A method of obtaining a recombinant human interferon alfa-2b, recombinant plasmid and strain producer for its implementation

 

The invention relates to genetic engineering, biotechnology, medicine, pharmacology. New recombinant multicamera plasmid DNA pSX50 encoding the synthesis of leukocyte alpha-2b interferon man, the expression of which is under the control of lactose and tryptophan promoter and terminator of transcription. In the transformation of cells of the recipient strain E. coli BL21 recombinant plasmid DNA pSX50 the resulting strain E. coli SX50 - producer of recombinant leukocyte alpha-2b interferon man with a productivity of up to 0.9-1.0 g of alpha-2b interferon with 1 l of culture medium. A method of obtaining a recombinant Alfa-2b interferon based on the use of the recombinant strain E. coli SX50 and providing for his deep cultivation on a nutrient medium with reduced levels of tryptophan with continuous addition of nutrient substrates in the process of biosynthesis, mechanical destruction of cells of the microorganism at high pressure, dissolution of aggregated protein in the concentrated solution of guanidine hydrochloride followed by denaturaciei interferon in physiological buffer solutions in the presence of chaotropic agents and clean, using the ion Cu+2, ion-exchange chromatography ion-exchange resins of the type CM Sephsrose Fast Flow and gel filtration chromatography on resin type Superdex 75. The method allows to obtain the substance interferon more than 99% purity according to electrophoresis in reducing and nereguliruemyi the conditions for staining gels with silver and more than 98% according to HPLC RF and does not contain pyrogens (LAL-test) in amounts not less than 400-800 mg from 1 l of culture medium. 3 BC and 3 C.p f-crystals, 6 ill.

The invention relates to genetically engineered drugs, obtained by means of biotechnology, and in particular to methods of industrial production of recombinant leukocyte interferon Alfa-2b human medical devices (hereinafter interferon), and rekombinantnymi strains producers of Escherichia coli (E. coli) and plasmid DNA coding for the synthesis of interferon.

Interferons are protein molecules with a molecular weight of from 15,000 to 21,000 daltons produced and secreted by cells in response to viral infection or other pathogens. There are three main groups of interferons: alpha, beta and gamma. By themselves, these groups are not homogeneous and may contain several different molecular species EES and are widely used in medicine as an antiviral, antiproliferative and immunomodulating drugs.

Known methods for producing leukocyte interferon man from leukocytes of donor human blood, induced by viruses and other inductors (SU1713591, EN 2066188, EN 2080873).

The main disadvantage of these methods for producing interferons are the likelihood of contamination of the final product by human viruses such as the virus of hepatitis b and C, human immunodeficiency virus and other

Currently more promising recognized as the method of producing interferon microbiological synthesis, which provides the ability to obtain the target product with a significantly higher yield from a relatively inexpensive starting material. The approaches allow to create optimal conditions for bacterial expression of variants of the structural gene and regulatory elements that control its expression.

As the source of microorganisms use different designs strains of Pichia pastoris, Pseudomonas putida and Escherichia coli.

The disadvantage of using P. pastoris as a producer of interferon (J. N. Garcia, J. A. Aguiar et. al. //High level expression of human IFN-2b in Pichia pastoris.//Biotecnologia Aplicada, 12(3),152-155, 1995), is extremely difficult conditions of fermentation of this type of yeast, Matcom use of strains of Ps. putida (SU1364343, SU1640996, SU1591484, RU1616143, RU2142508) is the complexity of the fermentation process with a low level of expression (10 mg interferon for 1 l culture medium). More productive is the use of strains of Escherichia coli (Semin. Oncol.,1997, Iun; 24 (3 Suppl. 9):S9-41-S9-51).

There are a large number of plasmids and created on the basis of strains of E. coli expressing interferon: the strains E. coli ATCC 31633 and 31644 with plasmids Z-pBR322 (>PST) HclF-11-206 or Z-pBR 322(Pstl)/HclN SN 35-AHL6 (SU 1764515), a strain of E. coli pINF-AP2 (SU 1312961), a strain of E. coli pINF-F-Pa (AU 1312962), a strain of E. Coli SG 20050 with plasmid p280/21FN (Kravchenko Century. Century. and other Bioorganic chemistry, 1987, T. 13, No. 9, S. 1186-1193), a strain of E. Coli SG 20050 with plasmid pINF14 (SU 1703691), a strain of E. coli SG 20050 with plasmid pINF16 (EN 2054041) and other Lack of technology based on the use of these strains is their instability, and lack of expression of interferon.

Along with specific strains of the efficiency of the process depends largely on the technology used in the isolation and purification of interferon.

A known method of producing interferon, comprising culturing cells of Ps. putida, the destruction of biomass processing polyethylenimine, fractionation seroquel the s and diafiltration, ion-exchange chromatography on cellulose DE-52, elution in a gradient of pH, ion-exchange chromatography eluent obtained on cellulose CM-52, concentration passing through the cartridge filters and gel-filtration on Sephadex G-100 (SU 1640996). The disadvantage of this method in addition to complex multi-stage fermentation is a multi-stage in obtaining the final product.

There is also known a method of producing interferon, comprising the cultivation of a strain of E. coli SG 20050/pIF16, in LB-broth in flasks in a temperature-controlled shaker, centrifuged biomass, the washing buffer solution and treated with ultrasound for cell disruption. The resulting lysate centrifuged, washed with 3M solution of urea in a buffer, dissolved in a solution of guanidine chloride in the buffer, treated with ultrasound, centrifuged, conduct oxidative sulfites, dialyzed against 8 M urea, renaturation and final two-step chromatography on CM-52 cellulose and Sephadex G-50 (EN 2054041). The disadvantages of this method is its relatively low performance of the main stages of the process of isolation and purification. This applies in particular to ultrasonic processing product, dialysis and oxidative sulfatase,umyshlennogo production of interferon.

As the closest analogue (prototype) can be mentioned a method of obtaining a leukocyte interferon of man, which lies in the cultivation of recombinant strains of E. coli, freezing the obtained biomass at a temperature not higher than -70°C, defrost, the destruction of the cells of the microorganism with lysozyme, the destruction of DNA and RNA introduction to the lysate dnaase and clears the currently selected insoluble forms of interferon washing buffer solution with a detergent, dissolving the precipitate of interferon in a solution of guanidine hydrochloride, renaturation and one-step purification ion exchange chromatography. As a producer uses a strain of E. coli SS5 obtained using recombinant plasmids pSS5 containing three promoter: PlacPt7and Ptrpand gene alpha-interferon with the introduced nucleotide substitutions.

Expression of interferon by E. coli strain SS5 containing this plasmid, is controlled by three promoters: PlacPt7and Ptrp. The expression level of interferon is about 800 mg per 1 l of cell suspension (EN 2165455).

The disadvantage of this method is low manufacturability using enzymatic cell disruption, DNA and RNA of the microorganism and the one chromatographica is of quality, and limiting the use of this scheme for the industrial production of interferon. The disadvantages of this plasmid and strain on its basis are the use of plasmid strong unregulated promoter of T7 phage in E. coli strain BL21 (DE3), in which the gene for T7 RNA polymerase is under the promoter of the lac operon and which is always "flows". Therefore, the cell continuously, the synthesis of interferon, which leads to dissociation of plasmid and decreased viability of cells of strain, resulting in the reduction of the yield of interferon.

The objective of the invention is to construct recombinant industrial producer strain E. coli using the new recombinant plasmid DNA with a high level of biosynthesis of interferon, and the development of efficient industrial technologies for substance interferon medical devices, corresponding to as "European Pharmacopoeia" for substance interferon Alfa-2b.

This problem was solved by creating a recombinant plasmid DNA pSX50 and Escherichia coli SX50 deposited in Russian national collection of industrial strains SSRI genetics, the number VKPM B-8550,

as well as a method of obtaining a recombinant Alfa-2b interferon, based on the use of recombinant strains of E. coli SX50 and providing for his globintel substrates in the process of biosynthesis, mechanical destruction of cells of the microorganism at high pressure, dissolution of aggregated protein in the concentrated solution of guanidine hydrochloride followed by denaturaciei interferon in physiological buffer solutions in the presence of chaotropic agents and three-stage chromatographic purification of interferon on the resin type Chelating Sepharose Fast Flow, immobilized ions si+2, ion-exchange chromatography ion-exchange resins of the type CM Sepharose Fast Flow and gel filtration chromatography on resin type Superdex 75.

According to the invention proposes a new recombinant multicamera plasmid DNA pSX50 encoding the synthesis of leukocyte alpha-2b interferon man, the expression of which is under the control of lactose and tryptophan promoter and terminator of transcription. Plasmid pSX50 has 3218 base pairs (p. O.), and is characterized by the presence of the following fragments:

Sequence 1 nucleotide 176 nucleotide (n) includes the DNA fragment size 176 p. O., containing the tryptophan promoter (Ptrp);

Sequence with 177 N. in 194 BC, includes a synthetic DNA fragment size 18 p. O., containing the sequence Shine Delgarno responsible for initiating etelnost interferon gene with the following nucleotide substitutions: 37 replacement And With, in position 39 replacing G by T at position 40 replacement And, in position 42 replacing G by T at position 67 replacement And, in position 69 replacing G by T at position 70 replacement And, in position 72 replacing And T, at position 96 replacing G with a at position 100 to replace And, in the position 102 replacement And T, at position 114 replacement And, in the position 120 replaced With G, in position 126 replacing G with a at position 129 replacing G with a at position 330 replaced With G, in position 339 replacing G with a at position 342 replacing G with a at position 487 replacement And, in position 489 replacement And T, at position 495 replacement of G on A;

Sequence with 696 N. in 713 ad includes a synthetic DNA fragment size 18 p. O. containing synthetic polylinker;

Sequence with 714 N. in 1138 ad includes the DNA fragment of the plasmid RCC-3 4129 N. on 4553 N. size 425 p. O., containing a sequence of strict transcription terminator rrnBT1T2;

Sequence with 1139 N. in 1229 ad includes the DNA fragment of the plasmid pUC19 with 2487 N. on 2577 N. size 91 p. O., containing the promoter of the gene-lactamase (resistance gene ampicillin AMRR);

Sequence with 1230 N. by 2045 N. includes DNA fragment from plasmid pUC4K with 720 N. in 1535 ad size 816 p. O., sod is 19 1625 453 N. size 1173 p. N., containing a sequence that is responsible for replication of the plasmid (ori) and the lac promoter (Plac).

In Fig.1-5 depict schematic design and physical map of the plasmid S50.

In Fig.6 presents installed for plasmids pSX50 complete nucleotide sequence.

The strain Escherichia coli SX50 obtained by transformation of Escherichia coli cells BL21 a plasmid pSX50 using conventional genetic engineering techniques. The strain of E. Coli SX50 has the following features.

Cultural morphological traits

Cells small, straight, thickened rod-shaped, gram-negative, risperadone. Cells grow well on simple nutrient media. During growth on agar "Difco" formed round, smooth, convex, dull, shiny, grey colonies with smooth edges. With the growth in liquid medium (minimal medium with glucose or in LB-broth) intensive form a smooth suspension.

Physical-biological characteristics

Aerobe. Temperature range for growth 4-42°C at the optimum pH of 6.5-7.5.

As the source of nitrogen used as a mineral salt in ammonium and nitrate forms, and organic compounds in the form of amino acids, peptone, tryptone, yeast extract and so on

The strain Escherichia coli H producing interferon.

The method, conditions and medium composition for the storage of strain

In L-arape with the addition of kanamycin to a concentration of 20 µg/ml under oil, in L-broth containing 15% glycerol and appropriate antibiotics in the ampoule at a temperature of minus 70°C in dried condition in the ampoule at a temperature of 4°C.

The strain Escherichia coli SX50 identified by the Identifier of Berga (1974) as the type strain of Escherichia coli.

A method for industrial preparation of alpha-2b interferon

Feature of the proposed method is to develop technologies that allow the separation of interferon from insoluble forms that accumulate during fermentation, which allows to significantly simplify the technological scheme of the process of selection and to increase the yield of the target product.

The method consists in the cultivation of Escherichia coli S50 in a nutrient medium, with the constant addition of nutrient substrates, preferably glucose and yeast extract in the process of biosynthesis, preferably with low content of tryptophan, mechanical destruction of cells of the microorganism at high pressure 700-900 bar, the dissolution of interferon in the buffer solution of guanidine hydrochloride, renaturation of different chromatographic purification of interferon on the resin type Chelating Sepharose Fast Flow, immobilized ions si+2, ion-exchange chromatography ion-exchange resins of the type CM Sepharose Fast Flow and gel filtration chromatography on resin type Superdex 75.

The optimal conditions of the individual stages receiving interferon are the following:

the fermentation was carried out under continuous addition of substrate throughout the process, which leads to high level expression of interferon;

- destruction of cells is carried out in the cage mill type Gaulin at a pressure of 900 bar;

- removal of soluble cellular components (DNA, RNA, proteins, lipopolysaccharides, and so on) produced by washing the insoluble forms of interferon buffer solutions containing detergents (Triton XI00, urea and so on);

- the precipitate containing interferon, dissolved in a buffer solution of 6 M guanidine hydrochloride;

- renaturation interferon is carried out in a physiological buffer solution containing chaotrope agents;

- three-stage chromatographic purification of interferon spend on a Chelating Sepharose Fast Flow, immobilized ions si+2on cation exchange resin CM Sepharose Fast Flow and gel filtration chromatography on resin type Superdex 75;

- after each chromatographic is interferon as a result of application of the described method is approximately 400-800 mg of interferon with 1 l of culture medium. The quality of the product meets the standards and requirements of the "European Pharmacopoeia" for substance alpha-2b interferon.

Significant differences of the proposed method from the prototype are:

- type strain, with higher performance, allowing you to get in the biosynthesis of more interferon with 1 l of culture medium;

- the use of effective mechanical destruction of the cell biomass, which allows to obtain a more pure extract insoluble forms of interferon in a shorter time, with fewer losses;

- using physiological buffer solutions at renaturation in the presence of chaotropic agents can improve the output correctly denaturirovannah forms of interferon;

- three-stage chromatographic purification of interferon allows you to get substance interferon more than 99% purity according to electrophoresis in reducing and nereguliruemyi the conditions for staining gels with silver and more than 98% according to HPLC RF and practically free of pyrogens (LAL-test).

The nature and advantages of the claimed group of inventions is illustrated by the following examples.

Example 1. Construction of recombinant plasmids S50

0;

1. construction of plasmids pSX3 (2641 p. O.)

2. construction of vector plasmids pSX10 (2553 p. O.)

construction of recombinant plasmids pSX41 (3218 p. O.);

construction of recombinant plasmids pSX43 (3218 p. O.);

construction of recombinant plasmids pSX45 (3218 p. O.);

construction of recombinant plasmids pSX50 (3218 p. O.).

Construction of vector plasmids pSX10

Vector plasmid pSX10 is a vector pUC19 in which the coding sequence of the gene beta lactamase, providing resistance to ampicillin, is replaced by the coding sequence of the gene cap and contains a transcription terminator from plasmid RCC-3.

Construction of vector plasmids pSS10 is carried out in two stages:

- obtaining plasmids pSX3 (2641 p. O.), representing the plasmid pUC19 in which the coding region AMR gene is replaced with the coding region of the gene kan;

- getting veternas plasmids pSX10 (2553 p. O.), which is a plasmid pSX3, in which the BamHI site inserted DNA fragment, encoding a transcription terminator ghwt1T2.

To obtain plasmids pSX3 spend five rounds of DNA amplification by PCR (polymerase chain reaction). During the first round, using DNA plasmas is':

This and subsequent PCR reaction is carried out in the following conditions: 20 mm Tis-HCl, pH 8.8, 10 mM (NH4)2SO4,10 mm KCl, 2 TM MgCl2, 0.1% Triton X100,0.1 mg/ml BSA, 0.2 mM of each dNTP, 1.25 units of Pfu DNA polymerase, 100 ng of DNA. The amplification process comprises the following steps: heating at 95°C 5 min, 35 cycles of PCR (30 sec 95°C, 30 sec 56°C, 2 min 72°C) and incubation of 10 min at 72°C. After amplification (and after further amplification) DNA fragment purified by electrophoresis in 1% agarose gel. In the second and third rounds, using DNA plasmid pUC4K as a matrix, perform the amplification of DNA fragment size 555 p. O. (fragment KM1-KM2) using the primers:

and amplification of DNA fragment size 258 p. O. (CMH-KM4) with primers

Further fragments (KM1-KM2) and (KM3 KM4) unite in the fourth round of amplification, using primers KM1 and KM4, get the DNA fragment size 813 p. O. (KM1-KM4), encoding the structural part of the gene kan.

In the fifth round of PCR performed combining fragments (PU1-PU2) and (KM1-KM4) under the following conditions: heating at 95°5 min, 5 cycles of PCR (30 sec 95°C, 30 sec 56°C, 10 min 72°C) is plated on Wednesday, LA, containing 20 μg/ml kanamycin. After incubation for 12 h at 37°With the clones Tsevaot, isolated plasmid DNA and carry out restriction analysis. The result is the plasmid S3 size 2641 p. O.

To obtain the vector plasmid pSX10 conduct three rounds of DNA amplification by PCR. During the first round, using DNA plasmids pSX3 as a matrix, perform the amplification of DNA fragment size 2025 p. O. (fragment 10.1-10.2) using the primers:

During the second round, using DNA plasmids RCC-3 as a matrix, perform the amplification of DNA fragment size 528 p. O. (fragment KK1-KK2) using the primers:

In the third round of PCR performed combining fragments (10.1-10.2) and (KK1-KK2) under the following conditions: heating at 95°5 min, 5 cycles of PCR (30 sec 95°C, 30 sec 56°C, 10 min 72°C) and incubation of 10 min at 72°C. the DNA obtained after the last PCR, directly transform cells of E. coli strain DH5 and plated on Wednesday, LA, containing 20 μg/ml kanamycin. After incubation for 12 h at 37°With the clones Tsevaot, isolated plasmid DNA and carry out restriction analysis. In achiev ntna plasmid pSX41 is a Hind III - BamHI DNA fragment from the vector plasmid pSX3 (2529 p. O.), Hind III - EcoRI DNA fragment size 168 p. O. encoding the promoter of tryptophan operon of E. coli (Ptrp), EcoRI-XbaI synthetic DNA fragment size 20 p. O. encoding SD sequence (Shine-Dalgarno) and XbaI-BamHI DNA fragment size 501 p. O., gene encoding alpha 2b interferon person.

To obtain the Hind III - Wamn the DNA fragment of the vector plasmid pSX3 (2529 p. O.) DNA plasmids pSX3 treated with restriction enzymes HindIII and BamHI, followed by electrophoretic cleaned in 1% agarose gel. Hind III EcoRI DNA fragment size 168 p. O. encoding the promoter of tryptophan operon (Ptrp) get by PCR using total DNA of E. coli as the template and primers TRP1 and PRP2 with the subsequent processing of the amplified fragment restrictase Hindlll and EcoRI:

To obtain the EcoRI-Xbal synthetic DNA fragment size 20 p. O. encoding SD sequence (Shine-Dalgarno), synthesized following complementary oligonucleotides:

XbaI-Am DNA fragment size 501 p. O., gene encoding alpha 2b interferon man, produced by PCR using total DNA as mA

Next electrophoretic purified fragments unite, are ligated enzyme ligase of phage T4 DNA transformed into cells of E. coli strain DH5 and plated on Wednesday LA containing 20 μg/ml kanamycin. After incubation for 12 h at 37°With the clones Tsevaot, isolated plasmid DNA, perform restriction analysis and determine the primary structure of DNA. The result is the plasmid pSX41 size 3218 p. O. Next, perform a stepwise mutagenesis of a gene of interferon to increase the expression level of the target product. Mutagenesis of the gene of interferon is to replace rare in E. coli triplets encoding the corresponding amino acids in common in E. coli triplets that encode the same amino acids. Mutagenesis of the DNA of the gene of interferon carried out by PCR method.

Construction of recombinant plasmids pSX43

To obtain recombinant plasmids pSX43 conduct one round of DNA amplification by PCR using DNA of plasmid pSX41 as template and the primers and IFN3 IFN4:

PCR is carried out in the following conditions: heating at 95°5 min, 20 cycles of PCR (30 sec 95°C, 30 sec 56°C, 10 min 72°C) and incubation of 20 min at 72°C. the DNA obtained after PCR, the right transactie 12 h at 37°With the clones Tsevaot, allocate plasmid DNA, perform restriction analysis and determine the primary structure of DNA. The result is the plasmid S43 size 3218 p. O.

Construction of recombinant plasmids pSX45

To obtain recombinant plasmids pSX45 conduct one round of DNA amplification by PCR using DNA of plasmid pSX43 as template and the primers IFN5 and IFN6:

PCR is carried out in the following conditions: heating at 95°5 min, 20 cycles of PCR (30 sec 95°C, 30 sec 56°C, 10 min 72°C) and incubation of 20 min at 72°C. the DNA obtained after PCR, directly transform cells of E. coli strain DH5 and plated on Wednesday LA containing 20 μg/ml kanamycin. After incubation for 12 h at 37°With the clones Tsevaot, isolated plasmid DNA, perform restriction analysis and determine the primary structure of DNA. The result is the plasmid pSX45 size 3218 p. O.

Construction of recombinant plasmids pSX50.

To obtain recombinant plasmids pSX50 conduct one round of DNA amplification by PCR using DNA of plasmid pSX45 as template and the primers IFN7 and IFN8:

PCR is carried out in the following conditions: heating at 95°5 min, 20 qi will form in the cells of the strain E. coli DH5 and plated on Wednesday, LA, containing 20 μg/ml kanamycin. After incubation for 12 h at 37°With the clones Tsevaot, isolated plasmid DNA, perform restriction analysis and determine the primary structure of DNA. The result is the plasmid pSX50 size 3218 p. O.

Example 2. Obtaining strain E. coli SX50 - producer of interferon

The producer strain of E. coli interferon SX50 is obtained by transformation of cells of the strain E. coli BL21 recombinant plasmid S50. Strain producer of interferon grown in a 30 l fermenter until the optical density 25.0-30.0 O. E. in the M9 medium containing 1% acid hydrolyzed casein (Difco), 1% glucose, 40 μg/ml kanamycin, at a temperature of 38-39°C. In the fermentation process carried out continuous addition of nutrient substrate using gravimetrically controller.

The content of interferon in the biomass of cells obtained from 1 l of culture, is based on a series of 0.9-1.0 g interferon.

Example 3. The method of selection of interferon from strain E. coli SX50

Receiving interferon was performed in 4 stages:

stage 1. Cultivation of a strain of E. coli SX50.

stage 2. Isolation and purification of insoluble forms of interferon.

stage 3. Dissolution and resaturate interferon.

stage 4. Chromatographic purification of interfrontal LB medium for 12 h at 26°With filled contribute to the fermenter, containing 27 l of sterile medium containing M9, 1% acid hydrolysate of casein, 1% glucose, 1 mm MgCl2, 0.1 mM CaCl2, 40 mg/ml kanamycin. Culturing in the fermenter is carried out at a temperature of 38-39°C, maintaining a pH of 7±0,15 by automatically Podarok 40% sodium hydroxide solution. The concentration of dissolved oxygen in the range of (50±10)% of the saturation support by changing the rpm of the stirrer from 100 to 800 rpm and air flow from 1 to 15 l/min the Concentration of substrates, in particular glucose and yeast extract, measured during fermentation and maintain their concentration by changing the feed rate of the concentrated solutions, via peristaltic pumps using gravimetric controller.

The accumulation of interferon in the form of insoluble forms control using phase-contrast microscopy, by electrophoresis in 15% polyacrylamide gel (SDS-PAAG) and method obetovannoi high-performance chromatography (HPLC RF). The fermentation is stopped after reaching the maximum optical density (~ 25-30 O. E.) and stop the synthesis of interferon. At the end of the fermentation culture liquid sephirot by centrifugation in running the rotor at speeds>p>stage 2. Isolation and purification of insoluble forms of interferon

300-400 grams of frozen biomass of the strain E. coli SX50 suspicious in 3000 ml of buffer 1 (20 mm Tris-HCl, pH 8.0, 10 mm EDTA, 0,1% Triton X100). The suspension is passed through a flow homogenizer type Gaulin support pressure 900 bar and centrifuged in running the rotor at 15 000 R/min the precipitate is washed under similar conditions sequentially with buffer 2 (20 mm Tris-HCl, pH 8.0, 1 mm EDTA, 3 M urea) and buffer 3 (20 mm Tris-HCl pH 8.0, 1 mm EDTA) and finally the precipitated interferon suspicious in 200 ml of buffer 3. At the same time, the isolation and purification of insoluble forms of interferon is not more than 5 hours.

stage 3. Dissolution and resaturate interferon

To the obtained in the previous step suspensions of insoluble forms of interferon add dry guanidine hydrochloride to a concentration of 6 M, add dithiothreitol to a concentration of 50 mm Tris-HCl pH 8.0 to a concentration of 50 mm NaCl to a concentration of 150 mm, Triton X100 to a concentration of 0.1%, incubated at room temperature for 2 hours, Nerastvorim material separated when sterilizing filtration through membranes with pore diameters of 0.22 micron.

Renaturation interferon is carried out by slow dilution of the resulting solution 100-200 remesiana for 12-15 hours at a temperature of 4-8°C. Next add magnesium sulfate to a concentration of 1 mm and the aggregate material removed sterilizing by filtration through a membrane filter with a pore diameter of 0.22 micron.

stage 4. Chromatographic purification of interferon

The chromatographic purification of interferon is carried out in three stages.

1. Received denaturirovannyj interferon in the first stage purified using affinity chromatography on resin type Chelating Sepharose Fast Flow (Amersham Biosciences), immobilized ions Cu+2. For this purpose a solution of interferon is applied to the column with Cu+2Chelating Sepharose Fast Flow and interferon elute buffer 0.1 M citric acid, pH 2.2.

2. In the second phase chromatographic purification solution interferon put on a cation-exchange resin type CM Sepharose Fast Flow (Amersham Biosciences) and interferon elute with a gradient of solutions of (0.0-0.5 M NaCl) in buffer 50 mm PA(CH3MEO) pH 5.5.

3. Purification of Monomeric forms of interferon from the remnants of polymeric forms of interferon carried out at the third stage of purification of the interferon gel-filtration resin type Superdex 75 (Amersham Biosciences). Chromatography is carried out in a buffer of 50 mm Na(CH3COO), pH 5.0, containing 0.15 M NaCl.

Described the isolation and purification of interferon allows you to get 4-8 g purified in the received interferon fully comply with the requirements of the "European Pharmacopoeia" for substance interferon Alfa-2b, namely:

The concentration of interferon for at least 2×108IU/ml;

Specific activity of interferon is not less than 2.0×108IU/mg;

- Electrophoretically purity of the drug at least 99% in reducing and not reducing conditions when staining the gels with silver;

The content correctly denaturirovannah form of interferon is not less than 98% according to HPLC RF;

- Isoelectric point of the selected interferon is in the region of pH 5.8-6.3;

Peptide map of selected interferon not fundamentally different from the peptide map for European standard interferon Alfa 2b CRS;

- Bacterial endotoxin content of not more than 100 ME 1 mg of interferon.

As follows from the above examples, the claimed group of inventions allows you to receive interferon Alfa-2b with high yield in a relatively simple and reliable technology.

Claims

1. Recombinant plasmid DNA pSX50 encoding the synthesis of recombinant human alpha-2b interferon, characterized in that it has a size 3218 base pairs (p. O.) and consists of the following fragments: the sequence from 1 to 176 of the nucleotide (n) includes the DNA fragment size 176 p. O., containing tryptopha., contains a sequence of Shine Delgarno responsible for the initiation of translation, the sequence with 195 in 695 ad includes the DNA fragment size 501 p. O., containing the gene for interferon Alfa-2b with nucleotide substitutions: 37 (And>C), 39 (G>T), 40 (a>C), 42 (G>T), 67 (>C), 69 (G>T), 70 (a>, 72 (A>T), 96 (G>A), 100 (>C), 102 (A>T), 114 (A>C), 120 (C>G),126 (G>A), 129 (G>A), 330 (C>G), 339 (G>A), 342 (G>A), 487 (A>C), 489 (>T), 495 (G>A), the sequence with 696 in 713 ad includes a synthetic DNA fragment size 18 p. O. containing synthetic polylinker, the sequence with 714 in 1138 ad includes the DNA fragment of the plasmid RCC-3 4129 at 4553 N. size 425 p. O., containing a sequence of strict transcription terminator rrnBT1T2the sequence with 1139 in 1229 ad includes the DNA fragment of the plasmid pUC19 with 2487 on 2577 N. size 91 p. O., containing the promoter of the gene-lactamase (resistance gene ampicillin-AmpR), the sequence with 1230 at 2045 N. includes DNA fragment from plasmid pUC4K with 720 N. in 1535 ad size 816 p. O., containing structural region of the gene kan, a sequence with 2046 N. on 3218 N. includes the DNA fragment of the plasmid pUC19 1625 453 N. size 1173 p. N., containing the sequence responsible for the replication plasmas on p. 1 - the producer of recombinant leukocyte interferon Alfa-2b human.

3. The method of producing interferon Alfa-2b human, including the cultivation of Escherichia coli SX5 under item 2 in the nutrient medium with the constant addition of nutrient substrates in the process of biosynthesis, mechanical destruction of cells of the microorganism at a pressure of 700-900 bar, dissolution of interferon in the buffer solution of guanidine hydrochloride, renaturation interferon in physiological buffer solutions in the presence of chaotropic agents, three-stage chromatographic purification of interferon on the resin type Chelating Sepharose Fast Flow, immobilized ions si+2, ion-exchange chromatography ion-exchange resins of the type CM Sepharose Fast Flow and gel-filtration chromatography on resin type Superdex 75.

4. The method according to p. 3, in which the cultivation is carried out in a nutrient medium with reduced levels of tryptophan with continuous addition of nutrient substrates, preferably glucose and yeast extract.

5. The method according to p. 3, in which the dissolution of interferon his atonement for removing soluble cellular components, including DNA, RNA, proteins, lipopolysaccharides, washing buffer solutions containing detergents type of the ith filtered through filters with a pore size of 0.22 μm.



 

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The invention relates to medicine, gynecology, and can be used for the treatment of uterine cervix using locoregionally citicentre

FIELD: medicine, hepatology.

SUBSTANCE: invention relates to a method for treatment of chronic hepatitis. Method involves applying granocyte that is administrated every day intravenously in the dose 1-10 mcg/kg of patient weight per 24 h for 7 days, not above, in combination with conventional complex used in treatment of this disease. Method provides normalization of activity of blood enzymes that indicates the liver function recovery and improvement of the liver structure indices.

EFFECT: improved method for treatment.

2 ex

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