Chapter 14 Part One Biotechnology and Industry: Microbes at Work

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1 Chapter 14 Part One Biotechnology and Industry: Microbes at Work Objectives: After reading Chapter 14, you should understand How biotechnology has resulted in numerous pharmaceutical products to help lessen human disease and suffering. The molecular basis of antisense molecules and DNA vaccines. The variety of industrially important microbes and the products they help to produce. Introduction: the hopes and products of biotechnology are only possible because we now understand the structure and function of DNA. This allows us to manipulate the DNA to do things like cloning, control gene expression and investigate potentially life-saving procedures (i.e. through stem cell research). Microbes and Gene Technology What is biotechnology? How have we benefited from biotechnology? Gene sequencing, drug production, vaccines, disease- or herbicide-resistant plants, genetic fingerprinting, etc. DNA is the central molecule that forms the foundation of biotechnology. 1

2 By manipulating DNA, many advances can be made in the fields of agriculture, medicine and industry. Example: How can we use cloning to get a bacterium to express human genes? Assume that an enzyme deficiency in humans could be treated by adding the enzyme to the diet of affected people. Let s call this enzyme protein A. Sounds simple you just obtain protein A and add it to the human diet. But there is one problem, where do you get the enzyme? This is called recombinant DNA or rdna. This type of technology can be used to help solve many medical problems 1. Diabetes Diabetics have limited production of the hormone insulin (what does insulin do?) Insulin was discovered in early 1920s, but its use was problematic because it was not always pure. From where was it isolated? 2

3 1970s Insulin became the first ever product of biotechnology used for human therapy (1980). How is it made? Genes that encode for the production and secretion of human insulin are inserted into a plasmid, which is then inserted into and expressed in E. coli. 3

4 2. Dwarfism Human growth hormone (HGH) is normally secreted by the pituitary gland to stimulate growth and reproduction of human cells. A lack of HGH production is one cause of dwarfism. From: Escamilla et al., 1966 Prior to 1980s, HGH was obtained from pituitary glands of cadavers. Problem: the HGH was often contaminated with bacteria and viruses (e.g. HIV) Now, HGH can be synthetically manufactured in what bacterium??????. 4

5 3. Dissolving blood clots Clots often form in response to disease conditions such as atherosclerosis, stroke, cancer and some complications during pregnancy. Doctors now use recombinant tissue plasminogen activator (tpa) as a powerful drug to help dissolve these clots and prevent much of the damage associated with loss of blood flow to vital organs. How do you think tpa is mass-produced? 4. Anemia (limited red blood cell production) The hormone erythropoietin (EPO) is produced by the kidneys and is responsible for stimulating stem cells in the bone marrow to mature into red blood cells. Increases the red blood cell count. Why might athletes choose to abuse EPO? 5

6 Antisense molecules Might someday be useful to treat viral diseases (not yet clinical). Specific to treating proviruses viral DNA (or reverse transcribed viral RNA) that is incorporated into the host genome (e.g. HIV, Ebola). An antisense molecule is a piece of RNA that combines with the mrna from the invading virus, limiting the ability of ribosomes in the infected cell to synthesize viral proteins from the mrna. 6

7 If the host cell cannot produce the viral proteins, then the virus cannot be replicated. In early 2006, scientists developed antisense technology to treat the Ebolavirus Electron micrograph of Ebolavirus. ~80 x 1000 nm Infection results in Ebola hemorrhagic fever (EHF). Ebola first emerged in 1976 in Zaire. The virus reservoir is thought to be bats and is transmitted between humans by bodily fluids. 7

8 Blood vessel walls become damaged and the platelets are unable to coagulate and patients succumb to hypovolemic shock. Experiments in which monkeys were infected with Ebolavirus and then treated with antisense drugs have shown a 75% recovery rate. The usual mortality rate for monkeys infected with Ebola is 100% Antisense molecules can also be used to interrupt oncogene activity (cancer inhibition) and the process of Chron s disease (intestinal inflammation). Biotech vaccines Traditional vaccines usually made from inactivated or attenuated viruses, microbial proteins or carbohydrates or whole microbes. As you already know, these are administered to elicit a mild immune response. What is a major (although rare) problem with traditional vaccines? Newer, biotech vaccines can help avoid these side effects. One success - Hepatitis B vaccine Key element of the vaccine is a protein found in the hepatitis B virus capsid. To produce the vaccine, the gene for the protein was cloned into yeast, which expressed the protein the intact virus never gets used. Therefore, the vaccine is composed of harmless capsid proteins. Many pediatricians recommend vaccination for newborns and it has been used to immunize millions of healthcare workers. 8

9 and one possibility - Vaccination against AIDS In 1984, after the confirmation of the etiological agent of AIDS by scientists at the U.S. National Institutes of Health and the Pasteur Institute, the United States Health and Human Services Secretary Margaret Heckler declared that a vaccine would be available within two years. This has not happened for two reasons: 1. HIV is highly mutable. Because of the virus' ability to rapidly respond to selective pressures imposed by the immune system, the population of virus in an infected individual typically evolves so that it can evade the two major arms of the adaptive immune system; humoral (antibody-mediated) and cellular (mediated by T cells) immunity. 2. HIV isolates are themselves highly variable. HIV can be categorized into multiple clades and subtypes with a high degree of genetic divergence. Therefore, the immune responses raised by any vaccine need to be broad enough to account for this variability. 9

10 Diversity and divergence of HIV-1 gp160 sequences. Euler Z. et al. J. Virol. 2012, 86: However, researchers have identified, cloned and expressed (in E. coli) two major envelope proteins of HIV called gp120 and gp41. Vaccines produced using gp120 and gp41 results in antibody production in animals. 10

11 Upon exposure to HIV, antibodies bind to gp120 and gp41 and prevent it from binding to the T cells. DNA vaccines DNA vaccines differ from traditional vaccines in that just the DNA coding for a specific protein produced by a disease-causing organism is injected into the body Vaccines consist of plasmids engineered to contain a gene that encodes a protein against which antibodies can be produced. Example: West Nile virus. Two WNV envelope protein genes are inserted into a plasmid, which is injected into a human. 11

12 Once inside the human, the genes encoded by the WNV DNA are expressed by human cells and an immediate immune response occurs. Antibodies are produced, and the person is protected from subsequent infections. DNA vaccines have been used experimentally to protect against influenza, salmonellosis, HIV, herpes simplex and hepatitis B. Advantages of DNA vaccines 1. Since, the actual production of the immunizing protein takes place in the vaccinated host, the risk of infection associated with some live and attenuated virus vaccines is eliminated. 2. Vaccines for multiple diseases can be given in a single inoculation. Currently, in the United States, the full course of childhood immunizations requires 18 visits to the doctor. 12

13 Disadvantages 3. They are extremely stable. DNA vaccines can be stored under a vast array of conditions either dried or in a solution. 1. Limited to protein antigens. This eliminates the need for the "cold chain". Can greatly improve the ability to deliver vaccines to remote areas in developing countries. Ineffective to raise antibodies against carbohydrate or lipid antigens. 2. Potential for atypical processing of injected DNA or resulting protein. 13

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