Application of Rdna Technology and genetic engineering in Production of Interferons:

1. SJM College of Pharmacy,
Chitradurga
Prepare By,
Adarsh Patil
Ass Professor(Pharmacognosy)
SJM College of Pharmacy
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PHARMACEUTICAL
BIOTECHNOLOGY

2. Application of Rdna Technology and genetic
engineering in Production of Interferons:
Introduction:
• Interferon* is an antiviral substance, and is the first line of
defense against viral attacks.
• The term interferon has originated from the interference of
this molecule on virus replication.
• It was originally discovered in 1957 by Alick Isaacs and Jean
Lindemann.
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3. • Interferon actually consists of a group of more than twenty substances with
molecular weights between 20,000-30,000 daltons.
• All the interferons are proteins in nature. Interferons are glycoprotein
cytokines secreted by host lymphocytes in response to pathogens.
• Interferons activate or up regulate immune cells by interacting with
receptors that activate signal transducer and activator of transcription
(STAT) signaling complexes.
• This ultimately leads to clearance of the pathogen or clearance of tumor
cells from the organism.
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4. • Classification:
Based on their structure and Function
1. Interferon-α
2. Interferon-β
3. Interferon-γ
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5. Mechanism of action:
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6. • Interferons are produced by mammalian cells when infected by viruses. As the virus
releases its nucleic acid into cellular cytoplasm, it stimulates the host DNA to produce
interferons.
• These interferons, secreted by the cells, bind to the adjacent cells.
• Here, they stimulate the cellular DNA to produce a series of antiviral enzymes.
• The so formed proteins inhibit viral replication and protect the cells.
• It is believed that the protective (enzymes) proteins bind to mRNA of viruses and block
their protein synthesis.
• The action of interferon appears to be species specific.
• Thus, human interferon operate in humans.
• Other animal (dog, mouse) interferons are ineffective in man.
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7. • Method of production of interferons:
1. Older days:
• BIood was the only source of interferons earlier.
• The procedure was very tedious and the
quantity of interferon isolated was very little. Thus, as much
as 50,000 L of human blood was
required to get just 100 mg of interferons. Therefore, it was
very difficult to conduct research or use interferons for
therapeutic purposes.
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8. 2. Production of recombinant interferons:
• The complementary DNA (CDNA) was synthesized from the mRNA of a specific
interferon.
• This is inserted to a vector (say plasmid) which is introduced into E. coli or other cells.
• The interferon can be isolated from the culture medium. This is the basic mechanism of
producing recombinant interferons.
• The production of interferons is relatively less in bacterial hosts, although E. coli was the
first to be used.
• This is mainly because most interferons are glycoproteins in nature and bacteria do not
possess the machinery for glycosylation of proteins.
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9. Steps involved in production of rDNA derived interferon:
• Isolation of mRNA coding interferon from human
• Construction of CDNA from MRNA using reverse transcriptase by
reverse transcription mechanism.
• Fragmentation of the double stranded DNA using Restriction
endonucleases
• Isolation of the desired DNA fragment
• Amplification of the gene of interest.
• Ligation of the DNA fragment into a suitable vector by the enzyme
DNA ligases.
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10. • Transfer of DNA into the host
cell
• Screening
• Culturing the host cells on a
suitable medium on a large scale
• Extraction of the desired
product
• Downstream processing of the
products
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11. Production of Interferons by yeast:
• The yeast Saccharomyces cervisiae is more suitable for the production of
recombinant Interferons.
• This is mainly because the yeast possesses the mechanism to carry out
glycosylation of proteins, similar to that occurs in mammallan cells.
• The DNA sequence coding for specific human interferon can be attached to
the yeast alcohol dehydrogenase gene in a plasmid and introduced into 4
yeast cells.
• The yleld of Interferons io several fold higher compared to E. coll.
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12. THERAPEUTIC APPLICATIONS OF INTERFERONS:
• Interferons-γ,β & α were respectively approved for therapeutic use in humans
in the years 1986, 1993 and 1990. A Swian blotechnology firm was the first to
market interferon-α with a trade name Intron.
• Interferons are used for the treatment of a large number of viral diseases and
cancers.
• The cancers include leukemia, kaposis sarcoma, bladder cancer, head and neck
cancer, renal cell carcinoma, skin cancer and multiple myeloma.
• The other diseases employing interferon therapy are AIDS, multiple sclerosis,
genital warts, hepatitis C,herpes zoster etc.
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13. • interferons are found to cause the death of cancerous cells.
This is brought out by stimulating the action of natural killer
(NK) cells, a specialised form of lymphocytes that can destroy
cancerous cells, Interferons either stimulate or inhibit
antibody production, inhibit delayed hypersensitivity, inhibit
transplantation immune response, inhibit blastogenesis and
DNA synthesis, potentiate killer Tcells, Potentiates ADCC*
activity.
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14. • Interferons show activity on macrophages. Potentiate phagocytosis,
potentiate adherence of macrophages to tumor cells, inhibits
intracellular bacterial proliferation, improves MIF* activity.
• Interferons also promote chemotaxis for neutrophils, potentiate
NBT* reduction in neutrophils, increases histamine release in
basophils, promotes differentiation of erythroblasts, potentiate the
expression of MHC class I and II antigens.
• Interferons are also employed in the treatment of common cold and
influenza. For this purpose, interferons can be used as nasal sprays.
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