Name:______

Activity 1.4.2: Vaccine Development

Introduction

The discovery of vaccination was one of the farthest reaching medical discoveries of all time. Vaccines have dramatically decreased the impact of infectious diseases which were once considered deadly. Although the overall goal of creating a weakened version of the antigen to kick-start the immune system is the same, various laboratory techniques are used to generate vaccines. Scientists must be concerned with safety of the vaccine, ease of the delivery system, and its overall effectiveness over time. Technology for producing vaccines continues to evolve. In this activity, you will explore the methods used to develop vaccines as well as simulate the use of recombinant DNA techniques to produce these powerful interventions.

Recombinant DNA technology is the process of cutting and recombining DNA fragments. Since DNA is comprised of the same nucleotides in all species, segments of DNA can be combined from two different people, or even two different species. In addition to one large chromosome, bacteria carry additional genes in rings of DNA called plasmids. Plasmids can be employed as an important tool in genetic engineering and can serve as vectors, vehicles for the movement of genetic information. Genes, segments of DNA that code for the production of a protein, can be inserted into plasmids and the plasmids can then be inserted into bacterial cells. Cells containing a new plasmid are cloned, the plasmid replicates and proteins are transcribed and translated in the host cell. Scientists can use tiny bacterial cells as factories for the production of proteins. These proteins can be used for various medications, including vaccines.

In this activity, you will research the various methods used to produce vaccines. You will then try your hand at genetic engineering and simulate the formation of a recombinant plasmid used to produce a vaccine for the Hepatitis B virus.

Equipment

·  Computer with Internet access and Inspiration® software

·  Activity 1.4.2 Student Resource Sheet

·  Paper plasmid DNA

·  Scissors

·  Scotch tape

·  Colored pencils

·  Laboratory journal

Procedure

Part I: Vaccine Basics

1.  Visit the NOVA Making Vaccines site at http://www.pbs.org/wgbh/nova/meningitis/vaccines.html. Complete the Making Vaccines activity.

  1. Instead of taking notes, you will create a concept map for vaccine production. Use Inspiration to complete your map.
  2. Summarize the different techniques used to manufacture vaccines. Use short phrases with major ideas in the bubbles. Make sure to use appropriate connecting terms on the lines.
  3. Start the concept map with a bubble that is labeled as “Vaccine Product Methods”. Then, list each type of vaccine production method under the main bubble.
  4. Include an example of a vaccine produced by each method.

2.  Answer Conclusion question 1 and 2.

Part II: Recombinant DNA Technology

Now that you have researched the types of vaccine preparations, you will simulate the production of a vaccine through recombinant DNA technology.

3.  Refer to the bacterial cell drawing you created in Lesson 1.2 and review the way in which bacteria store genetic information.

4.  Answer Conclusion question 3-4.

5.  Visit the Dolan DNA Learning Center DNA Interactive site at http://www.dnai.org/b/index.html.

6.  Choose the Revolution tab at the bottom of the page to learn more about how scientists are able to manipulate and work with particular genes.

7.  Choose the Problem tab at the top of the page. Read the information presented in this section using the small arrow at the bottom of the screen to advance the slides.

8.  On a sheet of paper, describe the two tools of molecular biology that are often described as the scissors and glue for manipulating DNA. Describe how these tools of molecular biology combined with plasmids can be used to make copies of an important gene.

9.  At the end of the Problem section, click on the Techniques tab at the bottom of the page.

10. Click on Cutting and Pasting.

11. Click on Cutting and Pasting DNA to view the 2D animation. Use the arrow at the bottom right-hand corner of the screen to advance the animation.

12. Click on the Recombinant DNA tab at the left side of the page to view the 3D animation. Watch the video and read the text below.

13. View the Access Excellence diagram Inserting a DNA Sample into a Plasmid at http://www.accessexcellence.org/RC/VL/GG/inserting.php and read the description found below the diagram. Connect concepts you learned from the DNA Interactive site to the process shown on the diagram.

14. Answer Conclusion question 5.

Bacterial plasmids can be used to generate protein products that are part of a disease antigen. These products can be used to trigger an immune response. In this activity, you will engineer a plasmid to produce a protein used as a vaccine against the virus, Hepatitis B. You will transfer a gene for a viral envelope protein for Hepatitis B into a bacterial plasmid. This protein resides on the surface of the virus and assists with identity and infection. Once copies of this protein are made in bacterial cells, the protein can be purified for use as a vaccine.

15. Recall that each restriction enzyme recognizes a specific DNA sequence called a restriction sequence or restriction site. When the enzyme encounters this sequence, it makes a distinct cut in the DNA. Refer to the chart below for the restriction sites of five common enzymes.

Restriction Enzyme / Restriction Site / Cut
BamHI / 5’ GGATCC
3’ CCTAGG / 5’ ---G GATCC--- 3’
3’ ---CCTAG G--- 5’
EcoRI / 5’ GAATTC
3’ CTTAAG / 5’ ---G AATTC--- 3’
3’ ---CTTAA G--- 5’
HaeIII / 5’ GGCC
3’ CCGG / 5’ ---GG CC--- 3’
3’ ---CC GG--- 5’
HindIII / 5’ AAGCTT
3’ TTCGAA / 5’ ---A AGCTT--- 3’
3’ ---TTCGA A--- 5’
PstI / 5’ CTGCAG
3’ GACGTC / 5’ ---CTGCA G--- 3’
3’ ---G ACGTC--- 5’

16. Answer Conclusion question 6.

17. Obtain a Student Resource Sheet, a paper plasmid, scissors, tape and colored pencils from your teacher.

18. Refer to the table of restriction enzymes and their restriction sequences shown above or on the Student Resource Sheet. Scan the plasmid DNA and locate the restriction sites for each enzyme. These sites have been shaded in or colored for you. Identify the enzyme that would target each designated site and create a color key (on the table above #16).

19. Scan the viral DNA and locate restriction sites for each enzyme. Using the same color key you designated in Step 20, shade each restriction site with the appropriate color using colored pencils.

20. Scan the plasmid DNA as well as the gene from the Hepatitis B virus to find an appropriate enzyme to engineer the plasmid. Your goal is to open the plasmid ring and insert a piece of the viral gene. Follow the guidelines listed below to determine the most efficient enzyme to complete your task.

o  Use an appropriate restriction enzyme to open the plasmid ring as well as cut out a piece of the viral gene. You do not want to use an enzyme that digests the plasmid into many tiny pieces.

o  Do not cleave more than twenty base pairs off either side of the viral DNA. The remaining base pairs are required for production of the protein of interest. Important promoters at the beginning of the sequence are vital to transcribing the genetic code.

o  Choose an enzyme that does not interrupt the antibiotic resistance gene in the plasmid or the origin of replication (ori), the sequence that initiates replication of the plasmid. The antibiotic resistance gene provides scientists with a way of determining if an engineered plasmid successfully made it into a target cell. If bacterial cells that were not antibiotic resistant now grow on medium with antibiotics present, they must contain the new plasmid.

o  Make sure you create sticky ends on both the plasmid and the viral DNA.

21. When you think you have located an appropriate enzyme, present your plan to the teacher. Be prepared to defend your recommendation with evidence on your DNA.

22. Use scissors and tape to create your recombinant plasmid. Be sure to make your cuts according to the pattern shown on the restriction enzyme table.

23. Examine your completed plasmid. Compare your final product to that of a classmate’s. Discuss any differences.

Activity 1.4.2: Vaccine Development Conclusion Questions

1.  Which types of vaccines described on the website are examples of live vaccines? Explain your reasoning.

2.  How does vaccination contribute to the development of herd immunity in a community?

3.  What are the similarities and differences between the organization and structure of DNA in bacterial cells and in human cells?

4.  Why do you think plasmids and bacterial cells are such important tools in genetic engineering?

5.  Explain why a plasmid and a segment of DNA to be inserted into a plasmid must be cut with the same restriction enzyme. Use the term sticky ends in your response.

6.  Explain why HaeIII is unlikely to be used to create recombinant DNA. How does this enzyme differ from the others described in the table?

7.  What molecular tools do scissors and tape represent in this activity? Explain.

8.  Edible vaccines, a more controversial approach to vaccine development, have been investigated by scientists. Plants can be genetically modified to produce viral proteins and when ingested, these proteins ignite the immune system of the consumer. What are the advantages to edible vaccines? What are some of the possible disadvantages?

9.  Jimmy has always been told that if you have chicken pox once, you will not get the disease again. He had chicken pox when he was eight, but he only had five to ten small pox on his skin. As an adult, Jimmy is again showing symptoms of chicken pox. Using your knowledge of the human immune system, explain how this is possible.

10. A new flu vaccine is formulated and released every year. Explain why one shot against influenza does not necessarily protect a person from year to year. Think about the genetic material of the virus.