Chimeric protein for non-viral transgenosis, including dna-binding domain ssbtne and signal of nuclear localisation of vird2

FIELD: medicine.

SUBSTANCE: protein is constructed, which includes DNA-binding domain SSBTne from thermophile microorganism Termatoga neapolitana, connected to C-end of domain VirD2 from Agrobacterium tumefaciens, which is a signal of nuclear localisation.

EFFECT: efficient transport of transgene into cell nucleus.

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I. description of the invention

One of the most important tasks of biotechnology is to improve the targeting of delivery and effectiveness of integration of foreign genes by transfection of eukaryotic cells.

It is clear that the vectors for transport genes must overcome numerous physiological barriers, such as intracellular barriers:

- inability to self-release the nucleic acid from endosome,

- degradation of nucleic acids in lysosomes due to their long stay in endosome,

- the inability of the nucleic acid to self-directed transport in the nucleus of the cell.

It is the presence of these barriers leads to a low efficiency of microinjection and low efficiency of transfection of cell cultures. Until recently, most of the protocols of the transport genes included the delivery of enzogenol through viral vectors.

However, a number of obvious disadvantages of viral vectors encouraged to seek alternative non-viral systems, which are currently most often used different liposomal agents. But liposomal delivery also has several disadvantages - liposomes do not solve the problem of the above barriers.

It is assumed that future non-viral vector systems for transport genes should combine as elementaires vectors, and non-viral systems, it will no longer go on viral or liposomal transport genes, but rather about virosomal delivery.

New opportunities in addressing these questions were obtained through active learning mechanisms Agrobacterium-mediated transformation and viral infection of cells. Most interesting from this point of view are two mechanisms, the obligatorily present in almost all viral and intracellular bacterial parasites, namely the presence of DNA binding proteins, is able to counteract the host cell nucleases, and the presence of nuclear localization signals (NLS), with carifolnai properties, i.e. the ability to address parasitic drag DNA in the nucleus of the host cell.

I.e. one option for overcoming the above barriers to delivery can be used for transfection, in addition to such systems as liposomes, viral and bacterial protein agents, that would allow, on the one hand, to protect aksogan, and, on the other hand, to address the insertions of the transgene in the target genome. Thus, our goal was the creation of a new non-viral vector systems for the delivery of enzogenol on the basis of the above mechanisms of the DNA-binding and nuclear signals of transport.

The invention is a chimeric recombinant baie is OK including the DNA binding domain SSBTne of Termatoga neapolitana attached to the C-end domain of VirD2 of Agrobacterium tumefaciens, which is a nuclear localization signal, which as the invention relates to the field of "genetic engineering" and "biotechnology".

The prototype of the present invention are DNA binding domain SSBTne and the nuclear localization signal VirD2 as a stand-alone protein structures [1; 2].

The distinctive essence of the invention lies in the fact that the designed protein VirD2-SSBTne (figure 1) has transfairusa capacity and the ability to increase the efficiency of liposomal transfection.

The technical result achieved by the present invention is that the resulting protein structure, which includes the DNA binding domain SSBTne and the nuclear localization signal VirD2, able to independently act as a vector by transfection of eukaryotic cells, and to increase the efficiency of liposomal transfection. Main objective: obtain highly purified native protein VirD2-SSBTne and study its basic properties.

To achieve the goal the following objectives:

- obtaining a gene construct that encodes a target protein,

- optimization of plasmid vectors, in which was inserted gene construct,

- time genes in preparative quantities in cloning vectors,

- p is the receiving purified target protein.

Sources of cloned material:

1) a Gene DNA-binding protein SSBTne from archeobacteria Termatoga neapolitana [1].

2) Gene signal nuclear localization or nuclear transport VirD2 of Agrobacterium Agrobacterium tumefaciens [2].

The cloning scheme in stages.

Gene SSBTne was obtained based on the known sequence DNA-binding protein SSBTne from archeobacteria Termatoga neapolitana by PCR [(94°C - 2 min; 60°C - 30 s; 72°C - 1 min) X 30; 72°C - 5 min] corrective primers (see table. No. 1). VirD2 gene was assembled based on the known sequence of the nuclear localization signal of the correcting oligonucleotides (see table. No. 1 of the sequence listing). Next, by ligature gene SSBTne was built into the plasmid vector pQE6 and VirD2 gene was embedded in classicly vector pQE16 company Quageen (see sequence listing No. 1; No. 2).

Legatione structures, hydrolyzed on the relevant restriction sites, was carried out on 4-component scheme: 10×buffer for latinoware, 250 mm KCl, 10×Neh, 10×ATP. The duration of legatione the ligase of phage T4 was 24 hours at a temperature of +4°C. Next, the competent cells of E. coli M15 (nals, strs, rifs, lac-, ara-gal-mtl-, F'-, recA+, uvr+lon+, pREP4 - kanamycin resistance 25 mg/ml) was transformed ligase mixtures by the method of electroporation with abuses mode: 2,5 kV, 25 mF for 4 s, after which they were inoculated on agar growth medium LB with the appropriate antibiotics (ampicillin; kanamycin for selection. On the following day was spent re-seeding of selected clones as sector agar nutrient medium and in liquid nutrient medium LB with appropriate antibiotics for subsequent screening of plasmid DNA, which was distinguished by the method of alkaline lysis. The correct Assembly of genes encoding chimeric protein constructs were confirmed by restriction analysis and sequencing on an automated sequencing machine ALF express II (Amersham Biosciences corp., USA) (sequence corresponded to theoretical). Were obtained plasmid construction: pQeSSBTne, pQeVirD2, pGD-1 (see sequence listing№3; №4; №5).

For protein expression was used expression strain .coll M15. Induction of expression was carried out by adding to the cell culture with an optical density of 0.9 to 1.2 (550 nm) of the inductor IPTG (isopropyl-β-D-thiogalactopyranoside) at a concentration of 0.1 mm. The induction time was 3.0 hours.

In preparative amounts of protein were separated by fractionation by the gel filtration method. 1.5 l of induced culture was deposited at 4000 rpm for 30 min, the precipitate was stored at -70°C day, then the precipitate was washed with 100 ml of N2O MQ and resuspendable 40 is l assay buffer Buf.B (Tris Cl, pH 7.4; 50 mM KCl.; EDTA 1 mM; Tween 20, 0.5%; the Triton X100, 0.5 percent).

Then lysozyme was added to olizane, the precipitate was aged for 15 min at 0°C. Then the solution was processed ULTRASOUND in soft mode in ice, and then added Tnkase and Mcasa (1 ml/40 ml lysate)and it was warming on a water bath at 75°C for 45 min, in this case, due to the presence of thermostable domain of the target protein is completely maintained their properties and were concentrated in the supernatant as a soluble form, while most stem proteins coagulate, which greatly facilitated its subsequent clearance by the gel filtration method. Next was made centrifugation (4000 rpm for 40 min 3°C) and the supernatant was transferred into a clean test tube through a filter (0.45 µm) (before it was taken 1.5 ml as a control after fit). Since the optimal ratio of the volume of the speakers to the volume of the applied sample is Vto/Vabout=40 [3], the product was concentrated by dialysis in Sefadex G-50 (Sigma, USA) to 7 cm3. Column V=300 cm3filled with sorbent Sefadex G-75 (Sigma, USA)were washed with 3 volumes of assay buffer at a flow rate of 9 cm3/min (P=0.15 MPa or 1.5 bar), after which it was balanced with 5 volumes of assay buffer. 7 cm3the sample was applied on the column. At a speed of elution of 19 cm3/h collected fractions, focusing on data recorder and spec is ratemeter 50 (nm). By electrophoresis in a 17% polyacrylamide gel (figure 2) it was proved that the target protein was released in the fractions No. X-XI.

Thus was obtained a highly purified chimeric duomenys protein VirD2-SSBTne (A.K. sequence, see sequence listing No. 6) with a molecular weight of 23 kDa and a concentration of 2 mg/ml, which will be used for further research to improve methods of non-viral transfection of eukaryotic cells.

In the next step were studied basic properties of the protein VirD2-SSBTne, these properties were determined is he able to be part of transfairusa reagent.

First studied DNA - binding properties of the proteins ViRD2-SSBTne method retardation agarose gel, as by binding to DNA, proteins, aggravate her.

Pre-proteins associated with the DNA fragments at a temperature of 94°C., to form a single-stranded forms of DNA.

On the gel (figure 3) is seen a clear shift DNA-protein complexes relative to naked DNA, the most clear retardation is observed on the 2nd track, the optimal molar ratio of nucleic acid and protein is 1:50, respectively.

Then they studied the ability of the obtained proteins protect DNA from exposure to Gnkazy. DNA was mixed at a temperature optimum with our protein, then add Dinkas the quantity 3 ml and the results were evaluated relative to the positive and negative controls in agarose gel (figure 4). It was obvious (at 1-m) tracks that DNA in the presence of our proteins are not subjected to the destructive influence of Gnkazy, i.e. our proteins had pronounced nucleoprotein properties. As a functional reporter gene was selected gene protein GFP under the control of the CMV promoter in the composition of the commercial Shuttle vector pCECMVGFP (sequence listing Annex 7).

Were developed and optimized reagents for transfection-based cationic liposomes (lipofectin) and the resulting protein structures. Ie was selected most physiological buffer, which was phosphate buffer PBS, was selected background ionic composition, which acted ions Mg2+ and Zn2+, due to its stabilizing effect on our whites, and it was determined the optimum volumetric ratio of the DNA-protein complex and lipofectin (1:1).

The final stage was carried out transfection of cell lines Cos-1 and ner-2 our transferirase reagents, then performed statistical analysis of transfection by fluorescence microscopy with subsequent photodetection, in the result, it was proved that the design of the VirD2-SSBTne itself has vector properties, identifying reliable output transfection at the 5%level, and determines a significant (P>0,999) increase in EF is aktivnosti liposomal transfection cells Cos-1 and ner-2 2 times (15-25% 30-50%) (figure 5).

Caption 2

The results of electrophoresis in 17% SDS page in the presence of SDS

1. The strain of E. coli M15 (VirD2-SSBTne) before IPTG induction (without warming up)

2. The strain of E.coli M15 {VirD2-SSBTne) induction of IPTG (without warming up)

3. The strain of E.coli Ml5 (VirD2-SSBTne) to individual IPTG thermolysis (75°C 45 min.)

4. The strain of E. coli M15 (VirD2-SSBTne) ind IPTG thermolysis (75°C, 45 min), the sample to gelfiltration)

5. The fraction of protein IX (after gelfiltration)

6. The protein fraction No. XI (after gelfiltration)

7. Marker molecular weight (30 kDa).

Caption 3

The retardation of the DNA-protein complex in a 1.5% agarose gel

1. DNA-free

2. DNA: Protein 1:50

3. DNA: Protein 1:25

4. DNA: Protein 1:12,5

Captions figure 4

Electrophoresis in 1.5% agarose gel (study nucleoprotein properties VirD2-SSBTne).

1. DNA+Tnkase+VirD2-SSBTne

2. token mol. weight lambda

3. DNA without Gnkazy

4. DNA+Tnkase

5. DNA+Tnkase (double)

Captions figure 5

Records of the results of transfection of the cell line ner-2 complex pCECMVGFP and [VirD2-SSB]-LP. a) pCECMVGFP+.PBS, b) pCECMVGFP+LP+.PBS,) pCECMVGFP+[VirD2-SSB], g) pCECMVGFP+[VirD2-SSB]+LP.

Caption 6

Records of the results of transfection of the cell line Cos-1 complex pCECMVGFP and [VirD2-SSB]-LP. a) pCECMVGFP+.PBS, b) pCECMVGFP+LP+.PBS,) pCECMVGFP+[VirD2-SSB], g) pCECMVGFP+[VirD2~SSB]+LP.

Sequence listing

Literature

1. Stewart A.M., Cotton M.D., M.S. Pratt, S.A. Phillips, D. Richardson, Heidelberg J., Sutton G.G., Fleischmann R.D., White O., Salzberg, S.L., Smith H.O., Venter J.C. and C.M. Fraser // Evidence for lateral gene transfer between Archaea and bacteria from genome sequence of Thermotoga maritime. - Nature, 1999. - P.399, 323-329.

2. Pawel Pelczar, Veronique Kalck, Divina Gomez & Barbara clear Hohn // Agrobacteriumproteins VirD2and - VirE2 mediate preciseintegration of synthetic T-DNAcomplexes in mammalian cells Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland. - Scientific Report, 2004. - P.632-637.

3. Osterman L.A. research Methods proteins and nucleic acids. - Izdat. "Nauka", 1981.

Chimeric recombinant protein for delivery of enzogenol by transfection of eukaryotic cells, including the DNA-binding domain SSBTne from thermophilic microorganism Termatoga neapolitana attached to the C-end domain of VirD2 of Agrobacterium tumefaciens, which is a nuclear localization signal, positioned where the protein has the amino acid sequence shown in sequence listing No. 6 description.



 

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