice bath 30min.The 42 ℃ of moving reaction of water-bath heat shock 45sec place 3min then on ice.Add the LB substratum 500 μ l of preheating, jolt 30min at 37 ℃ of following 250rpm.
6, get 200 μ lLB substratum bacterium liquid, on the coating Ampicillin LB culture plate, cultivated 16 hours down at 37 ℃.The picking positive monoclonal extracts plasmid.
7, digested plasmid is identified, carries out sequencing analysis, and confirmation hTERT promotor has been inserted among the carrier pGL33-TSTA-Luc called after pGL3-hTERT-TSTA-Luc plasmid.
(5) T-VISA-Luc plasmid clone
1, cuts pGL3-Luc-WPRE plasmid (on seeing) with the NotI/BglII enzyme; Cut pGL3-hTERT-TSTA-Luc plasmid (on seeing) with the NotI/BglII enzyme.Enzyme is cut system: 10X Buffer A2 μ l, NotI 1 μ l, BglII 1 μ l, plasmid 5 μ g, moisturizing to 20 μ l, 37 ℃ of water-baths 1 hour.
2, enzyme is cut product electrophoresis in 1XTAE Agarose gel, and 120v electrophoresis 30 minutes reclaims WPRE (590bp size) segment and GL3-hTERT-TSTA-Luc (7593bp size) segment.
3, enzyme is cut product and is reclaimed the test kit recovery with the DNA of Qiangen company (U.S.), and method is with reference to its specification sheets.
4, the WPRE product 6 μ l of Hui Shouing, the pGL3-hTERT-TSTA-Luc product 2 μ l of recovery, DNA ligase 1 μ l, buffer1 μ l, last moisturizing to 10 μ l, 4 ℃ of ligations 4 hours.
5, after 2 μ l connect product and 50 μ lE.coli DH5 competent cells mix, ice bath 30min.The 42 ℃ of moving reaction of water-bath heat shock 45sec place 3min then on ice.Add the LB substratum 500 μ l of preheating, jolt 30min at 37 ℃ of following 250rpm.
41, get 200 μ lLB substratum bacterium liquid, on the coating Ampicillin LB culture plate, cultivated 16 hours down at 37 ℃.The picking positive monoclonal extracts plasmid.
42, digested plasmid is identified, the confirmation of checking order, and called after pGL3-hTERT-TSTA-Luc-WPRE plasmid is called for short the pGL3-hTERT-VISA-Luc plasmid, i.e. the T-VISA-Luc plasmid.Find to have inserted luciferase Luciferase gene after the T-VISA-Luc plasmid is 3084 of sequence (its nucleotide sequence is shown in SEQ ID NO.1) at the T-VISA carrier through order-checking, its structural representation is shown in Figure 1A and C.
Three, effect experiment:
(1) with T-VISA-Luc plasmid transfection ovarian cancer cell line and normal cell system; After 48 hours; Collecting cell, cracking; Spectrometer luciferase is active; Its result by can finding out among Fig. 2 that the Luciferase gene expresses in ovarian cancer cell line, but expresses very low in normal ovarian clone as shown in Figure 2; And the CMV-Lus plasmid is expressed in ovarian cancer cell line, in normal ovarian clone, also expresses.The result shows that can be in the ovarian cancer cell efficient high specific of T-VISA carrier expresses target gene, also has the extremely low advantage of expressing in normal tissue cell.
Concrete grammar is following:
A, place 12 porocyte plates to cultivate ovarian cancer cell line and normal cell, when waiting cell density to reach 70% left and right sides, wash with PBS.
B, T-VISA-Luc plasmid (on seeing) or CMV-Luc (construction process is seen Chen et al (2004) .Cancer Gene Ther.740-747) plasmid 2 μ g join among OPTI-MEM substratum (Invtrogen company) the 100 μ l; Room temperature is placed 5min, obtains solution I; Lipofectame 2000 (Invtrogen company) 2 μ l join among OPTI-MEM substratum (Invtrogen company) the 100 μ l, and room temperature is placed 5min, obtain solution II; Solution I and solution II are mixed, and room temperature is placed 20min, obtains solution III.
C, get solution III and be added in the above-mentioned cell, added the DMEM culture medium culturing 43 hours after 5 hours.
D, remove substratum, PBS washing 3 times, active detection kit (Promega company) the lysate cracking 10min of luciferase receives cell collection lysate.
E, cell pyrolysis liquid 100 μ l, according to the active detection kit specification sheets of luciferase, it is active to detect luciferase with beam split range meter.
2, with T-VISA-Luc plasmid transfection breast cancer cell, three cloudy breast cancer cells, breast carcinoma stem cell system and normal cell system; After 48 hours; Collecting cell, cracking; Spectrometer luciferase is active; The result as shown in Figure 3; By finding out among Fig. 3, the luciferase gene has very high reactivity at ten breast cancer cell lines and three breast carcinoma stem cell cordings, but in normal cell system, expresses very low.In breast cancer cell line, breast carcinoma stem cell system and normal cell system, the activity of T-VISA is respectively 1.8 (1.7-4.3) of CMV, 2.6 (2.0-3.2) and 0.006 times.The result shows T-VISA at breast cancer cell, can express target gene by efficient high specific in three cloudy breast cancer cells and the breast carcinoma stem cell system, also has the extremely low advantage of expressing in normal tissue cell.
Concrete experimental technique is following:
A, place 12 porocyte plates to cultivate breast cancer cell, three cloudy breast cancer cells, breast carcinoma stem cell system and normal cell, when waiting cell density to reach 70% left and right sides, wash with PBS.
B, T-VISA-Luc plasmid (on seeing) or CMV-Luc plasmid 2 μ g join among OPTI-MEM substratum (Invtrogen company) the 100 μ l, and room temperature is placed 5min, obtain solution I; Lipofectame 2000 (Invtrogen company) 2 μ l join among OPTI-MEM substratum (Invtrogen company) the 100 μ l, and room temperature is placed 5min, obtain solution II; Solution I and solution II are mixed, and room temperature is placed 20min, obtains solution III.
C, get solution III and be added in the above-mentioned cell, first DMEM culture medium culturing is 43 hours after 5 hours.
D, remove substratum, PBS washing 3 times, active detection kit (Promega company) the lysate cracking 10min of luciferase receives cell collection lysate.
E, cell pyrolysis liquid 100 μ l, according to the active detection kit specification sheets of luciferase, it is active to detect luciferase with beam split range meter.
3, in-situ inoculating ovary cell line Hey8 in the Balb/c nude mice; After 2 weeks; The liposome that comprises the T-VISA-Luc plasmid through tail vein injection 50 μ g; Utilization Xenogen IVIS living imaging appearance dynamic observes target gene Luciferase; The result as shown in Figure 4, by finding out among Fig. 4, CMV-Luc causes Luciferase at mouse lung tissue, tumor tissues and whole body normal tissue expression; But T-VISA-Luc is high expression level in the mouse tumor tissue, and is very low at the whole body normal tissue expression.The result show T-VISA-Luc can be in the mouse body in the ovarian cancer cell efficient high specific express extremely low expression the in normal tissue cell.
Concrete experimental procedure is following:
The ovary cell line Hey8 cancer cells of logarithmic phase is collected in a, cultivation, and PBS washs counting.1X10
6Cell in-situ inoculation Balb/c nude mice (Shanghai animal center) became knurl after 14 days.
B, T-VISA-Luc plasmid (on seeing) or CMV-Luc plasmid 50 μ g join among 5% glucose solution, the 50 μ l, and room temperature is placed 5min, obtain solution I; 20mM HLDC liposome (this laboratory development) 20 μ l join among 5% glucose solution, the 30 μ l, and room temperature is placed 5min, obtain solution II; Solution I and solution II are mixed, and room temperature is placed 20min, obtains to comprise the liposome of T-VISA-Luc plasmid or CMV-Luc plasmid.
C, 100 μ l comprise the liposome of T-VISA-Luc plasmid or CMV-Luc plasmid, become knurl Balb/c nude mice through tail vein injection.
D, become after 2 days, dynamic observe into knurl Balb/c nude mice target gene Luciferase expression with Xenogen IVIS (Xenogen company) living imaging appearance.
E, put to death into knurl Balb/c nude mice, dissect, further observe confirmation with Xenogen IVIS (Xenogen company) living imaging.
F, computational analysis.
With the mouse that is left intact as blank (ctrl).
Described HLDC liposome makes up by the following method:
Lipid is taken out (DOTAP is stored in-20 ℃, cholesterol and is stored in-4 ℃) from refrigerator, return to room temperature.2 Rotary Evaporators of heating in water bath are respectively to 30 ℃, 50 ℃.Take by weighing the 68.75mg cholesterol, put into the 1000ml round-bottomed flask.In round-bottomed flask, add 100mg DOTAP and 25mg Choroform.Rotate flask, make its thorough mixing.Rotation round-bottomed flask 2min mixes it in 30 ℃ water bath, on flask walls, forms thin film.Open vacuum aspirator, 30 ℃ of following 30min.5% glucose solution that adds the 8.9ml preheating dissolves exsiccant film, under 50 ℃ with 105 commentaries on classics/min fast rotational 45min.Reduce temperature to 35 ℃ rotation 10min then.Seal flask with preservative film (or paraffin), spend the night lucifuge under the room temperature.Measurement volumes adds distilled water to 8.9ml.200W carried out supersound process 1-3 minute flask in 50 ℃ ultrasonic water bath case.Under 50 ℃, pass through 1.0 μ successively, 0.45 μ, 0.22 μ, (1.0 μ, 0.45 μ are necessary for the polysufone filter membrane of Whatman to the filter membrane of 0.1 μ, and article No. is respectively #6780-1310 and #6780-1304; 0.22 μ, 0.1 μ are necessary for the Anotop filter membrane of Whatman, article No. is respectively #6808-1122 and #6809-1112), filter the liposome that obtains at last and put into clean glass bottle.Filled test tube 10 seconds with nitrogen, liposome is stored in places 4 ℃ keep in Dark Place subsequent use (preventing air admission simultaneously) in the test tube.
To sum up, T-VISA carrier of the present invention efficient expression target gene of high specific in tumour cell, and in normal tissue cell extremely low the expression.
Claims (1)
1. the carrier T-VISA of an efficient high specifically expressing target gene in tumour cell is characterized in that, described carrier is a circular vectors, and its base sequence is shown in SEQ ID NO.1.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102716498A (en) * | 2012-03-12 | 2012-10-10 | 中山大学肿瘤防治中心 | Drug liposome capable of efficient and highly specific killing of P53 gene mutation type of breast cancer cells |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1997745A (en) * | 2004-04-02 | 2007-07-11 | 得克萨斯州大学系统董事会 | Cancer specific promoters |
-
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1997745A (en) * | 2004-04-02 | 2007-07-11 | 得克萨斯州大学系统董事会 | Cancer specific promoters |
Non-Patent Citations (3)
| Title |
|---|
| M.IYER ET AL.: "Non-invasive imaging of a transgenic mouse model using a prostate-specific two-step transcriptional amplification strategy", 《TRANSGENIC RESEARCH》 * |
| XIE X ET AL.: "Targeted Expression of BikDD Eradicates Pancreatic Tumors in Noninvasive Imaging Models", 《CANCER CELL》 * |
| 尹岚岚等.: "EIAV 基因转移载体的优化", 《中国预防兽医学报》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102716498A (en) * | 2012-03-12 | 2012-10-10 | 中山大学肿瘤防治中心 | Drug liposome capable of efficient and highly specific killing of P53 gene mutation type of breast cancer cells |
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Application publication date: 20120215 |