표제지
목차
초록 6
Abbreviation 10
I. 서론 11
II. 실험 재료 및 방법 17
1. 발현 Vector 및 숙주세포 17
2. 배양 및 배양액 19
3. 시약, 효소, 단백질 정제 20
4. ADAM 21 Cloning 22
4.1. Gene 합성 22
4.2. Polymerase Chain Reaction and ADAM 21 Cloning 24
5. Transformation to E.coli 29
6. ADAM 21 Truncation form Design and cloning 30
7. Transfection to HEK293 E cell 32
8. Signal Peptide Replacement 33
9. Western blot analysis 40
10. Signal Peptide Library Screening 41
11. Protein Purification 43
12. Protein Quantitation 44
III. 실험 결과 45
1. pcDNA 3.4 ADAM 21 SP ADAM 21 Extracellular Domain and 3type Truncation form 45
2. Disintegrin의 발현 분석 48
3. Effect of Signal Peptide on Expression to ADAM 21 49
4. Validation of Signal Peptide Library Screening 56
5. Protein Purification Volume Comparison 58
IV. 결론 60
V. 참고문헌 61
ABSTRACT 69
Table 1. Oligonucloetide used in PCR 26
Table 2. PCR Protocol for ADAM 21 Extracelluar domain 26
Table 3. Oligonucleotide for ADAM21 Signal Peptide Synthesis 27
Table 4. Oligonucleotide for ADAM21 Truncation form 31
Table 5. Signal Peptide Library List 1~300 34
Fig. 1. Cancer Tissue distribution of A Disintegrin And Metalloproteinase 21(ADAM21). 16
Fig. 2. pcDNA 3.4 TOPO vector. 18
Fig. 3. ADAM21 ORF. 23
Fig. 4. Schematic of ADAM21 Signal Peptide Synthesis. 28
Fig. 5. Condition of ADAM 21 Signal Peptide Synthesis. 28
Fig. 6. 3 Type of ADAM21 Truncation form. 31
Fig. 7. Quantification of Signal Peptide Library Screening 42
Fig. 8. Map of pcDNA 3.4 ADAM21 SP ADAM21 Extracellular Domain. 45
Fig. 9. Point mutarion of ADAM21 Extracellular Domain 46
Fig. 10. pcDNA 3.4 ADAM21 SP 3Type Truncation form 47
Fig. 11. pcDNA 3.4 ADAM21 SP Disintegrin Bradford assay result 48
Fig. 12. Signal Peptide Library Screening for Disintegrin protein 50
Fig. 13. Validation of Signal Peptide Library Screening 57
Fig. 14. Comparison of Protein Expression by Bradford Assay 59