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R dna seminar

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R dna seminar

  1. 1. In genetic engineering, recombinationcan also refer to artificial anddeliberate recombination of piecesof DNA, from different organisms,creating what is called recombinantDNA.
  2. 2.  Insulin is an important hormone whichregulates sugar metabolism An inability to produce insulin results in a formof diabetes, this disease can be treated bydaily injections of insulin Historically, insulin from pigs or cows is used,but known to produce immune reactions insome patients Challenge: how to make human insulin to beused as a drug in cell systems or microbes?Insulin - first recombinantprotein to be produced
  3. 3.  Idea: take the gene of human insulin, clone into aplasmid, introduce the plasmid into E. coli or cells,and use them E.coli as “Biological Factory” forinsulin production Amino acid sequence produced insulin (Contains 51amino acids) and is identical to that of the“natural human protein” and it will not cause anyimmune reactions Much more economical than attempts to produceinsulin by chemical synthesis So, how to do this?
  4. 4. Insulin crystals from the purification process
  5. 5. Owen Mumford Ltd., UK
  6. 6.  Secreted by the pituitary gland, andis responsible for normal body growth anddevelopment, by stimulating proteinproduction in muscle cells, energy releasefrom the breakdown of fats and stimulatesthe development of bones These processes together are responsiblefor longitudinal growth. Inadequateproduction of GH results in short stature,defined as a below normal height for agiven age
  7. 7.  In children and adolescents, the rate of growthin height is primarily determined by the rate atwhich endogenous GH is secreted The growth spurt during puberty is caused byincreased secretion of GH Under normal conditions, GH secretion andgrowth rate remain increased until final heightis reached, after which GH secretion is reducedto a steady state
  8. 8. Growth Hormone: 191 amino acids, single chainTeritiary structurePrimarystructureHormone binding to receptors
  9. 9. Production of recombinant GHIsolating and constructing hGH cDNAsConstructing expression cassette with hGH cDNAs insertsCultivating the recombinant clones in small scaleflask/bioreactorProducing the hGH in pilot scale bioreactorsDeveloping large scale purification procedure andprocess chromatography optimization (Affinitychromatography)
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  11. 11. Dr.T.V.Rao MD 17Historical picture of vaccination
  12. 12.  A vaccine is any preparationintended to produceimmunity to a disease bystimulating the production ofantibodies.Vaccines include,for example, suspensions ofkilled or attenuatedmicroorganisms, or productsor derivatives ofmicroorganisms.The most common methodof administering vaccines isby injection, but some aregiven by mouth or nasalspray.Dr.T.V.Rao MD 18
  13. 13. Dr.T.V.Rao MD 19Vaccination benefits ……. Vaccination intends to provide individualswith immunological protection before aninfection actually takes place. However, theimmune system is very complex, andimmunity against different infectiousagents is based on fine-tuned balancesbetween the various types of cells, signalsubstances and antibodies that make upthe total immune system
  14. 14.  Modern molecularbiology, recombinantDNA technology andgenetic engineeringhave opened theroad to a number ofalternative strategiesfor vaccineproduction,Trends in vaccine DevelopmentDr.T.V.Rao MD 20
  15. 15.  A preparation of directmanipulation of genes ofweakened or killedpathogen, such as abacterium or virus thatupon administrationstimulates antibodyproduction or cellularimmunity against thepathogen but isincapable of causingsevere infection.A genetically engineered vaccine is …21
  16. 16. 22DNA vaccines: They employ genes encoding proteins ofpathogens rather than using the proteinsthemselves, a live replicating vector, or anattenuated version of the pathogen itself.Theyconsist of a bacterial plasmid with a strong viralpromoter, the gene of interest, and apolyadenylation / transcriptional terminationsequence.The plasmid is grown in bacteria (e.coli), purified, dissolved in a saline solution, andthen simply injected into the host. In presentversions only very small amounts of antigens areproduced within the vaccinated individual.
  17. 17. Dr.T.V.Rao MD 23Recombinant (DNA) vaccines Made by isolation of DNA fragment(s) coding for theimmunogenic(s) of an infectious agent/cancer cell,followed by the insertion of the fragment(s) into vectorDNA molecules (i.e. plasmids or viruses) which canreplicate and conduct protein-expression withinbacterial, yeast, insect or mammalian cells.Theimmunogen(s) may then be completely purified bymodern separation techniques.The vaccines tend togive good antibody responses, but weakT-cellactivation.
  18. 18. Dr.T.V.Rao MD 24Naked DNA vaccines: They are engineered from general genetic shuttlevectors and constructed to break species barriers.They may persist much longer in the environmentthan commonly believed. Upon release or escapeto the wrong place at the wrong time. Horizontalgene transfer with unpredictable long- and short-term biological and ecological effects is a realhazard with such vaccines.There may be harmfuleffects due to random insertions of vaccineconstructs into cellular genomes in target or non-target species.
  19. 19.  They are produced by theinsertion of the DNAfragment(s) coding for animmunogen(s) intendedfor vaccination into thegenome of a non-dangerous virus orbacterium, the vector.Theinsertion is performed insuch a way that the vectoris still infectious live.Live vector vaccinesDr.T.V.Rao MD 25
  20. 20.  This involves the useof in vitro synthesisedRNA (a single-stranded relative ofDNA). RNA aredifferent from DNAvaccines in that thereis no risk ofchromosomalintegration of foreigngenetic material.RNA vaccinesDr.T.V.Rao MD 26
  21. 21. Dr.T.V.Rao MD 27Edible vaccines: These are produced by making transgenic,edible crop plants as the production anddelivery systems for subunit vaccines. Littleis known about the consequences ofreleasing such plants into the environment,but there are examples of transgenic plantsthat seriously alter their biologicalenvironment.A number of unpredicted andunwanted incidents have already takenplace with genetically engineered plants.
  22. 22. Dr.T.V.Rao MD 28Making DNAVaccines The gene for an antigenic determinant of apathogenic organism is inserted into aplasmid. This genetically engineered plasmidcomprises the DNA vaccine which is theninjected into the host. Within the host cells,the foreign gene can be expressed(transcribed and translated) from the plasmidDNA, and if sufficient amounts of the foreignprotein are produced, they will elicit animmune response
  23. 23. Dr.T.V.Rao MD 29Genetic Engineering a great tool indeveloping newer vaccines It is possible, using genetic engineering, tointroduce a gene coding for an immunogenicprotein from one organism into the genomeof another (such as vaccinia virus).Theorganism expressing a foreign gene is called arecombinant. Following injection into thesubject, the recombinant organism willreplicate and express sufficient amounts ofthe foreign protein to induce a specificimmune response to the protein.
  24. 24. Dr.T.V.Rao MD 30Advantages of DNA VaccinesOver Other Types of Vaccines Cheaper and easier to produce Safer Can elicit antibody and cellular immuneresponses Stable at a broad range of temperature (no cold-chain requirement) Can be designed and produced by geneticengineering to have only the desired antigens orantigenic sequences (epitopes) in the vaccine
  25. 25. Dr.T.V.Rao MD 31Hepatitis B a life threatinginfection Hepatitis B is one of the worlds most common blood-borne viruses. It infects some 200,000 people per yearin the U.S. alone.The virus is one hundred times morecontagious than the HIV virus; like HIV, it istransmitted through blood and sexual contact andcan be transmitted from mother to child at birth.Thevirus can exist in the bloodstream of a carrier for anentire lifetime. It is estimated that about 300 millionpeople worldwide are carriers, of whom about 25%will die from cirrhosis or cancer of the liver brought onby the disease.
  26. 26. Dr.T.V.Rao MD 32Genetically engineered vaccineTriumph of Biotechnology The worlds first genetically engineeredvaccine against a human disease--HepatitisB--is considered one of biotechnologysgreatest triumphs.The achievement standson the shoulders of pioneering work by UWgenetics professor Benjamin Hall and then-postdoctoral researcherGustav Ammerer todevelop genetic engineering techniques usingyeast cultures to produce proteins of interest.
  27. 27. • Manyrecombinantvaccines havebeen produced.These include liverecombinant,vector, subunit,and DNAVaccines.Genetically Engineered Vaccines33
  28. 28. Dr.T.V.Rao MD 34How hepatitis b engineered vaccineproduced Hall and Ammerer fused a segment of viralDNA specifying the surface antigen to the controlelements of a yeast gene.When they transferredthese hybrid genes into yeast cells, the resultingcultures produced Hepatitis B surface antigen.Serendipitously, these protein building blockswere found to clump together into the immunity-producing overcoat particles. With thatobservation, the key to a safe and effectivevaccine was in hand.
  29. 29. Dr.T.V.Rao MD 35
  30. 30.  Gene vaccines may berelatively new, buttheyre the logicaloutgrowth of twofamiliar strands ofmedical science. First isthe 200-year-oldpractice of vaccination,in which the body isinfected with aweakened form of adisease that preparesthe immune system for afuture encounter withthe real thing.Gene vaccinesDr.T.V.Rao MD 36
  31. 31. Genetically Engineered Vaccines afuture tool DNA vaccination is a technique for protectingan organism against disease by injecting it withgenetically engineered DNA to produce animmunological response. Nucleic acid vaccinesare still experimental, and have been applied toa number of viral, bacterial and parasitic modelsof disease, as well as to several tumour models.DNA vaccines have a number of advantagesover conventional vaccines, including the abilityto induce a wider range of immune responsetypes.
  32. 32. Dr.T.V.Rao MD 38Emerging trends in engineeredvaccines Some genetically engineered viral vaccines consist ofchimera viruses that combine aspects of two infective viralgenomes. One example is the live Flavivirus chimeravaccine against West Nile virus (WNV) in horses(PreveNile), registered in the United States in 2006.Thestructural genes of the attenuated yellow feverYF-17Dbackbone virus have been replaced with structural genesof the relatedWNV. Chimera avian influenza virus vaccineshave been produced on a backbone of an existing,attenuated Newcastle disease virus vaccine strain toprotection against wild-type influenza virus as well asagainst Newcastle disease virus