A method of obtaining a protein with the activity of gm-csf primates
(57) Abstract:The invention relates to genetic engineering. Developed a method of obtaining a protein with the activity granulocytemacrophage factor (DM - SF) primates. In the process of the method is carried out cultivation of yeast or prokaryotic cells after transformation with the recombinant vector with the gene coding for the polypeptide with the established amino acid sequence. Produced protein is a specified factor of the person (SF - or Th2 SF - Ile ) or Gibbon (SF - G). After cultivation protein isolate and purify. The obtained recombinant protein has a higher degree of purity and the level of biological activity. 6 C.p. f-crystals, 7 Il. The invention relates to the production of a protein having the ability to stimulate the growth and differentiation of primary hematopoietic progenitor cells, in particular factor stimulation colony (FGC). In one aspect the invention provides a method of obtaining FGC-protein by recombinant DNA technology, vectors containing the gene expression of the indicated protein, microorganisms and cell lines transformed by these vectors, and FSK protein obtained in this way. Vetoshnikov and thus purified FGC-protein, having a degree of purity and the level of activity higher than those that were described earlier.Found that many different types of blood cells originate from mnogoportsionnyh krovetvornymi cells of the spinal cord. Cells of the spinal cord serve two functions: /1/ they reproduce themselves, resulting supported a population of cells of the spinal cord in the body and /2/ they provide progeny cells, transmitting the differences in all types of Mature blood cells. Cell that transmits differences in specific hematopoietic path, called the cage predecessor. Precursor cells for T-lymphocytes, B-lymphocytes, granulocytes, red blood cells, platelets and eosinophils, as well as earlier predecessors, which can individually lead to an increase in some types of Mature cells, were experimentally studied in vivo and in vitro /Dexter, T. M., 1983, J. Patology 141, 415-433/. It has already been determined in vitro proliferation and/or differentiation of precursor every type of cell depends on certain factors, which originate from different sources. For instance, the precursors of red blood cells require a factor called eritropoyetina. Factors trebouxiophyceae granulocytes, monocytes and Mature macrophages, called factors stimulate colonies (FGC).SFK activity extensively studied in mice. Most organs of adult mice produces SFK activity. However, compositions containing SFK activity, which were obtained from different tissues and different methods differ in their biochemical characteristics. So, the structural relationship between the various factors remains unknown. In addition, SFK activity affects more than one stage of the development of granulocytes and macrophages, and again it is not known exactly, only if the factor is responsible for all the observed activity or a variety of factors are at each stage. / Burgess, A. and Metealf, D. 1980 Blood 56, 947-957/.Human SFK activity was obtained from the placenta of some fetal tissues, macrophages and stimulated T-cells. Line T-cells (Mo), which produces one or more powerful FGC activities, was established from a patient with variant T-cells "hairy cells leukemia (leukemia reticuloendotheliosis) /bolde et al, 1978, Blood, 52, 1068-1072).The ability of SC activity to stimulate the production of granulocytes and macrophages indicates that farmacia increased production of these types of /myeloid/ cells. Indeed, it is shown that some patients with extremely high levels of apparently normal circulating granulocytes have tumors that swrh-produce FGC. In one case, surgical removal of the tumor, the number of granulocytes quickly drops to a normal level, clearly confirming that FGC can be useful in regulating the quantities of circulating granulocytes. /Hockiug, W. Goodinau, J., and Golde, D., Blood, 61, 600 /1983//. In particular, FGC compositions are clinically useful for the treatment mielo-suppression caused by chemotherapy treatment for cancer or its treatment using radiation. In addition, FGC compositions are useful in the treatment of severe infections, because FGC may increase and/or activate a number of granulocytes and/or monocytes.There are various differentiated types are known FGC activities, including granulocytes. FGC /G-FSK/ macrophage-FGC/M-FSK/, granulocyte-macrophage FGC /GM-FSK/ and multi-FSK. The present invention particularly relates to GM-FSK. FGC-proteins are known from various animal sources. However, the present invention in particular relates to a noticeable FGC, more specifically to human FGC and monkey FUK.Previously developed techniques of molecular cloning made it possible to clone a nucleotide sequence that encodes a protein, and the receipt of such protein in sufficient quantities, using appropriate system host-vector, /T. Maniatis, "Molecular cloning - a Laboratory manual", cold spring Harbor to the Laboratory, cold Fife Harbor, 1982/. Then the protein can be extracted by known methods of isolation and purification. Cloning techniques used to date can be grouped into three main categories: /1/ methods based on the knowledge of protein structure, for example, its amino acid sequence, /2/ ways based on the identification of protein expressed the cloned gene using specific to to modify receipt of protein or activity, the encoded gene of interest.Each of these classes of methods becomes difficult to apply when the protein of interest, such as FGC-protein, available in very small quantities. Therefore, if it is difficult to get an adequate amount of purified protein, it is difficult to determine amino acid sequence or partial sequence of a protein. Similarly, identification of the expressed protein binding antibody is preferably carried out using a polyclonal antisera with high titer. This anticavity cannot be obtained in the absence of sufficient quantities of pure protein (antigen). Monoclonal antibody alternative attempt, but the desired antibody can be obtained only with great difficulty in the absence of a suitable antigen and this monoclonal antibody is not able to interact with the protein in the form in which protein downregulation available recombinant system host-vector. Finally, the translation of RNA species to obtain identifiable protein or activity requires that the desired RNA was the source of RNA in sufficient excess to get reliable protein or signal activebook usually encoded rare RNA.Monoclonal line was used as a starting material for the purification of human FGC, and to identify the corresponding messenger RNA. However, even with such a relatively good source of SFK activity was found to be very difficult to isolate the protein even for structural studies.In order to solve the problems inherent in the cloning of the nucleotide sequence that encodes a rare protein, such as FGC, using the above methods, developed a new way. This method requires only that the gene product or activity can be measured reliably. Suitable methods for determining the FGC described in example 2 below. In the second aspect, a method of cleaning which is suitable for separation and purification of the protein FGC from both recombinant and natural sources at the level of purification and activity is much higher than was previously possible.In its first aspect the present invention solves the problems of the prior art and provides a source of protein with SFK activity, when using the technology of recombinant DNA. In accordance with the present invention uses a new method of cloning, which required the present invention provides cDNA, encoding a protein having SFK activity /ie FGC/cDNA/, the microorganism or cell line, transformed with the recombinant vector containing such FGC/cDNA, and the method for the FGC protein by expression specified FGC/cDNA under cultivation of the microorganism or cell line. Because FGC protein is produced from a clone in accordance with the present invention, we can be sure that this protein, which has SFK activity. Further, the invention consists in the method of preparation and isolation of the vector transformation, containing FGC/cDNA, this method consists of obtaining RNA from cells producing FGC; receiving polyadenylated messenger RNA from the indicated RNA; obtain single-strand cDNA from a specific messenger RNA; conversion of single-strand cDNA in duntou cDNA; inserting denistone cDNA into vectors of transformation and the transformation of bacteria by the specified vector to form colonies; selection of pools from 200-500 colonies each and isolation of plasmid DNA from each pool; transfection of plasmid DNA in a suitable cell host for the expression of SFK protein; culturing transfected cells and testing the supernatant layer on SFK activity; selection FGC positive pool of SFK activity.SFK proteins of the present invention provide for the growth and differentiation of hormones to cells of the myeloid system. For example, it was stated on their clinical use in the treatment of mielosupression, especially (symptomatic) granulotsitopenii after chemotherapeutic treatment of cancer or after the radiation treatment.In Fig.1 shows the DNA sequence that encode FGC-protein in accordance with the present invention. The DNA sequence represented completely encodes one variant of human FGC, referred to as FGC-Thr. Another allele encodes an identical product with the exception that Thr at position 100 is replaced with Ile /FGC-Ile/. The changes shown above for the human sequence, represent differences in the DNA sequence that encodes a FGC Gibbon /FGC monkey Gibbon/ /SFK-G/. Also shown reduced amino acid sequence.In Fig. 2 schematically presents the receipt plasmids pTP from the plasmid pAd D26 SV pA /3/.In Fig. 3 schematically presents a continuation of Fig. 2 and illustrates the receiving plasmid p91023 of plasmids the ptpl.In Fig. 4 schematically presents a continuation of Fig. 3 and shows the plasmid p91023 /B/.
Get a free bone marrow. Separate spicules by extrusion through a needle 20, 22, and then 25 Caleb the as/ and layer Ficoll-pack /30 ml VM-FBI 6 ml Ficoll/. Stirred at 1500 rpm for 30 minutes at room temperature. Remove the fat and the FBI layer and discard. Pipetted layer of low density. Washed 2 times by the FBI and consider it. Have cells on the plate in RPMI /received from FLEXIBLE as PPMI 1640/ 10% HTFCS /heat inactivated NWT/ for 3 hours to remove the pasted cells.Environment for a plate is prepared fresh/:
0.3% agar dissolved in water, cooled to 40o< / BR>2x Eskova /1:1 volume/volume with agar/
1% P/S final concentration of 100 U/ml streptomycin, 100 μg/ml penicillin
10-4M alpha diglycerin 2x Iscove of 10-2M original Agar, cooled to 40o. Mix with other ingredients Cooling on a water bath until 37-38oand storage at this temperature.After 3 hours pipetted neprecejusies cells. Mix and count. Add 2.10 cells/ml seed medium and stored at a controlled temperature water bath 37-38oC. Add samples /for example, the medium from transfected cells, usually 10 μl of the sample/ in the first row of recesses plate microtitration in duplicate. Add 100 ál of cell suspension in each recess. Pripiat 50 μl of the solution from the first row to the next row and so on and continue dilution 1:3 across the plate. Wrap the plate parafilm. Incubated for 10-14 days at 10% CO2, 37oC in a fully humid atmosphere and count colonies.For counting colonies consider the total number of colonies that grew each recess. In each sample, several recesses sow without the inclusion of the sample /blank/ to obtain the basis for counting colonies. The average number of colonies that grew in the control cavities, is subtracted from the number of colonies found in each recess containing samples. One unit of the NWT is the number that will encourage the formation of one group more than the control level of 105cells human bone marrow /sown 105cells/ml when the concentration of the NWT is sub-saturated. Sub-saturated concentration is determined by dilution and by comparing the number of colonies at various dilutions to find concentrations below saturation.For this sample count colonies containing granulocytes, monocytes, or both types of cells. The types of cells in the colonies are determined by the selection of colonies and staining of individual cells.B. Sample KG-1 clitcauses 2105cells/ml Cells use for test only passage between 30-35. The sample is the same as described above for bone marrow, with the exception that KGI cells were seeded in agar mixture at 4103cells/ml.The number of colonies grown in each recess, determine and subtract the control account idle experience, as in the above-described experience with bone marrow. One KG-1 NWT unit/ml means, such concentration NWT, which will stimulate the half maximum number /saturation/ KG-1 colonies for growth. The maximum number obtained when the inclusion level of saturation NWT in several pits.Stage 3. Design vector p91023/B/.The vector transformation is a pAdD26SUpA /3/ described by Kaufman and others, Mol. Cell Biol. 2 /11/: 1304-1319 /1982/. It has the structure shown in Fig. 2. In short, this plasmid contains the mouse gene cDNA dihydrofolate reductase /DHFR/, which is the last major promoter of adenovirus 2 /Ad2/ under transcriptional control. 5' splice site is included in the DNA of adenovirus and 3' splice site derived from immunoglobulin gene, is located between the Ad2 major last the promoter and the sequence encoding DHFR. SV40 early sadinia pAd26SVpA /3/ from /P. Mellon, Parker V., Gluzman J. and Maniatis, T., 1981, Cell 27: 279-288/ and does not contain pBP322 sequences, known for inhibiting replication in mammalian cells /M. Lusky and Botchan, M., 1981, Mature (London) 293: 79-81/.pAdD26SUpA /3/ turn into a plasmid pCSV2, as shown in Fig. 2. pAdD26SVpA /3/ turn into a plasmid pAdD26SVpA /3/ by dividing one or two Pst sites in pAdD26SVpA /3/. This is partially digesting > PST /using the deficiency of enzyme activity, so that could be obtained subpopulation of linearized plasmid, in which split only one Pst website/, then spend processing on Kleinow, linking to recircularisation plasmid transformation of E. coli and screening for the division P I site, localized in 3'SV40 polyadenylation sequence.Tripartite leader of adenovirus genes associated viruses /VA genes/ insert into pAdG26SVpA /3/ /d/, as shown in Fig. 2. First pAdD26SVpA /3/ /d/ PVUII digest to make a linear molecule open within 3' of the site of the first of the three elements of three-leader. Then PJAW 43 /Zain et al. 1979, Cell 16, 851/ digested by Xho I, process for Kleinow, digest P II and isolate a fragment of 140 base pairs, containing the second and third part of the leaders of the 140 base pairs are then associated with PVUII digested pAdD26SVpA /3/ /d/. The product link is used for transformation of E. coli for resistance to tetracycline and sceneroot colonies using the procedure of Grunstein-Hogness with the use of32P-labeled probe hybridization to a fragment of 140 base pairs. Get DNA from positively hybridized colonies, to check whether the reconstructed PVUII site in the 5'-or 3'-ends of the inserted specific DNA 140 base pairs in the 2nd or 3rd leaders last adenovirus. When properly aligned, PVUII site is located on the 5'side of the insert 140 base pairs. This plasmid denote the ptpl in Fig. 2.AVaII D fragment SV 46 containing SV40 amplifying the sequence, get the digestion of SV40 DNA enzyme AVaII, zatuplenii all fragment Klenow Pol I binding Xho I linkers to the fragments, digestion Xho I to open the Xho I site and insulation fourth largest /D/ fragment gelelectrophoresis. Then this fragment is associated with the cut Xho I the ptpl, receiving plasmid pCVSVL2-TPL. The orientation of the SV40 D fragment in pCVSVL2-TPL will be such that the SV40 last promoter is in the same orientation as the last main promoter of adenovirus.For the introduction of genes associated virus /VA/ adenovirus in pCVSVL2-TPL with perevarivat Hind III fragment and isolate In after gelelectrophoresis. Then this fragment is inserted into pBR322, which is pre-digested Hind III. After transformation of E. coli for resistance to ampicillin sceneroot recombinate to insert the fragment Hind III and determine the orientation of the insert in the digestion of restrictionism enzyme. pB322 - AdHind III B contains a fragment Hind III B adenovirus type 2 in the orientation shown in Fig. 3.As shown in Fig. 3, VA genes is usually derived from the plasmid pBR322-Ad Hind III B in the digestion of HpaI, the addition of EcoRI linkers and digestion of EcoRI and the fragment of 1.4 square Fragment with EcoRI sticky ends, then bind EcoRI site pTRL /which was previously digested EcoRI/. After transformation of E. coli HBIOI and selection for resistance to tetracycline, colony sceneroot by hybridization of the filter with a specific probe DNA for VA genes. DNA obtained from the positively hybridized clones and characterized by digestion with restriction endonucleases. The obtained plasmid denote p91023.Remove both EcoRI site in p91023. p91023 cut completely EcoRI, to generate two DNA fragments, one of about 7 kV, and the other example 1.3 kV, containing VA genes. The ends of both fragments are filled using a fragment of Klenow PolI, and then both rnnow on 2 sites EcoRI, identified by screening of Grunstein - Hogness fragment VA gene and using traditional analysis of restriction enzymes cut sites.Then remove only website > PST in p91023 /A/ and replace the EcoRI site. p91023 /A/ cut to end > PST and then treated fragment Klenow PolI to generate full ends. EcoRI linkers are associated with blunted > PST site p91023 /A/. Linear p91023 /A/ with the EcoRI linkers attached to the blunt > PST site separated from the unattached of linkers, and digested to completion EcoRI, and then re-connect. Extract plasmid p91023 /B/ and identify to have a structure similar to p91023 /A/, but with EcoRI site located on the site of a previous > PST site.Stage 4. Obtaining cDNA Bank.Mo cells induce within 16-20 hours using NSS and PMA to enhance the production of lymphokine. These cells were seeded at 5105cells/ml in medium Iscove with 20% NWT,0,3% /volume/volume/ RNS and 5 ng/ml TPA. Cells are harvested by centrifugation. The ball of cells again suspended in 20 ml of chilled on ice hypotonic buffer /RSB buffer: 0.01 M Tris-HCl, pH of 7.4, 0.01 M KCl, 0,0015 M MgCl2, 1 μg/ml cycloheximide, 50 units/ml of RNA-Shin and 5 mm dithiothreitol/. The cells are allowed to swell on ice enat centrifuging at low speed /2000 rpm in a centrifuge Beckman J6/ to remove nuclei and deletirovannykh cells. The supernatant layer is kept on ice until such time as the ball of the cores is not re-suspended in 10 ml of BSB and again attentivegreat low speed. This second supernatant layer combined with the first and centrifuged United supernatant layers with low speed to remove residual contamination cores and deletirovanie cells. The supernatant layer of this operation was adjusted to 0.15 M KCl addition of 2M KCl, then centrifuging with high speed /25000 rpm, Beckman SW rotor for 30 minutes to get the ball out of the membranes. Diaphragm ball washed thoroughly with cold RSB, then re-suspended in 12 ml of RSB containing 2 M sucrose and 0.15 M KCl. Prepare two discontinuous gradient in the centrifuge tubes Beckman SW41 layup by 6 ml of membrane solution in 2 M sucrose 2 ml of RSB with 2.5 M sucrose and 0.15 M KCl. The tube is filled to the top by layering on top of 2.5 ml PSB containing 1.3 M sucrose and 0.15 M KCl. These gradients is stirred for 4 hours at 27000 rpm /Beckman, rotor SW 41/ 4oC. the Membrane layer at the phase boundary between 2.0 M and 1.3 M sucrose/ carefully remove the side using 18 gauge needle and syringe. Combine the membrane fraction of the two gradients and diluted with 1 volume of distilled water, Loy re-extracted with a mixture of 1:1 phenol and chloroform and, finally, an equal volume of chloroform. Finally, precipitated connected to the membrane of the RNA by the addition of sodium chloride to 0.25 M and 2.5 volumes of cold ethanol and incubated overnight at -20oC. the Precipitated RNA is collected by centrifugation /4000 rpm for 10 minutes in a centrifuge Beckman J-6/ and re-suspended in 1 ml of distilled water. From 2109cells receive approximately 1 mg of RNA. Messenger RNA /mRNA/ isolated from total RNA by chromatography on a column with 0.5 ml oligo-dT-cellulose. Briefly, RNA is heated for 5 minutes, rapidly cooled on ice, then diluted 5 times at room temperature in binding buffer /0,5 MLiCI 0.01 M Tris-HCl, pH 7,4, of 0.002 M EDTA, and 0.1% SDS/. RNA in binding buffer and transferred to a column in the oligo-dT-cellulose, equilibrated binding buffer at room temperature. The column was washed with 5 ml of binding buffer, followed by 5 μl of 0.15 MLiCI Cl, 0.01 M Tris-HCl, pH 7,4, of 0.002 M EDTA and 0.1% SDS. Finally, elute mRNA 2 ml of 0.01 M Tris-HCI, pH 7,4, of 0.002 M EDTA and 0.1% SDS mRNA is precipitated by adding sodium chloride to 0.25 M and 2.5 volumes of ethanol and incubated overnight at - 20oC. the Precipitated mRNA harvested by centrifugation /30000 rpm for 30 minutes in an SW55 rotor of Weckman/. The tube is carefully drained and the ball mRNA REP is using 1:1 phenol and chloroform: then 3 times with chloroform. Precipitated mRNA by adding 2.5 volumes of ethanol. The mixture was frozen and thawed several times in a bath of dry ice/ethanol, and then centrifuged for 15 minutes in an Eppendorf centrifuge. The tube is carefully drained and the ball mRNA re-suspended in 20 µl of distilled water. The final output is approximately 30 μg of mRNA.Prepare first strand cDNA using standard techniques. Briefly, 10 μg of membrane mRNA was diluted in 100 μl of reaction mixture for the synthesis of cDNA, containing 300 mm Tris, pH of 8.4, 140 mm KCl, 10 mm MgCl2, 10 mm - mercaptoethanol, 500 mm of each of the dATF, dGTP, dCTP and dTTP, 5 μg oligo-dT-/phosphorylated and with an average size 12-18 as primer, 150 Ku32dCTP /400 Ku/mg/ DL and 20 units of ribonuclease inhibitor RNA-sin. The reaction is initiated by addition of 100 units of reverse transcriptase and incubated for 30 minutes at 42oC. the Reaction is stopped by addition of EDTA to 40 mm and degrade RNA by incubation for 20 minutes at 65oC in 0.2 M NaOH. The base is neutralized by adding 20 μl Tris, pH of 7.4. The reaction mixture was then extracted with phenol/chloroform, once again extracted with 50 μl of 10 mm Tris, pH 7.5 1 mm EDTA /TE/ and combine the aqueous phase. Single-strand cDNA turn in duntou by incubation for 12 hours at 16what I a pH of 7.4, 2.3 mm DTT, 2-mercaptoethanol, 10 mm MgCl2, 150 moles of each of the four deoxynucleotides and 25 Ku32P dCTP. The reaction is stopped by extraction with phenol/chloroform and remove nevoshedshy triphosphates by passing the aqueous phase through the column with 1 ml Sephadex G-50. Derived fractions are combined and precipitated by ethanole.Ball cDNA is washed with cold ethanol, and then re-suspended in 200 μl of 20 mm Tris, pH 8.0, 1 mm EDTA 80 Molar 5-adenosyl-methionine and 300 units of EcoRI of methylase for 60 minutes at 37oC. the Reaction is stopped by excretia phenol/chloroform and collect methylated cDNA by ethanol precipitation.Ball cDNA breaking 70% ethanol, and then re-suspended in 200 ál Sl buffer/Maniatis and others / and incubated with 200 units of Sl nuclease at 30oC for 30 minutes. The reaction is stopped by excretia phenol/chloroform and collect cDNA by ethanol precipitation.Duntou cDNA blunt by incubation in 100 μl of 20 mm Tris, pH 7.4 in 50 mm NaCl, 10 mm 2-mercaptoethanol and 500 Molar of all four deoxynucleotides with 25 units Kleinow at room temperature for 30 minutes. The reaction is stopped by extraction with phenol/chloroform and collect cDNA saidlooking from new England Biolabs /sequence: pCGGAATTCCG/, using 2000 units of T4 ligase overnight at 16oC. the Reaction is stopped by insulinopenia at 70 for 20 minutes, then diluted to 300 μl so that the final salt concentration was 0.1 M NaCl, 10 mm MgCl2, 50 mm Tris-Cl, pH of 7.4. Then cDNA digested for 2 minutes at 37oC 700 units KcoRI. The reaction is stopped by extraction with phenol/chloroform and collect cDNA precipitation with ethanol. The ball re-suspended in 50 µl TE and passed through a column of 5 ml Cl-4B. Derived fractions are collected, pooled and precipitated with ethanol. The precipitated cDNA subjected to electrophoresis through 1% agarose gel in Tris-acetate buffer in the presence of 1 μg/ml of amidinopropane. Isolated from the helium cDNA with a size in the range of 500-4000 base pairs, using a standard technique with glass powder. Elyuirovaniya cDNA extracted with phenol/ chloroform, precipitated with ethanol and ball /after washing with ethanol/ re-suspended in 60 μl of TE. The final output is 100-500 ng of cDNA.Obtaining expression vector p91023/B/ described above. Link digested EcoRI and treated with phosphatase vector /500 ng/ 100 ng cDNA in 100 μl of reaction mixture /standard T4 ligase reaction mixture/ during the night when the 16oC. the Reaction Alenia 5 µg t-RNA as a carrier.Precipitated by ethanol, the DNA was washed with 70% ethanol, and then re-suspended in 100 ál of TE. This DNA is used in 4 ál of aliquots for transformation of E. coli MC 1061/4 ál 100 ál transformational environment/. Each of the 25 startformula transferred into a 150 mm Petri dishes containing 1% agar, L-broth and 10 μg/ml tetracycline /Tet plates/ and incubated overnight at 37oC. On each plate grows approximately 2000 colonies, the result is about 50 000 colonies. After reaching approximately 0.5 mm in diameter colonies transferred to nitrocellulose disks /137 mm/, gently placing a dry filter on the surface of the plate, then gently otscheplaut filter. All colonies on the plate is transferred onto the filter, which is then placed /colony side / Sideup/ fresh Tet-plate. After growing colonies within a few hours preparing a replica of each of the filters by placing fresh moist filter exactly on the original filter, pressing them to each other, arseplay them, then returning each filter on a fresh Tet-plate and inquira plates overnight at 37oC. Each replica is carefully labeled so that it can be compared with the original filter.Stage 5. Preparation of pooled plasmid DNA.Each of the filters 25-replica ośno original filter. Scraped colony from each section in 10 ml of L-broth. Bacteria are harvested by centrafricana /3000 rpm, 10 minutes, centrifuge, Beckman J-6/, re-suspended in 0.6 ml of 25% sucrose, 50 M Tris-HCl, pH 8.0, and transformed into protoplasts by the addition of 0.12 ml of 5 mg/ml lysozyme and incubated on ice for 5-10 minutes. The protoplasts again incubated at room temperature for 10 minutes, then add 0,125 ml of 0.5 M EDTA, and then are lysed with the addition of 0.12 ml of 10% SDS in 50 mm Tris-HCl, pH 8.0. The lysate gently mixed, incubated at room temperature for 15 minutes, then precipitated protein and chromosomal DNA by adding 0.3 ml of 5 M NaCl. After incubation on ice for 15 minutes, the lysate centrifugual on the Eppendorf centrifuge for 30 minutes in the cold. Carefully remove nadosadocnuu layer, leaving the bottom of the viscous bead, DNA/protein and diluted by adding 2.5 ml of water. The mixture is extracted with 1 ml of phenol, separate the layers by centrifugation /IOK for 10 minutes in the rotor Servall SS-34/ and remove the aqueous layer to a fresh tube. Precipitate DNA by adding 0.5 ml of 5 M NaCl and 7.5 ml of cold ethanol, freeze the mixture several times in a bath of dry ice/ ethanol. The precipitate is collected by centrifugation /IOK, 15 minutes in Sorvall SS-34/, re-suspended in 0.3 ml of 0.3 M sodium acetate and again m ilitary DNA by centrifugation/ 5 minutes in a centrifuge Eppendorf/ and finished the ball again suspended in 100 ál of sterile TE/10 mm Tris, pH 8.0, 1 mm EDTA/. Typical operations receive 5-10 μg of plasmid DNA. Each preparation contains DNA from 200-500 colonies on the original filter. Just cooked 200 DNA samples from 25 filters.Stage 6. Isolation of clone FGC.Each of the DNA samples from stage 5 separately stansfeld COS M6 cells of monkeys, as described below.M6 cells grown routine in the environment of the Needle, modified Dulbecco "DMA available from Gibco/ containing 10% heat inactivated serum embryo calf (HIFCS), split twice a week at a dilution of 1:6. 24 hours after separation M6 cells are ready for transfection. 24 hours before transfection were seeded 1,2108M6 cells /division 1:6/ Cell Factor / available from Nunc/ 1.5 L. the DMA + 10% HIFCS. Immediately before transfection plate is sucked off and washed twice 7 ml containing no serum the DMA. DNA was dissolved in 0.1 M Tris /pH 7.3/ and add in the DMA medium containing 2 mm glutamine, 100 μg/ml streptomycin, 100 U/ml penicillin, and 0.25 mg/ml DEAE Dextran, leading to a 4 ml solution of Tris-DNA. Add 4 ml of medium containing dissolved DNA on the plate containing COS M6 cells, and incubated for 12 hours.After incubation, the cells ol the mm glutamine and 0.1 mm of chloroquine cells and incubated for 2.5 hours.After 2.5 hours, washed once SF the DMA and add 10 ml of DMA + 10% HIFCS /plate. After 30 hours suck environment and serves 4 ml/plate the DMA + 10% HIFCS. Harvest, removing air-conditioned environment after 24-26 hours of additional incubation.Air-conditioned environment after each transfection check on SFK activity using KG-I sample. For each sample, positive on SFK activity must be identified clone on the original primary filter, responsible for SFK activity. For example, for one transfection, positive on SFK activity, all transplanted colonies from the initial section of the primary filter, which is a model of DNA transfection. Some of these 320 colonies transplanted into 3 ml of L-broth plus 10 μg/ml tetracycline. Crops grown during the night. 320 colonies are placed on a matrix of 18 x 18. Prepare pools of each horizontal row and vertical column of the matrix/ 36 pools/ /note: the last horizontal row has only 14 clones/. Prepare DNA samples from each pooled culture, and then used for transfection of COS cells. Check supernatant layers of these transfection, using a sample KG-1 colony. From this group Traore these pools contains FGC-clone.Isolated 12 individual clones from this culture and prepare miniprep DNA from 10 ml cultures in L-broth as described above. Samples of 10 µg DNA from these preparations EcoRI digest and analyze the resulting DNA fragments by electrophoresis on agarose gel. Nine of the twelve clones are inserted approximately 750 base pairs. DNA from four of these clones and the other three clone is introduced into COS M6 cells, as described above. The supernatant layers from these transpency check using KG-1 sample, and the sample on FGC with bone marrow. Four clones, each of which contains a fragment of 750 base pairs, all direct the expression COS M6 cells with a high level of SFK activity as defined in any sample, while the other three clone no. Therefore, the coding region for FGC should be localized inside the insert 750 base pairs.The DNA sequence encoding FSK, was removed from the vector transformation in the positive clone by digestion with EcoRI and was defined by its amino acid sequence using standard methods dideoxysequencing after sublimirovanny fragments into M13 vectors to obtain the sequence pakisamahan pCSF-1. This plasmid was deposited in culture Collections of the American type in the strain E. coli MC1061 number storage ATCC 39754 July 2, 1984.Stage 7. Expression of SFK protein.M6 COS cells monkeys, the transformed vector p91023/B/, containing FGC/cDNA, as highlighted in stage 6, grown as described in stage 6, for receiving the FSK-protein in the culture medium.Specifically, 1 mg of DNA /pC5F/ dissolved in 1 ml of 0.1 M Trio pH 7.3 and added to 600 ml of DMA, containing 2 mm glutamine, 100 11/ml streptomycin, 100 μg/ml penicillin /P/S/ and 0.25 mg/ml DEAE Dextran molecular weight of 500 000, obtained from Pharmacia/. 600 ml of the DNA solution, DEAE Dextran is added to COS M6 cells in the apparatus for the cultivation of cells and incubated at 37oC for 12 hours. After incubation the cells are washed once with 900 ml of SF the DMA, and then incubated for 2.5 hours with 600 ml of DMA, containing 0.1 mm of chloroquine, 10% HIFCS, 2 mm glutamine and P/S. After the extraction medium containing chloroquin, cells washed SF the DMA and serves 1500 ml DMA with 10% HIFCS. After 30 hours the cells are washed SF the DMA, the environment replace 800 ml SF the DMA and put transfected cells in culture conditions at 37oC for 24 hours. An elaborate air-conditioned environment is sucked off and replace the other 800 the Le collection concentrate the sample waste environment 20 times by ultrafiltration under pressure, using the camera Amicon 2.5 l with YM5 membrane/ 5000 MW cut/.Stage 8. Purification of recombinant FGC.200 ml of concentrated spent medium from 4 l of the initial material - phase 7/ brought to 30% saturation with ammonium sulfate by adding solid ammonium sulfate and remove the precipitated protein by centrifugation. The supernatant layer was adjusted to 80% saturation with ammonium sulfate by adding more solid ammonium sulfate and the precipitated protein is collected by centrifugation. The ball re-suspended in 5 ml of 20 mm sodium citrate, pH of 6.1, containing 1 M NaCl. Dissolved protein applied to the column 1.6 x 100 cm Ultragel AcA54, balanced those of the buffer, VSC activity, allerona with a column that has apparent molecular weight of 19 to Dalton or after approximately 90 ml was Observed that the gel-filtration with low ionic strength, SFK activity elute from the column in two positions with facile molecular mass of about 19 to Dalton and 38 to Dalton, confirming that GM-FSK can easily form dimers/. Combine active faction and bring to 0.15% TFWC /add 10% TFWC/ and applied to a column of the video recorder C4 /0,46 x 25 cm/, self-composed of 0.1% of TN. Column display with a linear gradient of 0-90% are the awn eluted between 39% and 43% acetonitrile/ /fractions 16-20/. Analyze the sample 20 ál of fraction 19 by electrophoresis on SDS-polyacrylamide gel/ 13.5% gel as described Laemmli, Nature 227, 680 /1970//. Observed a broad protein band with an apparent molecular mass of 18-26 to Dalton. The wider the stripe size for FGC is a common characteristic of glycoproteins and thins, reflecting extensive, but alternating the addition of carbohydrate. Protein of fraction 19 is subjected to decomposition in Adminu using gas-phase microsequencer Applied Biosystems. Of the approximately 20 µg protein caused get a sequence of the first 16 amino acids /A-P-A-R-S-P-S-P-S-T-Q-P-W-E-H/. The high output of this single protein sequence well confirms that FGC is a protein in fraction 19 was purified to homogeneity. The bioassays shows that the fraction 19 is 3107units on A280units of absorption. Because typical proteins in aqueous solution give extinction coefficients in the range of from 0.8 to 1.2 A280units of absorption at milligram of protein, the specific activity of the purified FGC is between about 1107and about 4107units/mg when tested using samples from human bone marrow.Example B (II). Clone FGC Gibbon
Stage 1. Pot for several weeks in RPMI 1640 /received from Flexible/ and 20% whey embryo calf /NWT/ as long unless 1109cells only. Cells to induce the production of high levels of FGC by activation within 24 hours in the presence of 10 ng/ml 12-O-tetradecanoylphorbol-13-acetate /TN/ in RPMI 1640 plus 1% of the NWT. Gather grown cells by centrifugation / 1000 rpm, 5 minutes, washed once with phosphate buffer saline /FBI/ and again collected by centrifugation.These cells are prepared associated with membrane polysomal /MBP/ mRNA using the same procedure as described in example A to obtain RNA Mo cells.Stage 2. The reaction single-strand cDNA.Dilute 6 MGK MBP mRNA from stage 1/ 50 ál reaction mixture DM cDNA synthesis /see example A - stage 4/ and initiate the reaction by adding reverse transcriptase. After 30 minutes incubation at 42oC the reaction is stopped by extraction EDTA to 50 mm and diluted with water to 100 μl. The mixture is extracted with phenol/chloroform and again extracted with chloroform. Separate the hybrids cDNA/RNA from unreacted triphosphates by chromatography on a column of Separate CL-4B. Derived fractions are combined and collect hybrids precipitation with ethanol. The final output is 570 ng.Stage 3. Response doutores threads in a standard reaction mixture with E. coli. Polymerase I, E. coli-ligase and RNase H. the Reaction mixture was incubated overnight at 16oC and then incolarum 1 hour at 37oC. the Reaction is stopped by adding EDTA and extracted with phenol/chloroform. Separate cDNA from unreacted triphosphates by chromatography on a column of Separate CL-4B derived fractions are combined and collect cDNA by ethanol precipitation.Stage 4. The preparation of recombinant cDNA.Again suspended ball cDNA /stage 3/ 75 μl of water. Homopolymer C "tails" attached to the ends of the cDNA by adding 10 μl of the cDNA solution to 25 ál standard reaction mixture with terminal transferase and incubated at 30oC for 5 minutes. The reaction is stopped by addition of EDTA to 40 mm and heat inactivation at 68owithin 10 minutes. Hardened 10 ng of this cDNA with tails with 50 ng G-tail pBR322 /HEH received from/ in 10 μl of 10 mm Tris, pH 7.5, 1 mm EDTA and 100 mm NaCl. Quenched the reaction mixture was incubated for 10 minutes at 68oC, and then 2 hours at 57oC.Stage 5. Transformation of bacteria.Grow E. coli strain MC1061 in L-broth, cooled on ice, collected by centrifugation and treated with CACl2to prepare them for Transfo the lead 50 such transformations, using all hardened cDNA, and put on the plate with 1% agar L-broth, size 15 cm, containing 10 μg/ml tetracycline. On each plate grows approximately 1000 colonies.Stage 6. Sowing replicas.Transplanted 10,000 colonies from each transformation with a toothpick, transferred to a fresh plate /500 on the plate in the grill/ and grown overnight at 37oC. Then the colony is removed from each plate, pressing a dry nitrocellulose filter tightly to the surface of the plate. For each of these basic filters preparing two filter-replica. The main source filters stored at 4oC, and filters replica is treated with a base and dried to prepare them for hybridization.Stage 7. Cooking32P-labeled hybridization probes.Isolate the cDNA insert of pCSF-1 by digestion with restriction enzymes EcoRI and electrophoresis on agarose gel with Tris-acetate and amidinopropane. The band containing the cDNA fragment is cut out from the gel and purified by the method of glass powder.Then add 300 ng of fragment cDNA 1 μl 10 × T4 DNA Polymerase buffer /0.33 M Tris-acetate, pH 7,9,0,66 M potassium acetate, 0.1 M magnesium acetate and 10 mm dithio-10 minutes at 37oC, this mixture is combined with 1 ál of 10 x T4 DNA Polymerase buffer; 1 μl of 2 mm solution of each of dCTP, dTTP, dGTP, 10 ál32PdATP/10 Ku ál 3000 Ku/mmol and 3 units of Tr DNA Polymerase. The reaction mixture was incubated for 20 minutes at 37oC. and Then added 1 μl of 2 mm dATP and the reaction mixture was incubated an additional 10 minutes at 37oC.Separate is not included triphosphates from labeled cDNA by chromatography on a column of Sephadex GIOO. Prepare a second probe from a synthetic oligonucleotide having the sequence:
ATC TGG CTG CAC AG,
which complementary aminobenzo region coding FGC. This oligonucletide Machen32P dATP at its 5'end using polynucleotide kinase reaction.Stage 8. Isolation of clones FGC cDNA.In the standard procedure of screening by hybridization hybridizat some 45 clones with T4 labeled pCSF-1 cDNA. Of these approximately 20 also hybridized with a labeled oligonucleotide probe. The coding region of one of them was sequencially and sequence data indicate the number of base substitutions, some of which are the result of the difference of amino acids in the expressed protein. These differences are shown in Fig. 1 licensing FGC from lymphocyte mRNA of peripheral blood flow.Stage 1. Obtaining mRNA from lymphocytes of peripheral blood flow.The lymphocytes of peripheral blood were obtained from four products plasmaphoresis "received from Ed Galina Vasilyevna/ fractionation on the gradient Ficoll-Hipac. Low density in RPMI 1640 in the presence of 5% serum embryo calf, 0,17% phytohemaglutinin and 10 ng/ml of formal myristate acetate /PMA/ at a density of 2106cells /ml/ just received 6109cells/. Collect cells by centrifugation /1000 rpm, 5 minutes, once washed with saline phosphate buffer /PBS/, and again collected by centrifugation. Prepare cytoplasmic RNA with a gentle lysis, in which the cells are again suspended in 50 ml of cold Titanoboa buffer for lysis /140 mm NaCl, 1.5 mm MgCl2, 10 mm Tris, pH 8,6, 0,5% Triton X-100/ s 10 mm dithiothreitol /DTT/ and 50 units/ml Rxin/ obtained from Biotec/. This lysate is divided into 2 equal parts and each part layered on 10 ml pillows lisanova buffer containing 20% sucrose. Remove the cores of the cells by centrifugation in the cold /4oC, 400 rpm for 5 minutes. Carefully remove the top layer /cytoplasmic extract/ and add sodium dodecyl sulphate /SDS to a final concentration of 1%. This solution two is ardanuy RNA is collected by centrifugation /15 minutes at 4000 rpm/ and again suspended in 0.01 M Tris, pH 7.5 1 mm EDTA, 0.25 M NaCl/TE buffer plus 0.25 M NaCl/ and again precipitated by adding 2.5 volumes of cold ethanol. Finally collect the RNA by centrifugation and again suspended in 5 ml of water. The final yield of 7.5 mg.Isolate messenger RNA from the entire cytoplasmic RNA selection on oligo-dT-cellulose. Heated 2.5 mg of total RNA to 65owithin 5 minutes. Add NaCl to 0.5 M and allow RNA to cool to room temperature. This RNA is passed through the column with 1 ml oligo-dT-cellulose, equilibrated in TE + 0.5 M NaCl/binding buffer/. Remove unbound RNA by extensive washing of the column binding buffer. Related messenger RNA elute 3 ml of water and precipitated by adding 0.2 ml of 4 M NaCl and 2.5 volumes of cold ethanol. Precipitated mRNA harvested by centrifugation /30 minutes at 25000 rpm/. The final ball /approximately 100 µg/ re-suspended in 50 µl of water.Stage 2. The reaction single-strand cDNA.Diluted with 20 µg PBL mRNA in 50 μl of the cDNA synthesis reaction containing 100 mm Tris, pH of 8.4, 140 mm KCl, 10 mm MgCl2, 10 mm 2-mercaptoethanol, 400 mcm each of dATP, dGTP, dCTP and dTTP, 5 μg oligo-dT /average size 12-18 as primer, 25 Ku32PdCTP /400 Ku/mol and 20 units of ribonuclease inhibitor RNA SYN. The reaction to initiate the live addition of EDTA to 40 mm and extracted with an equal volume of water-saturated phenol. Phenolic phase is again extracted with 50 μl of TE buffer. The aqueous phase unite. Separate the hybrids cDNA/RNA from unreacted triphosphates, passing the combined aqueous phase through the column with 5 ml Sepharose CL-4B. /obtained from Sigma/, balanced TE. Faction, which divert from the column are pooled, adjusted to 2500 mm NaCl and precipitated nucleic acids by adding 2.5 volumes of cold ethanol. Hybrids harvested by centrifugation for 30 minutes at 4000 rpm meters Final ball /2.5 µg cDNA/ again suspended in 50 μl of water.Stage 3. The reaction dvuhnitochnogo cDNA.Synthesize duntou cDNA under combined action of the enzymes of E. coli DNA Polymerase 1, E. coli DNA ligase and E. coli RNase H. The reaction mixture /50 μl/ containing 20 mm Tris, pH 8.0, 4 mm MgCl2, 1.2 mm EDTA, 25 M OVER 100 M each of dATP, dGTP, dCTP and dTTP, and 50 Ku32PdCTP/3000 Ku / mg/DL.Conduct the reaction by adding 3 units of DNA Polymerase 1.05 units of DNA ligase and 0.75 units of RNase H and incubating at 16owithin 18 hours, then 1 hour at 37oC and then stopped by the addition of THIS to 40 mm is extracted with an equal volume of phenol. Phenolic phase is again extracted with 50 μl of TE, combine the aqueous phase and to separate DNA from unreacted triphosphor32P, single-strand cDNA quantitatively converted into duntou form.Stage 4. The preparation of recombinant cDNA.Add homopolymer C "tails" to the end of the cDNA with gentle heating to 400 ng of cDNA in a 50 μl reaction mixture containing 1 mm 2-mercaptoethanol, 1 mm CoCl2and 9 units end deoxynucleotidyltransferase, 30oC for 5 minutes. The reaction is stopped by addition of EDTA to 40 mm and heated to 68oC for 10 minutes. Quenched with 200 ng of this tail cDNA from 500 ng G-tail pAT153 /received from Amersham/ 100 ál 10 mm Tris, pH 7.5, 1 mm EDTA and 100 mm NaCl. The hardening reaction is carried out at 57oC within 2 hours after 3 minutes pre-incubation at 68oC.Stage 5. Transformation of bacteria.The reaction product of hardening cDNA used directly to transform E. coli strain MC1061. Use fresh colony of bacterial cells for inoculation of 50 ml of L-broth and grown for several hours up until the optical density at 550 nm reaches of 0.25. Cells frozen on ice and harvested by centrifugation /2000 rpm for 10 minutes. The ball re-suspended in 10 ml of cold 0.1 m CaCl2and leave on ice for 10 min the. the ATEM 10 μl of the reaction mixture to quench the cDNA is incubated with 200 μl of processed CaCl2bacteria for 30 minutes on ice and then for 2 minutes at 37oC, then add 0.8 ml of L-broth and finally incubated for 30 minutes at 37oC.Carry out 20 of these transformations, using all hardened cDNA. Each of the transformed mixture is poured on a plate with 1% agar L-broth / 15 cm diameter) containing 10 μg/ml tetracycline. 20 transformations all 20 of these plates were poured and incubated overnight at 37oC. on average, about 1500 bacterially colonies grew on each plate, only 30 000 clones.Stage 6. Sowing replicas.The original colonies growing on each plate is transferred to 137 mm nitrocellulose filters by pressing the dry filter to the tops of the colonies and removing them from the plate. Prepare two identical replicas for each source filter according to the standard method of sowing replicas, in which each of the original filter carefully placed on the side of the colony with a sterile square of filter paper /Whatman 3MM/ leaving on a square piece of glass. New pre-wetted nitrocellulose filter gently place on top Snowmist with the first and second piece of glass. Numbered sandwich filters, and 3 holes of small diameter punch through them asymmetrically, so that you can accurately align in the future. Then remove the replica from the base and placed colony side of the new plate with L-broth agar containing tetracycline. Immediately prepare a second replica in the same way. Each main return filter on the plate and all plates are incubated at 37oC for several hours up until bacterial colonies reaches about 1 mm in diameter. The main source filters stored at 4oC and prepare reprice for hybridization as described below.Stage 7. Preparation of filters for hybridization.Each filter-replica /stage 6/ is placed on the side of the colonies on the filter paper Whatman 3 MM/ soaked in 0.5 M NaOH, 1.5 M NaCl for several minutes. The filters are transferred to neutralize on filter paper soaked in 1 m Tris, pH 7.5 1.5 M NaCl for 2 minutes, and then transferred to the second group of filters to neutralize within 5-10 minutes. Finally, the filters are placed on filters soaked SSC-buffer /0.015 M sodium citrate, 0.15 M NaCl, pH 7,4/ within 5 minutes, air-dried and heated in vacuum at 80oC for 1-2 hours.
PST / DNA /quick prep/ of these clones, indicating that 3 is close to full length. One of them was sekvenirovan. The sequence region that encodes a FGC, this clone is identical to the corresponding sequence pCSF-1 /and it has a T at position 365-CSF/Ile//.Example D (III). Cleaning FGC line Mo-cells.Incubated elaborate air-conditioned MO medium not containing serum /40 l/, when 55oC for 30 minutes to inactivate HTZV-II virus associated with cell line. The medium was concentrated by ultrafiltration under pressure using Pellicon the Gazette with PTGC membrane /1.5 square feet/, which has a segment with a molecular weight of 10000. Then the protein is concentrated by precipitation with ammonium sulfate /80% saturation/. Re-suspended the final ball of the protein /800 mg/ 100 ml 20 mm Tris /oximeter /aminomethane hydrochloride/Tris-HCl/ pH 7.4 and cialiswhat against the same buffer /3 times with 4 l of loading up every time/. Cialisovernight protein applied to the column 2.5 x 10 cm with DEAE /diethylamide activity 800 ml of 20 mm Trim-HCl, a pH of 7.4, containing 0.12 M NaCl. Collect fractions of 10 ml and tested for FGC. Combine active fractions /3/ and concentrate 6 times /up to 5 ml by ultrafiltration under pressure membrane Amicon UM, a segment with a molecular weight of 5000/. The concentrated sample from DEAE column applied to a column of 1.6 x 100 cm with AcA44 ultraglam/acrylamide-agarose ultragel with fractionation 10-130 to Dalton/, balanced 20 mm N-2-acetylpiperidine-N-2-econsultancy acid /HEPES / pH of 7.4, 50 mm NaCl and 0.01% polyethylene glycol /PEG-8000/. SFK activity elute from the column with an apparent molecular mass of 30 to Dalton. Combine active faction and lead up to 0.15 per cent /amount/volume/triperoxonane acid /TFWC/ add 10% TFUK and applied on a column of 1 x 25 cm with a video recorder C4 reverse phase. Column display with a linear gradient of 0-90% acetonitrile in 0.1% TFWC /the volume of/the amount of/ at 4 ml/min/1000 ml/. SFK activity eluted at approximately 47% volume/volume/acetonitrile/. The combined active fractions was adjusted to 0.05% volume/volume/heptacosanoic acid /HFMC/ adding half of the volume of 0.15 per cent/amount/volume/GMPC/ and put on a column /0,46 x 25 cm/ s video recorder C4, balanced 0,15% volume/volume/GFMC. Column display with a linear gradient of 0-90% /volume/volume/acetone is lonitrile. It was found that are active fractions 37-44 /1 ml each. Concentrate 0.15 ml fractions 40 to 4 times /using a SAVANT AIDS HAC Hub/ and add 40 μl of 2 × SDS gel sample buffer /0.125 M Tris-HCl, pH of 6.8, 4% SDS, 20% glycerol and 0,004% Bromphenol blue/. These samples boiled for 2 minutes and put on a 13.5% SDS gel /laemmli's method I., Nature 227, 680 /1970/ /see Fig.2/. It was determined that the fraction 40 has 110 000 units SFK bone marrow ml This corresponds to approximately 3,0107units on A280units of absorption. Because typical proteins have extinction coefficients in the range between 0.8 and 1.2 A280units/mg, purified FGC has a specific activity in the range of from about 1107to about 4107units/mg sample of bone marrow. Subjected to decomposition by Admino 1 mg sample of purified GM-FSK, using gas-phase microsequencer applied Biosystems. Was determined sequence of residues 3 to 5, which is the AIa Arg Ser.Example E (IV). Cotransformation and amplification FSK sequence in CHO cells. Plasmid p91023/B/-CSF, is introduced into CHO DHFR-deficient cells DICH-BII /Chasin and Urlaub PVAS 77: 4216, 1980/ by fusion of protoplasts, as described /Sandri-goldin et al. Mol. Cell Biol I, 743-752, 1981/. Growth and maintenance of CHO cells described /Kaufman and Si, containing 0.5% Kazimirovich acid, 0.5% glucose, 0,012% MgSO4, 5 μg/ml thiamine, and 10 μg/ml tetracycline until the absorption of 0.6 at 600 nm. Add chloramphenicol 250 ág/ml and incubate the culture at 37oC additional 16 hours in order to amplify the number of copies of the plasmid. Cells are centrifuged at 3000 g for 10 minutes at 4oC and suspended in 2.5 ml of chilled 20% sucrose in 50 mm Tris-Cl, pH 8.0. Add lysozyme /0.5 ml 5 mg/ml in 0.25 M Tris-Cl, pH 8.0 and the mixture was incubated on ice for 5 minutes. Add EDTA /1 ml of 0.025 M EDTA, pH 8.0/ and incubated on ice for another 5 minutes, then slowly added 1.0 ml of 0.05 M Tris-Cl, pH 8.0. Incubate the suspension for 15 minutes at 37oC up until the bacteria will not turn into protoplasts. Then, the suspension is slowly diluted with 20 ml of pre-warmed medium containing 10% sucrose and 10 mm MgCl2, and incubated for 15 minutes at 37oC. Solution of protoplasts /approximately 109/ml/ add to CHO DHFR deficient DTK-BII cells in the plate 6 holes /approximately 1106labels /dredging/ at a ratio of approximately 1-2104protoplasts/ cell and the protoplasts are collected in the ball into the cells by centrifugation at 2000 rpm for 8 minutedock layer suction. Add 2 ml of polyethylene glycol 50 g of PEO - 1450 /Baker Chem Co/ 50 ml of medium in each recess plate with 6 holes. The cells are again centrifuged at 2000 rpm for 90 seconds, remove solution of polyethylene glycol and the plates washed three times with 4 ml of medium/deepening. The cells are then trypsinized, suspended in 10 ml of medium containing 10% serum embryo calf, and centrifuged in a conical tube at 500 rpm in a clinical centrifuge. Balls of cells from the recesses 3 are combined and seeded into the Cup 10 cm tissue culture. To each plate, add fresh medium containing 100 μg/ml kanamycin, thymidine, adenosine, deoxyadenosine, penicillin and streptomycin and 10% cialisbuynow serum embryo calf. Kanamycin is injected to prevent the growth of any bacteria that have the ability to turn into protoplasts.After 2 days, cells will podculture 1:15 in alpha medium with 10% cialisbuynow calf serum, penicillin and streptomycin, but without nukes. The cells are then again served in the same selective environment /devoid of nucleoside/ 4-5 days.Colonies appear in 10-12 days after subculturing in the selective medium. Followed two selection schemes nasnavi expression of DHFR and grown consistently each clone under conditions for amplifying the number of copies of the above DNA, namely growth in increasing concentrations of methotrexate. In the second scheme isolate the pool from multiple independent transformants on the basis of DHFR expression and multiply together in amplification of the above DNA, namely the growth in increasing concentrations of methotrexate. Then isolate individual clones from the mass of selected populations and analyze GM-SFK expression. These clones showing the highest levels of expression, again grown in further amplification of the above DNA /namely growth in increasing concentrations of methotrexate in culture medium/.In one experiment, seven DHFR+transformed unite in alpha medium lacking nucleosides. These cells are sequentially grown while gradually increasing MTX concentrations ranging from 0.02 M, then the 0.1, 0.5 and 2,M MTX. When the sample of GM-SFK activity in KG-1 sample cells, these cells produce from 3000 to 12000 units/ml Selected population clone with 0.02 m MTX and 2.0 m MTX. The clones obtained in 0.5 m MTX O10, D2 and B6/ sequentially selects for growth in 2,M. MTX. When tested for GM-SFK activity in the sample in KG-1 cells, a cloned cell line produced from 15 000 to 300 000 units/ml GM-SFK activity is I FGC-Thr in Fig.1.Example G (X),
The expression of GM-FGC in E. coli.GM-FSK is expressed in E. coli from the vector pTALC.-185R, schematically described in Fig. 6. Sequence encoding a GM-FSK, begins with a synthetic sequence ATG.The CCA.The CCA.CCT.CCT.TCT. The CCA.TCT.The CCA.CTC.ACT, which defines the start II amino acid residues of the Mature GM-FSK. The rest of the sequence encoding GM-FSK, pTALC.-185R is identical sequence pCST-1, nucleotides 97-447 following sequence of TAR. TAR. TAG. Immediately after the triplet terminator has pUM-18 polylinker. The gene for resistance to tetracycline from pBP322 was inserted in the opposite orientation to SFK gene, 100 bases down from pUC-18 polylinker. The gene for resistance to tetracycline has its own view. Moving counterclockwise, you have the following gene-for-lactamase after pUC-18/CdZEI/ source replication.The final structural characteristic plasmids before returning to FGC sequences is the PL promoter. This promoter is essentially described Schatzmann and M. Rosenberg /in "Laboratory manual molecular cloning" /1982/, cold spring Harbor Lab, S. 419/. SFK expression is provided PL of promotoras design of all strains is a W 3110 IacIoL8 /R. Brent and M. Ptashne PNAS 78 /1981/ 4204-4208/.A fragment of DNA / nucleotides 34499 - 38214/ integrate into the chromosome W 3110 Iac1oL8 at IacL locus. The integration is performed with the use of vector integration, consisting of pBR325 sequences that carry genes for resistance to chloramphenicol and ampicillin and pBR322 replication source. /F. Bolivar, Gene, 4: /1978/, S. 121-136/. Insert DNA fragment in IacL gene, which is in the plasmid as a fragment extending from customers Bst Eilb LacI to customers IthillI down from lacL.Integration of DNA into the chromosomal copy of lacL is achieved by homologous recombination and find Iac+sensitive to ampicillin resistant to chloramphenicol colony. The second recombination case deletes all extra-plasmid sequences, but leads to the fact that the integrated DNA fragment screenrooms on lactose plates of Macconkey. In the second recombinant case, the original Iac+, amp, camPphenotype changes to Iac-, amps, camsthe phenotype. The resulting strain was named GZ400 and ispwhen 30oandswhen the 42oC. This phenotype demonstrates the existence of a functional x is SG20252 /lac. and 1169, ara 139 gr. sl lon , T h 10/ Th10 prepare skanirovaniya on Tetson the selective medium. /S. Maloy, W. Nunn, J. Bacterial. 145/1981/ 1110-1112/
The finished strain the owner called G. 1413 /Iac1oL8, LacZ / Cl, REX, N/, lon 100/.pTALC-185R, transported in G1413. The culture of this strain is grown overnight at 30oC in 5 ml of induction medium containing 7 μg/m tetracycline. Induction medium contains, per 1 liter: 20 g Kazimirovich acid, 6 g of Na2HPO47H2O, 3 g KH2PO4, 0.5 g NaCl, 1 g NH4Cl 1% glycerol, 2 mg of vitamin B1, 2 mg CaCl22H2O, 0.2 g MgCl26H2O.Inoculant 25 ml of this medium containing 7 μg/ml tetracycline, 125 µl of the overnight culture and shaken at 30oC in a water bath until then, until the culture reaches a density at A5500,5. Then quickly goes to the 40owater bath and shaken for a further 2 hours, to give the opportunity to the synthesis of GM-FSK. Harvest cells and test them on the content of FGC by electrophoresis on SDS-polyacrylamide gel. Under these conditions, GM-FSK accumulates to approximately 5% of the cellular protein.Example C (VI). The expression of GM-FSK Saceharomyus Cerevisial.A. Vector design.Construct a plasmid that contained the plasmid derives from any longer-the vip5 /Botstein et al, Gene, 8, S. S. 17-24 /1979// add fragment containing the source for replication from the 2 micron plasmid of yeast.B. Isolation of the gene for glyceraldehyde phosphate dehydrogenase /SPDH/.Isolated from the yeast two genes for GFDH /Holl and and Hol land Jarnal of Bidogical Chemistry 225, S. C. 2596-2605 /1980// Oligonucleotide probe synthesized from published sequences used for insulation GFDH gene from a plasmid library of yeast genomic DNA by standard methods. A plasmid containing a GAP491 gene was deposited earlier /ATCC N 39777/.C. Receiving promoter of glyceraldehyde phosphate dehydrogenase for the expression of heterologous gene.Construct a plasmid that allows the natural distribution of the GPDH promoter from the beginning of the target heterologous structural gene. This is done with the introduction of Kp I site in close proximity to the initiating machinenbau the codon GPDH structural gene. Then insert the promoter "cas sette in yeast expression vector YOpI.D. Isolation of the gene for factor.Of yeast isolated the gene for factor ripening pheromone /Kurjan and Herskowitz, Cell 30, S. S. 933-943 /1982//. Oligonucleotide probe synthesized from this ASS="ptx2">E. Obtaining the plasmid expression FGC.Design by standard methods of the above elements and the gene of human FGC expression vector /AJ 14, Fig. 7/. In this vector removed the natural leader sequence of FSK and inserted sequence encoding a Mature FGC, near the pre-Pro sequence of the factor. The connection between the GPDH promoter, pre-Pro sequence of the factor and the sequence of the Mature FGC updated /below/ and were confirmed deoxynucleotidyl sekvenirovanie. AAATAAACAAAATG.CGTTTTCCTTCA....... AAA AGA GAG GCG GAA GCT.GCA CCC GCC CGC TCG...F. Expression of GM-FSK
Plasmid AJ 14 transform into a strain Saccharowyces Cerevisial Cultured cells for the production of FGC. 1. A method of obtaining a protein with the activity of granulocytemacrophage colony stimulating factor (GM-CSF) primates, comprising culturing the host cells after transformation with the recombinant vector, which provides the expression in the selected cell type is embedded in the specified vector of the gene encoding GM-CSF primates, characterized in that for the transformation of host cells using recombinant vector with the gene coding for the polypeptide with the amino acid sequence below the first cell is a yeast cell.3. The method according to p. 2, characterized in that the said host cell is a prokaryotic cell.4. The method according to p. 1, characterized in that the said host cell is an E. coli cell.5. The method according to p. 1, characterized in that is included in a recombinant vector, a gene encoding GM-CSF primates with the amino acid sequence shown in paragraph 1 for CSF-Thr.6. The method according to p. 1, characterized in that is included in a recombinant vector gene encodes GM-CSF primates with the amino acid sequence shown in paragraph 1 for CSF-Ile.7. The method according to p. 1, characterized in that is included in a recombinant vector, a gene encoding GM-CSF primates with the amino acid sequence shown in paragraph 1 for CSF - G.
FIELD: genetic engineering, in particular production of human granulocyte colony-stimulating factor.
SUBSTANCE: Recombinant plasmid DNA pES3-7 with molecular weight of 3.63 MDa (5907 b.p.) is constructed. Said DNA consists DNA Ndel/Notl-fragment containing sequence of recombinant G-CSF artificial gene, β-lactamase gene; and plasmid pET22b(+) DNA Ndel/Notl-fragment containing promoter and terminator of T-RNA-polymerase transcription, amplifier of 17 phage 10 gene translation. Plasmid pES3-7 contains as genetic marker β-lactamase gene which determines resistance of E.coli cells transformed with plasmid pES3-7 to ampicillin, and unique restriction endonuclease recognition sites existing on the next distance to the right from Ndel-site: Xbal - 38 b.p.; Hpal - 1332 b.p.; Pstl - 4065 b.p.; Pvul - 4190 b.p.; Xhol - 5363 b.p. Obtained plasmid is used in transformation of Escherichia coli cells to produce strain E.coli BL21(DE3)/pES3-7 as subproducer of recombinant G-CSF. Method of present invention makes in possible to produce recombinant G-CSF with high yield (20-30 % based on total cell protein content).
EFFECT: simplified method for production of recombinant G-CSF with high yield.
2 cl, 2 dwg, 2 ex
FIELD: biotechnology, microbiology, genetic engineering.
SUBSTANCE: invention describes construction of recombinant plasmid DNA pFGM17 encoding constitutive synthesis of polypeptide of human granulocytic-macrophagal colony-stimulating factor (GM-CSF) and consisting of Kpn I/Eco RI-fragment of plasmid pSPF1 DNA and artificial DNA sequence encoding signal peptide of the protein Caf1 from Yersinia pestis, and also Kpn I/Eco RI-fragment of intermediate plasmid pSK-GM comprising the synthetic human gene GM-CSF. Escherichia coli cells are transformed with plasmid DNA pFGM17 and strain E. coli BL21(DE3)/pFGM17 is prepared that is a producer of human polypeptide GM-CSF. Invention provides enhancing technological effectiveness and economy of process for preparing recombinant FM-CSF due to excluding the induction stage in biosynthesis process and in simultaneous increasing the yield of the end product by 2 times. Invention can be used for preparing human granulocytic-macrophagal colony-stimulating factor.
EFFECT: valuable properties of plasmid DNA and microorganism strain.
2 cl, 4 dwg, 4 ex
FIELD: molecular biology.
SUBSTANCE: invention relates to isolated DNA fragment encoding horse GM-CSF, and isolated horse GM-CSF protein. Also disclosed are vectors and various compositions containing thereof. GM-CSF is useful as adjuvant for horse vaccination as well as non-specific immunity stimulator in veterinary.
EFFECT: new compositions for gorse vaccination.
18 cl, 2 dwg, 1 tbl, 7 ex
FIELD: medicine, endocrinology, pharmacy.
SUBSTANCE: invention proposes an agent eliciting antidiabetic activity. Agent represents nonglycosylated recombinant human granulocyte colony-stimulating factor. It differs from native preparation by absence of glycosyl group and the presence of additional methionine residue by its N-end. Effect of agent is associated with mobilization and migration of bone marrow mesenchymal stem cells, homing into pancreas. This results to reparation of insulin-producing activity of organ and normalization of the peripheral blood glucose level after monotherapy with recombinant human granulocyte colony-stimulating factor.
EFFECT: valuable medicinal property of agent.
3 tbl, 2 dwg, 1 ex
FIELD: gene engineering.
SUBSTANCE: invention can be used for production of recombinant polypeptide of human granulocyte colony-stimulating factor. Recombinant plasmid DNA is constructed in vitro. It includes synthetic gene of human granulocyte colony-stimulating factor, strong constitutive promoter A3 from the early stage of bacteriophage T7 and synthetic section - translation enhancer (TREN) of gene 10 in bacteriophage T7. This DNA in combination with high copy number of plasmid and optimisation of cultivation conditions ensures constitutive biosynthesis of target protein in transformed by this DNA race of Escherichia coli SGK25/pA3GF with high yield.
EFFECT: constitutive biosynthesis of target protein in cells; high yield.
2 cl, 4 dwg, 7 ex
SUBSTANCE: invention relates to field of biotechnology, namely, to obtaining genetically modified cell lines and can be applied in medicine for immunotherapy and immuno-prophylaxis in patients with malignant neoplasms. By means of recombinant method line of cells of human melanoma KG is obtained, which secretes recombinant granulocytic-macrofagal colony-stimulating human factor. Obtained line is deposited with Specialised cell culture collection of vertebrates of Russian cell culture collection under number RCCC ("П") 699"Д".
EFFECT: line of human melanoma cells KG possesses stable cultural, morphological and immunological characteristics and possesses ability to secrete recombinant human GM-CSF, remaining after cell inactivation with ionising irradiation.
SUBSTANCE: imiquimod or resiquimod is injected locally to a mammal over 12-26 hours after injection of nucleotide sequences encoding granulocyte-macrophage colony-stimulating factor and antigen peptide or protein.
EFFECT: significant amplification of immune response of mammal to antigen.
18 cl, 23 dwg, 1 tbl, 1 ex