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*
*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.