Biotechnology technique known as recombinant DNA technology are used in the large scale production of important biomolecules that can be used to improve the quality of life of humans. One of the important applications of biotechnology is the production of biomolecules like hormones such as insulin, growth stimulating hormone, somatotropin, blood products like clotting factor VII, albumin, tissue plasminogen activator and also this technology is used in the production of cytokines, interferons and also colony stimulating factor. Recombinant DNA technology can also be used in the production of vaccines, monoclonal antibodies and much more compounds.
Clotting Factor VIII:
Factor VIII is very important and essential blood clotting factor. This factor is also called as antihemophilic factor (AHF). In humans clotting factor VIII is encoded by a gene known as F8 gene. Malfunctioning of this gene results in disease hemophilia A. This condition is a recessive X-linked coagulation disorder that is blood clotting time is prolonged resulting in large amount of blood loss due to internal bleeding. Factor VIII is a glycoprotein procofactor, has been produced by the vascular, gomerular and tubular endothelium and also the sinusoidal cells of the liver. Then they are released into the bloodstream, to do their job.
Production of Recombinant Factor VIII:
1. Gene encoding for the production of Factor VII is located in X-chromosome of human. This gene is 186kb in size and organized in to 26 Exons and also many introns.
2. Recombinant DNA technology is used to produce mature mRNA coding for the production of Factor VIII is isolated by following method
a. First cDNA are produced complementary to the mature mRNA coding for Factor VIII.
b. This cDNA is then inserted into mammalian cell or hamster kidney cells
c. These cells are then cultured in suitable animal cell culture media and they produce Factor VIII
d. Recombinant Factor VIII is then isolated from the media and purified using appropriate technology.
Erythropoietin is a hormone produced by the kidney and this biomolecule stimulates the bone marrow stem cells to produce mature erythrocytes or red blood cells. There are two types of erythropoietin are present such as Erythropoietin a, and Erythropoietin b, both are produced in large quantities using animal cell culture and are used as therapeutic agents.
Erythropoietin a is used in the treatment of anaemia resulting from chemotherapy or by cancer. Erythropoietin b is used as therapeutic agent in treating anaemia secondary to kidney diseases.
Recombinant erythropoietin such as
EPOGEN is used in the treatment of anaemia.
PROCRIT act as natural hormone and stimulates the production of erythrocytes. This is used as alternate treatment to blood transfusion method. But this treatment with recombinant erythropoietin is very expensive one when compared to traditional blood transfusion method.
Vaccines are biomolecules produced from antigens (protein) of other animals which give immunity against a particular virus. Many types of vaccines are produced in large quantities using recombinant DNA technology or genetic engineering. Vaccines produced against foot and mouth disease is an example of using large scale cell culture. Several other vaccines such as polio vaccine, bovine leukaemia virus (BLV) vaccines, rabies vaccines, and many more are produced in large quantities using animal cell culture method.
Recombinant Vaccine Production against Hepatitis B Virus:
1. Recombinant vaccine against hepatitis B virus is produced by cloning hepatitis B virus surface antigen gene into yeast cells.
2. Yeast cells are used because they have got the machinery for glycosylation of protein and also they secrete them into the culture media.
3. Autonomously replicating plasmid is integrated with hepatitis B surface antigen gene near the yeast alcohol dehydrogenase promoter region.
4. This recombinant plasmid is then inserted in to yeast cells and cultured in tryptophan-free medium.
5. Transformed yeast cells are selected and cultured in large industrial fermenters.
6. Secreted hepatitis B vaccine is isolated from the culture media and purified using appropriate method.
7. Vaccine are then stored for further use at low temperature. Entire vaccine production must be carried out under strict quality controlled and sterilized area or unit.
8. Vaccines produced using recombinant DNA technology are purer, safer and more efficacious and are being used in clinical trials.
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