Recombinant DNA Technology in Vaccine and Insulin Production
Recombinant DNA Technology in Vaccine Manufacture
Steps for Recombinant DNA Preparation
1. Isolation of Genetic Material or DNA
Genetic material is present inside the cells. It has to be obtained in pure form without even the attached histones and other proteins. To get the desired DNA, cells are treated with lysozyme (for bacteria), cellulase and pectinase (for plant cells), and chitinase (for fungal cells).
- The treated cells are homogenized and centrifuged to rupture the cells as well as nuclear envelopes.
- The homogenized product is then treated with proteases (for digesting histones and other proteins) and ribonucleases (for digestion of RNAs).
2. Cutting of DNA at Specific Location and its Pasting
Purified DNA molecules are subjected to restriction enzymes. The progress of restriction enzyme digestion is checked through agarose gel electrophoresis.
The genetic material which is meant for transfer to another organism is called passenger DNA.
3. Amplification of Gene
Amplification of the gene of interest is done by PCR (Polymerase Chain Reaction).
- PCR is the in vitro synthesis of multiple copies of a gene or DNA segment.
4. Insertion of Recombinant DNA into Host Cell/Organism
Both direct and indirect methods are known to introduce the desired genes into host cells.
In a common method, the desired gene is ligated to a plasmid vector, and introduced into bacteria (E. coli).
Recombinant DNA Technology in Vaccine Manufacture
The Hepatitis B vaccine is the most common example of rDNA technology.
- The Recombivax HB is a Hepatitis B vaccine, one of the most significant developments in rDNA technology.
- Hepatitis causes a severe acute infection that ultimately results in chronic infection and permanent liver damage, caused by the Hepatitis B virus (HBV).
- Hepatitis B infection can be prevented with a vaccine made using recombinant DNA technology.
Steps for Production of Hepatitis B Vaccine
- Genetic material is extracted from hepatitis viruses.
- The gene is removed from viral DNA and inserted into a plasmid.
- Plasmids are inserted into yeast cells.
- Yeast is grown by fermentation.
- Cells reproduce and generate surface protein.
- After 48 hours, yeast cells are ruptured to release the free surface protein.
- The mixture is processed to extract and purify the surface protein.
- Surface proteins are combined with a preserving agent and other ingredients to make the vaccine.
PCR or Polymerase Chain Reaction
PCR is the in vitro synthesis of multiple copies of a gene or DNA segment.
- It uses two aspects: small chemically synthesized oligonucleotide primers and thermostable DNA polymerase like Taq Polymerase.
Three Basic Steps in PCR
The three basic steps in PCR are: Denaturation, Annealing, and Extension.
- Denaturation: The target DNA is heated to about 94°C. It separates the two strands which are now ready to act as templates.
- Annealing: The oligonucleotide primers anneal or hybridize to each of the single-stranded DNA template at its 3′ end. This step is performed at a lower temperature.
- Extension: Taq polymerase is added. The temperature required is 72°C. Taq polymerase synthesizes the DNA strands complementary to the template strand.
For starting the next cycle, DNA is again heated to 94°C for denaturation of newly synthesized DNAs. This is followed by annealing and extension.
Applications of PCR
- DNA amplification.
- Detecting mutation.
- Diagnosing genetic disorders.
- Producing in vitro mutations.
- Preparing DNA for sequencing.
- Analyzing genetic defects in single cells from humans.
- Identifying virus and bacteria in infectious disease assays.
- Characterization of genotypes.
Preparation of DNA by Recombinant DNA Technology
Human insulin was the first recombinant derived product, used for the treatment of diabetes.
- Two DNA sequences were prepared by Eli Lilly for the two chains, A (for 20 amino acids) and B (for 30 amino acids) of insulin by reverse transcription of their mRNAs.
- Plasmids of E. coli and the insulin gene are treated with the same restriction endonuclease. The two are joined together by DNA ligase.
- This produces rDNA in the form of plasmids carrying the genes.
- Insulin genes are attached in the vector plasmids adjacent to the $\beta$-galactosidase gene.
- A culture of plasmid-free E. coli is now inoculated with the recombinant plasmid.
- The genetically engineered bacteria are tested for the formation of a fusion polypeptide consisting of one insulin subunit and $\beta$-galactosidase sequence.
- As both insulin subunits are required, the bacteria are first multiplied and then introduced into a sterilized bioreactor having the growth medium.
- When the bacteria increase in number, a part of the population is harvested. Insulin is extracted from the harvested bacteria and treated with cyanogen bromide for separating the polypeptide of $\beta$-galactosidase and purifying insulin subunits.
- The latter are then mixed. Insulin is formed, which is exactly similar to human insulin; this is also called Humulin.
Biosensors
A biosensor is an analytical device containing immobilized biological material (enzyme, antibodies, nucleic acid, hormone) which specifically interacts with an analyte and produces physical, chemical, or electrical signals that can be measured. An analyte is a compound (e.g., glucose, urea, drug, pesticide) whose concentration has to be measured.
Components of Biosensors
Biosensors consist of two main components: the “sensing element” and the “transducer”.
- The “sensing element” may be either enzymes, antibodies, DNA, tissues, or whole cells.
- The “transducer” converts the biochemical reaction or response into electrical signals.
- The basic components of biosensors are: Analyte (Sample), sensing element, transducer.
Principle of Biosensor
The analyte, upon binding to the biological material, forms a bound analyte that produces a response. The analyte converts into a product, and changes associated with this product are transformed by the transducer into electrical signals that are amplified and measured.
Biosensor Types and Their Applications
The main types are:
- Electrochemical Biosensors
- Thermometric Biosensors
- Optical Biosensors
- Piezoelectric Biosensors
1. Electrochemical Biosensors
These are used to measure electronic current, ionic, or conductance changes carried by bio-electrodes. Common examples include:
- Glucose biosensors.
- Hydrogen peroxide (HRP) biosensors.
Applications:
- Clinical diagnosis such as measuring glucose, cholesterol, urea, etc.
- Food analysis.
- Environmental analysis.
2. Thermometric Biosensors
Most biological reactions are associated with the production of heat, and this forms the basis of thermometric biosensors. Example: calorimetric biosensor.
Applications:
- Determination of water content in food, jelly, and fish.
- It can be used as part of enzyme-linked immunoassay, and the new technique is referred to as Thermometric ELISA (T. ELISA).
3. Optical Biosensors
These work on the principle of optical measurements like fluorescence or absorbance. They use fiber optics and optoelectronic transducers.
Applications:
- Medical diagnostics like diagnosis of COVID-19, gout.
- TDM (Therapeutic Drug Monitoring).
- Used to detect organic contaminants in water and food.
4. Piezoelectric Biosensors
These are based on the principle of sound vibrations. They contain piezoelectric crystals, and the characteristic frequencies vibrate with the positively and negatively charged crystals. With the help of electronic devices, certain molecules on the crystal surface can be measured. For example, a biosensor for cocaine (in gas phase) works by attaching cocaine antibodies to the crystal surface.
Applications:
- Cancer detection.
- Biomarker detection associated with various diseases like Alzheimer’s, diabetes, etc.
- Environmental monitoring.
- Food safety analysis.
Enzyme Immobilization
Enzyme immobilization is the confinement or attachment of enzymes to a solid support matrix, restricting their movement to enhance stability, allow for easy separation from products, and enable repeated reuse in industrial processes.
Key Aspects of Enzyme Immobilization
- Purpose: To make enzymes more economical, stable, and reusable for industrial applications such as food production, pharmaceuticals, and environmental engineering.
- Components: The process involves the enzyme, the solid support (carrier), and the immobilization method.
- Advantages:
- Reusability: Enables continuous operation and multiple batch uses, reducing costs.
- Stability: Protects against harsh conditions like high temperatures and extreme pH.
- Separation: Simplifies purification, as the enzyme can be easily separated from the product.
Common Immobilization Techniques
- Physical Adsorption: Enzymes are attached to a support surface via weak interactions (hydrogen bonds, van der Waals forces, ionic links).
- Covalent Binding: Chemical bonds form between the enzyme’s functional groups and the support, ensuring strong attachment and reducing enzyme leaching.
- Entrapment: Enzymes are physically trapped within a polymer matrix (e.g., alginate-gelatin, gel fibers).
- Encapsulation/Membrane Separation: Similar to entrapment, but the enzyme is enclosed within a semi-permeable membrane.
- Cross-linking: Intermolecular cross-links are formed between enzyme molecules using reagents like glutaraldehyde, resulting in a stable, carrier-free catalyst.
Advantages and Limitations
While immobilization provides significant advantages in terms of stability and cost reduction, it can sometimes result in reduced enzyme activity due to conformational changes or diffusion limitations of the substrate to the enzyme’s active site.