๐ Medical Biotechnology
Study recombinant insulin, gene therapy and molecular diagnostics for CUET Agriculture. Humulin, ELISA, DNA vaccines and stem cell therapy.
Recombinant Pharmaceuticals
Biotechnology has revolutionized medicine by enabling the production of human proteins in microbial or cell culture systems.
1. Recombinant Insulin (Humulin)
- Insulin โ hormone produced by ฮฒ-cells of the Islets of Langerhans in the pancreas; regulates blood glucose.
- Diabetes mellitus โ caused by insulin deficiency (Type 1) or insulin resistance (Type 2).
- Before rDNA: insulin extracted from pig and cow pancreas โ allergic reactions in some patients (slight amino acid differences).
Recombinant Human Insulin:
- First produced by Eli Lilly (1983). Marketed as Humulin โ first commercially available recombinant pharmaceutical product.
- Structure: Human insulin has two polypeptide chains:
- A chain โ 21 amino acids.
- B chain โ 30 amino acids.
- Connected by disulfide bonds (A-A and A-B bridges).
How recombinant insulin is produced
**Method 1 (Original โ Separate chains):** 1. A-chain gene inserted into *E. coli* plasmid โ bacteria produce A chain. 2. B-chain gene inserted into separate *E. coli* plasmid โ bacteria produce B chain. 3. Both chains purified and **combined in vitro** with formation of disulfide bonds.Method 2 (Proinsulin method โ Current):
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Recombinant Pharmaceuticals
Biotechnology has revolutionized medicine by enabling the production of human proteins in microbial or cell culture systems.
1. Recombinant Insulin (Humulin)
- Insulin โ hormone produced by ฮฒ-cells of the Islets of Langerhans in the pancreas; regulates blood glucose.
- Diabetes mellitus โ caused by insulin deficiency (Type 1) or insulin resistance (Type 2).
- Before rDNA: insulin extracted from pig and cow pancreas โ allergic reactions in some patients (slight amino acid differences).
Recombinant Human Insulin:
- First produced by Eli Lilly (1983). Marketed as Humulin โ first commercially available recombinant pharmaceutical product.
- Structure: Human insulin has two polypeptide chains:
- A chain โ 21 amino acids.
- B chain โ 30 amino acids.
- Connected by disulfide bonds (A-A and A-B bridges).
How recombinant insulin is produced
**Method 1 (Original โ Separate chains):** 1. A-chain gene inserted into *E. coli* plasmid โ bacteria produce A chain. 2. B-chain gene inserted into separate *E. coli* plasmid โ bacteria produce B chain. 3. Both chains purified and **combined in vitro** with formation of disulfide bonds.Method 2 (Proinsulin method โ Current):
- Proinsulin gene (includes C-peptide connecting A and B chains, as in native preproinsulin) expressed in E. coli or yeast.
- Proinsulin produced.
- C-peptide removed enzymatically โ mature active insulin.
2. Other Key Recombinant Products
| Product | Function | Host System |
|---|---|---|
| Insulin (Humulin) | Blood glucose regulation | E. coli / Yeast |
| Human Growth Hormone (HGH / Somatotropin) | Growth; treats dwarfism (GH deficiency) | E. coli |
| Calcitonin | Calcium regulation | E. coli |
| Clotting Factor VIII | Hemophilia A treatment | CHO cells |
| Clotting Factor IX | Hemophilia B treatment | CHO cells |
| TPA (Tissue Plasminogen Activator) | Dissolves blood clots (heart attack treatment) | CHO cells |
| Erythropoietin (EPO) | Stimulates RBC production; treats anemia | CHO cells |
| Interferons (ฮฑ, ฮฒ, ฮณ) | Antiviral, anti-tumor | E. coli / Yeast |
| Hepatitis B vaccine | Immunization against Hepatitis B | Yeast (S. cerevisiae) โ surface antigen (HBsAg) expressed |
| Streptokinase | Blood clot dissolution | E. coli |
TIP
Simpler proteins (insulin, HGH, interferons) are produced in E. coli or yeast because they don't need complex post-translational modifications. Complex glycoproteins (Factor VIII, EPO, TPA) require CHO cells (Chinese Hamster Ovary cells) โ a eukaryotic expression system that can glycosylate proteins correctly.
Gene Therapy
Gene therapy = Treatment of a genetic disorder by replacing, altering, or supplementing a defective gene with a functional one.
First Successful Gene Therapy (1990)
- Disease: ADA deficiency (Adenosine Deaminase deficiency).
- ADA enzyme is essential for immune system function. Deficiency โ Severe Combined Immunodeficiency (SCID) โ the "bubble boy" disease โ patients live in sterile environments.
- Year: 1990.
Procedure:
- Blood drawn from patient โ lymphocytes isolated.
- Functional ADA gene cloned into retroviral vector.
- Retroviral vector infects lymphocytes in culture โ ADA gene integrates into lymphocyte genome.
- Modified lymphocytes (now producing functional ADA) multiplied in culture.
- Modified lymphocytes infused back into patient's bloodstream.
- Immune function partially restored.
Limitation: Lymphocytes have a limited lifespan โ needs repeated treatments (not permanent cure). Permanent cure: Bone marrow transplantation from a matched donor โ stem cells continuously produce immune cells with functional ADA.
Types of Gene Therapy
| Type | Target | Inheritance | Status |
|---|---|---|---|
| Somatic gene therapy | Body (somatic) cells | Not inherited by offspring | Currently practiced |
| Germline gene therapy | Germ cells or embryos | Inherited by all future generations | Ethically controversial; banned in most countries |
WARNING
Germline gene therapy is banned in most countries because permanent changes to the germ line are inherited by all future generations. The ethical implications of altering the human gene pool are enormous. In 2018, Chinese scientist He Jiankui created genome-edited babies (CCR5 knockout using CRISPR) โ widely condemned by the scientific community; he was convicted and imprisoned.
Molecular Diagnostics
ELISA (Enzyme-Linked Immunosorbent Assay)
- Based on antigen-antibody interaction โ highly specific.
- A specific antibody is linked to an enzyme (e.g., alkaline phosphatase, horseradish peroxidase).
- When the enzyme acts on its substrate โ color change โ measured by spectrophotometer.
- Types: Direct ELISA, Indirect ELISA, Sandwich ELISA, Competitive ELISA.
- Used to diagnose: HIV/AIDS, hepatitis B/C, pregnancy (hCG), allergies, autoimmune diseases.
PCR-based Diagnosis
- Amplifies specific DNA/RNA sequences from pathogens.
- Extremely sensitive โ can detect even a few copies of pathogen nucleic acid.
- Used for: HIV (viral load), tuberculosis, SARS-CoV-2 (RT-PCR), genetic disorders, cancer mutations.
DNA Probes
- Short, labeled DNA sequences complementary to target sequences.
- Used in hybridization-based detection (Southern blot, Northern blot, in situ hybridization).
Transgenic Animals
Animals that carry foreign genes (transgenes) stably integrated into their genome.
- First transgenic animal: Mouse (1980) โ the "Supermouse" created by Ralph Brinster and Richard Palmiter; rat growth hormone gene injected into mouse embryos โ mice grew significantly larger than normal (2-3ร body weight).
Applications of Transgenic Animals
| Application | Description | Example |
|---|---|---|
| Study of gene function and regulation | Understanding how genes are expressed and regulated | Transgenic mice with reporter genes |
| Disease models | Animals engineered to develop human diseases for drug testing | Oncomouse (Harvard mouse) โ carries activated oncogene โ develops cancer; used for cancer research and drug testing |
| Drug safety and efficacy testing | Testing toxicity before human trials | Transgenic mice expressing human drug-metabolizing enzymes |
| Vaccine safety testing | Testing vaccines before human trials | โ |
| Biological products (Pharming) | Producing human proteins in animal milk/eggs | Transgenic goat โ antithrombin III in milk; Transgenic sheep "Rosie" โ ฮฑ-1-antitrypsin in milk; Transgenic cow โ lactoferrin in milk |
| Knockout models | Specific genes removed | Knockout mice for studying gene function |
Genetically Engineered Microorganisms (GEMs)
| Microorganism | Products / Applications |
|---|---|
| E. coli | Insulin, HGH, interferons, clotting factors, vaccines |
| Saccharomyces cerevisiae (yeast) | Hepatitis B vaccine (HBsAg), insulin |
| Bacillus thuringiensis | Bt toxin (Cry proteins) for biopesticide |
| Pseudomonas putida | "Superbug" โ created by Ananda Mohan Chakrabarty (1971); degrades multiple hydrocarbons in oil spills (naphthalene, octane, camphor, xylene). First organism to be patented (US Supreme Court, 1980: Diamond v. Chakrabarty) โ landmark ruling opening the door to biotech patents. |
| Corynebacterium glutamicum | Amino acid production (lysine, glutamic acid) |
| Agrobacterium tumefaciens | Plant transformation vector (Ti plasmid) |
Key Points to Remember
- Recombinant insulin: Eli Lilly, 1983; Humulin; A chain (21 aa) + B chain (30 aa); produced in E. coli or yeast.
- First recombinant pharmaceutical: Humulin (insulin), 1983.
- Complex proteins (Factor VIII, EPO, TPA) require CHO cells for correct glycosylation.
- Gene therapy for ADA deficiency (SCID): First success, 1990; lymphocytes + retroviral vector; not permanent (lymphocytes die).
- Somatic gene therapy = not inherited; Germline = inherited โ banned in most countries.
- ELISA: antigen-antibody + enzyme label โ color change; used for HIV, hepatitis, pregnancy.
- First transgenic animal: Supermouse, 1980 (Brinster & Palmiter; rat GH gene).
- Oncomouse: Harvard mouse; carries oncogene; cancer research model.
- Pharming: transgenic goats/sheep/cows producing human proteins in milk.
- Chakrabarty's Pseudomonas putida (1971): oil-degrading superbug; first patented organism (Diamond v. Chakrabarty, 1980).
Summary Cheat Sheet
| Concept / Topic | Key Details / Explanation |
|---|---|
| Insulin source in body | Produced by ฮฒ-cells of Islets of Langerhans in the pancreas |
| Diabetes mellitus | Type 1: insulin deficiency; Type 2: insulin resistance |
| Before rDNA insulin | Extracted from pig and cow pancreas โ caused allergic reactions in some patients |
| Humulin | First commercially available recombinant pharmaceutical; produced by Eli Lilly (1983) |
| Insulin structure | A chain: 21 amino acids; B chain: 30 amino acids; connected by disulfide bonds |
| Insulin production (original) | A-chain and B-chain genes expressed separately in E. coli; chains combined in vitro |
| Insulin production (current) | Proinsulin gene expressed in E. coli/yeast; C-peptide removed enzymatically โ mature insulin |
| Simple proteins host | E. coli or yeast โ insulin, HGH, interferons (no complex post-translational modifications needed) |
| Complex glycoproteins host | CHO cells (Chinese Hamster Ovary) โ Factor VIII, EPO, TPA (require correct glycosylation) |
| Human Growth Hormone (HGH) | Treats dwarfism (GH deficiency); produced in E. coli |
| Clotting Factor VIII | Treats Hemophilia A; produced in CHO cells |
| Erythropoietin (EPO) | Stimulates RBC production; treats anemia; produced in CHO cells |
| Hepatitis B vaccine | HBsAg (surface antigen) expressed in yeast (S. cerevisiae); recombinant vaccine |
| Gene therapy (definition) | Treatment by replacing, altering, or supplementing a defective gene with a functional one |
| First gene therapy (1990) | Disease: ADA deficiency (Adenosine Deaminase deficiency) โ causes SCID ("bubble boy" disease) |
| ADA gene therapy procedure | Patient lymphocytes isolated โ functional ADA gene cloned into retroviral vector โ lymphocytes infected โ modified cells infused back |
| ADA gene therapy limitation | Lymphocytes have limited lifespan โ needs repeated treatments; permanent cure requires bone marrow transplant |
| Somatic gene therapy | Targets body cells; changes not inherited by offspring; currently practiced |
| Germline gene therapy | Targets germ cells/embryos; changes inherited by all future generations; banned in most countries |
| ELISA | Based on antigen-antibody interaction; antibody linked to enzyme โ acts on substrate โ color change; diagnoses HIV/AIDS, hepatitis, pregnancy |
| PCR-based diagnosis | Amplifies specific DNA/RNA sequences; detects even a few copies of pathogen nucleic acid; used for HIV, TB, SARS-CoV-2 (RT-PCR) |
| First transgenic animal | Mouse (1980) โ "Supermouse" by Ralph Brinster & Richard Palmiter; rat GH gene โ mice grew 2-3ร larger |
| Oncomouse | Harvard mouse carrying activated oncogene โ develops cancer; used for cancer research and drug testing |
| Pharming | Producing human proteins in transgenic animal milk/eggs Goat โ antithrombin III; Sheep "Rosie" โ ฮฑ-1-antitrypsin; Cow โ lactoferrin |
| Pseudomonas putida ("Superbug") | Engineered by Ananda Mohan Chakrabarty (1971); degrades multiple hydrocarbons in oil spills |
| First patented organism | Pseudomonas putida; Diamond v. Chakrabarty (US Supreme Court, 1980) โ landmark ruling for biotech patents |
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