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脊髓灰质炎病毒空斑实验 PowerPoint PPT Presentation


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脊髓灰质炎病毒空斑实验. 细胞培养. 试验前准备( 4 人 / 组) * 无菌操作:套脚套,洗手,个人防护器材佩戴,包括:衣帽、口罩、手套。掌握微生物学无菌操作技术。酒精棉球,无菌镊子,记号笔等。. 无菌操作过程中要经常用 75% 酒精消毒手。开瓶盖前也用酒精棉球消毒该区域。. 试管、瓶子的上 1/4 ~ 1/3 、移液管的下 2/3 部分不能触碰任何污染区域. 敏感宿主细胞. 一、传代细胞系培养 1 、优点: 1) 可以无限的传代。 2) 不少细胞系对病毒很敏感。 3) 某些传代细胞系能在悬浮培养条件下培养,适合病毒抗原的大量生产。

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脊髓灰质炎病毒空斑实验

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  • 4/

  • *


75%


1/4 1/32/3


  • 11)

  • 2)

  • 3)

  • 4)


  • 123105/ml(Hela 1:3 or 1:4)

  • 2pH pH7.07.4pH6.67.8

  • 3

  • 37

  • 28 30


*

  • 1%104U/ml10% 12ml100ml14mmol/L 7.5%pH7.2pH7.4


  • 7.5%pH7.4pH7.60.02%EDTA


*

  • 1

  • 22ml 0.02%EDTA(5ml 0.02%EDTA 1)37EDTA

  • 3,31:3*10ml15ml 375 7

*1:362.5ml3ml/3

15ml/3=45 ml/ 16ml


Hela

Vero





*Hanks 2MEM

  • Hanks1%104U/ml7.5%HankspH7.2pH7.4

  • Hanks1MEMPolio V 110-110-210-310-410-510-6


0.5ml

0.5ml

0.5ml

0.5ml

0.5ml

10min

5g

4.5ml

4.5ml

4.5ml

4.5ml

4.5ml

45ml

10-2

10-3

10-4

10-5

10-6

10-1

0.2ml

0.2ml

0.2ml

371.52.0 h15 min1

2ml

2ml

2ml

2


  • 2MEM45 1%104U/ml12ml100ml14mmol/L 2%7.5%pH7.2pH7.4

  • 2MEM2%1%45


PolioV

  • 1

  • 2 0.2 ml/ 0.2 ml/

  • 337 1.52 h15

  • 445 0.5%2 ml/30 min

  • 537 4872 h


*

  • 2 ml20 min

  • 1%10 min


PFU (plaque-forming units)

P

n

v


  • End


f2


  • (Coliphage) f2 1 (Coliphage) f2RNA pH234


  • f2285


  • 1

PFU

2ID50 TCID50

50%


  • 3/


  • 285 121020 ml37 1012 h

  • 2%50 1020 ml/


  • 1%45

  • f210-110-210-310-410-510-610-710-8

  • 3


  • f21 ml2850.2 ml45 1%5 ml

  • 37 1624 h


f21 ml

0.2 ml

45 1%5 ml


Plaque


PFU (plaque-forming units)

P

n

v


  • End


1ml

1ml

1ml

1ml

1ml

10min

10g

9ml

9ml

9ml

9ml

9ml

90ml

10-2

10-3

10-4

10-5

10-6

10-1

0.1ml

0.1ml

0.1ml

20ml

20ml

20ml

2


CPE


CPE


PAGERotavirus

  • 1/41/3-1/2PAGE

  • RNA11RNApHRNARNARNARNARNARNA


  • a)RNA (PAGE)

  • PBS10%0.25 g2.25 ml PBS

  • 10%250l1.5 ml50l 3

  • 3730 min

1345


4. 4300l 3

5. 12000/ min2 min

6.

7. 300l 3

8. 12000/ min2 min

9.

  • 4912

  • -20500l5 M NaCl 5l3-20-70 2




12.12000/ min1520 min

13. 1530 min

14.50lRNA-20PAGERNA


b) PAGE

  • 1 g100 ml


3.

0.75 M Tris-HCl (pH 8.8) 20 ml

0.1 M EDTA 0.4 ml

13.3 ml

4.7 ml

  • 3%1.6 mlTEMED 70 l11.5 cm1h

1ml 50 l TEMED10 min

20101.4 ml + 60 l


  • Tris (Figure 3) is used to remove ammonia and amine contaminants and to keep the gel at a constant pH.

Figure 3 - Tris Molecule


Figure 6 - BIS Molecule

Figure 5 - Acrylamide Molecule

Figure 7 - Ammonium Persulfate (APS) Molecule

Figure 8 - TEMED Molecule


Figure 9 - Polyacrylamide Matrix



4.

0.5 M Tris-HCl (pH 6.8) 5.0 ml

0.1 M EDTA 0.2 ml

2.0 ml

12.4 ml

  • 3%0.4 mlTEMED 20 l

3 ml1ml 70 l TEMED

20100.8 ml + 40 l


5.

10 lRNA 10 l

6.

1820 mA,1640 mA, 161 cm

669.96 D

Figure 12 - Bromophenol Blue Molecule



c)

  • AgNO31 h,342ml1020 min34


  • End


  • SDS (Sodium dodecylsulfate) is a detergent (Figure 4) that binds to proteins to give them an overall negative charge, allowing proteins to migrate in one direction only. Thus, they can be easily separated according to size.

Figure 4 - SDS Molecule


Figure 11 - Beta-Mercaptoethanol (B-ME) Molecule


Figure 13 - Movement of Proteins


ELISA

  • ELISAimmunoassayIA


ELISA

ELISA


* 2mol/L H2SO4 ** 2 mol/L


mg/ml5~10g/mlng/mlHBsAg0.1ng/ml


ELISA

ELISA1""immunosorbent2""conjugate3ELISA


1



  • A15


  • 1124

  • 250

  • 3l

  • 41

  • 51

  • 6A

  • 7B


11(50)

21(50)

310110

4AB5



  • End


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