Cultured endothelial cells expressing the protein, with ib-activity, a dna sequence encoding a protein, recombinant design dna, the method of obtaining cells, the protein having ib-activity

 

(57) Abstract:

The invention relates to medicine. Described endothelial cells that Express a protein having the activity of B resulting in NFB - dependent activation of these cells is completely suppressed. Cells can be obtained by changing the genetic material of these cells, for example, by introduction of a heterologous sequences encoding the activity of IB or promoter, in which the activity of IB is expressed structurally, directly after the action of stimuli that activate endothelial cells, or if necessary, in response to a predetermined external stimulus. The technical result - the creation of a transfer material, suitable for use in transplantation, such as xenotransplantation, because they are characterized by favorable, less chance of rejection. 7 C. and 9 C.p. f-crystals, 2 Il.

The invention concerns of xenotransplantation, in particular endothelial cells, which change so that the containing fabric becomes less likely to be rejected by the recipient in xenotransplantation. More specifically, the present invention concerns a factor for the selected cells, and suppression of the inflammatory process.

The main problem of the successful transplantation of organs between discordant species is Verhoture rejection of the authority concerned. The two main components involved in the phenomenon sverhmoschnogo exclusion, are the presence of antibodies specific to xenogenic cells, as well as the consistent recording and activation of the complement system in the recipient. It is known [1] the solution of this problem, in which the transgenic animals Express the factors that inhibit complement in the body. Thus, if the recipient of the transplanted organ from a transgenic animal, activation pathway of complement is blocked specified inhibiting factor.

However, cells of the donor organ also cause rejection, stimulating blood clotting in the recipient, as well as causing changes in the endothelium of the specified donor organ. During the inflammatory process in endothelial cells increases the expression of a certain number of different genes, including genes encoding interleukins, transcription factors, adhesion molecules and components of the blood coagulation system. In the transcription of many of utive is expressed in the cell cytoplasm. It has been suggested that the induction of gene transcription through protein, similar to the NFB is the result of post-transcriptional modifications, including the movement of preexisting transcription factor from the cytoplasm to the nucleus. The specified movement is controlled through the modification of the inhibitory protein called IB, which binds and forms a complex with NFB, thereby retaining it in the cytoplasm. Stimulation of these cells defined signals leads to a modification of IB, which in turn causes the dissociation of its complex with NFB.

Consider that binding of the protein IB with NFB masks the nuclear localization signals (SAL) NFB. During stimulation of cells of specific agents, which depend on cell type and stage of cell development, IB is modified so that this modification disrupts its binding to the NFB, resulting NFB disconnected from IB. Consider that the signals leading to specified modifications are involved in the generation of oxygen radicals and lead to the phosphorylation NFB on specific sites. As a result of this SAL be open and NFB moves into the nucleus where it binds to sites join NFB. This is ion factor NFB was originally isolated from Mature B-cells, he is associated with the motif, representing declarou sequence in the light chain enhancer . Although NFB initially considered specific for a given cell type and the stage of cell development, similar to the NFB proteins were subsequently detected in a large number of cell types, and, as noted above, it was demonstrated that NFB more widely involved in the induction of gene transcription. In the future, this fact was confirmed by the detection of functionally active binding sites NFB in various inducible genes [2].

NFB is a heterodimeric protein consisting of subunits with a molecular mass of 50 KD (p50) and a subunit with a molecular mass of 65 KD (p65). Were cloned cDNA corresponding to p50 and p65, they have been shown to have homology throughout site 300 amino acids. The p50 subunit has significant homology with the products of the proto-oncogene c-rel, isolated from mammals and birds, as well as with the product of the gene dorsal in Drosophila. Recently another member of the family NFB, relB gene, was cloned from fibroblasts stimulated with serum as early gene response.

As p50 and p65 are capable of forming homodimers, which, however, is great for obny to TRANS-activation of transcription, homodimer p65 can only weakly bind to DNA, but is able to TRANS-activation. p50 is synthesized in the form of amino-terminal part of the precursor with a molecular weight of 110 KD (p110), which does not possess DNA-binding or promote dimerization activity. Carboxy-terminal part of the specified predecessor contains eight anchirinah repeats, representing a motif found in several proteins involved in the control of cell cycle and differentiation. Cloning short (2,6 etc., ad) RNA, occurring in conjunction with RNA precursor of p50, which has a length of 4, etc., ad, showed that the C-terminal part of the protein with a molecular weight of 110 KD can also be expressed by either alternative splicing or the use of differential promoters.

Cloned three protein similar to IB: pp40 identified in transformed lymphoid cells of the chicken as associated with rel of phosphoprotein [3]; RL/IF-1, representing the factor 1 suppression of liver regeneration [4]; and MAD-3, which is the product of a gene induced in human macrophages during attachment to plastic surfaces [5].

All three Russia in regenerative furnace for 30 min after hepatectomy. Studies using deletion mutagenesis showed the necessity of having four of the five anchirinah repeats in pp40 to suppress the DNA-binding activity and Association with c-rel, and the necessity of its C-terminal region for the implementation of these effects. Studies using monospecific antibodies that are attached to the p50 precursor with a molecular weight of 110 KD, showed that the C-terminal part (the part with the activity IB) masks the nuclear localization signal (SEL) located in the amino-terminal region p50.

Unexpectedly, we found that the expression of IB is induced in endothelial cells (EC) using the same stimuli that activate EC, albeit with slower kinetics compared with the rapid post-transcriptional activation of NFB. Thus, it appears that the expression of IB is a natural feedback mechanism that suppresses the activation of EC. We used this fact to develop a further strategy to prevent graft rejection.

Accordingly, the first aspect of the present invention provides an endothelial cell, which expre which I am quite depressed.

In the present description the term "protein having the activity of IB" means a protein that is able to communicate with NFB and prevent its migration into the nucleus and/or the NFB binding sites of its recognition in the genetic material of the cell, thereby preventing NFB-dependent induction of transcription of the gene. This protein may be a natural protein, similar to IB, part thereof, or an analogue or variant of the proteins having the above-mentioned NFB-binding activity and the activity, inhibiting the induction of gene transcription. In the case of the preferred embodiment of the present invention this protein may represent a variant comprising one or more signal sequence, intended for the direction of this protein in a specific cell compartment, such as the cell nucleus.

In the preferred case, endothelial cells provided by the present invention that Express a protein having the activity of IB, and this expression is constitutive (i.e. permanently, and in the absence of delayed kinetics compared with the expression NFB), directly after the action of stimulus, activating endothelialisation the present invention endothelial cells receive as a result of the modification of the genetic material of these cells. This modification involves the introduction of heterologous DNA that encodes the expression of a protein having the activity of IB, and modification or replacement of the signals controlling the above expression and located in a part of the native sequences encoding IB.

Thus, one of the incarnations of endothelial cells of the present invention provides heterologous DNA sequence, which is expressed constitutively specified cell and encodes the expression of a protein having the activity of IB.

The sequence encoding a protein having the activity of IB, expeditiously attached to the promoter sequence, which usually also is heterologous in relation to the above cells, resulting in this protein is expressed constitutively.

In an alternate embodiment of endothelial cells corresponding to the present invention contain a heterologous inducible promoter DNA sequence attached to a DNA sequence that encodes the expression of a protein having the activity of IB, resulting in this protein is expressed directly predetermined external stimulus.

The specified encoding a protein sequence, expeditiously attached to heterologous sequences inducible promoter, can be a native or heterologous sequence encoding IB, and can also include a sequence encoding an appropriate signal sequence, such as specific signal sequence, nuclear localization. The above incentives, activating endothelial cells, can be any incentives that lead to changes in the endothelium of donor tissues or organs, as well as stimulating blood clotting in the recipient that leads to exclusion. The specified predefined stimulus can serve as availability of drugs, cytokine or other inducing agent that stimulates the expression with the specified inducible promoter. In particular, transplant, the patient may be after transplantation subjected to processing by an inducing agent such as a drug, in order to induce the expression of IB and, thus, prevented the activation of endothelial cells in the donor organ, as well as subsequent rejection of the body.

May be what they are aspect of the present invention is a method for endothelial cells, relevant to the present invention, involving the transfection of endothelial cells by heterologous DNA sequence, which is expressed constitutively these cells and encodes the expression of a protein having the activity of IB.

In addition, another aspect of the present invention is a method for endothelial cells of the present invention, providing the operative connection of a heterologous DNA sequence, which is an inducible promoter, to the native DNA sequence that encodes the expression of a protein having the activity , resulting in this protein is expressed directly following a stimulus that activates endothelial cells, or if necessary, in response to a predetermined external stimulus.

In the preferred case, these heterologous DNA sequences include gene endothelial cells. In the alternative case, these heterologous sequences can be maintained in endothelial cell extrachromosomal or consistently, or for a limited period.

In accordance with this and the AK TNF and IL-1, was identified previously unknown protein similar to IB and denoted in the future ECI-6. This protein and its parts, which has active IB, and their equivalents fall within the scope of the present invention. Amino acid sequence ECI-6 and encodes its natural DNA sequence is represented as a sequence of N 1 (the sequence listing is provided at the end of the description). This protein, which has active IB, may include ECI-6 or its active part, or active analogs or variants.

Thus, in case your preferred embodiment, the heterologous sequence encoding a protein having the activity of IB, encodes a protein that comprises a sequence characterized by at least 70%, in the preferred case, at least 80%, more preferred is at least 90% homology with the protein sequence represented by Sequence No. 1 or No. 2 from position 1 to position 280, or includes part of the specified protein active IB.

In a particularly preferred variant of an embodiment of the specified heterologic itov phosphorylation (amino acids 30-36, amino acids 39-47, and amino acids 260-264 in Sequence No. 1). These changes may include the replacement of the Central Ser residue located in the first or third of the above sequences, Gly, and replacement of Tyr, located in the second above-mentioned sequence, Phe.

This protein, with similar IB activity, prevents migration of the NFB in the core and/or suppresses the accession of the NFB to the binding sites NFB found in the genetic material of cells. Inhibition of NFB binding can be detected using standard biotesting methods. So, in order to register the inhibition of binding of the NFB in endothelial cells, you can use the test on the change in electrophoretic mobility. A concrete example of such a test is the following.

Endothelial cells in the aorta of pigs grown in a culture medium, representing modified by Dulbecco Wednesday Needle containing 10% fetal calf serum. To stimulate these cells for 2 h using 100 ng of bacterial lipopolysaccharide, and then extracted nuclear proteins according to [7]. Then in vitro transcribing RNA from a plasmid (Bluescript II, recip is syshestvyut in accordance with the Protocol of the manufacturer, except, what type of 0.25 nm m7G(5')ppp(5')G (Boehriger).

The obtained RNA is subsequently used for in vitro translation in wheat germ extract or lysate of rabbit reticulocytes (both of them receive from Promega Corporation, Madison, WI, USA) according to the manufacturer's Protocol. Then perform EMSA, preparing labeled 16-dimensional BS-2 oligonucleotide (represented as a sequence of N 3), which is the binding site NFB. The binding reaction is carried out at a temperature of 20oC and a pH of 7.9 for 15 min in a total volume of 15 μl containing 12% glycerol, 12 mm HEPES, 4 mm Tris, 60 mm KCl, 1 mm EDTA, 1 mm DTT, 200 μg/ml poly(dl-dC), 300 μg/ml bovine serum albumin, 0.2 ng labeled BS-2 (100,000 flashes/min), 5 μg of nuclear proteins and different amounts specified translated protein. Inhibition of binding of NFB with the oligonucleotide BS-2 can be detected by comparing the control and wheat germ extract or lysate of rabbit reticulocytes.

Another aspect of the present invention provides a donor tissue or donor organ containing the endothelial cells. This donor material may be xenogeneic with respect to the recipient or it can be taken is Specified donor material can be obtained from the transgenic animal. In the alternative case, in endothelial cells of the donor organ can be inserted heterologous DNA using traditional methods of gene therapy. Suitable methods are described in [8].

Another aspect of the present invention provides for transgenic mammals other than human, and having the above-described endothelial cells.

Heterologous DNA can be entered in the specified animal or an ancestor of the specified animal located at the unicellular stage or at the stage of early morula. The preferred stage is the single-celled stage, although the process of introducing DNA can be established between two-celled and eight cells stages. The DNA sequence can be micronational in cells in accordance with traditional methods, or you can use retroviral or similar system [9].

In the preferred case, the specified mammal is a pig.

The present invention also provides donor organ obtained from the indicated transgenic mammal.

Along with the creation of the donor organ and tissue, the present invention can also be used to create geneticist what W, in particular, in connection with such diseases endothelial cells, which can be alleviated by blocking mediated NFB induction of the expression.

Another aspect of the present invention provides a nucleotide sequence that encodes a protein having the activity of IB, and includes an amino acid sequence represented as a sequence of N 1, starting from amino acid position 1 and ending with amino acid position 280; or contains amino acid sequences that are substantially, i.e. at least 95%, homologous to the above; and is intended for use in transformed endothelial cells. The corresponding transformed endothelial cells can be used for transplantation or to control the expression in smooth muscle cells.

These nucleotide sequences can also include codons encoding amino acids 281 314 on from the sequence shown in Sequence No. 1. In prior cases, these nucleotide sequences encode a protein, in which this sequence is modified to violation of one or Bo the major changes preferably lead to the replacement of the Central Ser, in the first or third of the above-mentioned sequence, Gly, as well as to the replacement of Tyr, located in the second above-mentioned sequence, Phe.

Another aspect of the present invention provides a DNA sequence which encodes a protein having activity of IB, and includes a heterologous signal sequence.

Usually specified signal sequence is a signal sequence that is used for directing the protein to a specific cell compartment, in the preferred case, the signal sequence-specific nuclear localization.

Another aspect of the present invention also provides a recombinant construct DNA comprising a DNA sequence which encodes a protein having activity of IB, and operational attached to

1) inducible promoter or

2) the specific promoter of endothelial cells,

moreover, the specified promoter differs from the natural promoter of the above-mentioned DNA sequence which encodes a protein having activity of IB.

In the preferred case occasisionally DNA sequence, which encodes a protein having the activity of IB is a heterologous sequence, and may represent the above sequence, including the signal sequence or the corresponding Sequence N 1.

The present invention also provides an expression vector containing the above-described nucleotide sequence or a recombinant construct DNA.

In the preferred case, the vector is a retroviral vector, an adenoviral vector, complex nucleotide sequence and transferrin-poly-conjugate, and the plasmid contained in the liposome.

Acceptable retroviral vector described in [10]. Acceptable adenoviral vector described in [11]. Acceptable transferrin-poly-complexes described in [12]. Liposomal complexes described in [13].

Another aspect of the present invention provides a protein having IB-like activity and comprising the amino acid sequence presented in the Sequence N 1, starting from amino acid position 1 and ending with amino acid position 280.

The specified protein lnasty PEST, which shows that in other proteins they are responsible for the instability of the protein [14]. In the preferred case, these amino acids are not present in the specified protein that can be done by introducing a stop codon after the codon corresponding to the amino acid 280. This result can be achieved by mutation engine codon corresponding to Proline, located at amino acid position 281, using polymerase chain reaction.

In the preferred case, the amino acid sequence of the specified protein change to violate one or more phosphorylation sites (amino acids 30-36, amino acids 39-47, and amino acids 260-264).

These changes may include the replacement of the Central Ser located in the first or third of the above-mentioned sequence, Gly, as well as to the replacement of Tyr, located on the second above-mentioned sequence, Phe.

Another aspect of the present invention provides a protein having the activity of IB and includes a heterologous signal sequence, such as the signal sequence-specific nuclear localization.

It should be noted that the above endothelially xenotransplantation, because this initial rejection is Verhoture rejection. In this regard, usually genetic material in the cells of the donor organ are also changing, resulting in the activation pathway of complement in the recipient is suppressed. In accordance with [1] this can be achieved using transgenic animals expressing specific to the type of recipient factors that inhibit the complement. Endothelial cells of donor organs obtained from such animal can be modified using the methods of gene therapy to create the above endothelial cells. Alternatively, in the specified transgenic animal is located at the unicellular stage or early stage of morula, may be introduced above the vector. Thus obtained transgenic animal should Express these factors that inhibit complement, and should be above the endothelial cells.

Thus, another aspect of the present invention provides for matching the above description is different from human endothelial cells, tissue, donor agencies and transgenic animals expressing one or more facto the notches of the aorta of a pig (ECAS), stimulated inflammatory agents TNF and IL-1. Was revealed induction of RNA ECI-6, carried out within 2 h after stimulation of these inflammatory agents and weakened after 4 o'clock in Addition, a small amount of it was found in the "unstimulated" cells. The addition of cycloheximide with LPS for 6 h led to the strengthening of the specified signal, suggesting that induction does not require the synthesis of protein. Specified RNA was detected using the method of differential screening using activated endothelial cells against unactivated.

IB-protein activity ECI-6 were tested using the method of DNA binding in vitro; it has been shown that it is specific, and depending on the dose inhibits the activity of connecting the NFB and present in nuclear fractions ECAS stimulated by lipopolysaccharide (LAS). Similarly, endogenous NFB present in the lysate of rabbit reticulocytes were also reduced by adding to the above lysates RNA ECI-6, but not control lysates. The NFB binding was specific, since it could enter into a competitive relationship with its oligonucleotide representing sites svyazyvanie reavley very high homology (>90%) with protein IB-protein MAD3, as well as high homology with proteins pp40 and RL/IF-1.

Further embodiments of the present invention is described only by means of examples with reference to the drawings, in which:

Fig. 1 shows a polyacrylamide gel illustrating the inhibition of binding of nuclear proteins with binding sites NFB in the presence of ECI-6, translated in vitro in wheat germ extract,

Fig. 2 shows polyacrylamide gel illustrating the inhibition of binding of endogenous NFB with the binding site IB in the lysate of rabbit reticulocytes.

Examples

Example 1: Cloning of induced genes

A. obtaining a cDNA library stimulated endothelial cells in the aorta of pigs (ECAS)

Post-confluent ECAS is treated with a combination of recombinant TNF and IL-1 person (obtained from Genzyme), and the concentration of each 100 u/ml for 4 and 9 o'clock Cells collected in both time using the method described in [15] , is extracted and allocate total cellular RNA for each time point. Then combine the obtained RNA for each time. In accordance with the Protocol of the manufacturer prepare 5 μg poly A+ RNA with oligo-dT-cellulose (Boehringer Mannheim).

Using set for when ofage Zap II. Then carry out the packaging of the resulting vectors using the system Gigipack Gold (Stratagene, La Jolla, California). Receive approximately 2.2 million primary clones. Sceneroot approximately 5000 plaques.

B. Receiving probes and screening

Prepare two probes: one of the RNA, which is obtained from reinducing ECAS, and one of ECAS induced as described in paragraph A. Specified neindutsirovannom probe get through reverse transcription of 1 μg RNA using reverse transcriptase (Gibco-BRL) according to the manufacturer's Protocol. In this reaction using 50 µci 32P-CTP. Induced probe receives the same way, however in the future it twice subjected to purification using 10 µg biotinylating neindutsirovannom RNA. The above-mentioned biotinylation and purification carried out using the Subtractor I (InVitrogen, San Diego, California) according to the manufacturer's Protocol.

Sceneroot two replicas from the library ECAS, one - neindutsirovannom probe, and the other induced by the probe under standard conditions of hybridization at 65oC [15]. Phages, which gave a stronger signal when using the induced probe compared to neindutsirovannom were subcloned way is the standard way [15] . As a probe using the fragment EcoRI-Xho1 length of 1.5 of TPN-induced clone labeled with the set to obtain a random oligonucleotide primers (Stratagene) according to the manufacturer's Protocol.

Then determine the nucleotide sequence of the obtained cDNA clones using the termination circuit using dideoxy using the Sequenase kit (US Biochemicals, Cleceland, Ohio) according to the manufacturer's Protocol. The obtained nucleotide sequence and predicted based on amino acid sequence presented in the Sequence No. 1. The above five anchirinah repetitions are amino acid positions 73-99, 110-136, 143-169, 182-208 and 216-242, and three potential site of phosphorylation CK-TYR II and PKC are amino acid positions 30-36, 39-47 and 260-264, respectively. 5'-end of the sequence (position 1-270) obtained from a genomic clone and attached to the above-mentioned cDNA by Xhol site (position 285) with a functional clone for translation in vitro.

Further, comparing the specified protein sequence with sequences MAD-3, RL/IF-1 and pp40 using the program PILEUP of the software development>Example 2: in vitro Translation

The linearized vector (10 μg; Bluescript), carrying a box, which contains the nucleotide sequence corresponding to positions 126-1605 Sequence N 1, transcribing into RNA using T7 polymerase (Stratagene) in a total volume of 100 μl following the manufacturer's recommendations, but with the inclusion also of 0.25 mm m7G(5')ppp(5')G. further obtained caerbannog mRNA extracted with phenol and chloroform and precipitated in ethanol. 1/50 of the obtained product is used for in vitro translation in wheat germ extract or lysate of rabbit reticulocytes, following the manufacturer's recommendations (Promega). For analysis of changes in electrophoretic mobility (AIAN) used different concentrations corresponding to 5, 1, and 0.2 and 0.04 ál system broadcast in vitro.

Example 3: AIN

Used Protocol AIAN described in [16]. Endothelial cells (EC) were cultured in the medium Needle, modified by Dulbecco and containing 10% fetal calf serum. These endothelial cells are stimulated with 100 ng/ml of bacterial lipopolysaccharide (DPS) for 2 h, then extracted nuclear proteins according to [7]. 0.2 ng of double-stranded 26-dimensional oligonucleotide BS-2 (which I loose ends with klenovsky fragment of DNA polymerase I in the presence of radioactive nucleoside triphosphates. Further the obtained oligonucleotide incubated in 15 μl of buffer for binding, containing 12% glycerol, 12 mm HEPES, 4 mm Tris, 60 mm KCl, 1 mm EDTA, 1 mm DTT, 200 μg/ml poly(dl-DC) and 300 μg/ml bovine serum albumin. Then add 15 μg of nuclear proteins, as well as 5, 1, and 0.2 or 0.04 ál translated protein ECI-6. The binding reaction is carried out at a temperature of 30oC and a pH of 7.9 for 15 minutes Suppression binding NFB with the oligonucleotide BS-2 can be detected by comparing the obtained results with the control and wheat germ extract or lysate of rabbit reticulocytes.

This procedure is repeated with the lysate of rabbit reticulocytes containing or nesteriak RNA ECI-6, but without the use of the nuclear extract.

Create competitive conditions binding or cold BS-2 or BS-1 (the sequence of which is presented below as a sequence of N 5), or with ELB (the binding site of the promoter ELAM-1 pig, the sequence of which is presented below as a sequence of N-6), or with IgB (the binding site NFB of enhancer Kappa light-chain immunoglobulin, the sequence of which is presented below as a sequence of N 7 In tests on competitive binding using a 500-fold molar excess of its oligonucleotides. The resulting complexes were separated on 5% polyacrylamide gel, as described in [7]. Protein ECI-6 inhibits the binding of NFB with the binding site IB-2.

Example 4: Experiments on transient expression

Minimum NFB-dependent promoter construct in the vector UBT.Luc [17], which contains the reporter gene luciferase, using standard molecular biology methods. Briefly, double-stranded oligonucleotide representing a fragment of a promoter thymidine kinase length 37 p. O. and bearing at its 5' and 3' ends of the additional plots characteristic of restricted EcoRI and HindIII, respectively, clone in plasmid pKSM13 (Stragene) to obtain the vector pKSM13-TK. Two copies of the double-stranded oligonucleotide, which carries additional areas specific to restrictase EcoRI, and represents a binding site NFB, clone in plasmid pKSM13-TK obtaining vector pKSM13-2xIgkB-TK. The correctness of the design is confirmed using DNA sequencing. The resulting fragment 2xIgkB-TK cut through DNA cleavage by restrictase NotI and HindIII (site NotI recognition localized upstream (5') relative to the EcoRI site). The resulting fragment are ligated in the vector UBT.Luc, pre resplend below and in a sequence of N 9:

The specified vector using transfection introduced into NIH3T3 cells using Lipofectin (BRL, see attached Protocol). Test for luciferase activity is carried out in accordance with [18] . Do not reveal excess of luciferase expression compared to the background. In that case, if exercised by co-transfection of the vector 2xIgkBTK. Luc together with the vector determining the expression of subunit p65 protein NFB (CMV.p65), reveal a high level of luciferase activity. In that case, if together with vectors 2xIgkBTK.Luc and CMV. p65 perform a transfection vector that determines the expression of ECI-6/IkBa, the levels of luciferase activity decreases to the background level. As an internal control during all transpency use the expression vector ESV. -galactosidase.

The aforementioned mutant carrying a deletion of the district of PEST, is obtained using PCR, introducing a stop codon after amino acid Leucine-280.

Example 5: Construction of recombinant retroviruses for stable transduction of primary ECAS cDNA ECI-6/IB

5 retroviral vectors evaluated in terms of production of recombinant retroviruses capable of highly efficient transduction ECAS (endothelial cells of the aorta of a pig) with the MOU as well as the same high expression in the pool living ECAS, as in the case of vectors, in which a given gene is transferred through the 5'viral LTR. On the basis of this research, to construct a recombinant retrovirus, designed for the expression of cDNA ECI-6/IB in ECAS, choose the vector pNTK-2. The corresponding recombinant provirus design, inserting the portion of the Xho1 fragment-Sal1 length of approximately 1, etc., of O., including cDNA ECI-6, unique Xho1 site, located in the provirus pNTK-2 downstream relative to the tk-promoter. Purified recombinant products that contain the specified cDNA in sense and antisense orientation relative to the tk-promoter and can be distinguished by cleavage with restriction enzyme BamHI, identified in the E. coli strain XL-1 blue. Received antisense construct which is used as a control. To achieve maximum efficiency transfection of the packaging cell line PA317 high-purity plasmid isolated from bacterial cultures using the Qiagen. To minimize the likelihood of integration, incompatible with the production of the virus, sense and antisense constructs (10 μg) linearized with restriction the compulsory extraction of phenol-chloroform and chloroform, and precipitation with ethanol and dissolved in 100 μl of buffer for electroporation (PBSA + 20 mm HEPES buffer, pH 7.0) overnight at a temperature of 4oC. Re-dissolution is confirmed through a previous transfection of agarose gel electrophoresis. Packaging cell PA317 transferout or pseudo-transferout (in the absence of DNA) using a kit of Biorad electroporation at 0.25 kV and 500 microfarad that determines the duration of the exposure equal to 13.7 MS. Surviving cells resuspended in 20 ml of fresh culture medium and incubated over night before adding selective agent (0.8 mg/ml G418). After 7 days of selection get resistant to the antibiotic colonies of cells resulting from transfection as sense and antisense design, while the control cells (transfected in the absence of DNA) completely killed.

The obtained colonies unite and seeded in flasks T-75 in the medium containing G41B. In log-phase and beginning to stick the cells from each flask freeze in the form of an aliquot in LN2(master Stoke-on cells). Cells intended for producing virus is grown to a state of sticking together and at 24 h, harvested virus in 20 ml paleogenetic cells, and also for storage at a temperature of -80oC. Then spend two additional collection of virus. Examine cells on the expression of IB, as well as testing them on suppression associated with NFB induction of the expression.

Literature

1. WO 91/05855.

2. Baeuerle P. (1991), BBA 1072, 63-80.

3. Davies et al. (1991), Science 253, 1268-1271.

4. Tewarl et al. (1992), Mol. Cell. Biol. 12(6), 2898-2908.

5. Haskill et al. (1991) Cell 65, 1281-1289.

6. Gosen and Bujard (1992), PNAS 89, 5547-5551.

7. Dignam et al. (1983), Nucl. Acids Res. 11, 1475-1489.

8. Miller, A. D. (1992) Nature 357, 455-460.

8. Miller, A. D. and Rosman, G. T. (1989), Biotechniques 7(9), 980-990.

10. WO 92/07573.

11. Rosenfeld, M. A. (1991), Science 252, 432.

12. Wagner et al. (1991), PNAS 88, 4255-4259.

13. Stewart et al. (1992), Human Gene Therapy 3, 267-275.

14. Rogers et al. (1986), Science 234, 364-368.

15. Maniatis et al. (1989), Molecular Cloning, Cold Spring Harbor, 7-10.

16. Ausubel I. and Frederick M. / Eds. (1987), "Current Protocols in Molecular Biology", J. Willey & Sons, section 12-2.

17. de Martin et al. (1993), Gene 124, 137-138.

18. de Wet et al. (1987), Mol. Cell. Biol. 7, 725-737.

1. Cultured endothelial cells expressing a protein having the activity of IB, resulting in totally suppressed NFB - dependent activation of the specified cell.

2. Cultured endothelial cells, estimulo, activating endothelial cells, or if necessary, in response to a predetermined external stimulus.

3. Cultured endothelial cells under item 2, characterized in that they contain a sequence of heterologous DNA, which is expressed constitutively these cells and encodes the expression of a protein having the activity of IB.

4. Cultured endothelial cells under item 2, characterized in that contain the DNA sequence representing a heterologous inducible promoter, which is functionally attached to a DNA sequence that encodes a protein having activity of IB, resulting in this protein is expressed directly after exposure to stimuli that activate endothelial cells, or if necessary, in response to a predetermined external stimulus.

5. Cultured endothelial cells under item 4, characterized in that the DNA sequence which encodes a protein having activity of IB, is also a heterologous sequence.

6. Cultured endothelial cells in PP.2 to 5, characterized in that said DNA sequence, which caderea 70% homologous to the sequence presented in a sequence of 1, starting with position 1 and ending at position 280, or a different sequence, which has active IB.

7. Cultured endothelial cells under item 6, characterized in that the DNA sequence which encodes a protein having activity of IB modified to disrupt one or more sites of phosphorylation.

8. The DNA sequence which encodes a protein having activity of IB, and having the amino acid sequence represented as a sequence of 1, starting from amino acid position 1 and ending with amino acid position 280.

9. The DNA sequence under item 8, wherein the specified sequence of DNA which encodes a protein having activity of IB, changed to nerolite one or more sites of phosphorylation.

10. The DNA sequence which encodes a protein having activity of IB, characterized in that it contains a heterologous signal sequence.

11. The DNA sequence under item 10, characterized in that the signal sequence is a specific signal placenta is holding inducible promoter, which is functionally attached to a DNA sequence that encodes a protein having activity of IB on PP.8 to 11, with the indicated promoter differs from the native promoter of such a DNA sequence.

13. Recombinant design DNA under item 12, characterized in that the DNA sequence which encodes a protein having activity of IB, is a DNA sequence under item 10.

14. The method of obtaining endothelial cells under item 4, which consists in the fact that the heterologous DNA sequence, which represents an inducible promoter, functionally attached to the native sequence of DNA which encodes a protein having activity of IB, the resulting recombinant construct DNA is introduced into endothelial cells and selecting cells that Express this protein directly after the action of stimuli that activate endothelial cells, or if necessary, in response to a predetermined external stimulus.

15. The protein having the activity of IB, characterized in that it comprises the amino acid sequence represented as a sequence of 1, starting with amino acid inoculate sequence change for to violate one or more sites of phosphorylation: amino acids 30 - 36, amino acids 39 to 47, and amino acids 260 - 264.

 

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The invention relates to molecular biology and can be used for the diagnosis of genetic diseases

The invention relates to chemical-pharmaceutical industry, namely to antiviral drugs exhibiting in-vitro high activity against human immunodeficiency virus, against herpes virus and cytomegalovirus

The invention relates to the field of Bioorganic chemistry, in particular to a method for the preparation of 3'-phosphate, N,P - unprotected phosphothioate analogues oligodeoxyribonucleotides General formula I, where In residue thymine, cytosine, adenine or guanine; n = 1 to 20, which can be used as starting compounds to obtain phosphothioate oligonucleotide reagents for biotechnological purposes
The invention relates to biotechnology, in particular genetic engineering
The invention relates to the field of biochemistry and can be used in applied biochemistry for the production of medical biological preparations

The invention relates to molecular biology, diagnosis and medicinal

The invention relates to a recombinant adenovirus expression vectors, characterized by partial or complete deletion of the DNA fragment of adenovirus encoding the protein IX, and containing the gene of a foreign protein, or a functional fragment or mutant form

The invention relates to biotechnology, immunology and medicine and can be used to direct cellular immune response to an infectious agent

The invention relates to biotechnology and can be used for the regulation of cell proliferation

The invention relates to biotechnology and can be used for selective destruction of cells infected with hepatitis C virus (HCV) or infectious RNA

The invention relates to medicine and can be used for selective destruction of cells infected with RNA of hepatitis C virus(HCV)

The invention relates to medicine, namely to the use of inhibitors of proliferation of stem cells

The invention relates to medicine, namely to immunology and immunology, and concerns the problem of vaccination against tumor cells and vaccine therapy of cancer
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