Expression of N-terminal seven amino acids peptide of fibrin (β–peptide) on surface of M13KO7 phage

1. ‫الرحمن‬ ‫هللا‬ ‫بسم‬
‫الرحیم‬
1

2. Expression of N-terminal seven
amino acids peptide of fibrin
(β–peptide) on surface of M13KO7
phage
Supervisor: Dr. Hossein Khanahmad
Aref Farrokhi Fard
aarreeff@ymail.com
2

3. Introduction
3

4. Vascular accidents
• Acute myocardial infarction
• Cerebrovascular accident (CVA) or acute
cerebral infarction
• Pulmonary thromboembolism
4

5. 5

6. Fibrinolytic drugs for therapy
 Urokinase (UK)
 Streptokinase (SK)
 Recombinant Tissue plasminogen activator
(rTPA)
Side effects:
• Low efficiency
• Hemorrhage
6

7. Clot targeting for preventing side
effects and elevating the fibrinolysis
efficiency
Local enrichment using fibrin targeting : Specific anti fibrin
antibody(MA-15C5), 59D8):
 Chemical conjugation
 Fusion protein(Ab-E)
Nanobody
Scfv FabWhole Ab
Fv
fragment
7

8. Material and methods
8

9. 9

10. fibrinogen beta chain isoform 1
preproprotein [Homo sapiens]
Beta-peptide as an antigen
Gly-His-Arg-Pro-Leu-Asp-Lys
FSARGHRPLDKKREE
45-52
NCBI Reference Sequence: NP_005132.2
10

11. 11

12. Bam HI
2220
BsrG I
10211119 bp
12
7550 bp
GTCTGTACACCGTTCATCTGTCC
TGGATCCTCATTAAAGCCAGAA

13. 1301..1522
1579..2853
gene="V"
CDS 843..1106
13

14. After overlap PCR
1. Agarose gel electrophoresis of PCR product
2. Gel extraction
3. Ligation between ABC fragment and plasmid
backbone
1179 bp
8729 bp
14
7550 bp

15. 15

16. After transformation to top10
Plasmid extraction from transformed top10 bacteria
Transformation of phage genome to TG1
PCR on recombinant phage genome
Confirmation by sequencing
Obtaining phages from culture for SDS PAGE
16

17. SDS PAGE
17

18. 18

19. 19

20. Thank you for your attention
20

21. 21

22. 22