CN102327624A

CN102327624A - Novel liposome capable of transfecting genes efficiently in vivo and in vitro and preparation method thereof

Info

Publication number: CN102327624A
Application number: CN201110300920A
Authority: CN
Inventors: 谢小明; 郭姣丽; 谢新华; 李来胜; 孔亚楠
Original assignee: TUMOR PREVENTION AND THERAPY CENTER ZHONGSHAN UNIV
Current assignee: TUMOR PREVENTION AND THERAPY CENTER ZHONGSHAN UNIV; Sun Yat Sen University Cancer Center
Priority date: 2011-09-28
Filing date: 2011-09-28
Publication date: 2012-01-25

Abstract

The invention discloses a novel liposome capable of transfecting genes efficiently in vivo and in vitro and a preparation method thereof. The preparation method comprises the following steps of: weighing phosphatidylcholine and cholesterol in a molar ratio of (1-2):1, dissolving the phosphatidylcholine and the cholesterol in chloroform serving as a solvent in an amount which is 1/8 to 1/2 the mass of the phosphatidylcholine, putting the solution into a rotary flask, mixing uniformly, and sucking the solvent dry by using a vacuum aspirator to obtain a layer of lipid membrane which is attached to the wall of the flask; adding 5 mass percent glucose solution in amount which is 1/15 to 1/2 ml/g of phosphatidylcholine, dissolving the dried lipid membrane, and performing rotary drying under vacuum; and adding double distilled water until the volume of the double distilled water is equal to that of 5 mass percent glucose solution, performing ultrasonic treatment in an ultrasonic water bath tank at the temperature of 50 DEG C for 1 to 3 minutes, and collecting the liposome passing through a 0.1-micrometer polycarbonate filter membrane at the temperature of 50 DEG C to obtain the novel liposome capable of transfecting the genes efficiently in vivo and in vitro. The novel liposome capable of transfecting the genes efficiently in vivo and in vitro has the advantages of high transfection efficiency and wide transfection spectrum and particularly has the high-efficiency tumor targeting properties.

Description

A kind of can be efficiently novel lipide of rotaring redyeing gene and preparation method thereof in vivo and in vitro

Technical field:

The invention belongs to biomedicine field, be specifically related to a kind of can be efficiently novel lipide of rotaring redyeing gene and preparation method thereof in vivo and in vitro.

Background technology:

Rotaring redyeing gene is exactly a kind of method that imports to exogenous gene active somatic cell, is the regulation and control of research protein gene, one of most important basic links such as signal conduction and gene therapy.

Transfection method commonly used at present has methods such as calcium phosphorus infection protocol, electroporation, microinjection and liposome method.Calcium phosphorus infection protocol is to mix with normal gene DNA (and copy) and charged species and calcium phosphate, DEAE-glucose or with some lipids, forms sedimentary DNA subparticle, in the direct impouring culture medium with the transfection method of cells contacting.This method is simple, but efficient is extremely low, and the DNA that gets into cell has only 1%-5% can get into nucleus, wherein only can integrate with cell DNA less than 1% DNA, and its repeatability is not good in addition, is subject to influences such as pH value, calcium ion concentration.Electroporation is that cell is placed high-pressure pulse electric, makes cell produce reversible perforation through electric shock, and the DNA in the substrate can infilter cell on every side.This method transfection efficiency is higher, but needs expensive instrument and equipment (electroporation apparatus), and different cells need the different operation program, and cell injury is also comparatively serious, causes cell death easily, and transfection in the inapplicable body can't be applied to the vivo gene treatment.Microinjection is under the microscope direct-view, direct injection exogenous gene in nucleus, and this method is effective; But once can only inject a cell; Work consumption power is time-consuming, and when this method was used for sexual cell, effective percentage was higher; But directly be used for very difficulty of somatic cell, can not be used for transfection and gene therapy in the body.Liposome method is a using artificial liposome packing exogenous gene, merges quiding gene with target cell again, is the method comparatively widely of using at present.

Liposome transports carrier as newtype drug and has many-sided advantage, after medicine is sealed, can reduce drug toxicity, reduces drug dose, can carry out target administration, improves curative effect of medication.The Liposomal formulation of circulation is more in the market, but transfection efficiency is lower in some tumor cell or normal cell, and effect is poorer in vivo; Difficulty is applied to transfection research in the animal body; And as carrier, the liposome targeting that uses at present distributes undesirable, less stable.

Summary of the invention:

It is high to the purpose of this invention is to provide a kind of transfection efficiency, and the transfectional cell spectrum is wide, and transfection efficiency is high in the body, have especially efficient cancer target characteristic can be efficiently novel lipide of rotaring redyeing gene and preparation method thereof in vivo and in vitro.

Of the present invention can be efficiently in vivo and in vitro the novel lipide of rotaring redyeing gene prepare through following method, this method may further comprise the steps:

The molar ratio of by phospholipase phatidylcholine and cholesterol is 1-2: 1 takes by weighing phosphatidylcholine and cholesterol; With the chloroform of phosphatidylcholine quality 1/8～1/2 as dissolution with solvents phosphatidylcholine and cholesterol; Above-mentioned solution is placed rotary flask; Rotary flask makes its mix homogeneously, and the reuse vacuum aspirator blots solvent, obtains one deck attached to the adipose membrane on the flask walls; And then to use amount by 1/15～1/2ml/g phosphatidylcholine to add mass fraction be 5% the exsiccant adipose membrane of glucose solution dissolving; The rotation vacuum drying; And then add the volume that distilled water to above-mentioned mass fraction is 5% glucose solution; In 50 ℃ ultrasonic water bath case, carry out supersound process 1～3min, again at 50 ℃ of liposomees of collecting down through the polycarbonate membrane filter membrane of 0.1 μ m, be can be efficiently the novel lipide of rotaring redyeing gene in vivo and in vitro.

Preferably; The molar ratio of described phosphatidylcholine and cholesterol is preferably 1.7: 1; The consumption of described chloroform is 1/4 of a phosphatidylcholine quality, described adding mass fraction be the amount of 5% glucose solution for by the 8.9/100ml/g phosphatidylcholine, described is successively through 1.0 μ m under 50 ℃ at 50 ℃ of liposomees of collecting the polycarbonate membrane filter membrane through 0.1 μ m down again; 0.45 μ m; 0.22 μ m, the filter membrane of 0.1 μ m is collected the liposome through the polycarbonate membrane filter membrane of 0.1 μ m at last.

Can with of the present invention can be efficiently in vivo and in vitro the novel lipide of rotaring redyeing gene put into clean glass bottle, fill test tube with nitrogen, liposome be stored in place 4 ℃ to keep in Dark Place in the test tube.

The inventor is through experimental exploring repeatedly; The molar ratio that utilizes phosphatidylcholine and cholesterol is 1～2: 1 ratio as the phospholipid and the cholesterol of liposome; Anthropomorphic dummy's somatic cell membrane lipid body and cholesterol ratio make this liposome have better biocompatibility, again through method preparation of the present invention; Utilize ultrasonoscope to handle and push and make liposome pass through the fixedly polycarbonate membrane of particle diameter, obtain the liposome of appropriate particle size.This liposome diameter is suitable, and average diameter is about 180nm, allows that it penetrates superfine blood capillary and by cellular uptake.Behind liposome and gene or the plasmid parcel, average diameter is 250 (200～300) nm.External, but various human bodies of high efficiency transfection and mouse cell, and in 293 cells, its transfection efficiency is up to 80%-90%.In breast cancer cell line, ovarian cancer cell line and pancreatic cancer cell system etc., its transfection efficiency can reach 30%-50%.In vivo, be prone to pass the tumor capillary wall, can make it produce high efficiency transfection at the tumor locus enriched target to tumor cell.Therefore, of the present invention can be efficiently in vivo and in vitro the novel lipide of rotaring redyeing gene have the transfection efficiency height, the transfectional cell spectrum is wide; Transfection efficiency is high in the body; The characteristic that has efficient tumor-targeting especially makes its application more extensive, is particularly suitable for being applied to research such as transfection and gene therapy in the body.

Description of drawings:

Fig. 1 is the efficiently novel lipide particle diameter and the distribution thereof of rotaring redyeing gene in vivo and in vitro of the present invention that dynamic light scattering is surveyed; Wherein liposome promptly refer to can be efficiently the novel lipide of rotaring redyeing gene in vivo and in vitro, liposome+pEGFP-N1 be meant with can be efficiently in vivo and in vitro the novel lipide of rotaring redyeing gene wrapped up the plasmid that contains GFP;

Fig. 2 be utilize of the present invention can be efficiently in vivo and in vitro the parcel of the novel lipide of rotaring redyeing gene contain the figure of plasmid transfection 293 cells of GFP;

Fig. 3 be utilize of the present invention can be efficiently in vivo and in vitro the parcel of the novel lipide of rotaring redyeing gene contain the figure of the plasmid transfection breast cancer cell MCF-7 of GFP;

Fig. 4 be utilize of the present invention can be efficiently in vivo and in vitro the parcel of the novel lipide of rotaring redyeing gene contain the figure of the plasmid transfection breast cancer cell MDA-MB-468 of GFP;

Fig. 5 be utilize of the present invention can be efficiently in vivo and in vitro the parcel of the novel lipide of rotaring redyeing gene contain the figure of the plasmid transfection ovarian cancer cell OVCA-420 of GFP;

Fig. 6 be utilize of the present invention can be efficiently in vivo and in vitro the parcel of the novel lipide of rotaring redyeing gene contain the figure of the plasmid transfection pancreatic cancer cell Aspc-1 of GFP;

Among above-mentioned Fig. 2-6, A: of the present invention can be efficiently after the novel lipide of rotaring redyeing gene parcel contains the plasmid transfection cell of GFP in vivo and in vitro, detect successfully GFP-transfected cell (being green fluorescence) with fluorescence microscope; B: of the present invention can be efficiently after the novel lipide of rotaring redyeing gene parcel contains the plasmid transfection cell of GFP in vivo and in vitro, under the same visual field of the GFP-transfected cell of detected success, take full cell state;

Fig. 7 be T-VISA-Luc in the mice body in the ovarian cancer cell efficient high characteristic express target gene Luciferase; In-situ inoculating ovary cell line Hey8 in the Balb/c nude mice; After 2 weeks; Through tail vein injection 50 μ g wrapped up the T-VISA-Luc plasmid can be efficiently the novel lipide of rotaring redyeing gene in vivo and in vitro, utilization living imaging appearance dynamic observes the figure that target gene Luciferase expresses, wherein with CMV-Luc and be left intact (Ctrl) as contrasting;

Fig. 8 is a plasmid map; Wherein Fig. 8 A is the T-VISA-Luc plasmid map that inserts reporter gene Luciferase; Fig. 8 B is a CMV-Luc plasmid structural representation, and wherein CMV is meant cytomegalovirus promoter, is that present activity does not have specificity promoter the most by force; Fig. 8 C is a T-VISA-Luc plasmid structural representation, is the luciferase expression plasmid that is made up of hTERT (human telomerase promoter) and VISA (integration amplification system).

The specific embodiment:

Following examples are to further specify of the present invention, rather than limitation of the present invention.

Embodiment 1:

One, the efficiently preparation of the novel lipide of rotaring redyeing gene in vivo and in vitro

1. 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 ℃.

2. take by weighing the 68.75mg cholesterol, put into the 1000ml round-bottomed flask.

3. in round-bottomed flask, add 100mg DOTAP and 25mg chloroform (Choroform).

4. the rotation flask fully mixes it.

5. rotation round-bottomed flask 2min in 30 ℃ water bath makes its mix homogeneously, on flask walls, forms thin film.

6. open vacuum aspirator, 30 ℃ of following 30min.

7. the exsiccant thin film of 5% (mass fraction) glucose solution dissolving that adds the 8.9ml preheating rotates 45min with 105 commentariess on classics/min fast vacuums under 50 ℃.Reduce temperature to 35 ℃ rotation 10min then.

8. seal flask with preservative film (or paraffin), spend the night lucifuge under the room temperature.

9. measurement volumes adds distilled water to 8.9ml.

10. in 50 ℃ ultrasonic water bath case (200w), flask is carried out supersound process 1min.

11. under 50 ℃, pass through 1.0 μ m successively, 0.45 μ m, 0.22 μ m, the filter membrane of 0.1 μ m (1.0 μ m, what 0.45 μ m used is the polysufone filter membrane of Whatman, article No. is respectively #6780-1310 and #6780-1304; 0.22 μ m; 0.1 what μ m used is the Anotop filter membrane of Whatman; Article No. is respectively #6808-1122 and #6809-1112), put into clean glass bottle through the liposome of 0.1 μ m at last, prepare therefrom present embodiment can be efficiently the novel lipide of rotaring redyeing gene in vivo and in vitro.

12. filled test tube 10 seconds with nitrogen, can be efficiently in vivo and in vitro the novel lipide of rotaring redyeing gene be stored in the test tube, place 4 ℃ keep in Dark Place subsequent use (preventing air admission simultaneously).

Two, can be efficiently the property testing of the novel lipide of rotaring redyeing gene in vivo and in vitro

1, can be efficiently the size of the novel lipide of rotaring redyeing gene in vivo and in vitro:

As shown in Figure 1, present embodiment can be efficiently the liposome particle diameter surveyed through dynamic light scattering of the novel lipide of rotaring redyeing gene is about 180nm in vivo and in vitro, particle diameter was about 200nm～300nm after parcel contained the plasmid of GFP gene.

2, the efficiently transfection of the novel lipide of rotaring redyeing gene in vivo and in vitro:

2.1: in-vitro transfection:

The refer step of in-vitro transfection is following:

1. reagent such as liposome, plasmid room temperature is placed 15-20min again to room temperature.

2. cell grows to about 70% density.

3. mix by a certain percentage liposome and plasmid (liposome concentration is 8mM, and the recommendation ratio is liposome (μ l): plasmid (ug)=0.5-2: 1), be example with 12 orifice plates, labelling pipe 1: add 1.5ug DNA mixing in the 50 μ l serum-free mediums; Labelling pipe 2: add 1 μ l 8mM liposome mixing in the 50 μ l serum-free mediums.To manage in 1 liquid fast and join pipe 2, blow and beat 2 times up and down after, leave standstill 20min under the room temperature.

4. add 100 μ l serum-free mediums in pipe 2, obtain 200 μ l mixed liquors.

5. the complete medium in the culture plate is removed, add 200 μ l mixed liquors (1.5ug plasmid/8mM liposome/2x10 to the hole of gained in the 4th step ⁵Cell).

6.37 ℃, 5%CO ₂After hatching 3-7 hour, changed culture medium into blood serum medium, confirmed expression time according to the experiment needs.

The present invention with prepare among the above-mentioned preparation embodiment can be efficiently in vivo and in vitro the novel lipide of rotaring redyeing gene parcel plasmid transfection 293 cells, breast cancer cell MCF-7, breast cancer cell MDA-MB-468, ovarian cancer cell OVCA-420, the pancreatic cancer cell Aspc-1 that contain the GFP gene test of the present invention can be efficiently the transfection efficiency of the above-mentioned cell of novel lipide in-vitro transfection of rotaring redyeing gene in vivo and in vitro.

Concrete steps are following:

1. can be efficiently in vivo and in vitro the novel lipide of rotaring redyeing gene, pEGFP-N1 plasmid (available from Clontech company, Cat#:6085-1) wait reagent from refrigerator, to take out, room temperature is placed 15-20min, and is multiple to room temperature.

With 293 cells, breast cancer cell MCF-7, breast cancer cell MDA-MB-468, ovarian cancer cell OVCA-420, pancreatic cancer cell Aspc-1 as treating cells transfected.In 12 orifice plates, behind the above-mentioned cell of inoculation, cultivate by conventional cell culture processes respectively, be cultured to 70% density with the DMEM culture medium routine that contains 10% hyclone.

3.12 the every hole of orifice plate 1.5ug pEGFP-N1 plasmid, labelling pipe 1: add 1.5ug plasmid pEGFP-N1 mixing in the 50 μ l serum-free mediums; Labelling pipe 2: add 1 μ l 0.8mM liposome mixing in the 50 μ l serum-free mediums.To manage in 1 liquid fast and join pipe 2, blow and beat 2 times up and down after, leave standstill 20min under the room temperature.

4. add 100 μ l serum-free mediums in pipe 2, obtain 200 μ l mixed liquors.

5. the complete medium in the culture plate is removed, the 200 μ l mixed liquors that add gained in the 4th step are to the hole

6.37 ℃, 5%CO2 has changed culture medium into blood serum medium after hatching 3-7 hour, cultivates under fluorescence microscope, to observe behind the 24h and take pictures.Confirm expression time according to the experiment needs, general 12-72 hour.

The result is like Fig. 2,3,4, shown in 5 and 6, measure according to the method described above of the present invention can be efficiently in vivo and in vitro the novel lipide of rotaring redyeing gene parcel contain plasmid transfection 293 cells of GFP gene, its transfection efficiency can be up to 80%-90% (Fig. 2); Transfection breast cancer cell MCF-7, its transfection efficiency are about 50% (Fig. 3); Transfection breast cancer cell MDA-MB-468, its transfection efficiency are about 30% (Fig. 4); Transfection ovarian cancer cell OVCA-420, its transfection efficiency are about 30% (Fig. 5); Transfection pancreatic cancer cell Aspc-1, its transfection efficiency are about 45% (Fig. 6).

22: transfection in the body:

The refer step of in-vitro transfection is following:

1. can be efficiently in vivo and in vitro reagent room temperatures such as the novel lipide of rotaring redyeing gene, plasmid place the 15-20min rewarmings.

2. mix liposome and plasmid (the recommendation ratio is 1: 1, and the definition of this place's ratio is the same with the definition of above-mentioned in-vitro transfection) by a certain percentage.

3. measure DNA-liposome mixture OD400 value: add in 5 μ l mixture to the 95 μ l water, detect its OD value in the 400nm wavelength, its OD value should be greater than 0.8, and minimum should reach 0.4, and the visible solution of naked eyes is even muddy shape but does not have deposition.

4. squeeze in the animal body after obtaining testing the concentration that needs with 5% glucose solution diluted mixture thing, luciferase detects in the general body needs 50 μ g/ mices.

Present embodiment with above-mentioned preparation embodiment obtain can be efficiently the novel lipide of rotaring redyeing gene parcel T-VISA-Luc plasmid in vivo and in vitro; Transfection mice in the body; Through detecting the expression of Luciferase in mice, detect of the present invention can be efficiently the novel lipide of rotaring redyeing gene transfection efficiency in vivo in vivo and in vitro.

Concrete grammar is following:

In-situ inoculating ovary cell line Hey8 in the Balb/c nude mice; After 2 weeks; After 50 μ l 0.8M liposomees and 50ug T-VISA-Luc plasmid (1 μ g/ μ l, 50 μ l) fully mix, through tail vein injection 100 μ l T-VISA-Luc liposomees; Utilization living imaging appearance dynamic observes the expression of target gene Luciferase, with CMV-Luc (construction method is seen Chen et al (2004) .Cancer Gene Ther.740-7) as contrasting.The result is as shown in Figure 7, and as can beappreciated from fig. 7 CMV-Luc causes Luciferase at mouse lung tissue, tumor tissues and whole body normal tissue expression, and is nonspecific because the CMV promoter is a cell.But T-VISA-Luc is high expressed in the mouse tumor tissue, and is very low at the whole body normal tissue expression, and T-VISA is the efficient promoter of in tumor cell, expressing genes of interest, through immunohistochemical analysis, the cell transfection rate about 20% can be arranged in the body.The result prove of the present invention can be efficiently in vivo and in vitro the novel lipide of rotaring redyeing gene can produce high transfection efficiency in the body, have the characteristic of efficient tumor-targeting.

The construction method of above-mentioned used T-VISA-Luc plasmid is following:

Clone's construction step of pGL3.T-VISA-Luc (being the T-VISA-Luc plasmid) plasmid

(1) pCRII-TOPO-hTERT plasmid clone

1, conventional method is extracted the DNA of breast carcinoma 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′-ctcgggttaccccacagcctaggcc-3′；

Reverse：5′-cccacgtgcgcagcaggacgcagcg-3′。

3, be template with breast carcinoma MCF-7 cell DNA, hTERT PCR primer (Forward:5 '-ctcgggttaccccacagcctaggcc-3 '; Reverse:5 '-cccacgtgcgcagcaggacgcagcg-3 '), Pfu DNA polymerase, dNTP and PCR reactant liquor, amplification obtains hTERT promoter (380 to+60) product, its sequence is shown in SEQ ID NO.2.Its PCR reaction system: 10 * PCR buffer, 5 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 condition: 94 ℃ of thermal denaturation 5min, 94 ℃ of degeneration 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 Qiangen company (U.S.) pcr dna, and method is with reference to its description.

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 coupled reaction 10min.

6,2 μ l are connected product and 50 μ l E.coli 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 culture medium 500 μ l of preheating, jolt 30min at 37 ℃ of following 250rpm.

7, get 200 μ l LB culture medium antibacterial liquid, on the coating ampicillin LB 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 promoter (its sequence is shown in SEQ ID NO.2) has been inserted among the cloning vehicle pCRII-TOPO called after pCRII-TOPO-hTERT.

(2) pGL3-hTERT-Luc plasmid clone

1, with KpnI/Xho1 enzyme action pCRII-TOPO-hTERT plasmid, (Promega company, Madison WI), contain luciferase Luciferase gene in this plasmid with KpnI/Xho1 enzyme action pGL-3-basic plasmid.Enzyme action system: 10 * BufferA 2 μ l, KpnI/Xho1 1 μ l, plasmid 5 μ g, moisturizing to 20 μ l, 37 ℃ of water-baths 1 hour.

2, enzyme action product electrophoresis in 1 * TAE Agarose gel, 120v electrophoresis 30 minutes.

3, the enzyme action product reclaims the test kit recovery with the PCR DNA of Qiangen company (U.S.), and method is with reference to its description.

4, the hTERT product 6 μ l that reclaim, 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 coupled reactions 4 hours.

5,2 μ l are connected product and 50 μ l E.coli 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 culture medium 500 μ l of preheating, jolt 30min at 37 ℃ of following 250rpm.

6, get 200 μ l LB culture medium antibacterial 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 promoter 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 with Asp718/SalI enzyme action pGEM-3Z-WPRE plasmid; With SmalI enzyme action pGL-3-basic plasmid (Promega company, Madison, WI).Enzyme action system: 10X Buffer A 2 μ l, Asp718 1 μ l/SalI 1 μ l, plasmid 5 μ g, moisturizing to 20 μ l, 37 ℃ of water-baths 1 hour.

2, enzyme action product electrophoresis in 1XTAE Agarose gel, 120v electrophoresis 30 minutes.

3, (U.S. PCR DNA reclaims test kit and reclaims the enzyme action product, and method is with reference to its description with Qiangen company.

4, the WPRE product 6 μ l that reclaim, the pGL-3-basic product 2 μ l of recovery, DNA ligase 2 μ l, buffer 1 μ l, last moisturizing to 10 μ l, 4 ℃ of coupled reactions 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 culture medium 500 μ l of preheating, jolt 30min at 37 ℃ of following 250rpm.

7, digested plasmid is identified, carries out sequencing analysis, confirms that WPRE (its sequence is shown in SEQ ID NO.3) has inserted among the pGL-3-basic called after pGL3-Luc-WPRE plasmid.

(4) pGL3-hTERT-TSTA-Luc plasmid clone

1,, uses DNA polymerase flush end then with HindIII/NotI enzyme action pCRII-TOPO-hTERT plasmid (on seeing); (Invitrogen, Carlsbad CA), use DNA polymerase flush end then with MSCI/NheI enzyme action pGL33-TSTA-Luc plasmid.Enzyme action system: 10X Buffer A 2 μ l, HindIII/NotI or MSCI/NheI1 μ l, plasmid 5 μ g, moisturizing to 20 μ l, 37 ℃ of water-baths 1 hour.

4, the hTERT product 6 μ l that reclaim, 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 coupled reactions 4 hours.

7, digested plasmid is identified, carries out sequencing analysis, and confirmation hTERT promoter has been inserted among the carrier pGL33-TSTA-Luc called after pGL3-hTERT-TSTA-Luc plasmid.

(5) T-VISA-Luc plasmid clone

1, with NotI/BglII enzyme action pGL3-Luc-WPRE plasmid (on seeing); With NotI/BglII enzyme action pGL3-hTERT-TSTA-Luc plasmid (on seeing).Enzyme action system: 10X Buffer A 2 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.

3, the enzyme action product reclaims the test kit recovery with the DNA of Qiangen company (U.S.), and method is with reference to its description.

4, the WPRE product 6 μ l that reclaim, 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 coupled reactions 4 hours.

7, 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.The sequence of finding the T-VISA-Luc plasmid through checking order is shown in SEQ ID NO.1, and wherein 3085-4737 is a luciferase Luciferase gene, and the structure of this plasmid is shown in Fig. 8 A and C.

Claims

One kind can be efficiently the method for preparing of the novel lipide of rotaring redyeing gene in vivo and in vitro, it is characterized in that, may further comprise the steps:

The molar ratio of by phospholipase phatidylcholine and cholesterol is to weigh phosphatidylcholine and cholesterol at 1～2: 1; With the chloroform of phosphatidylcholine quality 1/8～1/2 as dissolution with solvents phosphatidylcholine and cholesterol; Above-mentioned solution is placed rotary flask; Rotary flask makes its mix homogeneously, and the reuse vacuum aspirator blots solvent, obtains one deck attached to the adipose membrane on the flask walls; And then to use amount by 1/15～1/2ml/g phosphatidylcholine to add mass fraction be 5% the exsiccant adipose membrane of glucose solution dissolving; The rotation vacuum drying; And then add the volume that distilled water to above-mentioned mass fraction is 5% glucose solution; In 50 ℃ ultrasonic water bath case, carry out supersound process 1～3min, again at 50 ℃ of liposomees of collecting down through the polycarbonate membrane filter membrane of 0.1 μ m, be can be efficiently the novel lipide of rotaring redyeing gene in vivo and in vitro.
2. according to claim 1 can be efficiently the method for preparing of the novel lipide of rotaring redyeing gene in vivo and in vitro; It is characterized in that the molar ratio of described phosphatidylcholine and cholesterol is 1.7: 1, the consumption of described chloroform is 1/4 of a phosphatidylcholine quality; Described adding mass fraction is that the amount of 5% glucose solution is for by the 8.9/100ml/g phosphatidylcholine; Described again 50 ℃ of liposomees of collecting down the polycarbonate membrane filter membrane through 0.1 μ m be under 50 ℃ successively through 1.0 μ m, 0.45 μ m, 0.22 μ m; 0.1 the filter membrane of μ m is collected the liposome through the polycarbonate membrane filter membrane of 0.1 μ m at last.
One kind according to claim 1 or 2 described can be efficiently in vivo and in vitro the method for preparing of the novel lipide of rotaring redyeing gene prepare can be efficiently the novel lipide of rotaring redyeing gene in vivo and in vitro.