Recombinant plasmid dna encoding tul4spcbd recombinant protein synthesis, m15[prep4, ptul4spcbd] escherichia coli strain as producer of tul4spcbd recombinant protein, tul4spcbd recombinant protein, method for production thereof, and method for production of specific antibody against tul4spcbd protein

FIELD: biotechnology, gene engineering, microbiology.

SUBSTANCE: invention relates to TUL4spCBD recombinant protein comprising TUL4 Francisella tulergenis protein, Gly-Ser spacer and cellulose-binding domain of CelD endoglucanase gen from Anaerocellum thermophilus. Said protein has TUL protein antigen properties and is capable of spontaneous binding to cellulose-containing sorbents. Described is recombinant plasmid pTUL4spCBD DNA of 4388 n.p. length, encoding TUL4spCBD recombinant protein. Abovementioned plasmid contains: artificial bacterial operon of TUL4spCBD recombinant protein, including promoter region of N5 bacteriophage earlier promoter (7-87 n.p.); TUL4spCBD recombinant protein gene (115-1113 n.p.); transcription terminator (1134-1230 n.p.); beta-lactamase bacterial operon (4183-3323 n.p. of complementary chain)), providing ampicillin resistance; ColE1-type bacterial site of replication initiation, providing plasmid replication in E.coli strain (4388 n.p.). Also discoised is M15[pREP4, pTUL4spCBD] Escherichia coli strain as producer of TUL4spCBD recombinant protein and method for production of purified, concentrated and immobilized TUL4spCBD recombinant protein from said strain by treatment of cell hydrolyzate supernatant from M15[pREP4, pTUL4spCBD] Escherichia coli with cellulose-containing sorbent. Method for production of specific antibodies against TUL4spCBD protein also is described. Said method includes animal immunization with TUL4spCBD recombinant protein immobilized onto cellulose.

EFFECT: method for high yield production of TUL4spCBD recombinant protein; simplified method for purification, concentration and immobilization of said protein.

5 cl, 1 dwg, 4 ex

 

The invention relates to biotechnology, genetic engineering, Microbiology, and can be used to produce recombinant plasmid that encodes the synthesis of the immunodominant antigen TUL4, merged with Gly-Ser spacer with cellulosebased domain endonuclease gene, recombinant protein and receiving based subunit vaccines against tularemia.

Now for the prevention of tularemia in the Russian Federation use the live tularemia vaccine. Annual vaccination and revaccination against tularemia is subjected to two to three million people living in enzootic areas, as well as risk. The only producer of live tularemia vaccine is attentionally strain F.tularensis No. 15 NIIEG (or LVS in the terminology of foreign authors), obtained in 40-ies of the last century. The strain is susceptible to dissociation and to save valuable immunobiological properties requires constant monitoring and, if necessary, restore lost during storage protective activity. Received in recent years, data on the study of the genome of a strain of F. tularensis indicate the presence of the main factors of pathogenicity of the pathogen, which restricts the use of the live tularemia vaccine for a certain contingent of persons.

E is th link is especially relevant to the problem of creating vaccines of new generation, in particular the design of subunit safe vaccines based on protective antigen of F. tularensis.

It is known that the major immunodominant antigens of F. tularensis proteins are outer membrane (the main and most studied - TUL4 with a molecular mass of 17 kDa), and lipopolysaccharide.

It is shown that TUL4 protein conserved in different strains of F. tularensis (Sjostedt, A., Kuoppa, K., Johansson T., Sandstrom G. The 17 kDa lipoprotein and encoding gene of F. tularensis LVS are conserved in strains of F. tularensis. Microb. Pathog., 1992, V.13 (3), R-249), is recognized by T-cells of individuals vaccinated with F. tularensis, some T-cell determinants TUL4 protein identified (Sjostedt, A., Sandstrom G., Tarnvik, A., Jaurin Century Nucleotide sequence and T cell epitopes of a membrane protein of Francisella tularensis. J. ImmunoL, 1990, V.145 (1), P.311-317).

Gene TUL4 protein was cloned and expressed in strain Salmonella enterica, serovar Typhimurium (Sjostedt, A., Sandstrom G., Tarnvik A. Humoral and cell-mediated immunity in mice to a 17-kilodalton lipoprotein of Francisella tularensis expressed by Salmonella typhimurium. Infect. Immun., 1992, V.60 (7), R-2862), but the expression was very low and only proven method of Western blot turns. Gene TUL4 protein prolongirovanne and expressed in E. coli. In this case, was also received minor expression (less than 1% of the total protein of the cells), shown only by Western blot turns (Sjostedt, A., Sandstrom G., Tarnvik, A., Jaurin C. Molecular cloning and expression of a T-cell stimulating membrane protein of Francisella tularensis. Microb. Pathog., 1989, V.6 (6), R-414).

Well-known drug, representing the Wallpaper lipopolysaccharide-protein complex of the outer membrane Francisella tularensis (Khlebnikov V.S., Golovliov I.R., Kulevatsky D.P., Tokhtamysheva N.V., Averin S.F., Zhemchugov V.E., Pchelintsev S.Y., Afanasiev S.S., Shcherbakov G.Y. Outer membranes of a lipopolysaccharide-protein complex (LPS-17 kDa protein) as chemical tularemia vaccines. FEMS Immunol. Med. Environ., 1996, V.13 (3), R-233), which can be used to create subcellular vaccines against tularemia.

However, the lipopolysaccharide-protein complex is a complex mixture of proteins, lipopolysaccharides, fatty acids and other components of the cells of F. tularensis. The composition of the drug is difficult to standardize, which is a significant limitation in its use as a vaccine against tularemia.

Known drug OM"C"representing a set of outer membranes isolated from vaccine strain 15 F. tularensis (patent application Zhemchugova and other No. 2003102089, 2003). Immunization with this drug protects mice against subcutaneous infection with virulent strains of Francisella tularensis. The drug may serve as a basis for creating a subcellular vaccine against F. tularensis.

However, the drug OM"WITH", as well as the lipopolysaccharide-protein complex is a complex and difficult standard mixture component cells of F. tularensis. In this regard its use as a vaccine against tularemia in humans is less favorable compared with products individual proteins and/or lipopolysaccharides or mixtures thereof.

The closest in technical SunOS and the present invention is a drug representing recombinant or purified protein TUL4, encapsulated in immunostimulating complexes (ISCOMs) (Golovliov I., Ericsson, M., L. Akerblom, G. Sandstrom, Tarnvik, A., Sjostedt A. Adjuvanticity of ISCOMs incorporating a T cell-reactive lipoprotein of the facultative intracellular pathogen Francisella tularensis. Vaccine, 1995, V.13 (3), R-267). Mice vaccinated with this drug had reduced resistance to infection with F. tularensis LVS.

However, recombinant or purified protein TUL4, encapsulated in immunostimulating complexes (ISCOMs)are rather complicated and time-consuming way. In addition, the specific concentration of immunogenic TUL4 protein in the product is low, which can degrade the protective properties of this drug. The impossibility of increasing the specific TUL4 protein concentration in this preparation is determined by the toxicity of the main component ISCOMs, adjuvant Quil A.

The objective of the invention is to construct the recombinant plasmid DNA that encodes the synthesis of the recombinant protein TUL4spCBD, Escherichia coli strain M15 [pREP4, pTUL4spCBD] - producer of recombinant protein TUL4spCBD, the design of recombinant protein TUL4spCBD and a method of obtaining specific antibodies to protein TUL4spCBD, providing a high level production of recombinant protein TUL4, simplifying the process of its purification, concentration and immobilization on cellulose-containing sorbent.

usnot invention is that recombinant plasmid DNA pTUL4spCBD encodes the synthesis of the recombinant protein TUL4spCBD consisting of TUL4 protein Francisella tularensis, Gly-Ser Spencer, cellulose-binding domain gene endoglucanase CelD from Anaerocellum thermophilum, size 4388 i.e. having a nucleotide sequence No. 1 and containing:

artificial bacterial operon recombinant protein TUL4spCBD, including:

the promotor region of the early promoter of bacteriophage T5 (7-87 n),

gene recombinant protein TUL4spCBD (115-1113 n),

the terminator of transcription (1134-1230, NP);

bacterial operon beta-lactamase, providing resistance to ampicillin (4183-3323 i.e. complementary chain);

bacterial a site of initiation of replication of ColE1 type that provides replication of plasmids in strains of E. coli.

The Escherichia coli strain M15 [pREP4, pTUL4spCBD], deposited in Russian national Collection of Industrial Microorganisms (VKPM) under number B-with 8,728 from 18.05.2004, is a producer of recombinant protein TUL4spCBD.

A method of obtaining a recombinant protein TUL4spCBD comprises growing cells of Escherichia coli strain M15 [pREP4, pTUL4spCBD], obtaining a supernatant hydrolysate cells, the treatment of cellulose-containing supernatant sorbent, followed by separation of the target product.

Recombinant protein TUL4spCBD antigenic properties of the protein TUL4 and ability to self-associate is I with cellulose sorbents, consists of TUL4 protein Francisella tularensis with a sequence of amino acids No. 2, Gly-Ser spacer with the sequence of amino acids No. 3 and cellulose-binding domain gene endoglucanase CelD from Anaerocellum thermophilum with the amino acid sequence of No. 4.

In the method of obtaining specific antibodies to protein TUL4spCBD immunized animals spend immobilized on cellulose recombinant protein TUL4spCBD.

The use of the invention allows to obtain the following technical result.

The Escherichia coli strain obtained by transformation of the cells of Escherichia coli M15 [pREP4] the plasmid pTUL4spCBD, provides a high level production of recombinant protein TUL4spCBD, simplifying the process of its purification, concentration and immobilization of the protein on cellulose-containing sorbent.

The technical result is achieved by constructing recombinant protein TUL4spCBD able to spontaneously communicate with cellulose-containing sorbent.

The effect of increased synthesis of recombinant protein TUL4spCBD is achieved due to the introduction of the recombinant protein cellulose-binding domain from endoglucanase CelD from Anaerocellum thermophilum, and also due to the possibility of single-stage high-efficiency purification of recombinant protein TUL4 containing cellulose-binding domain on cellulose-containing sorbent.

The drawing shows a flow diagram of recombi is based plasmid DNA pTUL4spCBD.

The invention is illustrated by the following examples.

Example 1. Obtaining plasmids pTUL4spCBD.

a) Chemical synthesis of oligodeoxyribonucleotides.

Oligodeoxyribonucleotides were synthesized by solid-phase method was used with a synthesizer AFM 100-2 (Novosibirsk) and purified by electrophoresis in a 12%PAA gel.

b) obtaining the TUL4 gene with its subsequent cloning.

The TUL4 gene was obtained by polymerase chain reaction, which were selected primers corresponding to the beginning and end of this region of DNA. Using as a DNA template the chromosomal DNA of F. tularensis, by PCR using oligonucleotide primers composition (underlined sites restricts BamHI, Kpn2I and BglII)

1. 5'-gcgagcggatccactctagggttaggtggctctgatgatg-3'

2. 5'-atatttccggaaattaagatcttcttgaatcagaagcgattacttctttgtc-3',

the corresponding initial and the terminal part of the DNA, 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 (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-DNA-polymerase. Reaction amplification was performed under vaseline oil: 1 cycle 94°C - 5 min; 5 cycles: 94°C - 2 min, 60°C - 30 to 72°C - 2 min; 35 cycles: 94°C - 1 min, 65°S - 30 s and 72° C - 1 min; then 65°C - 5 min, and 72°C - 10 min amplification Product size 412 N.P. was treated with chloroform, perioadele ethanol and dissolved in water. For cloning of this product resulting DNA PCR TUL4, and DNA plasmids pQE16 (Quagene, USA) hydrolyzed restriction endonucleases BamHI and Kpn2I at 37°C in buffer containing 66 mm Tris-acetate (pH of 7.9 at 37°C), 20 mm magnesium acetate, 132 mm potassium acetate and 0.2 mg/ml BSA for 1.5 hours. Isolated from the gel of restriction fragments: a fragment of DNA corresponding to the gene TUL4, size 395 n, and a fragment of a plasmid pQE16 size 3028 i.e. ligated using DNA ligase of bacteriophage T4. Received legirovannoi mixture was used to transform cells of E. coli M15 [pREP4] the method of electroporation. Transformed cells were selected on agar LB medium with antibiotics ampicillin and kanamycin. Plasmid DNA was isolated by the method of alkaline lysis was analyzed using restricted BamHI and Kpn2I and restricts inside the cloned fragments, and selected clones pTUL4, plasmid DNA (size 3423 n) contains the TUL4 gene sequence:

ATGAGAGGATCCACTCTAGGGTTAGGTGGCTCTGATGATGCAAAAGCTTCA

GCTAAAGATACTGCTGCTGCTCAGACAGCTACTACTGAGCAAGCTGCTGCTG

TATCTAAGCCAACTGCAAAAGTAAGTTTAAATAAACTTGGTCAGGATAAAA

TAAAAGCAACTGTATATACAGCATACAATAATAACCCACAAGGAATGTAAG

ATTACAATGGCAGGCTCCAGAAGGTTCTAAGTGCCATGATACAAGCTTCCCA

ATTACTAAGTATGCTGAGAAGAACGATAAAACTTGGGCAACTGTAACAGTT

AAGCAAGGTAATAACTTCTGTAGCGGTAAGTGGACAGCTAATTAGTTTAT

GACAAAGAAGTAATCGCTTCTGATTCAATAAGATCTTAA

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

Amino acid sequence TUL4 (sequence No. 2)

MRGSTLGLGGSDDAKASAKDTAAAQTATTEQAAAVSKPTAKVSLNKLGQDKI

KATVYTYNNNPQGSVRLQWQAPEGSKCHDTSFPITKYAEKNDKTWATVTVKQ

GNNFCSGKWTANVVYDKEVIASDSIRS

b) obtaining a sequence that encodes a Gly-Ser spacer, with subsequent cloning.

To obtain the sequence of the spacer, consisting of replays amino acid residues Gly-Ser, were synthesized two complementary oligonucleotide of the following composition (underlined site of the restriction enzyme BglII, under the nucleotide sequence of the first oligonucleotide shows the encoded amino acid sequence (sequence No. 3):

1. 5'-gatcc ccg ggt tct ggc tcc ggc tct ggt tcc ggt tct ggc gcc aga tct a - 3'

P G S G S G S G S G S G A

2. 5'-agcttagatctggcgccagaaccggaaccagagccggagccagaacccggg-3'.

To obtain double-stranded DNA fragment was mixed for 20 RMB each of the oligonucleotide, warmed up the mixture 10 min at 95°and cooled within 4 hours to 25°C. To clone this product DNA plasmids pQE13 hydrolyzed restriction endonucleases BamHI and HindIII at 37°C in buffer containing 66 mm Tris-acetate (pH of 7.9 at 37°C), 20 mm magnesium acetate, 132 mm potassium acetate and 0.2 mg/ml BSA for 1.5 hours Selected from a gel of restriction fragment of plasmid pQE13 size 3420 n / a United educationin the DNA fragment, encoding a spacer, and ligated using DNA ligase of bacteriophage T4. Received legirovannoi mixture was used to transform cells of E. coli M15 [pREP4] the method of electroporation. Transformed cells were selected on agar LB medium with antibiotics ampicillin and kanamycin. Plasmid DNA was isolated by the method of alkaline lysis was analyzed using restricted BamHI and HindIII, and restricts inside the cloned fragments, and selected clones pRsp, plasmid DNA (size 3471 n) contains the sequence number 3 Gly-Ser spacer. The primary structure of the resulting plasmid was confirmed by sequencing according to the method of Sanger.

g) Obtaining CelD gene containing the sequence of the cellulose-binding domain (CBD), with its subsequent cloning in plasmid design pRsp.

A copy of the CelD gene was obtained by polymerase chain reaction, which were selected primers corresponding to the beginning and end of this region of DNA. Using as a DNA template chromosomal DNA Anaerocellum thermophilum, by PCR with oligonucleotide primers composition (underlined sites restricts BamHI and HindIII and BglII):

1. 5'-aaagaaggatccaatgcacctttaggcg-3' and

2. 5'-cctcaaaaaagctttaggtagatctaacattatctatatac-3',

the corresponding initial and terminal parts of the DNA, the amplified nucleotide sequence. The reaction was carried out in 25 µl of reaction the th mixture, containing 2 μg DNA, 10 RMB each primer, 67 mm Tris-HCl (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-DNA-polymerase. Reaction amplification was performed under vaseline oil: 5 cycles: 93°C - 2 min, 57°C - 5 min, and 72°C - 30 s; 30 cycles: 93°C - 1 min, 63°C - 1 min, and 72°C - 30 s; then 63°C - 5 min, and 72°C - 10 min amplification Product size 583 N.P. was treated with chloroform was perioadele ethanol and dissolved in water. For cloning of this product resulting DNA PCRCBD hydrolyzed restriction endonucleases BamHI and HindIII at 37°C in buffer containing 66 mm Tris-acetate (pH of 7.9 at 37°C), 20 mm magnesium acetate, 132 mm potassium acetate and 0.2 mg/ml BSA for 1.5 h DNA plasmids pRsp hydrolyzed restriction endonucleases BglII and HindIII. Isolated from the gel of restriction fragments: a fragment of DNA corresponding to the gene CelD size 563 N.P., and a fragment of a plasmid pRsp size 3465 gel containing a selective marker, the site of replication initiation, promoter region and the sequence encoding a Gly-Ser Spencer, was in the lead with the help of DNA ligase of phage T4. Received legirovannoi mixture was used to transform cells of E. coli M15 [pREP4] the method of electroporation. Transformed cells were selected on agarita the Anna LB medium with antibiotics ampicillin and kanamycin. Plasmid DNA was isolated by the method of alkaline lysis was analyzed using restricted BamHI and HindIII, and restricts inside the cloned fragments, and selected clones pspCBD (size 4028 n), plasmid DNA containing the sequence portion of a gene CelD with a spacer at the N-end, consisting of Gly-Ser repeats. The primary structure of the resulting plasmid was confirmed by sequencing. The nucleotide sequence of the CBD gene CelD:

AGATCCAATGCACCTTTAGGCGAAAAAGTCTTACCATCCACGTTTGAAGATG

ACACTCGTCAGGGCTGGGATTGGGATGGACCATCTGGTGTGAAAGGTCCTA

TTACTATCGAAAGTGCGAATGGTTCAAAAGCGCTATCTTTTAATGTTGAGTA

TCCAGAGAAAAAACCACAAGATGGCTGGGCAACAGCTGCAAGGCTTATACT

TAAAGACATAAATGTAGAAAGGGGAAATAATAAATATTTGGCTTTTGATTTT

TATTTGAAACCAGATAGGGCTTCAAAAGGTATGATTCAGATGTTTTTAGCTT

TTTCACCACCTTCCTTAGGTTACTGGGCTCAGGTACAAGACAGTTTTAATATT

GACCTTGGCAAAACTGTCAAGTGCAAAAAAGATAGAAGAACAGAAGTTTAT

AAGTTCAATGTATTTTTTGACTTAGACAAGATACAAGATAATAAAGTACTGA

GTCCAGACACACTCTTGAGAGATATAATAGTAGTCATAGCAGATGGCAATA

GCGATTTTAAGGGGAAAATGTATATAGATAATGTTAGATCTACCTAA

Amino acid sequence of the CBD CelD gene (sequence No. 4)

RSNAPLGEKVLPSTFEDDTRQGWDWDGPSGVKGPITIESANGSKALSFNVEYPE

KKPQDGWATAARLILKDINVERGNNKYLAFDFYLKPDRASKGMIQMFLAFSPP

SLGYWAQVQDSFNIDLGKTVKCKKDRRTEVYKFNVFFDLDKIQDNKVLSPDTL

LRDIIVVIADGNSDFKGKMYIDNVRSYRST

e) Obtaining a plasmid containing the TUL4 gene sequence, Gly-Ser spacer, and a cellulose-binding domain CBD.

To obtain plasmids containing the TUL4 gene sequence, Gly-Ser spacer, and a cellulose-binding domain CBD, plasmid pTUL4 hydrolyzed DNA restriction endonuclease BglI and BglII at 3° 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 BSA for 1 h, and plasmid pspCBD hydrolyzed restriction endonucleases BamHI and BglI 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 h were isolated from gel restriction fragments: a fragment of DNA plasmids pTUL4, including the TUL4 gene size 1377 n, and the DNA fragment of the plasmid pspCBD, including Gly-Ser spacer and the sequence of the cellulose-binding domain CBD, size 3011 n, ligated using DNA ligase of bacteriophage T4. Received legirovannoi mixture was used to transform cells of E. coli M15 [pREP4] the method of electroporation. Transformed cells were selected on agar LB medium with antibiotics ampicillin and kanamycin. Plasmid DNA was isolated by the method of alkaline lysis was analyzed using restricted BamHI, BglI and restricts inside the cloned fragments, and selected clones pTUL4spCBD (size 4388 n), plasmid DNA which contains the TUL4 gene sequence, Gly-Ser spacer, and a cellulose-binding domain CBD. The primary structure of the resulting plasmid was confirmed by sequencing.

The nucleotide sequence of the recombinant plasmid DNA pTUL4spCBD represented in sequence No. 1.

Example 2. The floor is giving strain E. coli - producer of recombinant protein TUL4spCBD consisting of TUL4, Gly-Ser spacer, cellulose-binding domain.

To obtain a strain of E. coli producer of recombinant protein TUL4spCBD, cells of E. coli strain M15 [pREP4] was transformed with plasmid pTUL4spCBD.

The resulting strain E. coli M15 [pREP4, pTUL4spCBD] is characterized by the following features.

Morphological features. Cells small, short rod-shaped, gram-negative, risperadone, 1×3.5 µm, motile.

Cultural characteristics. During growth on agar LB medium colonies are round, smooth, translucent, shiny, grey. The edge is smooth, the diameter of the colonies 1-3 mm thick, pasty consistency. Growth in liquid media (LB, minimum environment M-15 with glucose) is characterized by smooth turbidity, sediment easily sedimentary.

Physiological and biochemical characteristics. Cells grow at temperatures 4-42°C, the optimum pH of 6.8 to 7.6. As the source of nitrogen used as mineral salts and organic compounds: amino acids, peptone, tripton, yeast extract. As a carbon source during growth on minimal medium using glycerol, carbohydrates, amino acids.

Resistance to antibiotics. The cells of the producer strain are resistant to ampicillin (300 mg/ml), due to the presence of plasmids pTUL4spCBD gene β-lactamase (bla), and kanamycin (30 mg/m is), due to the presence of the plasmid pREP4 gene encoding resistance to kanamycin (kan).

The resulting strain deposited in Russian national Collection of Industrial Microorganisms under the number B-with 8,728.

Transformed cells were grown in 500 ml LB medium at 37°C to an optical density corresponding to 1 unit of absorbance at a wavelength of 600 nm., induced 150 ál of 0.1 M solution of isopropyl-beta-D-thiogalactopyranoside and grown for 8 hours

To control the production of recombinant protein TUL4spCBD the cells of E. coli M15 [pREP4, pTUL4spCBD] used the method of polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Separation of proteins was performed in 12% polyacrylamide gel in the standard system buffers (electrode buffer: 25 mm Tris-HCl, 192 mm glycine, 0.1% sodium dodecyl sulphate, pH 8.3; buffer for gel: 375 mm Tris-chloride buffer, pH 8.8). After electrophoresis the gels were stained with 0.15 percent solution of Kumasi G250 25% ISO-propanol and 10% acetic acid and washed in 10% acetic acid. When comparing the spectrum of proteins in strains of E. coli M15 [pREP4] E. coli M15 [pREP4, pTUL4spCBD] found the appearance of an additional protein band with a molecular mass of at 36.1 kDa, which corresponds to the molecular mass of the recombinant protein TUL4spCBD.

The level of protein synthesis pTUL4spCBD was determined by comparing the intensity of staining bands recombinant protein with half the Soi of the corresponding protein the molecular mass standard.

According to the data obtained, the cells of E. coli strain M15 [pREP4, pTUL4spCBD] synthesize about 75 mg immunoreactive TUL4spCBD protein per liter of cell culture at a concentration of 109cells per ml

Example 3. Method of obtaining protein drug TUL4spCBD immobilized on cellulose and mortar TUL4spCBD.

Cells of E. coli M15 [pREP4], transformed with plasmid pTUL4spCBD, were grown in 3.5 ml of LB medium with ampicillin and kanamycin at 37°C to an optical density corresponding to 1 unit of absorbance at a wavelength of 600 nm, was added 3 μl of 0.1 M solution of isopropyl-beta-D-thiogalactopyranoside and were grown for 4 h, the Culture was diluted to an optical density of 0.7 at 530 nm was selected 400 μl of the suspension were besieging the cells by centrifugation at 5000 rpm for 10 min the Cells resuspendable in 500 μl of phosphate-citrate buffer (FCB, 50 mm, pH 6.0), was literally ultrasound, cell debris was removed by centrifugation at 16000 rpm for 10 minutes

For immobilization to the supernatant was added to 2 ml granulated cellulose. Cellulose with bound peroxidase protein 5 times washed from the cell lysate in a 10-fold volume of 1% solution of Triton X-100.

To obtain the protein solution TUL4spCBD cellulose with immobilized protein was introduced into the column, filmed protein 100% formamide and were dialyzed overnight against a solution steriade is about 10 mm Tris-HCl, pH 7.0 and 150 mm NaCl.

According to the results of electrophoresis in SDS page in the presence of LTOs protein concentration was 3 mg per 1 ml of the sorbent for immobilized protein TUL4spCBD, 0.3 mg/ml protein solution TULspCBD. The purity of all products was not less than 95%.

Example 4. The method of obtaining specific antibodies to protein TUL4spCBD.

To obtain specific antibodies against the antigen TUL4spCBD immobilized on cellulose, used rabbits of the chinchilla breed weighing 2.5 to 3.0 kg, which were immunized subcutaneously on a specially designed circuit. Immunization consisted of 5 cycles, in each of which the dose of injected antigen was 0.15 mg/ml TUL4spCBD immobilized on cellulose; in the first and second cycles are additionally used complete and incomplete adjuvant Freund's, respectively. The interval between cycles of immunization was three or five weeks. The result is a hyperimmune rabbit serum containing antibodies.

The antibody specificity was determined by the method of Western blot turns. For this he received the bacterial lysates of the strains of F. tularensis 15. To obtain lysates used 18-24 hour culture of cells grown at 37°on testinside.com agar with hemoglobin (CHA+Hg, Difco, USA). Washed with saline bacterial cells (pH 7.0 to 7.2) resuspendable in a solution of 62.5 mm Tris-HCl (pH 6.8) 2% LTOs, 5% 2-Mer is aftetnoon, 10% glycerol to a concentration of 2,5x109CFU per 1 ml, and incubated in a boiling water bath for 6 minutes

The resulting lysates and purified protein TUL4spCBD subjected LTO-PAG electrophoresis in 12% gel, followed by transfer of proteins to nitrocellulose filters. Antigen detection TUL4 as antibodies used rabbit serum by protein TUL4spCBD, then spent processing the protein conjugates with horseradish peroxidase. The results of Western blot turns demonstrated the specific interaction of serum proteins, the corresponding mobility TUL4 protein in the cell lysate of strain F. tularensis 15, and recombinant protein TUL4spCBD.

1. Recombinant plasmid DNA pTUL4spCBD encoding the synthesis of the recombinant protein TUL4spCBD consisting of TUL4 protein Francisella tularensis, Gly-Ser spacer, cellulose-binding domain gene endoglucanase CelD from Anaerocellum thermophilum, size 4388 i.e. having a nucleotide sequence No. 1 and containing

bacterial artificial operon recombinant protein TUL4spCBD, including the promotor region of the early promoter of bacteriophage T5 (7-87 N.P.), gene recombinant protein TUL4spCBD (115-1113 N.P.), the terminator of transcription (1134-1230, NP);

bacterial operanet-lactamase, providing resistance to ampicillin (4183-3323 i.e. complementary chain);

bacterial site of initiation of replication of ColE1 type, providing a replication of the plasmid in E. coli strains.

2. The Escherichia coli strain M15 [pREP4, pTUL4spCBD], deposited in Russian national Collection of Industrial Microorganisms under the number B-with 8,728, the producer of recombinant protein TUL4spCBD.

3. A method of obtaining a recombinant protein TUL4spCBD comprising growing cells of Escherichia coli strain M15 [pREP4, pTUL4spCBD] according to claim 2, obtaining the supernatant hydrolysate cells, the treatment of cellulose-containing supernatant sorbent, followed by separation of the target product.

4. Recombinant protein TUL4spCBD possessing antigenic properties of the protein TUL4 and the ability to spontaneously contact with cellulose sorbents consisting of TUL4 protein Francisella tularensis with a sequence of amino acids No. 2, Gly-Ser spacer with the sequence of amino acids No. 3 and cellulose-containing domain gene endoglucanase CelD from Anaerocellum thermophilum with the amino acid sequence of No. 4.

5. The method of obtaining specific antibodies to protein TUL4spCBD, characterized in that the immunized animals spend immobilized on cellulose recombinant protein TUL4spCBD according to claim 4.



 

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FIELD: biotechnology, in particular mammalian cell cultivation and protein production from mammalian cells.

SUBSTANCE: claimed medium represents synthetic medium containing soy hydrolyzate in amount of 0.1-100 g/l, wherein at least 40 % of said hydrolyzate has molecular weight 500 D or less. Optionally medium contains buffer, antioxidant, etc. Cell cultivation in claimed medium provides both increased yield of recombinant cells and productivity thereof (increased protein yield).

EFFECT: universal medium for method selection for mammalian cell cultivation.

14 cl, 5 dwg, 7 tbl, 9 ex

FIELD: biotechnology, virology.

SUBSTANCE: invention relates to preparing a new strain of hybrid cells of Mus musculus L., NIIMB-280 (9E2), as a producer of monoclonal antibodies to the West Nile virus (WNV) protein E. West Nile virus (strain WNV/LEIV-VIg99-27889) is isolated in Volgograd district in 1999 year from a patient. Producing monoclonal antibodies can be used effectively for detection of the strain WNV/LEIV-VIg99-27880 of WNV that causes human diseases in Russia territory. New hybrid strain of cells is obtained by fusion of murine myeloma cells p3-X63/Ag8.653 (NS0/1) with murine splenocytes BALB/c immunized with the purified and inactivated preparation WNV (strain WNV/LEIV-VIg99-27889). The strain of hybrid cells Mus musculus L., NIIMB-280 (9E2), is deposited in Collection of cellular cultures of NII cellular cultures GNTS VB "VEKTOR" at number № NIIMB-280. Author's name of hybridoma cellular strain is 9E2. Using hybridoma allows preparing specific monoclonal antibodies raised to the West Nile virus protein E that, in turn, gives a possibility for identification of WNV and to standardize the content of protein E in immunodiagnostics.

EFFECT: valuable properties of strain.

1 dwg, 3 ex

FIELD: biotechnology, hybridoma technology.

SUBSTANCE: hybridoma strain is prepared by fusion of murine plasmocytoma Sp2/0-Ag.8 and B-lymphocytes of murine spleen of the inbred strain BALB/c immunized with protein-polysaccharide complex from Y. enterocolitica. Hybridoma produces monoclonal antibodies of isotype IgG to Y. enterocolitica O3 and O9 serovars used as components of IFA-test-system for identification of indicated serovars that are isolated most often in European areas from sick humans, agricultural animals and from objects of environment. The usage of monoclonal antibodies producing by hybridoma allows carrying out the identification of Y. enterocolitica strains of indicated serovars representing the most epidemic danger among other intestine-persistent microorganisms. Invention can be used in the development of diagnostic test-systems for identification of Y. enterocolitica strains O3 and O9 serovars for aims laboratory diagnosis in the public health, veterinary science and in carrying out scientific investigations.

EFFECT: valuable properties of strain.

1 tbl, 2 ex

FIELD: biotechnology, medicine, oncology, peptides.

SUBSTANCE: invention relates to a method based on phage display for preparing peptides interacting specifically with mammary Ehrlich tumor and can be used in therapy and diagnosis of malignant neoplasm. Peptides are prepared by affinity selection from phage peptide libraries comprising ten millions of different peptides of size 15 amino acid residues, the group of nine peptides wherein each peptide shows ability for accumulation in Ehrlich tumor. For practice using mimetic-peptides selected by such manner can be prepared by chemical synthesis and to use for preparing conjugates on their basis with the known cytotoxic preparations, radioactive isotopes and they can be incorporated in the composition of liposomal preparations for visualization of tumor neoplasm also.

EFFECT: valuable medicinal properties of peptides.

2 dwg, 2 ex

FIELD: biotechnology.

SUBSTANCE: heterologous protein is obtained by cultivation of strain Corynebacterium glutamicus AJ 12036, which does not produce cell surface protein and contains gene expressing construct wherein nucleic acid sequence encoding signal peptide of cell surface protein of Corynebacterium glutamicus or C. ammoniagenes is bound in direct direction to promoter sequence, and nucleic acid sequence encoding heterologous protein is bound in direct direction to abovementioned nucleic acid sequence encoding signal peptide. Further heterologous protein secreted from cells is isolated.

EFFECT: high effective method for heterologous protein production.

6 cl, 8 tbl, 10 ex

FIELD: medicine, allergology, toxins, pharmacy.

SUBSTANCE: invention relates to recombinant allergens of insect venom and to specific methods for their preparing, in particular, antigen 5 of wasp venom allergen. Recombinant antigen 5 is prepared in bacterial cells as insoluble aggregates followed by their denaturation and transfer to a soluble monomeric allergen. Transfer is carried out by dialysis using acid buffer solution (pH = 3.5-6.5) that can comprise guanidine hydrochloride, or by using a cysteine-containing solvent. Based on describes methods the practically pure recombinant antigen 5 of wasp venom allergen is isolated and used in pharmaceutical composition for hyposensibilization of body to wasp venom allergen. Invention provides preparing protein with reduced reaction capability JgE owing to it can be used in immunotherapy in treatment of allergy.

EFFECT: improved preparing method, valuable properties of allergen.

8 cl, 1 dwg

FIELD: immunology; treatment of mediated diseases IL-1 and failures.

SUBSTANCE: bonding molecule IL-1β which is antibody to human IL-1β and especially human antibody to human IL-1β where hypervariable sections CDRs of heavy and light chains have definite amino acid sequences. Antibody may be used for treatment of mediated disease IL-1, for example osteoarthritis, osteoporosis and other inflammatory processes of bones of rheumatism or podagra nature. Constructions of deoxyribonucleic acid are described which code heavy and light chains or their fragments and expressive vectors which may be replicated in cells including deoxyribonucleic acid constructions. Method of obtaining bonding molecule IL-1β by means of cell transformed by vector is described. Proposed antibody may be used both in prophylactic and treatment of diseases.

EFFECT: enhanced efficiency.

15 cl, 3 dwg, 5 ex

FIELD: medicine, immunobiology, pharmacy.

SUBSTANCE: humanized monoclonal antibody (monAb) or its fragments comprises heavy and/or light chain with the binding rate constant with AILIM 1.0 x 103 (1/M x s) and above, and the dissociation rate constant between monAb and AILIM 1.0 x 10-3 (1/s) or less. MonAb shows also a nucleotide sequence encoding variable region of light and/or heavy chain and corresponding amino acid sequences. Invention relates to DNA and it part encoding monAb or its fragments, and vectors comprising nucleotide sequences encoding antibody or its fragments. The humanized monAb can be prepared by using a genetically recombinant host. MonAb is comprised as a component of pharmaceutical compositions used for inhibition or induction of AILIM-mediated transfer of signal into cell for induction of antibody-dependent cytotoxicity against AILIM-expressing cell and others. Invention can be effective in treatment of different autoimmune diseases associated with AILIM-mediated transfer of co-stimulating signal. Invention can be used in medicine for treatment of diseases associated with AILIM-mediated transfer of co-stimulating signal.

EFFECT: valuable medicinal properties of antibody.

75 cl, 78 dwg, 14 ex

FIELD: molecular biology, biochemistry, microbiology, medicine.

SUBSTANCE: method involves the simultaneous amplification of DNA and RNA targets and E. coli plasmid fragment or E. coli 16S-RNA as universal standard. For amplification two pairs of primers with the identical annealing temperature are used being primers of the first pair are specific to the target nucleotide sequence and primers of the second pairs of primers - to nucleotide sequence of the internal standard. A number of target copies are determined by data of electrophoretic separation of target amplicons and the universal internal standard. Using the invention allows carrying out the determination of amount of copies of DNA and RNA targets.

EFFECT: improved assay method.

4 dwg, 1 tbl

FIELD: biotechnology, biochemistry, genetic engineering.

SUBSTANCE: invention proposes a method for construction of genetically modified strains of microorganisms able to destroy steroids. These strains comprise multiple inactivated genes, for example, genes encoding enzymes steroid dehydrogenases implicated in destroying the steroid ring. The gene kstD1 is an example of such genes. Strains comprising the multiple amount of inactivated genes encoding enzymes destroying steroids provides the enhanced effectiveness with respect to accumulation of intermediate steroid compounds. The preferable product of steroid accumulation if 9α-hydroxy-4-androstene-3,17-dione.

EFFECT: improved method for construction of strain.

8 cl, 5 dwg, 7 ex

FIELD: molecular biology, criminology, genetic medicinal trials.

SUBSTANCE: the present innovation deals with new markers , the method for their obtaining and applying to identify one's sex in DNA-containing human biological samples. The innovation suggested enables to detect chromosomal abnormalities by sex more accurately and at high sensitivity.

EFFECT: higher accuracy and efficiency of identification.

4 cl, 3 dwg, 3 ex, 2 tbl

FIELD: biotechnology, biochemistry, amino acids.

SUBSTANCE: invention describes a polynucleotide showing activity of glucose-6-phosphate isomerase and comprising polynucleotide sequence taken among the group including: a) polynucleotide encoding polypeptide that comprises amino acid sequence identical at least by 90% with amino acid sequence represented in SEQ ID NO:2; b) polynucleotide that is complementary with polynucleotides given in sub-paragraph a). Also, invention describes a method for enhancing the metabolism intensity in pentose phosphate cycle by attenuation of pgi gene and a method for preparing L-amino acids. Invention provides preparing L-amino acids with the high effectiveness degree.

EFFECT: improved preparing method, valuable properties of polynucleotide.

16 cl, 7 dwg, 3 tbl, 6 ex

FIELD: biotechnology.

SUBSTANCE: invention relates to polynucleotide encoding zwal gene product containing polynucleotide sequence selected from group including a) polynucleotide encoding polupeptide with amino acid sequence with at least 90 % identity to amino acid sequence represented in SEQ ID NO:2; b) polynucleotide which is complementary to polynucleotides from a), as well as primer representing polynucleotide containing at least 15 sequential base pairs of abovementioned polynucleotide.

EFFECT: new zwal gene encoding ionic zwal product.

6 cl, 1 dwg, 1 tbl, 5 ex

FIELD: gene engineering, in particular purification and isolation of polynucleotides.

SUBSTANCE: invention relates to purification and isolation of polynucleotides regulating mammalian gene transcription and is useful in regulation of heterologous polynucleotide expression, obtaining transgene animals, and identification of affined regulatory DNA sequences. DNA containing transcriptional regulatory DNA of hamster gene EF-1α was isolated by screening of genome library to Chinese hamster ovary (CHO-K1). Chimeric polynucleotide including isolated regulatory DNA of hamster gene EF-1α operably bonded to gene sequence encoding target protein product other than protein encoded by hamster gene EF-1α was constructed. Obtained chimeric polynucleotide is used as component of expression plasmid for transformation or transfection of host cell. To increase target gene transcription in host cell DNA containing regulatory DNA of hamster gene EF-1α was integrated into host cell genome DNA in site operably bonded to target gene. Method of present invention make it possible to increase mRNA expression level for operably bonded heterologous polynucleotides by 3-11 times.

EFFECT: increased mRNA expression of operably bonded heterologous polynucleotides.

31 cl, 3 tbl, 7 ex

FIELD: biochemistry.

SUBSTANCE: the present innovation deals with an anti-sense oligonucleotide or one of its derivatives which can inhibit expression of human eg5 protein being relative to kinesin of motor proteins. The oligonucleotide has got a sequence being correspondent to that of nucleic acid coding certain part of human eg5. This innovation deals with the way to obtain the above-mentioned oligonucleotides, pharmaceutical composition for inhibiting human eg5 and its application. Advantage of the innovation deals with developing e new preparation to be applied for inhibiting cell proliferation.

EFFECT: higher efficiency of inhibition.

11 cl, 1 dwg, 2 ex, 3 tbl

FIELD: biotechnology, molecular biology, biochemistry.

SUBSTANCE: invention relates to regulatory sequences. Method involves isolation of DNA molecule with nucleotide sequence SEQIDNO:2 or SEQIDNO:3 that is necessary for expression of the required encoding sequence. Then vector comprising any of indicated sequences and the required sequence is constructed followed by transformation a plant with the prepared vector. Invention provides preparing transgenic plants with regulating expression of the required gene.

EFFECT: improved preparing method.

19 cl, 1 tbl, 6 ex

The invention relates to molecular biology and genetic engineering, specifically to the creation of synthetic polyepitope vaccines against HIV-1

FIELD: biotechnology, biochemistry, amino acids.

SUBSTANCE: invention relates to a method for producing L-amino acids, for example, L-isoleucine and L-leucine prepared by culturing a microorganisms in medium and able to produce L-amino acid. This microorganism carries the avtA gene encoding enzyme alanine-valine transaminase (transaminase C) and possesses the enhanced activity of this enzyme as compared with it's the parent strain. Accumulated L-isoleucine and L-leucine are isolated from the cultural fluid. Invention provides preparing L-isoleucine and L-leucine with high effectiveness degree by diminishing the amount of amino acid as a by-side product that results to complications in the process of purification of indicated L-amino acids.

EFFECT: improved producing method of amino acids.

8 cl, 1 dwg, 2 tbl, 3 ex

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