DISEASE TREATMENT

PRODUCTS FROM

RECOMBINANT

ORGANISMS

RECOMBINANT ORGANISM

• An organism in which the DNA has been made by joining together segments of DNA using the techniques of genetic modification .

• Has developed out of RDNA technology or recombinent DNA technology 

• Recombinant DNA Technology Creates Recombinant Animals

• RECOMBINANT DNA TECHNOLOGY IS A MAJOR DNA-BASED TOOL .THIS TECHNOLOGY ALLOWS SCIENTISTS TO FIND INDIVIDUAL GENES, CUT THEM OUT, AND INSERT THEM INTO THE GENOME OF ANOTHER ORGANISM. 

• RECOMBINANT DNA TECHNOLOGY HAS APPLICATIONS IN HEALTH AND NUTRITION.

GENETICALLY ENGINEERED MICRO ORGANISMS• Human gene has been known to encode a large number of pharmaceutically

important proteins. These are cloned and expressed in micro organisms for increased production.

• Microbes are used as hosts in cloning purposes.

• E. Coli, yeast are some of the most common examples of microbes which are used as hosts. 

• Eg: recombinant insulin used in the treatment of diabetes, human growth hormone for dwarfism, interferon, interleukin, granulocyte macrophage colony stimulating factor, etc.

• Diabetics are unable to produce satisfactory amounts of insulin, which facilitates the processing of sugars from food into energy that the body can use.

• In the past, diabetics needed to take insulin purified from pigs and cows to fulfill their insulin requirement.

• Non-human insulin causes allergic reactions in many diabetics.

• Recombinant dna technology provided a way for scientists to produce human insulin in the laboratory.

CREATING HUMAN INSULIN

• Insulin consists of two chains known as a and b which are interlinked by two disulphide bridges

• The gene coding was integrated separately to a host cell, expressed and modified to produce functional insulin. 

STEPS OF PREPARATION1. ISOLATE GENE

The gene for producing HUMAN insulin protein is isolated. The gene is part of the DNA in a human chromosome. The gene can be isolated and then copied so that many insulin genes are available to work with.

2. PREPARE TARGET DNA

 A circular piece of DNA called a plasmid is removed from a bacterial cell. Restriction enzymes are used to cut the plasmid ring open.

3. INSERT DNA INTO PLASMID

With the plasmid ring open, the gene for insulin is inserted into the plasmid ring and the ring is closed. The human insulin gene is now recombined with the bacterial DNA plasmid.

4. INSERT PLASMID BACK INTO CELL

The bacterial DNA now contains the human insulin gene and is inserted into a bacteria. Scientists use very small needle syringes to move the recombined plasmid through the bacterial cell membrane.

5. PLASMID MULTIPLY

Many plasmids with the insulin gene are inserted into many bacterial cells. The cells need nutrients in order to grow, divide, and live. While they live, the bacterial cell processes turn on the gene for human insulin and the insulin is produced in the cell. When the bacterial cells reproduce by dividing, the human insulin gene is also reproduced in the newly created cells.

6. TARGET CELLS REPRODUCE

Human insulin protein molecules produced by bacteria are gathered and purified. The process of purifying and producing cow and pig insulin has been greatly reduced or eliminated.

7. CELLS PRODUCE PROTEINS

Millions of people with diabetes now take human insulin produced by bacteria or yeast (biosynthetic insulin) that is genetically compatible with their bodies, just like the perfect insulin produced naturally in your body.

• THE DOUBLE STRAND OF THE ELEVENTH CHROMOSOME OF DNA DIVIDES IN TWO, EXPOSING UNPAIRED NITROGEN BASES WHICH ARE SPECIFIC TO INSULIN PRODUCTION

Unravelling strand of the DNA of chromosome 11, with the exposed

nucleotides coding for the B chain of Insulin.

• USING ONE OF THE EXPOSED DNA STRANDS AS A TEMPLATE, MESSENGER RNA FORMS IN THE PROCESS OF TRANSCRIPTION

A single strand of DNA coding for Insulin chain B The mRNA strand. 

• The role of the mrna strand, on which the nitrogen base thymine is replaced by uracil, is to carry genetic information, such as that pertaining to insulin,from the nucleus into the cytoplasm, where it attaches to a ribosome

Process of translation at the Ribosome.

• The nitrogen bases on the mrna are grouped into threes, known as codons. Transfer RNA (trna) molecules, three unpaired nitrogen bases bound to a specific amino acid, collectively known as an anti-codon pair with complementary bases (the codons) on the mrna. 

• Animal cell cultures are also used for expression of human genes encoding pharmaceutically valuable proteins. 

•  The main proteins produced by this way are erythropoietin and blood clotting factor viii. 

•  Plant cells producing recombinant proteins are high in demand as many ethical issues related to animal cell culture do not exist with the transgenic plants

 

ERYTHROPOIETIN PRODUCTION• Erythropoiietin is a glycoprotein hormone that controls erythropoiesis, or red

blood cell production.

• Recombinant dna technology was used to insert a fetal liver genomic library apa i fragment encoding for human erythropoietin (epo) into bowes melanoma cells. 

• The cells expressed the erythropoietin gene was secreted into the culture medium together with the normally-secreted tissue plasminogen activator.

BLOOD CLOTTING FACTOR 8 PRODUCTION• Factor VIII (FVIII) is an essential blood-clotting protein, also known as anti-hemophilic

factor (AHF)

• In humans, factor VIII is encoded by the F8 gene.

• Defects in this gene results in hemophilia a, a recessive x-linked coagulation disorder.

• Antihemophilic factor (recombinant) is produced by chinese hamster ovary cells that have been modified by recombinant dna technology to introduce the gene for human factor viii and express factor viii. 

•  The antihemophilic factor (recombinant) is highly purified by several steps involving biotechnology processes. 

TRANSGENIC PLANTS

•   In the case of transgenic plants, the process of retrieval of recombinant proteins from the parts of plant cells is comparatively easy

• The most relevant example of transgenic plants aiding in the disease treatment is production of a polypeptide called hirudin.

• Hirudin is a protein that prevents blood clotting.

• Its gene was chemically synthesized and was transferred into brassica napus where hirudin accumulates in seeds.

• The hirudin is extracted and purified and used as medicine.

• This is produced by a synthetic gene expressed in the plant Brassica napus.

• Produced as a fusion protein with oleisn  which is an oil body protein.

• Upon successful integration and extraction, the hirudin is extracted with water and later centrifuged to separate our protein from the rest of the proteins produced with the help of oil body. 

• The pure hirudin is separated from olesin by subjecting the obtained oil moisture to proteolytic cleavage.

HIRUDIN PRODUCTION

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