Recombinant vascular endothelial growth factor (vegf) secreting escherichia coli bl21 (pvegf-a165) cell strain

FIELD: chemistry; biochemistry.

SUBSTANCE: invention pertains to biotechnology. In particular, the invention relates to an Escherichia coli BL21 (pVEGF-A165) strain and can be used to produce a vascular endothelial growth factor - GST-VEGF-A165 protein. A novel Escherichia coli BL21 (pVEGF-A165) cell strain is obtained, which is transformed by the pGEX-VEGF-A165 plasmid. This strain produces a recombinant GST-VEGF-A165 protein.

EFFECT: invention enables to obtain a Escherichia coli BL21 (pVEGF-A165) strain which is stably transformed by plasmid which codes VEGF, and which secrete this factor in extracellular space when cultured in vitro.

3 dwg, 4 ex

 

The technical field of the present invention

The invention relates to the field of Microbiology, molecular biology and biochemistry, in particular the production of a genetically modified strain of a microorganism used for the production of recombinant factor vascular endothelial growth (VEGF).

The prior art of the present invention

The presence of factors that stimulate the growth of blood vessels and thereby attracting newly formed vessels to supply the growing tumor, it was postulated several decades ago, although the identification and selection of these factors has long remained elusive. Currently, the growth factor vascular endothelial (VEGF), which was identified in the 1980-ies, is a key regulator of normal and abnormal growth of blood vessels. In 1993 it was shown that monoclonal antibodies against VEGF contribute to the dramatic suppression of tumor growth in vivo. This observation was the basis for the development of anticancer tools bevacizumab (Avastin (Avastin; Genentech)), gumanitarnogo version of this antibody against VEGF. The recent approval of bevacizumab US FDA as a therapeutic agent of the first line for metastatic colorectal cancer confirms the notion that VEGF is a key mediator of tumor angiogenesis and that blocking angiogenesis is an effective strategy for the treatment of malignant tumors in humans.

The observation that tumor growth may be accompanied by enhanced vascularization, was published over a century ago (an overview can be found in reference (1). However, only in 1939 Ide et al. first postulated the presence of a tumor factor stimulating the growth of blood vessels, encouraging the blood supply to the growing tumor (2). A few years later, based on the observation that rapid tumor growth is preceded by an increase in the local density of the vascular network, Algire et al. suggested that the rapid growth of tumor grafts depends on the formation of branched vascular network (3). Then new messages in this area of technology did not appear until the 1960's, when the experiments Greenblatt and Applications (4) and Ehrmann and Knoth (5) were obtained the first evidence that tumor angiogenesis is mediated capable of diffusion factors produced by tumor cells.

In 1971, Folkman hypothesized that inhibition of angiogenesis could be an effective anticancer strategy (6). On the basis of this revolutionary hypothesis in the early 1970-ies Folkman et al. undertook efforts on the selection of tumor angiogenesis factor" of tumors of humans and animals (7). In 1978 Gullino also suggested that blocking angiogenesis can prevent the development of zlocesto the Neu tumors (8). Were subsequently described angiogenic effects of many factors (e.g. epidermal growth factor (EGF), transforming growth factor (TGF)-α, TGF-β, factor-α tumor necrosis (TNF-α) and angiogenin) (9). However, although these factors increased angiogenesis in the course of some biological analyses, none of them acted physiologically (10).

The lion's share of attention was paid to the two widespread and highly effective mitogen of endothelial cells and angiogenic factors, acidic and basic fibroblast growth factors (aFGF and bFGF). In the mid 1980-ies have been conducted clean-up state of homogeneity, sequencing and cDNA cloning of these FGF (11). An unexpected finding was that the genes as aFGF and bFGF does not encode standard secretory signal peptide. However, as noted above, earlier studies suggested that tumor angiogenesis is mediated able to diffusion of molecules (4, 5). In addition, some studies have attested to the fact that immunoneutralization FGF had no effect or had a weak effect on tumor angiogenesis (12, 13), which suggests that the key regulators of angiogenesis have not yet been identified.

It is now known that one such key regulator of angiogenesis is an endothelial growth factor is Asadov (VEGF, also called VEGF-A), and the role of the VEGF gene family in the regulation of angiogenesis studied intensively for over a decade (1). The VEGF family includes the prototype member of the VEGF-A, placental growth factor (P1GF) (14), VEGF-B (15), VEGF-C (16) and VEGF-D (17). Convincing evidence suggests that while the creation and maturation of the vascular wall are highly complex processes that require concerted effects angiopoietins, platelet-derived growth factor B (PDGF-B) and other factors (18), the effect of VEGF-A is a limiting speed stage of normal and abnormal growth of blood vessels (19). Importantly, VEGF-C and VEGF-D regulate angiogenesis lymphatic vessels (20), which emphasizes the unique role of this family of genes in controlling the growth and differentiation of some anatomical components of the vascular system.

In 1983, Senger et al. reported partial allocation of the environment, air-conditioned tumor cell line of the Guinea pig, "factor vascular permeability" (VPF), a protein that is induced increase in vascular permeability of the skin (21). However, since the VPF has not been isolated and sequenced, at that time, the molecular structure factor remained unknown. In 1989 it was reported that the allocation of the environment, air-conditioned follicular and cells of the pituitary gland of the cow, "factor vascular endothelial growth" (VEGF), specific endothelial cells mitogen (22). Aminobenzene amino acid sequence of VEGF did not coincide with any known protein in the available databases (22). Then Connolly et al., made on the basis of Senger et al., independently led to the isolation and sequencing VPF (23). Cloning of cDNA of VEGF (24) and VPF (25) showed that VEGF and VPF are one and the same molecule. This result was unexpected, given that other mitogen of endothelial cells, such as FGF, do not increase the permeability of blood vessels.

VEGF is characterized by significant homology with chains and PDGF (24). The gene encoding VEGF-A person, consists of eight exons separated by seven introns (26, 27). Alternative splicing of exons leads to the formation of four major isoforms - VEGF121, VEGF165, VEGF189 and VEGF206), made up after removal of the signal sequence, respectively, of 121, 165, 189 and 206 amino acids (24). Alternative splicing regulates the bioavailability of VEGF (28, 29). By this time gathered a lot of evidence that VEGF165 is the most physiologically competent isoform (30, 31). Also in the regulation of the bioavailability of VEGF plays an important role in extracellular proteolysis. The plasmin is able to cleave VEGF165 and VEGF 189 and release biologically active product comprising the C first 110 aminobenzene amino acids (32). Given the importance of the plasminogen activation during physiological and pathological angiogenesis (33), this mechanism may be particularly important for regulating the activity and bioavailability of VEGF in the development of remodeling in response to signals from the microenvironment. In addition, some tumors VEGF proteolysis mediated matrixes metalloproteinase-9 (MMR), may be responsible for triggering angiogenesis (34).

Reliably established by action of VEGF is to accelerate the growth of endothelial cells of arteries, veins and lymphatic vessels (an overview is given in reference 35). VEGF induces potent angiogenic response in a variety of models in vivo (24, 36). In addition, as mentioned above, VEGF increases vascular permeability, and this property underlies the important role of this molecule in inflammation and other pathological processes (37). In this context, VEGF also induces the expression of the endothelial some adhesion molecules that regulate leukocyte adhesion during inflammation (38).

In vitro VEGF prevents apoptosis of endothelial cells induced by depletion of serum that is mediated by a cascade of phosphatidylinositol 3'-kinase (PI3K)/Akt (39). Also, VEGF induces the expression in endothelial cells antiapoptotic proteins BCL-2 and A1 (40). Dependence on VEGF has been shown in endothelial cells of newly formed, but not fully formed blood vessels the tumor is th (41, 42). Education pavements by pericyte, presumably, is one of the key events leading to the loss of endothelial cells based on VEGF (42).

It should be emphasized that although endothelial cells are the main targets VEGF, several studies have shown its effects on mitotic activity/survival of some endothelially cell types, including neurons (43).

VEGF is crucial for normal embryonic vasculogenesis and angiogenesis. Inactivation of a single VEGF allele in mice leads to embryonic mortality (44, 45). Inhibition of VEGF in the early neonatal period results in the cessation of growth, apoptosis of endothelial cells and lethal outcome is mainly the result of renal failure (46, 47). VEGF is also necessary for vnutridomovogo of ossification, the fundamental mechanism of growth of tubular bones. Inhibitors of VEGF inhibit these processes in rodents and primates (48, 49). It is important to note that this effect is completely reversible upon cessation of anti-VEGF treatment (48, 49). Angiogenesis is a key aspect of normal ovarian-menstrual cycle and functioning endometrium (50). The expression of VEGF mRNA is associated in time and space with the proliferation of blood vessels in the ovaries of many species (51, 52). Injection of VEGF inhibitors slows down the development of the follicular the crystals (53) and inhibits angiogenesis in yellow rodents (54) and primates (49, 55, 56).

Research in situ hybridization suggest that VEGF mRNA expression in many human cancers, including carcinomas of the lung (57), breast cancer (58), gastrointestinal tract (59), kidneys (60) and ovarian cancer (61). However, the expression of VEGF varies not only between different types of tumors, but also within a single tumor. In the case of polymorphic glioblastoma and other tumors with significant necrotic component of the expression of VEGF mRNA is most intense in hypoxic tumor cells adjacent to areas of necrosis (62, 63). Tumor with particularly strong expression of VEGF is renal cell carcinoma. Positive regulation of VEGF expression may be related to a number of factors, including hypoxia and some mutations.

Clinical applicability of VEGF inhibition in Oncology are not limited to solid tumors. There is growing evidence that VEGF and its receptors are expressed by many malignant tumors of blood, suggesting the prospect of inhibiting VEGF or signal transmission VEGFR for the treatment of such conditions (64).

The disclosure of the present invention

The essence of the present invention is the creation of a new strain of Escherichia coli cells BL21 (pVEGF-A165)producing recombinant growth factor vascular endothelial - protein ST-VEGF-A165. The ability of cells of Escherichia coli BL21 (pVEGF-A165) to secrete recombinant VEGF is achieved by genetic modification of Escherichia coli cells with plasmid pGEX-VEGF-A165 (SEQ ID NO: 1).

The technical result is to obtain a strain of Escherichia coli cells BL21 (pVEGF-A165), stably transfected with a plasmid that encodes a VEGF, and secreting this factor in the extracellular space during cultivation in vitro.

The aim of the present invention is an expanding collection of unique cell strains that can be used for VEGF. This task is particularly relevant for modern pharmaceutical biotechnology. Accumulated the specified strain VEGF can be used in many areas of biological and medical research, in particular in the creation and study of drugs and other compounds that are agonists or antagonists of VEGF.

The problem is solved in that the new strain of Escherichia coli cells BL21 (pVEGF-A165), transformed with the plasmid pGEX-VEGF-A165 (SEQ ID NO: 1) and producing a recombinant protein GST-VEGF-A165 in the amount of not less than 2 µg of purified recombinant protein per 1 ml of growth medium for 24 hours (according to the ELISA). The resulting strain has a stable cultural and morphological properties and the genetic modification has the ability to secrete re ominantly growth factor vascular endothelium. The strain of Escherichia coli cells BL21 (pVEGF-A165) deposited in Russian national collection of industrial microorganisms (VKPM) FSUE gosniigenetika number VKPM B-10042.

Brief description of drawings

Figure 1 shows the genetic map of the plasmid pGEX-VEGF-A165. Figure 2 shows the result of analysis of fractions collected during purification of the recombinant protein GST-VEGF-A165. Proportionate amounts of protein from each fraction was analyzed using LTO-PAGE 10%acrylamide gel, followed by staining of the gel dye Coomassie Brilliant Blue (Serva). For estimation of molecular weight of proteins used marker of molecular weight Unstained Protein Molecular Weight Marker production (Fermentas), extreme right track. The arrow indicates the position in the gel of the recombinant protein GST-VEGF-A165. The apparent molecular mass of the protein GST-VEGF-A165 is approximately 45 kDa and corresponds to theoretically calculated (45526 Yes).

Figure 3 shows the result of analysis of the stability of recombinant protein GST-VEGF-A165 in the process of dialysis and freeze-thawing. Proportionate amounts of protein from each fraction was analyzed using LTO-PAGE 10%acrylamide gel, followed by staining of the gel dye Coomassie Brilliant Blue (Serva). For estimation of molecular weight of proteins used marker of molecular weight Unstained Protein Molecular Weight Marker production (Fermentas), the outermost track to the left. P the LCA indicates the position in the gel of the recombinant protein GST-VEGF-A165. Protein GST-VEGF-A165 stable during dialysis and can withstand freezing in liquid nitrogen and subsequent thawing.

The embodiments of the present invention

Example 1. The procedure for preparation of recombinant plasmid DNA that encodes a protein cDNA of VEGF-A165, fused with protein glutathione-8-transferase (GST-VEGF-A165)

cDNA human VEGF amplified by PCR using as template the first chain cDNA from human placenta. The primers were selected so that of the VEGF cDNA was deleted signal peptide (amino acids 1-26), and 5'-primer [5'-CAT GGA TCC GCT GCA CCC ATG GCA GAA GGA-3'] and 3'-primer [5'-GTC ACC CGG GTC ACC GCC TCG GCT-3'] contained the recognition sites of the restriction endonucleases BamHI and SmaI, respectively. Next BamHI-SmaI fragment cloned in the corresponding sites in the plasmid vector pGEX-4T2 (Amersham) so that the cDNA of the protein VEGF-A165 were in the same reading frame with the cDNA of the protein GST.

The obtained plasmid construction was about the size 5462 gel (figure 1). Recombinant protein GST-VEGF-A165 encoded by this plasmid had the size of 392 amino acids and a calculated molecular mass 45526 Yes.

Example 2. The procedure of getting a bacterial strain E. coli BL21 producing recombinant protein GST-VEGF-A165

The plasmid vector pGEX-VEGF-A165 transformed into E. coli strain BL21 using the standard methods of transformation of bacteria (65) and were sown on LB agar (0171 M NaCl, 1% tripton, 0,5% yeast extract, 1.5% agar)containing selective antibiotic ampicillin at a concentration of 50 μg/ml, and grown for 16-18 hours at 37°C. Single colonies of bacteria growing on the selective medium was perseval on fresh selective Cup and grown for 16-18 hours at 37°C. Petri dishes bacterial loop took a single colony of bacteria and inoculable 10 ml of liquid growth medium LB (0,171 M NaCl, 1% tripton, 0,5% yeast extract)containing the selective antibiotic ampicillin at a concentration of 50 μg/ml, and grown for 16-18 hours at 37°C and constant stirring. Take 2 ml of bacterial suspension, the cells were besieged by centrifugation, the supernatant was removed, and the residue resuspendable 1.5 ml of medium for freezing (liquid LB medium containing 15% glycerol). The resulting strain-producer kept at a temperature of -80°C.

Example 3. The procedure for obtaining the recombinant protein GST-VEGF-A165 in the bacterial expression system

The strain producing the recombinant protein GST-VEGF-A165 were sown stroke on a Petri dish containing LB agar containing the selective antibiotic ampicillin at a concentration of 50 μg/ml, and grown for 16-18 hours at 37°C. Petri dishes took a single colony of bacteria was inoculable 10 ml liquid LB medium containing 50 μg/ml ampicillin, and grown in the pic is a constant state of alert stirring and at a temperature of 37°C for 16-18 hours.

5 ml overnight culture of the producer strain was transferred with a sterile pipette into two 2-liter flasks containing 200 ml of liquid LB medium, and grown under constant stirring and at a temperature of 37°C for 2.5 hours, after which induced the expression of the recombinant protein by adding isopropyl-β-D-thiogalactoside (Fermentas) to a final concentration of 0.02 mm and the bacteria were grown at 28°C for 1.5 hours.

Cells were besieged by centrifugation at 4000 rpm and 4°C for 10 minutes, supernatant was removed, and the residue was washed with 40 ml of phosphate-saline buffer (0.01 M sodium phosphate, was 0.138 M NaCl, 0.0027 M KCl, pH 7.4), pre-cooled to 4°C, and then the cells were besieged by centrifugation at 4000 rpm and 4°C for 10 minutes. The supernatant was removed, and the precipitated cells resuspendable in 10 ml of buffer (0.01 M sodium phosphate, was 0.138 M NaCl, 0.0027 M KCl, 2.5 mm MgCl2, 1 mm EDTA, 0.05% of NP-40, pH 7.4) and added crystalline lysozyme (Sigma) to a final concentration of 10 μg/ml of the bacterial suspension cells were frozen in liquid nitrogen and kept at a temperature of -80°C until protein purification stage.

A suspension of bacterial cells was slowly thawed on ice and subjected to ultrasound at a temperature of 0°C by means of the device Vibracell 72434 (Scientific bioblock is used) until such time as the suspension is not shone and wescast have not disappeared (the duration of the ultrasonic pulse was 60 seconds between pulses had to take breaks at least 60 seconds; conduct at least 3 pulses).

Bacterial lysate was transferred into a chilled microcentrifuge tubes (Costar) with a capacity of 1.5 ml and centrifuged in a bench top microcentrifuge at 13,000 Rev/min and 4°C for 15 minutes. The clarified lysate was collected and mixed with 500 µl 4Fast Flow glutathione sepharose (Amersham Biosciences)pre-equilibrated chilled phosphate-saline buffer.

Then for 1.5 h at 4°C and stirring was performed binding of the recombinant protein GST-VEGF-A165 with glutathione by separate. After binding the lysate at 4°C was applied on the column, mechanically inhibiting glutathione-sepharose, and allow the lysate to flow through the column under gravity. Then at a temperature of 4°C. glutathione-sepharose once washed with 10 ml of buffer (0.01 M sodium phosphate, was 0.138 M NaCl, 0.0027 M KCl, 2.5 mm MgCl2, 1 mm EDTA, 0.05% of NP-40, pH 7.4) and twice with 5 ml of phosphate-saline buffer.

Contacting glutathione-separate recombinant protein GST-VEGF-A165 three suirable 500 ál of buffer for elution (50 mm Tris, pH 8.0, 10 mm restored glutathione), incubare glutathione-sepharose in the buffer for elution for 10 minutes at room temperature and constant stirring using Atomics (10 rpm).

At every stage of purification protein fractions were collected and analysis is Aravali using LTO-PAGE 10%acrylamide gel (66). The result of the analysis of fractions collected during purification of the recombinant protein GST-VEGF-A165, shown in figure 2.

Suirvey recombinant protein were dialyzed at 4°C against phosphate-saline buffer, frozen in liquid nitrogen and kept at -80°C in the aliquot. To check the stability of the protein GST-VEGF-A165 an aliquot was thawed and the concentration of recombinant protein was analyzed by the method of the LTO-PAGE. The result of the analysis of the stability of the recombinant protein GST-VEGF-A165 in the process of dialysis and freeze-thawing is shown in figure 3.

Example 4. The procedure for determining the concentration of the recombinant protein GST-VEGF-A165.

The concentration of recombinant protein GST-VEGF-A165 were determined by enzyme-linked immunosorbent assay (ELISA) using the set for ELISA determination of protein VEGF to Human VEGF ELISA Development kit production PeproTech in accordance with the Protocol of the manufacturer.

Antibody binding was diluted in phosphate-buffered saline to a final concentration of 0.5 μg/ml, 100 µl of this solution was added to the wells of the 96-hole tablet ELISA plate, and incubated at room temperature for 16-18 hours. The antibody solution was removed and the wells washed four times with 300 μl of buffer for washing (phosphate-saline buffer containing 0.05% Tween-20). The remains of the buffer for rinsing thoroughly removed, the wells were added 300 μl of buffer for blocking (1% BSA in ostatni-buffered saline) and the plate incubated at room temperature for 1 hour. The solution is to block was removed and the wells washed four times with 300 μl of buffer for washing.

Protein standards (in a concentration of from 0 to 4 ng/ml) and samples of 100 μl were added to wells on 3 samples in parallel and incubated at room temperature for 2 hours.

Next, the samples were removed and the wells washed four times with 300 μl of buffer for washing. Antibodies for detection were diluted in buffer for cultivation (phosphate-saline buffer containing 0.05% Tween-20 and 0.1% BSA) to a final concentration of 0.25 μg/ml, 100 μl was added to wells and incubated at room temperature for 2 hours.

The antibody solution was removed, and the wells were washed four times with 300 μl of buffer for washing. 6 ál avicenniaceae bred in the buffer for dilution rate of 1:2000, to each well was added 100 μl of this solution and incubated at room temperature for 30 minutes.

The solution avicenniaceae was removed, and the wells were washed four times with 300 μl of buffer for washing. To the wells were added with 100 μl of liquid ABTS substrate and incubated at room temperature until color development.

Measurement of the optical density of the samples in the wells was carried out at a wavelength of 405 nm and a correction wavelength, 650 nm, using a microplate reader (Multiscan EX (Labsystems).

The main characteristics of the cells (according to passport)strain/p>

1. Generic and species name of the strain of the host (recipient): Escherichia coli

2. The number or name of strain: BL21 (pVEGF-A165)

3. The method of obtaining strain: obtained by the transformation.

4. The product synthesized by the strain: GST-VEGF-A165.

5. Activity (productivity) strain: 2 µg of purified recombinant protein per 1 ml of growth medium according to the ELISA.

6. The method, conditions and medium composition for long-term storage of strain: Wednesday LB+15% glycerol in vials at -80°C.

7. The method, conditions and medium composition for growth of strain: the LB medium containing 50 μg/ml ampicillin).

8. Group level risk strain: I

9. Genetic strain:

A. The genotype of the host strain (mutations, deletions, inversion, the presence of plasmids or Protasov, resistance (sensitivity) to antibiotics, phages, etc., other genetic characteristics):

[F-, ompT, hsdSB (r-B, m-B), dcm, gal].

B. Description recombinant plasmids:

The name of plasmids: pGEX-VEGF-A165.

The size of plasmids: 5462 pairs.

C. Information about the vector, on the basis of which the constructed plasmid:

The name of the vector pGEX-4T2.

The size of the vector: 4970 BP

The complete nucleotide sequence of the vector pGEX-4T2: SEQ ID NO: 2.

The origin of the vector and its major genetic elements:

the vector contains

- gene resistance to ampicillin (bla);

- the gene encoding the enzyme glutathione S-t is ansfers from Schistosoma japonicum,

gene lad repressor from E. coli;

- the replication origin of the plasmid pBR322 from E. coli.

, Information about the cloned DNA:

The species of the donor organism: Homo sapiens

The size of the cloned fragment and included genes: 501 P.N.;

a fragment of the VEGF cDNA (GenBank No. NM_003376).

Literature

The strain of Escherichia coli cells BL21 (pVEGF-A165), transformed with the plasmid pGEX-VEGF-A165 (SEQ ID NO: 1) and producing a recombinant protein GST-VEGF-A165, deposited in Russian national collection of industrial microorganisms (VKPM) under number VKPM B-10042.



 

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FIELD: microbiology.

SUBSTANCE: glia cells are extracted, seeded, and cell is cultivated to produce monomolecular layer. Then cells are extracted and washed. Total RNA is extracted from them, reverse transcription is carried out, as well as amplification of produced DNA. Genes expression is assessed under action of tested compound.

EFFECT: invention makes it possible to accelerate screening of compounds, to search for new compounds with highest activity, which are potential neuroprotectors.

4 cl, 4 dwg, 1 tbl, 4 ex

FIELD: medicine.

SUBSTANCE: there is described method for preparing a polymorphous region of gene PAR1 which can contain T to C replacement in position 3090 and/or A to C replacement in position 3329 of the polynucleotide sequence of wild type gene (NM_001992) with applying a pair of specific primers, and also the method for observing said region prepared of a DNA-containing biological sample for the presence or absence of the specified replacements. There are offered complete sets of the components application of which provides both amplification of the polymorphous region of gene PAR1 under the invention, and if needed, further analysis for genetic modifications in positions 3090 and/or 3329.

EFFECT: higher accuracy of estimating risk of cardiovascular diseases.

7 cl, 17 dwg, 1 ex

FIELD: biotechnologies.

SUBSTANCE: invention is related to the field of biotechnology and immunology. Separated and cleaned DNA is presented, which codes receptor CTLA-4 (CD 152) of cat. The following is also suggested - diagnostic oligonucleotide, cloning vector, vaccine, methods of induction, strengthening and suppression of immune response in cat.

EFFECT: creation of model cat for research of retroviral infection.

24 cl, 10 dwg, 6 tbl, 8 ex

FIELD: medicine.

SUBSTANCE: genetically modified microorganisms having DNA encoding polypeptide involved in biosynthesis of macrolide compound of pladienolida, and DNA encoding polypeptide having hydroxylase activity aimed at pladyenolides hydroxylation in 16-position are proposed. The invention enables to produce effectively macrolide compound hydroxylated in the 16-position, directly in a single microorganism.

EFFECT: obtained compound has a high antitumor activity and can be used to develop anticancer drugs.

17 cl, 4 dwg, 7 tbl, 18 ex

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