* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Kretsu Anna PFUR
Biochemical cascade wikipedia , lookup
Biosynthesis wikipedia , lookup
Gene therapy wikipedia , lookup
Biochemistry wikipedia , lookup
Molecular cloning wikipedia , lookup
Signal transduction wikipedia , lookup
Gene expression wikipedia , lookup
Protein–protein interaction wikipedia , lookup
Transformation (genetics) wikipedia , lookup
Gene therapy of the human retina wikipedia , lookup
Paracrine signalling wikipedia , lookup
Silencer (genetics) wikipedia , lookup
Gene regulatory network wikipedia , lookup
Point mutation wikipedia , lookup
Expression vector wikipedia , lookup
Endogenous retrovirus wikipedia , lookup
Vectors in gene therapy wikipedia , lookup
Genetic engineering wikipedia , lookup
Two-hybrid screening wikipedia , lookup
* *Genetic Engineering is a set of methods, techniques and technologies for producing recombinant RNA and DNA isolation of genes from an organism (cell), the manipulation of genes and introducing them into other organisms. Genetic engineering is used to obtain the desired qualities of a variable organism. *Red biotechnology [Gr. bios - life, techne - art, skill and logos - teaching] - the use of biotechnological processes and techniques in medicine, for example, creating in biopharmaceuticals (proteins, enzymes, antibodies), as well as gene and cell therapy. The term “RB" was proposed in 2003 at the World Forum on the biological sciences. *Protein engineering [French. engenier - engineer, from Lat. ingenium - the ability, ingenuity] is a set of genetic engineering and biochemical techniques to help creating recombinant proteins and carry out the modification of physico-chemical and biological properties of natural proteins in order to improve their quality. Replacing individual amino acids or a combination of large blocks of polypeptide chains (domains) of different proteins are used in biotechnology for the production of proteins with new properties. Examples of methods used in BI is mutagenesis in vitro, allowing to change the ability of a gene encoding a specific location (eg, within a region encoding active site of the protein). * * Protein of Plasmodium falciparum * Protein shell NIV-1 * Capsid protein NIV * The protein of hepatitis C virus * Antigens NIV -1 * Antigen anthrax * Antigen dengue virus type I DIAGNOSTICS DRUG SUBSTANCE * Epidermal growth factor * insulin * Insulin-like growth factor * Platelet-derived growth factor * Fibroblast Growth Factor * Factor 13 blood clotting * interferons * erythropoietin * somatotropin * Bacillius brevis * Bacillius subtilis * Corinebacterium spp. * Erwinia spp. * Escherichia coli * Pseudomonas spp. * Streptomyces spp. * Saccharomyces cerevisiae * * 1.Microorganism-recipient should possess pathogenicity and toxigenicity. * 2. Safety of genetically engineer derivatives. * 3. Quickly breeding strains. * 4. Wanted growth on simple nutrient media. * 5. Ability to get suspensions of high density. * For introduction of foreign DNA into microorganisms require special carriers. * * 1)plasmids; * 2) the different types of bacteriophages (in particular this ( bacteriophages and M13); * 3) cosmids; * 4) phagemids, phasmids; * 5) the variety of Ultra-vectors (the so-called "artificial“ chromosomes (AC) * Y A C B A C P A C - P 1 Yeast Bacterial Phaqe-derived Artificial Artificial Chromosomes Artificial Chromosomes Chromosomes - yeast bacterial artificial chromosome - artificially produced artificially created bacteriophage chromosome mini-chromosome a M n d " e x t A C Mammilian Artificial Chromosomes – animals’ artificial chromosomes r a - l H o n g " : A C Human Artificial Chromosomes - artificially produced human chromosome * * Additional factors of heredity, located outside of the chromosomes in the cells and is a ring (closed) or linear DNA molecule. * * 1) must be big enough to contain stretches of foreign DNA; * 2) should be replicated in a host cell; * 3) must have a recognition site (the site) for the enzyme - a restriction enzyme; * 4) should be any mark (marker), allowing further away recombinant microorganisms (e.g., an antibiotic resistance gene). * * * 1.Restriction * Plasmid is cut with restriction enzyme. Restrictase is an endonuclease cutting two-chained DNA in the field of well-defined sequence of nucleotides. The same enzyme cuts the gene. Since the enzyme acted with the same substrate specificity at the ends of the plasmid and the gene formed complementary sequences ('sticky ends"). * 1) class I restrictases DNA break at arbitrary points I and III restriction enzyme classes have methylasing and endonuclease activity. * 2) class II enzymes, which are used in genetic engineering, consisting of two separate proteins: restriction endonuclease and methylase modifier. * more than 400 different restriction enzymes are currently used . These enzymes are synthesized by a variety of microorganisms. * For their cultivation optimum conditions (temperature, composition and pH, oxygen concentration, etc.) is required. * 2. Introduction and ligation. * Conduct joint incubation cut plasmids and gene. Due to the "sticky ends" gene can be incorporated into a plasmid with the formation of noncovalent hydrogen bonds between complementary bases. Add the enzyme DNA ligase, it crosslinks between a gene and a plasmid to form a covalent phospho-ester bonds. The recombinant plasmid (plasmid vector). All cloning systems must meet two basic requirements: *1) Presence of multiple sites for cloning; *2) Possibility of simple identification of cells with recombinant DNA. All routine procedures for molecular cloning in E.coli is widely used as a host cell. * * • Insulin is a peptide hormone secreted by the Langengars β- cells. * • It consists of two peptide chains: A chain - 21 amino acid residues. B-chain containing 30 amino acid residues * • The two chains are bonded by disulphide-SS-bonds which provide the spatial structure of the protein insulin. * • Insulin precursor is proinsulin. * • Proinsulin consists of A-chain, Bchain and the C peptide consisting of 35 amino acid residues. * • C-peptide is cleaved by the action of trypsin and carboxypeptidase and proinsulin transits into the active insulin. Insulin was first medicament recombinant preparation obtained on an industrial scale in 1982 is known, insulin is widely used in the treatment of insulin dependent forms of diabetes. * 1) Lack of livestock; * 2) Storage and transportation of raw materials; * 3) Difficulty of isolation and purification of the hormone; * 4) Possibility of allergic reactions. * * * 1) Modification of porcine insulin by synthetic-enzymatic method Porcine insulin differs from human insulin by replacement of a Cterminus of the B-chain Ala30Thr. Replacement of threonine by alanine is carried out by enzyme-catalyzed cleavage of alanine and protected connection instead of the carboxyl group of threonine residue present in the reaction mixture in a large excess. After deprotection of O-tert-butyl group human insulin is obtained. * 2) Genetic engineering (2.1) it is carried out separately (by various producer strains) receiving both chains followed by folding of the molecule (the formation of disulfide bridges) and separation of isoforms. (2.2) insulin is received in the form of precursor (proinsulin) followed by enzymatic digestion with trypsin and carboxypeptidase B to form the active hormone. In 1975 U. Gilbert proposed the following scheme for the synthesis of insulin: * • Insulin mRNA is secreted from the pancreas tumor cells. * • Using reverse transcriptase of mRNA cDNA is produced. * • The resulting cDNA is inserted into plasmid pBR322 E. Coli in the middle part of the gene of penicillinidase. * • Recombinant plasmid contains information about the structure of proinsulin. * • Result of translation of the mRNA is synthesized in the cells a hybrid protein comprising the sequence penicillinidase and proinsulin. * • Proinsulin is cleaved from the protein by trypsin. * • From the proinsulin insulin is released. * * Using E. coli two chains of human insulin were synthesized. Then they were linked to the molecule of biologically active hormone. To a singlecelled organism can synthesize on its ribosomes insulin molecule, it is necessary to provide the desired program, then he has to enter the hormone gene. * By chemical method the gene of the insulin precursor biosynthesis programming or two genes separately programmed in the biosynthesis of A and B chains of insulin is produced. * The next step is the inclusion of the insulin precursor gene (or genes chains separately) into the genome of E. coli - a special strain of E. coli grown in the laboratory. This task is performed by genetic engineering. * Plasmid is isolated from E. coli by appropriate restriction enzyme. Synthetic gene is incorporated into a plasmid (cloning a functionally active C-terminal portion of β-galactosidase E. coli). As a result E. coli acquires the ability to synthesize a protein chain consisting of insulin and galactosidase. The synthesized polypeptides are cleaved from the enzyme chemically, and then are cleaned. Approximately 100,000 molecules of insulin are synthesized in the bacterial cell. Escherichia coli At the Institute of Bioorganic Chemistry, Russian Academy of Sciences recombinant insulin (insuran) was obtained using genetically engineered strains of E.coli. From the grown biomass is allocated precursor fusion protein is expressed in an amount of 40% of total cell protein containing preproinsulin. Turning it into insulin in vitro is carried out in the same sequence as in vivo - split off leading polypeptide preproinsulins converted into insulin through the oxidative sulfitolise followed by reductive closure of three disulfide bonds and enzymatic isolation of the binding of C-peptide. After several chromatographic purifications, including ion exchange, gel and HPLC to give human insulin purity and natural activity. * "+": * • the composition is identical to human insulin → No allergic reactions. * • more economical as compared with an animal insulin (1 kg insulin can be obtained by fermenting vat 25 by using E. coli, or to 35 thousand units a / c animals. "-": * • Careful monitoring of isolation and purification, as admixture of microbial lipo-and glycoproteins have pyrogenic properties * * is secreted by the anterior pituitary and contains 191 amino acid residues. In humans, this hormone is essential for the growth of bone. In utero it is not needed, however, his failure dramatically manifested in late childhood and leads to dwarfism. * The synthesis and secretion of GH are made by highly specialized cells of the pituitary gland - somatotrophes. GH secretion is regulated by hypothalamic structures by releasing hormones to be stimulated (Somatoliberin) or inhibitory (somatostatin) effect. Recently a new physiological system, which controls heart rate secretion of GH releasing peptides– is discovered. The secretion of hypothalamic hormones, in turn, is controlled by several neurotransmitter systems (cateholaminergic, serotonergic, dopaminergic), has a stimulating or inhibiting effect. * 1 Fermentation * 2 primary purification of the protein * 3 chromatographic purification * 4 Formulation * 5 analysis of the quality of API. Recombinant protein factors of innate immunity Types of interferons: * 1.L-group (leukocyte interferon) * 2.B-group (fibroblast interferon) * 3.G-group (immune interferon T-lymphocytes) * Until recently, interferon from human cells have been available only in small quantities. As a medical drug used leukocyte interferon. It served as a source of blood, obtained from the maternity hospitals. Currently gene of leukocyte interferon is produced by chemical synthesis. Then it was incorporated into plasmid, which in turn, was introduced into E. coli cells and yeast cells became producers of human leukocyte interferon. * Hematopoietic growth factor, is a glycoprotein with the properties of hormone physiological role of which consists in regulating the production of red blood cells depending on the need for oxygen. A method for production of recombinant erythropoietin has an important feature - the human erythropoietin gene is not incorporated into microbial, and animal cells (chinese hamster ovary), where the protein may be glycosylated. At the same time, a producer of erythropoietin is monolayer culture of these cells. * * Comprise antigens of pathogens produced using genetic engineering techniques and include only highly immunogenic components contributing to the formation of protective immunity. Ways to create genetically engineered vaccines. • Including of virulence genes into weak-virulentor avirulent microorganisms. • Including of virulence genes of unrelated microorganisms followed by isolation of antigene and its use as an immunogen. • Artificial removal of virulence genes and the use of modified organisms in the form of corpuscular vaccine. * * Several modern antiviral vaccines are constructed by introducing the genes encoding main antigene pathogenic viruses and bacteria in the vaccinia virus genome (HBsAg hepatitis B virus (surface antigens of hepatitis B virus (Hepatitis in surface antigen - HBsAg) - antigen constituting the outer shell of the hepatitis B virus (HBV .)) and nonpathogenic Salmonella to humans (HBsAg Hepatitis B and A toxin tetanus bacillus.) Another example is the introduction of genes of Mycobacterium tuberculosis into BCG vaccine strain, which gives it a great activity as divergent vaccine. Such drugs are known as vector vaccines. * Cytokines are peptides or glycoproteins having a molecular weight of from 6 to 60 kDa. They act as humoral regulators at nanomolar and picomolar concentrations at modulating normal or pathological states of the organism functional activities of individual cells and tissues. The most important biological properties of cytokines include: - Inducibility - cytokine production is carried out in response to stimulating factors; - Local action; - Redundancy - each type of cell can produce several cytokines, cytokine and each can be produced by several cell types; - The interconnection and interaction - some cytokines can induce the production of other or increase the expression of the receptors. * * - Interleukins involved in the processes of interaction of leukocytes; * - Interferons with antiviral activity; * - Tumor necrosis factors; * - Colony stimulating factors - hematopoietic cytokines that act on hematopoietic cells from stem level; * - Chemokines - chemotactic cytokines involved in immune and inflammatory responses; * - Growth factors that modulate cell growth and differentiation. * * Cell-free systems are designed to simulate the biochemical processes that occur in vivo, and reproduce some of the features of cell activity. In Genetic Engineering acellular protein synthesis system used to study mechanisms and coding potential expression of cloned genes in vitro, and in the intermediate stages of constructing the recombinant gene to identify mRNA or DNA fragments of the encoded proteins. * * availability of components for the experimental conditions. These systems allow to investigate the influence of exogenous factors on their functioning (ionic conditions, pH, inhibitors and activators, etc.). In cell-free system we can replace individual components or directly affect them in an isolated state and then by the reaction system to know their functional significance. Most of the results were obtained using cell-free systems, it would be impossible to obtain with the use of living cells, killed by inappropriate homeostasis. Advantages of cell-free systems at the same time are their weak point, because after the destruction of the cells multiple interactions between their components, through which one can distinguish the living cell from the cell-free extract disappear. * * * Non-cellular extracts of bacterial or eukaryotic cell is placed in a cell, covered on both sides with semipermeable membranes. The pore size can pass through the membrane together with the fluid flow and low molecular weight chemicals small proteins. With components of the cell to the cell-free translation was incubated at ambient temperature. On one side of the cell-reactor at a rate of about 1 mL / h continuously receives ingredients spent during biosynthesis protein (amino acids, ATP, GTP), on the other hand are located synthesized protein products (if the molecular weight and the lack of ability to allow to pass aggregation through the membrane pores.) * Biosynthesis of the protein in the system can continue running continuously for several hours, with one molecule broadcast hundreds of copies of mRNA synthesized polypeptide chains of proteins, total yield can reach 200 micrograms or more per 1 ml of cell-free extract.