Chapter II Genetic Engineering Pharmacy
Section 1 Overview
Section II Production Process of Genetically Engineered Drugs
Section III Acquisition of Target Genes
Section IV Gene Expression
Section 5 Growth and Metabolic Characteristics of Genetically Engineered Bacteria
Section 6 Stability of Genetically Engineered Bacteria
Section 7 Pilot Test of Genetically Engineered Bacteria
Section 8 Cultivation of Genetically Engineered Bacteria
Section 9 High-density Fermentation
Section 10 Separation and Purification of Genetically Engineered Drugs
Renaturation of denatured protein in section 1 1
Section 12 Quality Control of Genetically Engineered Drugs
Section 13 Manufacturing Examples of Genetically Engineered Bacteria Drugs
Section 1 Overview
The role of genetic engineer in pharmacy
Main categories of genetically engineered drugs
Advantages of genetic engineering in drug production
Brief introduction to the development of genetic engineering drugs at home and abroad
The gap with foreign advanced level
Main categories of genetically engineered drugs
1. Hormones: insulin, growth hormone
2. Immune proteins: monoclonal antibodies and vaccines.
3. Cytokines: interferon and interleukin
4. Enzymes: urokinase, superoxide dismutase
Advantages of genetic engineering in drug production
1. Make great gains and serve the society more effectively.
2. The production efficiency is higher
3. Further improve pharmacological activities, such as protein Project.
4. Promoting access to new drugs: screening new compounds.
5..？
Genetic engineering (genetic engineering):
Consciously transfer the useful target gene in one organism to another organism, so that the latter can obtain new genetic traits or products needed for expression.
Rare and precious protein medicine
1982, the US Food and Drug Administration approved the first genetically engineered product-human insulin-which marked the official commercialization of genetically engineered products.
Human growth hormone, epidermal growth factor, tumor necrosis factor, interferon -A, cellulase, anti-hemophilia factor, erythropoietin, prourokinase, interleukin -2, colony stimulating factor, hepatitis B vaccine, etc.
Animal husbandry application
Animal vaccine, growth hormone, etc.
Example: tPA, a medicine for treating heart disease, is extracted from the goat's milk of transgenic sheep.
Application in planting industry
Plant protoplasts were transformed with Agrobacterium Ti plasmid carrying foreign genes, so that the foreign DNA was integrated with plant chromosome DNA, differentiated into callus through protoplast culture, and finally developed into a complete plant with new characteristics-transgenic plants.
Application in planting industry
Chemical herbicide resistance gene
Transgenic tomato
Nitrogen-fixing enzyme gene
Human DNA
……
Environmental protection and so on.
Section 2+3
Recombinant DNA technology
1 recombinant DNA technology is the core technology of genetic engineering.
2. Obtain the desired target gene (foreign gene)
3. Construction of recombinant plasmid and gene cloning
Screening of transformation receptor cells and transformants
Analysis of five transformants -Southern hybridization
The breakthrough of recombinant DNA technology led to the rise of modern biotechnology, and soon produced many high-tech industries of life sciences.
Recombinant DNA technology, also known as gene or molecular cloning technology, is the core technology of genetic engineering. This technology includes a series of molecular biology operation steps.
1 recombinant DNA technology is the core technology of genetic engineering.
General steps of recombinant DNA operation:
(1) to obtain the target gene;
(2) connecting with a cloning vector to form a new recombinant DNA molecule;
(3) transforming recipient cells with recombinant DNA molecules, and replicating and inheriting in the recipient cells;
(4) screening and identifying transformants;
(5) Culture cells or organisms with foreign genes to obtain the required genetic traits or products for expression.
(1) Construct a gene library, and then call the target gene from it;
(2) Using mRNA as a template, complementary DNA fragments were synthesized by reverse transcription;
(3) PCR amplification of the target gene fragment.
(4) Modification of old genes
(5) chemical synthesis of (short) genes
2. Basic methods of obtaining the target gene
Extraction and isolation of intracellular total DNA and construction of gene library
Extraction and separation methods of cell total DNA
Construction of gene library
Genomic fragments contained in total DNA are cloned on plasmid or phage vectors respectively to form a biological gene library.
Synthesis of complementary DNA by reverse transcription
The problem of constructing gene bank to obtain target gene—
Take time and trouble.
Intron sequence
Advantages of synthesizing complementary DNA by reverse transcription—
The obtained DNA fragments are often target genes with specific functions.
Polymerase chain reaction
PCR is a technique for selectively amplifying (including isolating) a target gene through enzymatic reaction in vitro.
Add four substances:
(1) as the DNA sequence of the template;
(2) DNA primers (short DNA single strand of about 20 bases) complementary to the 5' terminal sequences of the two strands of the isolated target gene;
(3)TaqDNA polymerase;
(4)dNTP(dATP, dTTP, dGTP and dCTP).
Polymerase chain reaction
Trilogy of denaturation, annealing and extension
Denaturation: Double-stranded DNA melts into single-stranded DNA.
Annealing: Some primers are paired with the specific complementary part of the template single-stranded DNA.
Extension: synthesis of complementary new DNA strands using target gene as template.
Polymerase chain reaction
Each round of polymerase chain reaction can double the target gene fragment.
After 30 cycles, -230 (1.07× 109) gene fragments were obtained.
Basic method for obtaining target gene (continue)
4. Transforming old genes-protein project
5. Chemical synthesis of (short) genes
The most important tools for gene recombination and cloning are restriction enzymes, vectors and host bacteria.
A small number of target genes must be cloned to obtain a large number of copies, and then further recombination, transformation and expression can be realized.
3. Construction of recombinant plasmid and gene cloning
restriction enzyme
Restriction endonuclease is an endonuclease isolated and purified from bacteria, which can recognize and cut a specific site of nucleic acid sequence-molecular scalpel.
Arber, Smith and Nathans won the 1978 Nobel Prize for their pioneering work in discovering restriction endonucleases.
restriction enzyme
More than 200 species have been discovered and identified.
EcoRI specifically recognizes double-stranded fragments composed of GAATTC and its complementary bases.
An unhappy ending
T4 ligase
carrier
Vector is a tool to transport the target gene fragment into the host cell. At present, the most commonly used vectors are bacterial plasmid, L phage, cosmid plasmid and so on.
Plasmid is a kind of circular DNA molecule, which naturally exists outside the chromosome of bacterial cells and can replicate independently. Plasmids entering host cells can greatly increase their copy number.
A. Plasmids are relatively small, and longer DNA fragments can be inserted.
B. After entering the host bacterial cells, pUC 18 can replicate in each cell to form about 500 copies.
C There is an artificially designed short sequence with various restriction sites in pUC 18, that is, multiple cloning sites.
Bacterial plasmid pUC 18
The multiple cloning sites of pUC 1 18 plasmid were integrated into lacZ gene. LacZ gene can be expressed if no foreign target gene is inserted into this site. Galactosidase, if the plate medium contains IPTG and X-gal, what will X-gal do? Galactosidase is hydrolyzed to blue, and Escherichia coli forms blue clones.
Inserting foreign target gene into multiple cloning sites destroyed the structure of lacZ gene, and E.coli formed a white clone.
D. inactivating D.lacZ gene insertion to screen recombinant plasmid.
E puc 18 also carries ampicillin resistance gene, which can be used to screen recombinant plasmid.
Lactose (4-D- glucose? -galactopyranoside) and allolactose
Gene cloning of Escherichia coli as host bacteria
The operation steps of cloning the target gene into E.coli cells:
A, obtaining a target gene and a plasmid vector;
B, forming a recombinant plasmid;
C, preparing competent cells, and transforming Escherichia coli cells with recombinant plasmids;
D, culturing Escherichia coli to form a large number of copies of recombinant plasmids and exogenous target genes;
E, screening Escherichia coli cells containing the recombinant plasmid, and checking or identifying.
Detection and identification methods of common cloned genes
Isolation and identification of enzymatic fragments with different sizes by agarose gel electrophoresis;
Phosphate groups are negatively charged.
The enzymolysis fragment moves to the anode.
Electric field driving force and gel resistance
-Different liquidity
Molecular weight standard reference
Enzymatic digestion and electrophoresis methods
32P labeled DNA molecular probe
hybridize
radioautography
Direct identification of DNA hybridization
After a large number of target genes are obtained by gene cloning, they need to be expressed in suitable host cells to produce the required gene expression products or make the host organism have the required characteristics, and the target genes can be stably inherited in the host cells. This process is gene transformation.
If the cloned gene needs to express and produce a large number of encoded protein, transformed E.coli can be cultured to express and accumulate a large number of target genes. The desired product can be obtained by separating and purifying the expressed product.
The recombinant plasmid constructed by DNA in vitro recombination technology can also be directly used to transform prokaryotes, such as cyanobacteria or other protozoa.
Screening of transformation receptor cells and transformants
Common methods of genetic transformation
Vector transformation-Agrobacterium-mediated transformation
Direct gene transfer
(1) high-voltage electrical pulse electrical stimulation perforation
(2) Gene marksmanship
(3) microinjection method
In memory of the inventor Edward South
(1) total DNA was extracted.
(2) enzymolysis
(3) electrophoresis
(4) transfer to a filter membrane
(5) denaturation and melting
(6)DNA probe and hybridization
(7) elution
(8) Autoradiography
(9) Comparative analysis
Analysis of five transformants -Southern hybridization
Examples of Southern hybridization analysis
A. DNA recombination experiment in vitro
B, screening transformed cells with antibiotics
C. culturing mutant cells.
D.southern hybridization results showed that the foreign target gene had been transformed into mutant cells.
During the period of 1973, the research team led by Professor Cohen of Stanford University and Professor Boyer of the University of California completed DNA recombination in vitro almost at the same time, which opened the door to genetic engineering.
Section IV Gene Expression
Selection of host cells
Gene expression in Escherichia coli
Gene expression in yeast
Gene expression in animal cells
1. Expression host bacteria
Prerequisites for host cells: 7 points
Gene expression host bacteria can be divided into two categories.
Common host bacteria
1. Prokaryotes: 3 species.
2. Fungi: 2 species
What are the characteristics of the above hosts?
Secondly, the expression of foreign genes in E.coli.
Six basic characteristics of eukaryotic genes in 1. Escherichia coli expression vector
2. Two expression vectors-PBV 220&; PET system
3. Five factors affecting the expression of target gene
4. Three expression forms of eukaryotic genes in E.coli.
Six basic characteristics of Escherichia coli expression vector
1. Independent replicon
2. Multiple cloning sites
3. Strong promoter
4. Strong Terminator
5. Repressor
6.Shine-Delgarno sequence & August
Five factors affecting the expression of target gene in Escherichia coli
1. gene dose
2. Expression efficiency
3. Stability of the expressed product:
A transcription intensity, b translation efficiency (ribosome binding, SD sequence, codon preference)
4. Metabolic load of host Escherichia coli
5. Cultivation conditions of engineering bacteria
Three expression form of eukaryotic genes in E. coli
1. fusion protein
2. Secretory expression
3. Ordinary expressions
3. Expression of foreign genes in Saccharomyces cerevisiae
1. Carrier:
Four categories, YEp, YRp, YCp, YIp.
Cloning Vector and shuttle vector
Expression vectors: common expression vectors and accurate expression vectors.
2. Factors affecting the expression of target gene in yeast
1. Exogenous gene dose
2. Expression efficiency of foreign genes
① the source of promoter
② Validity of terminator
③ Efficiency of signal secretion.
3. Glycosylation of foreign proteins
4. Influence of host strain
Section 5 Growth and Metabolism of Genetically Engineered Strain
Relationship between cell growth and energy
Keywords: oxygen supply/energy/by-products/cell growth
Relationship between cell growth and premise supply
Keywords: prerequisites/
Instability of genetically engineered strains
Stability of strains and plasmids
Six methods to improve plasmid stability
Section VI Instability of Genetic Engineering Strains
Section 7 Pilot Test of Genetic Engineering Strains
Purpose of the pilot test
Pilot process
Section 8 Cultivation of Genetic Engineering Strains
1. Cultivation (fermentation) mode of genetically engineered strains
Seven elements of fermentation technology of genetically engineered bacteria
Fermentation equipment of genetically engineered strains
Section 9 High-density Fermentation
High density: concept and function
Factors affecting high-density fermentation
How to realize high-density fermentation
Necessity of establishing separation and purification process
Basic steps of separation and purification
Separation and purification technology
1. How to choose a suitable separation and purification process?
2. Cell fragmentation and solid-liquid separation
3. Separation and purification of target product
Basis for selecting separation and purification process
1. According to the expression form of the product.
2. According to the connection between separation units
3. According to the basic requirements of separation and purification process
Section 10 Separation and Purification of Genetically Engineered Drugs
Renaturation of denatured protein in section 1 1
Inclusion body and its formation reasons
Decomposition and dissolution of inclusions
Renaturation method of inclusion body
Quality control of raw materials
Quality control in the process of cultivation
Quality control in purification process
Quality control of target products
1. Product identification
2. Purity analysis
3. Determination of biological activity
4. Stability
5. Product consistency
Preservation of products
Section 12 Quality Control of Genetically Engineered Drugs
Preparation of Interferon-Human Interferon α2b
Human granulocyte colony stimulating factor
Human interleukin-2
Section 13 examples of manufacturing genetically engineered drugs