1. Ch27 The Immune System Yuki Juan NTU May 26, 2003

2. Outline Introduction Antibody-based or humoral immunity Activation of B cells to produce antibodies T cells and cell-mediated immunity Why does the human immune system so fiercely reject foreign human cells Monoclonal antibodies

3. Outline Introduction Antibody-based or humoral immunity Activation of B cells to produce antibodies T cells and cell-mediated immunity Why does the human immune system so fiercely reject foreign human cells Monoclonal antibodies

4. Terms Autoimmune reactions: –The immune system would attack the components of the body Autoimmune disease

5. The Cells Involved In The Immune System B cells: –Multiply and undergo their primary maturation in bone marrow T cells: –Multiple and mature in thymus glands –Helper T cells and cytotoxic killer T cells

6. Hemopoiesis

7. Two Immune Protective Systems Humoral immunity Cell-mediated immunity

8. Humoral Immunity Antibody: –Combine with the foreign antigens Antigen: –Antibody generation

9. Cell-mediated Immunity Cytotoxic T cells or killer T cells recognize abnormal cells in the body. The killer cell attaches to the virus-infected cell and destroys it.

10. The Differences Between Humoral And Cell-mediated Immunity The humoral system: – is part of the defence against virus in the blood or in the mucous membrane before it infects a cell. Cell-mediated immunity –Destroys the host cell after the latter has become infected and in doing so aborts virus replication in that cell.

11. Outline Introduction Antibody-based or humoral immunity Activation of B cells to produce antibodies T cells and cell-mediated immunity Why does the human immune system so fiercely reject foreign human cells Monoclonal antibodies

12. Antibodies Immunoglobulins (Igs) Y-shaped protein –Two identical light polypeptide chains –Two identical heavy chains

13. Structure of Antibody

14. Terms Epitope –The specific part of the antigen which is recognized by an antibody Clone –A collection of identical cells arising from multiplication of a single cell

15. Different Classes of Antibodies IgM –When the body responds to an antigen, the first produced antibody –Multi-subunit or polymeric –Form of antibody with 10 antigen combining sites IgG –More prolonged challenge by the same antigen IgA –Of importance as a first of defence

16. Generation Of Antiboy Diversity 300 V gene fragments 4 J gene segments C gene segment

17. The Process of Rearrangment Leading to a Functional L Chain Immunoglobulin Gene

18. Outline Introduction Antibody-based or humoral immunity Activation of B cells to produce antibodies T cells and cell-mediated immunity Why does the human immune system so fiercely reject foreign human cells Monoclonal antibodies

19. The Principle of Clonal Selection Macfarlane Burnet

20. Conversion of A Virgin B Cell to An Activated State, But not yet to an Antibody- producing Plasma Cell MHC classes II proteins: on the surface of the B cells

21. Comparison of Receptors on B and T Cells B Cell receptors T Cell receptors Are formed from antibodies displayed in the cell membrane Special receptor proteins in the membrane Specific receptors for each cell generated randomly Bind to antigen molecules in solution. Receptor recognizes the antigen itself Cannot bind to antigens in solution. Antigen must be internalized by special APC which degrades it and displays peptides in MHC proteins. The receptor recognises the complex of peptide-MGC protein on the APC, not either component alone

22. Activation of a Helper T Cell By An Antigen-presenting Cell (APC) APC: A special phagocytic white cell Cell surface markers known as clusters of differentiation

23. A Helper T Cell Activating a B Cell to Become A Clone of Plasma Cells Secreting Antibody

24. Summary Scheme of Antibody Production by B Cells

25. Term Memory cells –They are the basis of long-term immunity from a repeat infection

26. Outline Introduction Antibody-based or humoral immunity Activation of B cells to produce antibodies T cells and cell-mediated immunity Why does the human immune system so fiercely reject foreign human cells Monoclonal antibodies

27. Class II MHCs and Class I MHCs Helper T cells –recognize displayed peptides on class II MHCs –Have CD4 proteins Killer T cells –require display on class I MHCs –Have CD8 proteins

28. Sequence of Events In Cell-mediated Immune Reaction to a Foreign Antigen Synthesized Inside Cells

29. Outline Introduction Antibody-based or humoral immunity Activation of B cells to produce antibodies T cells and cell-mediated immunity Why does the human immune system so fiercely reject foreign human cells Monoclonal antibodies

30. Why Does the Human Immune System So Fiercely Reject Foreign Human Cells The main cause of rejection of foreign tissue grafts in the MHC molecules on cells.

31. Outline Introduction Antibody-based or humoral immunity Activation of B cells to produce antibodies T cells and cell-mediated immunity Why does the human immune system so fiercely reject foreign human cells Monoclonal antibodies

32. Monoclonal Antibodies The achievement of this earned Cesar Milstein and Georges Kohler in Cambridge a Nobel Prize

33. 單株抗體 由單一細胞株所製造 出來的抗體

34. 多株抗體 抗血清是多株抗體, 這 些抗體是由各種不同 種 B 細胞所產生抗體之 混合物

35. 單株抗體的特株性質 它只和抗原中的某種抗原決定位置結合, 不 會與不同之抗原決定位置相結合, 所以有很 高的專一性 它和抗原決定位置結合的強度及性質完全 相同, 所以容易用它來純化抗原

36. 單株抗體的製造方法 正常細胞藉由轉型 (transform) 變成癌細胞 ; 也就是使正常細胞表現出致癌基因 (oncogene) 如何使抗體分泌細胞變成癌細胞 – 用致癌物質或病毒使此類細胞內表現致癌基因 – 細胞融合技術 致癌細胞之致癌基因融合到抗體產分泌細胞中  融合 瘤細胞 (hybridoma)

37. 融合瘤細胞 (Hybridoma) 之製造 1975, Milstein and Kohler 發表  1984, 諾貝 爾生理醫學獎 老鼠的融合瘤細胞 脾臟 B 細胞 + 骨髓瘤細胞  以特殊溶液處理，分離融合細胞  篩選具有特殊抗體的細胞株  自該細胞的組織培養中，收集所需抗體

38. 單株抗體的應用 檢驗試劑的開發製造醫學治療生技產品之純化農業及畜牧業基礎研究

39. 單株抗體的應用 - 檢驗試劑的開發製造 荷爾蒙組織及細胞抗原藥物監控藥物濫用微生物抗體及過敏原酵素生長因子

40. 單株抗體的應用 - 醫學治療 器官移植腫瘤影像抗癌藥

41. Ch 29 Gene Cloning, Recombinant DNA Technology, Genetic Engineering Yuki Juan NTU May 27, 2003

42. Outline The first step-cutting the DNA with restriction endonucleases Gene cloning, or how genes are isolated Isolation of a genomic clone of a human gene Cloning a human cDNA What can be done with the cloned DNA Knockout mice of gene targeting

43. Outline The first step-cutting the DNA with restriction endonucleases Gene cloning, or how genes are isolated Isolation of a genomic clone of a human gene Cloning a human cDNA What can be done with the cloned DNA Knockout mice of gene targeting

44. DNases and Restriction Enzymes DNases –The digestive enzymes of pancreatic juice –Cut the phosphodiester bonds –Cut DNA randomly Restriction enzymes –Don’t cut DNA randomly –Each recognizes a specific short sequence of bases so as to make a cut at a precision point in the sequence

45. 基因選殖 –1970, Smith 發現第二類核酸 限切酵素 ， 可以很準確分割 DNA –1973, Boyer-Cohen-Chang 完 成第一基因選殖的工作 The first to be isolated from E. coli strain R

46. Outline The first step-cutting the DNA with restriction endonucleases Gene cloning, or how genes are isolated Isolation of a genomic clone of a human gene Cloning a human cDNA What can be done with the cloned DNA Knockout mice of gene targeting

47. Clone Refers to multiple identical copies produced from a single origin Genomic cloning and cDNA cloning

48. 寄主細胞 蛋白質表現系統主要有 – 大腸桿菌 : 操作方便、便宜 缺點 : – 沒有修飾蛋白質的能力 – 無法正確摺疊 – 會產生毒素 – 酵母菌 – 昆蟲細胞 : 操作較複雜、成本高 – 哺乳類細胞 : 操作較複雜、成本高

49. Outline The first step-cutting the DNA with restriction endonucleases Gene cloning, or how genes are isolated Isolation of a genomic clone of a human gene Cloning a human cDNA What can be done with the cloned DNA Knockout mice of gene targeting

50. 第二類核酸限切酵素 鈍端 (blunt end ) 黏端 (sticky end)

51. 連接酵素 (ligase)

52. Sequence of Steps In Producing in  Bacteriophage a Recombinant DNA Library

53. Illustration of Gene Cloning Using Bacteriophage Lambda Phage as The Cloning Vehicle

54. Outline The first step-cutting the DNA with restriction endonucleases Gene cloning, or how genes are isolated Isolation of a genomic clone of a human gene Cloning a human cDNA What can be done with the cloned DNA Knockout mice of gene targeting

55. cDNA Complementary DNA: the double-stranded DNA copy of mRNA Genomic DNA: contains introns

56. Outline The first step-cutting the DNA with restriction endonucleases Gene cloning, or how genes are isolated Isolation of a genomic clone of a human gene Cloning a human cDNA What can be done with the cloned DNA Knockout mice of gene targeting

57. Transferring a DNA Insert Into A Plasmid

58. The Structures of dATP and ddATP

59. An Autoradiograph Sequencing Gel Dideoxy DNA- sequencing Technique

60. Photograph of an Actual Sequencing Ladder

61. DNA 定序 Maxam-Gilbert Method G Reaction –ATTGACTTAGCC –ATTGACTTA –ATT A and G Reaction –ATTGACTTAGCC –ATTGACTTA –ATTGACTT –ATTG –ATT T and C Reaction –ATTGACTTAGCC –ATTGACTTAGC –ATTGACTTAG –ATTGACT –ATTGAC –ATTGA –AT –A C Reaction –ATTGACTTAGCC –ATTGACTTAGC –ATTGACTTAG –ATTGA CCGATTCAGTTCCGATTCAGTT

62. The Basic Principle Underlying Amplification of A DNA Section by PCR

63. 聚合酵素連鎖反應

64. 聚合酵素連鎖反應 (1)

65. 聚合酵素連鎖反應 (2)

66. 聚合酵素連鎖反應 (3)

67. Terms Transgenesis: –Genes can be inserted into animals and plants to give different phenotypes Gene therapy –Involves correction of a gene deficiency by inserting a normal gene into cells

68. Outline The first step-cutting the DNA with restriction endonucleases Gene cloning, or how genes are isolated Isolation of a genomic clone of a human gene Cloning a human cDNA What can be done with the cloned DNA Knockout mice of gene targeting

69. Terms Knockout mice –Mice in which a specific gene has been functionally eliminated Stem cells –Cells can divide without limit Embryonic stem cells –Different from other stem cells and differentiated cells –They give rise every type of cell in the body –Totipotent

70. Procedure For Obtaining Knockout Mice By Gene-targeted Mutation

71. One Method of Gene Targeting by Homolgous Recombination in ES Cells