پایان نامه بیان و تخلیص پروتئین نوترکیب FGFR2b و بررسی تغییرات ساختاری آن بر اثر برهمکنش با فلزات سمی

فهرست:

فصل اول     مقدمه و اهمیت موضوع. 3

1-1- مقدمه. 4

1-1-1- فاکتور رشد فیبروبلاستی.. 4

1-1-2- گیرنده های فاکتور رشد فیبروبلاست.. 4

1-1-3- گیرنده های فاکتور رشد فیبروبلاست نوع 2. 5

1-1-4- گیرنده های فاکتور رشد فیبروبلاستی تیروزین کینازی.. 6

1-1-5- فعال سازی کینازها از طریق ایجاد موتاسیون.. 8

1-2- اهمیت موضوع وضرورت انجام تحقیق. 9

1-3- اهداف طرح. 9

1-3-1- هدف اصلی.. 9

1-3-2- اهداف فرعی.. 9

1-3-3- اهداف کاربردی.. 10

1-3-4- فرضیات.. 10

1-3-5- متغییرها.. 10

فصل دوم      مروری بر متون گذشته. 11

2-1- پروتئین. 12

2-1-1- ساختمان پروتئین ها.. 13

2-1-2- گیرنده های تیروزین کینازی.. 17

2-1-3- فاکتورهای رشد فیبروبلاستی.. 18

2-1-4- گیرنده های فاکتور رشد فیبروبلاستی.. 20

2-1-5- گیرنده های فاکتورهای رشد فیبروبلاستی و اختلالات پاتولوژیکی.. 22

2-1-6- مسیر پیام رسانی سلولی فاکتورهای رشد فیبروبلاستی.. 25

2-1-7- تنظیم مسیر پیام رسانی فاکتور های رشد فیبروبلاستی.. 29

2-2- فلز چیست؟. 31

2-2-1- فلزات سمی.. 31

2-2-3- سرب.. 32

2-2-4-کادمیوم.. 32

2-2-5- نیکل.. 33

2-2-6-آلومینیوم.. 33

2-7- تاثیر فلزات بر مسیرهای سیگنالینگ. 34

2-7-1-ROS. 34

2-7-2- MAPK.. 35

2-7-3- PI3K/Akt 36

2-7-4- HIF.. 37

2-7-5- NF-kB.. 38

2-7-6- NFAT. 39

2-7-7- AP.. 40

2-8- اثر ترکیبات فلزی بر مسیرهای سیگنالینگ و بیان ژن. 41

2-8-1- سرب.. 41

2-8-2- کادمیوم.. 43

2-8-3- نیکل.. 46

2-8-4- آلومینیوم.. 48

2-9- پیشگویی ساختمان پروتئین ها. 50

2-9-1- بررسی ساختار پروتئین ها.. 50

2-9-2- مطالعه ی ساختاری پروتئین ها.. 51

2-9-3- تکنیک های مطالعه ی ساختار پروتئین  ها.. 52

2-9-4- تکنیک فلوئورسانس اسپکتروسکوپی.. 53

2-9-5- تکنیک دورنگ نمایی حلقوی(CD) 56

2-9-6- تکنیک ها و عوامل دناتوراسیون.. 57

2-10- تکنیک های تولید و تخلیص پروتئین های نوترکیب. 60

2-10-1- کاربرد پروتئین های نوترکیب. 60

2-10-2- پروتئین های نوترکیب (Recombinant proteins ) 60

2-10-3- تولید پروتئین های نوترکیب در گیاهان.. 63

2-10-4- استفاده ازایشریشیاکلی به عنوان یک ارگانیسم میزبان برای تولید پروتئین   64

2-10-5- خالص سازی پروتئین های نوترکیب.. 64

فصل سوم   مواد و روش ها. 66

3-1- مواد، تجهیزات و متغیر ها ی آزمایش. 67

3-1-1- مواد مورد استفاده در آزمایش.. 67

3-1-2- دستگاه ها و تجهزات مورد استفاده در آزمایش.. 68

3-2- محلول ها و بافرها. 69

3-2-1- تهیه ی استوک آمپی سیلین.. 69

3-2-2- تهیه ی استوک IPTG.. 70

3-2-3- تهیه ی بافر لیز کننده سلول جهت بررسی حلالیت پروتئین.. 70

3-2-4- تهیه ی بافر لیز کننده  سلول جهت تخلیص پروتئین.. 70

3-2-5- تهیه ی محلول ژل پلی آکریل آمید 4%... 70

3-2-6- تهیه ی محلول ژل پلی آکریل آمید 12%... 71

3-2-7- تهیه ی محلول  APS10%... 71

3-2-8- تهیه ی بافر الکترود x10  (Running buffer) 71

3-2-9- تهیه ی بافر نمونه Sample Buffer)) 71

3-2-10- تهیه ی محلول Tris-Hcl 5/0 مولار.. 72

3-2-11- تهیه ی محلول Tris-Hcl 5/1 مولار.. 72

3-2-12- تهیه ی Staining Buffer. 72

3-2-13- تهیه ی Destaining Buffer. 73

3-2-14- تهیه ی محلول آکریل آمید- بیس آکریل آمید.. 73

3-2-15- تهیه بافر SDS. 73

3-2-16- تهیه بافرA (Washing Buffer) 73

3-2-17- تهیه بافر B (   (Eluting Buffer. 74

3-2-18- تهیه بافر دیالیز.. 74

3-2-19- تهیه استوک گوانیدین هیدروکلراید (GnHCl) 74

3-2-20- تهیه استوک فلزات.. 74

3-2-21- آماده سازی محیط های کشت باکتری.. 74

3-3- روش انجام کار. 75

3-3-1- نمایش ساختار پلاسمید نوترکیب pLEICS-01 و توالی ژنی ناحیه تیروزین کینازی پروتئین نوترکیب FGFR2b. 75

3-3-2- مراحل تولید پروتئین نوترکیب.. 77

3-3-3- تعیین غلظت پروتئین.. 83

3-3-4- مطالعات اسپکتروسکوپی فلوئورسانس.. 83

3-3-5- مطالعات اسپکتروسکوپی دورنگ نمایی حلقوی CD)) 85

3-3-6- مطالعات اسپکتروسکوپی FTIR.. 86

3-3-7- مطالعات دناتوراسیون شیمیایی با استفاده از اسپکتروسکوپی فلوئورسانس   87

         فصل چهارم یافته ها و نتایج................................................................................................................................................. ................................................................................................................................................. 88

4-1- بررسی بیان پروتئین در دمای 37 درجه سانتی گراد. 88

4-2- بررسی بیان پروتئین در دمای20 درجه سانتی گراد. 89

4-3- بررسی  حلالیت پروتئین بیان شده دردو دمای20 و 37 درجه سانتی گراد. 90

4-4- بررسی میزان خلوص پروتئین محلول شده. 91

4-5- آنالیز SDS-PAGE بعد از دیالیز. 92

4-6- بررسی عملکرد پروتئین خالص شده. 93

4-7- بررسی ساختار سوم ناحیه‏ی تیروزین کینازی پروتئین نوترکیب FGFR2b 94

4-8- بررسی اثر دناتوراسیون شیمیایی بر طول موج ماکزیمم نشر فلوئورسنس ذاتی ناحیه‏ی تیروزین کینازی پروتئین نوترکیب FGFR2b 98

4-9- بررسی اثردناتوراسیون شیمیایی بر شدت نشر فلوئورسانس ذاتی ناحیه‏ی تیروزین کینازی پروتئین نوترکیب FGFR2b 101

4-10- بررسی اثر سرب، کادمیوم، آلومینیوم و نیکل، بر روی ساختار دوم ناحیه‏ی تیروزین کینازی پروتئین نوترکیب FGFR2b 104

فصل پنجم      بحث و نتیجه گیری.. 109

5-1- بحث و نتیجه گیری. 110

فهرست منابع: 116

….………………………………………………………………………………..Abstract

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