Recombinant plasmid expressing module polypeptide for delivery of photosensitizer and strain escherichia coli vkpm b-8356 - producer of module polypeptide

FIELD: biotechnology, microbiology, genetic engineering.

SUBSTANCE: invention proposes a new recombinant plasmid pR752 (5269 pair bases) comprising genetic construction under control of bacteriophage T5 promoter and encoding a module polypeptide consisting of 6 histidine residues, hemoglobin-like protein of E. coli, modified fragment of large T-antigen SV-40, translocation domain of diphtheria toxin, spacer sequence (Gly-Ser)5 and human epidermal growth factor (6 His-HMP-NLS-Dtox-(Gly-Ser)5-EGF) and designated for the directed transfer of photosensitizers into target-cell nuclei. By transformation of the strain E. coli M15 (rep 4) with plasmid pR752 the recombinant strain E. coli VKPM B-8356 as a producer of new polypeptide vector is prepared. The usage of this new strain is able to enhance the effectiveness of effect of photosensitizers by some orders. Invention can be used in medicinal-biological industry in preparing agents providing the directed transport of photosensitizing agents into tumor cell nuclei.

EFFECT: valuable biological and medicinal properties of polypeptide.

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The invention relates to biotechnology, genetic engineering, and medicine and is a plasmid pR752 encoding modular recombinant polypeptide and the strain E. coli - M15 (Rep4, pR752) - producer of modular peptide.

Known composition for photodynamic damage to target cells, consisting of a photosensitizer, a component carrier, a photosensitizer, a component for recognition of target cells and transport of the photosensitizer into the target cells through specific receptor-mediated endocytosis and the component with the ability to direct transport of the photosensitizer within the target cells. On the basis of this composition, a method of photodynamic damage to cells, which consists of adding the proposed composition to the cells and maintaining them at a temperature of, normal functioning of the cells. Upon irradiation of the cells with light is a photochemical reaction in which the target cells are killed (RF Patent No. 2066552, 1996)Poluchenie such multicomponent transport compositions for use in clinical practice is too expensive and labor-intensive procedure. A more appropriate way to achieve the same transport media includes designing, developments in E-coli and purification modular recombinant transport is Turov (MRI) for photosensitizers (PS), bearing:

ligand module, optimized large T-antigen of the virus SV-40 as a nuclear localization signal, module-carrier - hemoglobinopathy protein NMR E-coli, endosomolytic module - amphipatic polypeptide. These MRI deliver FS in the kernel melanoma cell targets and greatly increase photocytotoxicity action FS (Genetics, 2003, vol 39, No. 2, S-268). Described in this work, MRI is not sufficient to effectively deliver the FS in the nucleus of cells.

The objective of the invention is to obtain a recombinant plasmid encoding a modular polypeptide, which will increase the efficiency of delivery of a photosensitizer, and the strain - producer of modular polypeptide.

The problem is solved by obtaining the plasmid pR752, coding modular recombinant polypeptide (MCI), consisting of 6 his-tag residues (6His), hemoglobinopathies E. coli protein (NMR), a modified fragment of the large T-antigen of the virus SV-40 (NLS), a fragment of diphtheria toxin, containing a translocation domain and a private cross-domain spacer (DTox), as well as having additional spacer - (Gly-Ser)5and epidermal growth factor (human) (EGP), and the strain of E. coli M15 (Rep4, pR752) - producer of modular peptide obtained by transformation of strain M15 (Rep4) plasmid pR752.

The invention consists in creating a new to the decomposition of recombinant plasmids pR752 to encode modular polypeptide (SEQ ID No. 1) length 694, M m 76306 D, having the following structure: 6His-HMP-NLS-DTox-(Gly-Ser)5-EGF in which AA 5-10 - 6His, aa 14-409 - NMR, AA 439-624 - DTox 198-384 with substitutions, AA 628-638 (Gly-Ser)5, 642-694 EGF.

Plasmid pR752 has a size 5269 bases (SEQ ID No. 2) and contains the following regulatory region and coding sections:

artificial bacterial operon, containing the promoter region of the bacteriophage T5 and the gene encoding a multidomain protein 6His-HMP-NLS-DTox- (Gly-Ser)5-ECP,(the coding region 115-2199, NP);

- bacterial bla operon, encoding a protein of beta-lactamase, which is a selective marker for transformation of E. coli strains (the coding region 5064-4204 i.e. in a complementary chain);

bacterial plot of replication initiation type Col E1 3206-4196, for example, by providing a replication of the plasmid in E. coli strains.

To obtain the modular polypeptide serves strain E. coli - M15 (Rep4, pR752), collection number VKPM B-8356 obtained by transformation of strain M15 (Rep4) plasmid pR752.

The strain deposited at the all-Union Collection of Industrial Microorganisms (VKPM), FGUP GosNII Genetika 17.07.2002,

Significant difference between the proposed plasmids pR752 is the presence of a gene encoding a multidomain protein 6His-HMP-NLS-DTox-(Gly-Ser)5EGF.

The cultivation of recombinant strains of E. coli - M15 (Rep4, pR752) receive modular recombinant polypeptide (MCI) to deliver photosin is debilitator, different from the well-known presence in its structure of the spacer (Gly-Ser)5separating the ligand from the bulk of the overall design, thereby ensuring efficient interaction of the ligand with the receptor on the cell surface, and before module NMR sequence 6His, to simplify the process of allocating MRI.

Characteristics of the strain E. coli - M15 (Rep4, pR752)

The strain obtained by transformation with a plasmid pR752 strain M15 (Rep4)provided by the company QIAGEN.

Cultural and morphological characteristics of the strain

When growing strain on LB agar (1 l - 10 g bacteriophora, 5 g backdragging extract, 10 g NaCl, 15 g baktagir, pH 7, 5 - NaOH) for 18 hours at 37°get:

- the colonies are round in shape,

- colorless,

opaque

- the surface is smooth,

profile convex;

the edge of the colony - wavy,

the structure is homogeneous,

- the consistency of a viscous,

- ability to emulsification - uniform,

the pigment in the environment does not emit.

The product synthesized by the strain - modular recombinant 6His polypeptide-HMP-NLS-DTox-(Gly-Ser)5EGF

The activity of the strain: the level of expression of modular recombinant 6His polypeptide-HMP-NLS-Dtox-(Gly-Ser)5EGF is 32% of the total protein, the solubility of 70%.

Genetic strain: resistant to ampicillin and kanamycin, metabol the ical markers can brivati lactose, arabinose, galactose and mannitol.

A brief description of the drawings.

Figure 1 Map of plasmid pR752 indicating the restriction sites.

Figure 2 the dependence of the survival rate of cells A431 on the concentration of added chlorin e6and its conjugate with MCI, the irradiation dose of 288 kJ/m2without filters.

3 Dependence of the survival rate of cells A431 concentration bacteriochlorin p6and its conjugate with 6His-HMP-NLS-Dtox-(Gly-Ser)5EGF irradiated at a dose of 75 kJ/m2with combination filter for transmission in the field 730-800 nm.

Examples of carrying out the invention

All standard geoingegneria and microbiological manipulation, and amplification and sequencing of DNA was performed according to known methods (Maniatis T., Fritsch E., Sambrook J., Molecular cloning, M.: Mir, 1984; DNA Cloning. Methods. Ed. by D. Glover, TRANS. from English., Moscow, Mir, 1988; The QIAexpressionist. QUIAGEN Inc., 1991; The QIAexpressionist. Second edition. QUIAGEN Inc., 1992).

Example 1. The method of obtaining the plasmid pR752

a) Chemical synthesis of oligodeoxyribonucleotides

Oligodeoxyribonucleotides were synthesized by solid-phase aminophosphines method using a synthesizer AFM 100-2 (Novosibirsk) and purified by electrophoresis in 12%SDS page gel.

b) Cloning of the site, providing a photosensitizer (porphyrin)-binding activity/p>

As the photosensitizer-binding protein was taken hemoglobinopathy protein NMR E. coli. For cloning NMR were designed primers corresponding to the ends of the coding region of the gene. The product was prolongirovanne in E. coli so that before the protein was 6His for possible selection of products expression using affinity chromatography on Ni-agarose.

Using as a DNA template the chromosomal DNA of E. coli containing the gene NMR, by polymerase chain reaction using oligonucleotide primers composition (sites restricts BamHI, XbaI and BgIII underlined)

5'-gcaaaaaaagggatcccatatgcttgacgctc

5'-ccggcaactctagatctcagcaccttatgcg

the corresponding initial and the terminal part of the DNA that encodes NMR, the amplified nucleotide sequence. The reaction was carried out in 25 µl reaction mixture containing 2 µg DNA, 10 RMB each primer, 67 mm Tris-HCl buffer (pH 8.8 at 25° (C), 15 mm ammonium sulfate, 2.5 mm magnesium chloride, 0.01% tween-20, the mixture of deoxynucleotides (dATP, dCTP, dTTP, DSTF 2.5 mm) and 1 unit of Taq polymerase. Reaction amplification was performed under paraffin oil for 5 cycles: 93°C - 1 min, 45°C - 1 min, and 72°C - 2 min, then 30 cycles: 93°C - 1 min, 55°C - 1 min, and 72°C - 2 min. amplification Products were separated by electrophoresis in 1% agarose gel, stained with a solution of ethidium bromide is a DNA fragment size 1213, NP, containing the gene NMR, cut out and isolated from the gel by centrifugation through glass wool. DNA was besieged three volumes of ethanol and dissolved in bidistilled water. The resulting DNA was labeled pCRHMP and consisted of 1213 i.e. With the aim of creating a vector expressing NMR, the obtained DNA pCRHMP, as well as DNA plasmids pQE13 hydrolyzed restriction endonucleases BamHI and XbaI at 37°C in buffer containing 33 mm Tris-acetate (pH of 7.9 at 37° (C), 10 mm magnesium acetate, 66 mm potassium acetate for 1.5 hours. Food restriction were separated by electrophoresis in a gel of 1% agarose, stained with a solution of ethidium bromide and the DNA fragments corresponding gene NMR size 1199 N. and fragment of plasmid pQE13 size 2443 n / a, carved, was isolated from the gel by centrifugation through glass wool and mixed. The mixture of restriction fragments ligated using DNA ligase of phage T4 in buffer containing 40 mm Tris-HCl (pH of 7.8 at 25° (C), 10 mm MgCl2, 10 mm dithiotreitol (DTT) and 5 mm ATP. Legirovannoi DNA was used to transform cells of E. coli M15[REP4] (Nals, Strs, rifs, lac-, ara-, gal-, mtl-, F-, recA+, uvr+) by means of electroporation. Transformed cells were selected on agar LB medium with ampicillin (100 μg/ml) and kanamycin (25 μg/ml). Plasmid DNA from antibiotic-resistant colonies were isolated by the method of shelo the aqueous lysis, were analyzed using restricted BamHI and XbaI, and restricted sites within the cloned fragment, and selected clones, DNA which contain the gene NMR. This plasmid was named pR507.

C) Cloning of the site, providing a ligand-specific activity

As a specific ligand was used epidermal growth factor, epidermal growth factor (EGF). The gene encoding this peptide was isolated by PCR from a plasmid provided to us Vertabim J.V. Using as a DNA template plasmid containing the cloned gene EGF, by polymerase chain reaction using oligonucleotide primers composition (sites restricts BamHI and CRP underlined)

5'-gggggcccgggatccaattccgatagcgagtgtcctc

5'-caaggagatggatcccaacagtcctccggacacggggcc

corresponding to the beginning of the EGF gene and DNA amplificare plasmids after its end, the amplified nucleotide sequence. The reaction is carried out in 25 µl reaction mixture containing 2 µg DNA, 10 RMB each primer, 67 mm Tris-HCl buffer (pH 8.8 at 25° (C), 15 mm ammonium sulfate, 2.5 mm magnesium chloride, 0.01% tween-20, the mixture of deoxynucleotides (dATP, dCTP, dTTP, DSTF to 2.5 mm) and 1 unit of Taq polymerase. Reaction amplification was performed under paraffin oil for 30 cycles: 93°C - 1 min, 65°C - 1 min, and 72°C - 30 sec. Products amp is eficacia were separated by electrophoresis in 1% agarose gel, stained with a solution of ethidium bromide and the DNA fragment size 355 i.e. cut out and isolated from the gel by centrifugation through glass wool. DNA was besieged three volumes of ethanol and dissolved in bidistilled water. The resulting DNA is labeled as pCREGF and consists of 355 n / a

To create a vector expressing EGF obtained DNA pCREGF, as well as DNA plasmids pQE16 hydrolyzed restriction endonucleases BamHI and CRP at 37°C in buffer containing 33 mm Tris-acetate (pH of 7.9 at 37° (C), 10 mm magnesium acetate, 66 mm potassium acetate for 1.5 hours. Food restriction were separated by electrophoresis in a gel of 1% agarose, stained with a solution of ethidium bromide and the DNA fragments, the fragment containing the gene EGF size 340 n, and a fragment of a plasmid pQE16 size 3052 i.e. cut, was isolated from the gel by centrifugation through glass wool and mixed. The mixture of restriction fragments ligated using DNA ligase of phage T4 in buffer containing 40 mm Tris-HCl (pH of 7.8 at 25° (C), 10 mm MgCl2, 10 mm DTT and 5 mm ATP. Legirovannoi DNA was used to transform cells of E. coli M15[REP4] (Nals, Strsrirs, lac-, ara-, gal-, mtl-, F-, recA+, uvr+) by means of electroporation. Transformed cells were selected on agar LB medium with ampicillin (100 μg/ml) and kanamycin (25 μg/ml). Plasmid DNA from resistant to antibiotics is of alone were isolated by the method of alkaline lysis, were analyzed using restricted BamHI and CRP and restricted sites within the cloned fragment, and selected clones whose DNA contained in the composition of the EGF gene. The obtained plasmid was named pR700.

d) Cloning of the site, providing endosomolytic activity (Dtox).

As endosomolytic module was used translocation domain (T) of diphtheria toxin with adjacent natural spacer separating T and L domains of native diphtheria toxin. For cloning were designed primers corresponding to the ends of the coding region of the T-domain, but the N-end of the T-domain modified199Leu is replaced by Gly, a201Cys - Ser.

Using as a DNA template plasmid containing the cloned gene, diphtheria toxin, by polymerase chain reaction using oligonucleotide primers composition (sites restricts BamHI and CRP underlined)

5'-gtaggtggatccgggtcatccataaatcttgattgg

5'-cccgtcatccggaaatggttaagatctatgccccgg

the corresponding initial and the terminal part of the DNA that encodes the T-domain of the diphtheria toxin gene, the amplified nucleotide sequence. The reaction was carried out in 25 µl reaction mixture containing 2 µg DNA, 10 RMB each primer, 67 mm Tris-HCl buffer (pH 8.8 at 25° (C), 15 mm ammonium sulfate, 2.5 mm magnesium chloride, 0.01% tween-20, the mixture is of toxinologists (dATP, dCTP, dTTP, DSTF 2.5 mm) and 1 unit of Taq polymerase. Reaction amplification was performed under paraffin oil for 5 cycles: 93°C - 1 min 30 sec, 45°C - 2 min, and 72°C - 40 seconds, and then 30 cycles: 93°C - 1 min, 55°C - 1 min, and 72°C - 40 sec. Amplification products were separated by electrophoresis in 1% agarose gel, stained with a solution of ethidium bromide and the DNA fragment (pCRDTox) size 599 nppvariable and was isolated from the gel by centrifugation through glass wool. DNA was besieged three volumes of ethanol and dissolved in bidistilled water. To create a vector expressing the T domain of diphtheria toxin, DNA pCRDTox, as well as DNA plasmids pQE16 hydrolyzed restriction endonucleases BamHI and CRP at 37°C in buffer containing 33 mm Tris-acetate (pH of 7.9 at 37° (C), 10 mm magnesium acetate, 66 mm potassium acetate for 1.5 hours. Food restriction were separated by electrophoresis in a gel of 1% agarose, stained with a solution of ethidium bromide and the DNA fragments corresponding to the diphtheria toxin gene size 584 NP and DNA plasmids pQE16 size 3052 i.e. cut, was isolated from the gel by centrifugation through glass wool and mixed. The mixture of restriction fragments ligated using DNA ligase of phage T4 in buffer containing 40 mm Tris-HCl (pH of 7.8 at 25° (C), 10 mm MgCl2, 10 mm DTT and 5 mm ATP. Levirowan the Yu DNA was used to transform cells of E. coli M15[REP4] (Nal s, Strs, rifs, lac-, ara-, gal-, mtl-, F-, recA+, uvr+) by means of electroporation. Transformed cells were selected on agar LB medium with ampicillin (100 μg/ml) and kanamycin (25 μg/ml). Plasmid DNA from antibiotic-resistant colonies were isolated by the method of alkaline lysis was analyzed using restricted BamHI and CRP and restricted sites within the cloned fragment, and selected clones, DNA which contain the gene of the T domain of diphtheria toxin. The obtained plasmid was named pR670.

d) Cloning of the site, including the nuclear localization signal (NLS)

As a nuclear localization signal, was used to sequence large T-antigen of the virus SV-40. As the matrix used plasmid P10, kindly provided to us by Dr. D. by Jans. To standardize the NLS sequence in accordance with the concept of module primers were designed to introduce the BgIII site in the region encoding the C-end structure and producing replacement in the insignificant region Asp Glu. This modification done to remove site acid hydrolysis of Asp-Pro.

Using as a DNA template plasmid containing the cloned gene NLS, by PCR using oligonucleotide primers composition

5'-gtgagatctgggttcttctacctttctcttc

5'-gtgagatctgcgcgtaatgagctccttgcaaac

appropriate consequences the successive matrix of plasmid DNA genome before the NLS and the late gene NLS, amplificatori the nucleotide sequence. The site restrictase BgIII, located in the reverse primer is underlined. The site restrictase BamHI contained in a plasmid, which is the matrix, and direct primer for cloning facilities due to the small size of the NLS (25 amino acids or 75 BP) was removed more than 700 BP Reaction was performed in 25 µl reaction mixture containing 2 µg DNA, 10 RMB each primer, 67 mm Tris-HCl buffer (pH 8.8 at 25° (C), 15 mm ammonium sulfate, 2.5 mm magnesium chloride, 0.01% tween-20, the mixture of deoxynucleotides (dATP, dCTP, dTTP, DSTF on 2.5 mm) and 1 unit of Taq polymerase (Deep Vent). Reaction amplification was performed under paraffin oil for 30 cycles: 94°C - 1 min, 57°C - 1 min, and 72°s - 1 min amplification Products were separated by electrophoresis in 1% agarose gel, stained with a solution of ethidium bromide and the DNA fragment (pCRNLS) size 809 i.e. cut out and isolated from the gel by centrifugation through glass wool. DNA was besieged three volumes of ethanol and dissolved in bidistilled water. The resulting DNA pCRNLS ligated with plasmid pGEM4Z, hydrolyzed with the restriction enzyme HincII at 37°C in buffer containing 33 mm Tris-acetate (pH of 7.9 at 37° (C), 10 mm magnesium acetate, 66 mm potassium acetate for 1.5 hours. Ligation was performed using DNA ligase of phage T4 in buffer containing 40 mm Tris-HCl (pH of 7.8 at 25°is), 10 mm MgCl2, 10 mm DTT and 5 mm ATP. Legirovannoi DNA was used to transform cells of E. coli DH5α [F-(φ80dlacZ) recA1 endA1 gyrA96 thi-1 hsdR17 (rk-mk+) supE44 relA1 deoR Δ(lacZYA-argF) U169] the method of electroporation. Transformed cells were selected on agar LB medium with ampicillin (100 µg/ml). Were selected recombinant clones with direct orientation of the cloned fragment, confirmed by restriction mapping of the plasmid pR402dir). With the aim of creating a vector expressing NLS, DNA plasmids pR402dir and pQE16 hydrolyzed restriction ectonucleoside BamHI and Bg1I at 37°C in buffer containing 33 mm Tris-acetate (pH of 7.9 at 37° (C), 10 mm magnesium acetate, 66 mm potassium acetate for 1.5 hours. Food restriction were separated by electrophoresis in a gel of 1% agarose, stained with a solution of ethidium bromide and the DNA fragment containing the gene NLS size 1507 n, and the DNA fragment of the plasmid pQE16 size 993 i.e. cut, was isolated from the gel by centrifugation through glass wool and mixed. The mixture of restriction fragments ligated using DNA ligase of phage T4 in buffer containing 40 mm Tris-HCl (pH of 7.8 at 25° (C), 10 mm MgCl2, 10 mm DTT and 5 mm ATP. Legirovannoi DNA was used to transform cells of E. coli M15 [REP4] (Nals, Strs, rifs, lac-, ara-, gal-, mtl-, F-, recA+, uvr+) method electropo the promotion. Transformed cells were selected on agar LB medium with ampicillin (100 μg/ml) and kanamycin (25 μg/ml). Plasmid DNA from antibiotic-resistant colonies were isolated by the method of alkaline lysis was analyzed using restricted BamHI and Bg1I and restricted sites within the cloned fragment, and selected clones, DNA which contain the gene NLS. The obtained plasmid was named pR407.

e) Cloning of the spacer (Gly-Ser)5

The spacer (Gly-Ser)5designed for spatial separation of the ligand from the bulk of the overall design to ensure the interaction of the ligand with the receptor on the cell surface, which can be located in the depth of the membrane. This sequence was obtained from chemically synthesized oligonucleotides.

Sequence flanked by pulpitami of restricted BamHI and HindIII, To the end before the HindIII site provided the site restrictase Bg1II and sites restricts SmaI and EheI for ease of testing (All sites restricts underlined). Each of the chains chemically synthesized oligonucleotides

5'-gatccccgggttctggctccggctctggttccggttctggcgccagatcta

5'-agcttagatctggcgccagaaccggaaccagagccggagccagaacccggg

was fosforilirovanii separately. The reaction was carried out in 10 ál of buffer Tris-HCl pH to 9.5, containing 10 mm magnesium chloride, 50 mm DTT, 1 mm ATP, 100 pmol of the oligonucleotide, 1 epoicocladius phage T4, for 30 min at 37°C. After the reaction the samples were combined, warmed up at 90°C for 10 min and slowly cooled to 25°C. To create a vector that expresses the spacer (Gly-Ser)5DNA plasmids pQE13 hydrolyzed restriction ectonucleoside BamHI and HindIII at 37°C in buffer containing 33 mm Tris-acetate (pH of 7.9 at 37° (C), 10 mm magnesium acetate, 66 mm potassium acetate in Bg1II. Food restriction were separated by electrophoresis in a gel of 1% agarose, stained with a solution of ethidium bromide and the DNA fragment of the plasmid pQE13 size 3420 i.e. cut, was isolated from the gel by centrifugation through glass wool and combined with a mixture generowanych oligonucleotides. The resulting mixture was ligated using DNA ligase of phage T4 in buffer containing 40 mm Tris-HCl (pH of 7.8 at 25° (C), 10 mm MgCl2, 10 mm DTT and 5 mm ATP. Legirovannoi DNA was used to transform cells of E. coli M15 [REP4] (Nals, Strs, rifs, lac-, ara, gal-, mtl-, F-, recA+, uvr+) by means of electroporation. Transformed cells were selected on agar LB medium with ampicillin (100 μg/ml) and kanamycin (25 μg/ml). Plasmid DNA from antibiotic-resistant colonies were isolated by the method of alkaline lysis was analyzed using restricted BamHI and HindIII, and restricted sites within the cloned fragment, and selected clones, plasmid DNA which is gained in its composition gene peptide (Gly-Ser) 5. The obtained plasmid was named pR703

f) obtaining the plasmid pR689 (block modules NMR-NLS)

To obtain plasmids expressing MRI, containing modules HMP-NLS DNA plasmids pR407 hydrolyzed restriction endonucleases BamHI, Esoo, and DNA plasmids pR507 hydrolyzed restriction endonucleases BglII, Esoo at 37°C in buffer containing 33 mm Tris-acetate (pH of 7.9 at 37° (C), 10 mm magnesium acetate, 66 mm potassium acetate for 1 hour. Isolated from the gel of restriction fragments: a fragment of DNA plasmids pR407, including the NLS module size 2363 NP, and the DNA fragment of the plasmid RR,including gene NMR size 1358 i.e. ligated using DNA ligase of phage T4. Received legirovannoi mixture was used to transform cells of E. coli M15 [REP4] the method of electroporation. Transformed cells were selected on agar LB medium with antibiotics ampicillin (100 μg/ml) and kanamycin (25 μg/ml). Plasmid DNA was isolated by the method of alkaline lysis was analyzed using restricted BamHI, Bg1II and Esoo and restricted sites within the cloned fragment, and selected clones (pR689), plasmid DNA which contains in its composition of modules NMR-NLS.

C) Obtaining plasmids pR711v (block modules Dtox-(Gly-Ser)5)

To obtain plasmids pR711v containing block modules Dtox-(Gly-Ser)5DNA plasmids pR703 hydrolyzed in restriction and endonucleases BamHI and Pvul at 37° With buffer 66 mm Tris-acetate (pH of 7.9 at 37°C), 20 mm magnesium acetate, 132 mm potassium acetate and 0.2 mg/ml bovine serum albumin (BSA) for 1 h, and DNA plasmids pR670 hydrolyzed Bg1II restriction endonucleases and Pvul at 37°C in buffer containing 50 mm Tris-HCl (pH 7.5 at 37° (C), 10 mm magnesium chloride, 100 mm sodium chloride and 0.1 mg/ml BSA for 1 hour. Isolated from the gel of restriction fragments: a fragment of DNA plasmids pR703 that includes a module (Gly-Ser)5size 2704 N., and the DNA fragment of the plasmid pR670, including module Dtox size 1307 n, ligated using DNA ligase of phage T4. Received legirovannoi mixture was used to transform cells of E. coli M15 [REP4] the method of electroporation. Transformed cells were selected on agar LB medium with antibiotics ampicillin (100 μg/ml) and kanamycin (25 μg/ml). Plasmid DNA was isolated by the method of alkaline lysis was analyzed using restricted Pvul, BamHI and Bg1II, and restricted sites inside the cloned fragments, and selected clones pR711v (size 4011 n), plasmid DNA containing the block modules Dtox-(Gly-Ser)5.

and Getting a plasmid pR727v (block modules Dtox-(Gly-Ser)5-EGF)

To obtain plasmids expressing MRI, containing modules Dtox-(Gly-Ser)5DNA plasmids and pR700 hydrolyzed restrictions endonucleases BamHI, Bg1I, and DNA plasmids pR711v Hydra who was litovali restrictions endonucleases Bg1II and Bg1I at 37° C in buffer containing 33 mm Tris-acetate (pH of 7.9 at 37° (C), 10 mm magnesium acetate, 66 mm potassium acetate for 1 hour. Isolated from the gel of restriction fragments: a fragment of DNA plasmids pR700, including EGF gene size 2365 NP, and the DNA fragment of the plasmid pR711v, including block modules Dtox-(Gly-Ser)5size 1602 i.e. ligated using DNA ligase of phage T4. Received legirovannoi mixture was used to transform cells of E. coli M15 [REP4] the method of electroporation. Transformed cells were selected on agar LB medium with antibiotics ampicillin (100 μg/ml) and kanamycin (25 μg/ml). Plasmid DNA was isolated by the method of alkaline lysis was analyzed using restricted BamHI, Bg1I and Bg1II and restricted sites inside the cloned fragments, and selected clones (pR727v), plasmid DNA which contains in its composition of modules Dtox-(Gly-Ser)5EGF.

to Obtain plasmids pR752, coding MRI

To obtain a plasmid containing the block modules Dtox-(Gly-Ser)5EGF (pR 727v) hydrolyzed in restrictase XhoI and BamHI (in buffer containing 10 mm Tris-HCl, pH 8.5 at 37°C, 10 mm MgCl2, 100 mm KS1, 0.1 mg/ml BSA), and the plasmids containing block modules HMP-NLS (pR689) - restrictable XhoI and Bg1II (in buffer containing 50 mm Tris-HCl (pH 7.5), 10 mm MgCl2, 100 mm NaCl, 0.1 mg/ml BSA) for 1 hour at 37°C. the Products of the enzyme were separated using electrophoresis the gel of 1% agarose, stained with a solution of ethidium bromide and the DNA fragment of the plasmid pR727v, including block modules Dtox-(Gly-Ser)5EGF size 3847 n, and the fragment of the plasmid pR689, including block modules HMP-NLS size 1422 n, carved, was isolated from the gel by centrifugation through glass wool and mixed. The mixture of restriction fragments ligated using DNA ligase of phage T4 in buffer containing 40 mm Tris-HCl (pH of 7.8 at 25° (C), 10 mm MgCl2, 10 mm DTT and 5 mm ATP. Legirovannoi DNA was used to transform cells of E. coli M15 [REP4] (Nals, Strs, rifs, lac-, ara-, gal-, mtl-, F-, recA+, uvr+) by means of electroporation. Transformed cells were selected on agar LB medium with ampicillin (100 μg/ml) and kanamycin (25 μg/ml). Plasmid DNA (pR752) of antibiotic-resistant colonies were isolated by the method of alkaline lysis was analyzed using restricted XhoI, BamHI and Bg1II and restricts inside the cloned fragments, and selected clones, the DNA which contains the desired sequence. Thus was obtained a plasmid pR 752 encoding MRI 6His-NMR-NLS-Dtox-(Gly-Ser)5EGF.

The primary structure of the resulting plasmid was confirmed by sequencing dimethoxymethane Sanger.

Example 2. Getting a producer strain MCI 6His-HMP-NLS-Dtox-(Gly-Ser)5EGF and microbiological synthesis of MCI.

Cells of E. coli M15 (RP 4) (Nal s, Strs, rifs, lac-, ara-, gal-, mtl-, F-, recA+, uvr+) transformed with plasmid pR752 method of electroporation. Transformed cells were selected on agar LB medium with ampicillin (100 μg/ml) and kanamycin (25 μg/ml).

The induction of the synthesis of MRI was performed in accordance with the standard Protocol of the firm QUIAGEN. Wednesday LB with ampicillin (100 μg/ml) and kanamycin (25 µg/mg) was inoculable fresh overnight culture of cells of strain M15 [REP4]carrying a plasmid encoding MRI, and were grown at 37°With aeration, determining the optical density at 600 nm every hour until reaching a density of 0.9-1.2. The cell suspension was added field isopropylthioxanthone (IPTG) to a concentration of 0.2 mm and grown for 5 hours at 30°With aeration. The cells were collected by centrifugation for 15 min at 5000 g; the precipitate was stored at -20°C. the Level of expression of MRI 6His-HMP-NLS-Dtox-(Gly-Ser)5EGF encoded by plasmid pR752 was 32%, the solubility is about 65%.

Example 3. Clearing MCI

Cells, grown, usually in 1 liter of medium, literally in the buffer of the following composition: 10 mm HEPES-Na buffer, pH 7.5, 0.1 mm EDTA, 1 mm DTT, 3 mm phenylmethylsulfonyl, 10 μg/ml lysozyme. The number of the buffer was determined by the ratio of 10 ml buffer per 10 g of cells. After ultrasonic treatment, the suspension was centrifuged for 25 min at 17000 rpm (rotor JA-20 to centrifuge "Beckman J2-21). Soup is mutant subjected to further cleaning sorbent Blue Sepharose CL-6B (Pharmacia"). Was suirable a protein concentration gradient of NaCl From 0.2 To 1.6 M Fraction Of 0.7-1.6 M NaCl contained the desired drug is 96%purity. The output of 20 mg per 1 g of biomass. Homogeneity and molecular weight MRI was evaluated from the results of the electrophoresis system Laemmly.

Example 4. Determination of the efficiency of photodynamic conjugates with MCI FS

To assess the interaction of modular recombinant polypeptide cancer cells study of photodynamic conjugates MCI with photosensitizers (PS) cells in culture. As cancer cells were used for cell epidermoid carcinoma person line A431, as FS - chlorin e6with donovanosis peak absorption in the region of 660 nm and bacteriochlorin p6with the long-wavelength peak absorption in the area at 760 nm.

Synthesis and purification of conjugates of MCI with photosensitizers - chlorin e6and bacteriochlorin p6

To a solution of chlorin e6or bacteriochlorin p6in 5 mm Na-phosphate buffer (pH 8.0) was added to the solution of a bifunctional cross-linking agent 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (30 mg/ml) in water and a solution of N-hydroxysuccinimide (100 mg/ml) in tetrahydrofuran (ratio of porphyrin:carbodiimide: N-hydroxysuccinimide 1:30:30). The reaction mixture was carefully mixed and incubated for 3 hours at anatoy temperature. All operations with FS was performed in the dark in an atmosphere of inert gas. To obtain the conjugate MCI-FS to the protein solution in 0.1 M borate buffer (pH 9,0) solution was added activated FS in the ratio of 1:15, and incubated on ice for 19 hours with constant stirring. For purification of the conjugate from unbound FS and cross-linking agents used gel filtration (Sephadex G-50). For this purpose, 1 ml of conjugate solution, pre-preincubating for 30 minutes at a temperature of 0°With 3 M guanidine with the addition of 1 mm EDTA was applied on the column diameter 1 cm, length 10 cm Purification was performed in buffer containing 50 mm Na-phosphate, 150 mm NaCl, 3 M guanidine chloride, 1 mm EDTA, pH 8.0. Fractions containing protein were immediately cialisbuy against buffer containing 5 mm Na-phosphate, 150 mm NaCl, 1 mm EDTA, pH 8.0, last dialysis fought against buffer containing EDTA.

Conducted thus the synthesis of conjugates of 6His-HMP-NLS-Dtox-(Gly-Ser)5-EGF with FS - chlorin e6allowed to obtain the product with a molar ratio of covalently attached FS to MCI 1.5 to 1, the conjugate with bacteriochlorin p6obtained with a molar ratio of covalently attached FS to MCI as 1 to 1.

Study of photodynamic FS and conjugates MCI-FS

The A431 cells were scattered in the 48-hole die 5 thousand cells per well in DMEM medium containing 10% fetal the second serum after 24 hours the cells were added various concentrations of free FS or conjugates MCI-FCS in DMEM medium containing 2 mg/ml BSA, and incubated for 20 hours. After incubation, the cells 3 times washed free from FS or conjugates with Hanks solution, was added to the DMEM medium containing 10% fetal serum, and incubated for 2 hours. After incubation, the cells washed three times with Hanks solution, was added to the DMEM for air and irradiated the right amount of light using the overhead projector Bearing-2400 (Russia). When irradiated cells incubated with bacteriochlorin p6and the corresponding conjugate, we used a combination of filters (KS17+SS) with transmission in the range 730-800 nm. Cells incubated with chlorin e6and its conjugate with protein, were irradiated in the same way without a filter. Next, cells were incubated in DMEM medium containing 10% fetal serum for 48 hours in CO2-incubator at 37°C and 5% CO2. The cells are then washed three times with Hanks solution, was added a 0.5% solution of methylene blue in 50% ethanol and incubated at room temperature for 30 minutes. Excess dye was washed with distilled water absorbed by the cells, the dye was extracted with 0.5 mm solution of HCl in 50% ethanol and measured the optical density on a Multiscan spectrophotometer (Labsystem, Finland) at a wavelength of 650 nm.

Studied the e photodynamic action of photosensitizers and their conjugates 6His-HMP-NLS-Dtox-(Gly-Ser) 5-EGF through 22 hours after addition of the active component was shown that the inclusion of the FC part of MCI increases their efficiency by 3 orders. The results of the experiments are shown in figure 2 and figure 3. Figure 2 shows the dependence of the survival rate of cells A431 on the concentration of added chlorin e6and its conjugate with MCI, the irradiation dose of 288 kJ/m2without filters, where 1 is the conjugate 6His-HMP-NLS-Dtox-(Gly-Ser)5-EGF with FS - chlorin e62 - chlorin e6. Figure 3 presents a similar dependence for conjugate 6His-HMP-NLS-Dtox-(Gly-Ser)5-EGF with bacteriochlorin p6upon irradiation at a dose of 75 kJ/m2with combination filter for transmission in the field 730-800 nm, where 1 is the conjugate 6His-HMP-NLS-Dtox-(Gly-Ser)5-EGF with bacteriochlorin p62 - bacteriochlorin p6.

Thus, testing of conjugates of 6His-HMP-NLS-Dtox-(Gly-Ser)5-EGF with FS-chlorin e6and bacteriochlorin p6showed that covalent joining of the Federal Assembly of the proposed MCI provides increased efficiency of the Federal Assembly of the cell culture A431 3 order. This is a good prerequisite for the creation of tools that can be used in photodynamic therapy of malignant tumors.

Urelominantly plasmid pR752 expressing modular recombinant polypeptide, for delivery of a photosensitizer, which has a size 5269 grounds, characterized by nucleotide sequence SEQ ID No. 2 and contains the following regulatory region and coding sections:

bacterial artificial operon containing the promoter region of the bacteriophage T5 and the nucleotide sequence encoding a multidomain protein 6 His-HMP-NLS-DTox-(Gly-Ser)5-EGF with SEQ ID No. 1;

bacterial bla operon, encoding protein beta-lactamase that is used as a selective marker;

bacterial site of replication initiation type Col E1, provides replication of plasmids in strains of E. coli.

2. The Escherichia coli strain VKPM B-8356 - producer of modular recombinant polypeptide for delivery of the photosensitizer.



 

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