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ABBREVIATIONS 7

I. 서론 8

I. 이론적 배경 8

1. 세포 내 유전자 및 단백질 도입기술 8

2. 단백질도입도메인(PTD; protein transduction domain) 11

3. PTD를 이용한 다양한 신약후보물질 개발 현황 13

4. 기존 단백질 도입 도메인의 문제점 14

5. Vector를 이용한 단백질 발현의 장단점 14

II. 연구방법 및 재료 15

1. 인간유전인자정보검색 15

2. Material and Drugs 15

3. Preparation of Competent cells 15

4. E. coli Transformation 16

5. Isolation of plasmid DNA 16

6. PCR(Polymerase chain reaction) 17

7. Agarose gel electrophorisis and EtBr staining 17

8. DNA sequencing 18

9. Oligonucleotide construction and annealing 18

10. Subcloning of PCR product and ligation 18

11. 융합단백질의 분리 및 정제 (Protein induction and Ni-NTA binding, purification) 19

12. Sample preparation 20

13. SDS-PAGE 20

14. Gel Electrophoresis and Electrotransfer 21

15. Commassie blue staining 21

16. Western blot 22

17. 세포주 및 세포주 배양 (Cell culture) 22

18. Analysis of cDNA(software) 23

19. Data statistical analysis 23

III. 결과 24

1. Drosophila antenapedia 유전인자 서열을 이용한 인간유전인자 검색 24

2. Annealing of HOXA5-PTD oligonucleotide and its ligation with pET28b and pET21b 24

3. HOXA5-PTD를 함유한 pET28b/HOXA5-PTD과 pET21b/HOXA5-PTD의 서열확인 25

4. pET28b/HOXA5-PTD에 EGFP 유전인자의 Subcloning 25

5. pET28b/HOXA5-PTD-EGFP과 pET21b/HOXA5-PTD-EGFP의 서열확인 26

6. HOXA5-PTD-EGFP 단백의 발현 및 정제 26

7. Western blot을 이용한 HOXA5-PTD-EGFP융합단백의 세포 내 도입확인 27

8. HOXA5-PTD-EGFP융합단백의 세포 내 도입효율성 비교 28

9. HOXA5-PTD-EGFP융합단백의 세포 내 도입 시 온도의 영향 28

10. HOXA5-PTD-Ref-1 발현벡터의 제조 29

11. HOXA5-PTD-Ref-1 융합단백의 항염증 효과 29

12. HOXA5-PTD 단백질 발현 벡터의 개발 30

IV. 고찰 45

V. 결론 49

Reference 52

Abstract 56

그림목차

Fig. 1. Homology search using Drosophila Antenapedia protein... 31

Fig. 2. Construction of Amino-terminal His-tag, HOXA5-PTD... 32

Fig. 3. Construction of 6His-HOXA5-PTD-EGFP fusion protein... 34

Fig. 4. Purification and western blot analysis of HOXA5-PTD-EGFP... 35

Fig. 5. The cellular uptake of HOXA5-PTD-EGFP in the HUVEC. 36

Fig. 6. Dose dependent cellular uptake of HOXA5-PTD-EGFP in the... 37

Fig. 7. Time dependent cellular uptake of HOXA5-PTD-EGFP in the... 38

Fig. 8. Cellular Transduction of HOXA5-PTD-EGFP and TAT-GFP in... 39

Fig. 9. Effect of temperature on the cellular transduction of HOXA5-... 40

Fig. 10. Construction of 6His- HOXA5-PTD-APE1/Ref-1 fusion protein... 41

Fig. 11. Effect of HOXA5-PTD-APE1/ref-1 on the TNF-α-induced... 42

Fig. 12. Construction of HOXA5-PTD containing E.coli expression vector. 44

초록보기

 Various physical and chemical techniques have been developed to deliver exogenous molecules such as peptide and proteins into living cells. To circumvent of immunogenicity problem of viral origin PTD such as Tat or VP22, utilization of human PTDs might be beneficial for the progress of gene therapy research. Protein transduction domains (PTDs), such as Tat-PTD, have been extensively utilized for intracellular delivery of biologically active macromolecules in vitro and in vivo. Homeoproteins belong to a family of transcriptional factors that bind dsDNA through a highly conserved structure, 60 amino acids in length, called the homeodomain. Antennapedia homeodomain has a 60 amino acid homeodomain, which is shown the membrane transducing ability of the protein by an energy-independent mechanism and receptor-independent. A 16-amino acid long poly peptide corresponding of the third helix of the DNA binding domain(homeodomain) of Antennapedia, a Drosophilia Antennapedia homeoprotein. Previously it was reported that the human Hoxa-5 homeoprotein (33kda) is efficiently internalized by fibroblasts and neurons. Especially, the third helix of the HOXA5 homeoprotein is similar with 100% homology with that of Antennapedia.

Construction, expression, and purification of HOXA5-PTD-EGFP. We investigated the cell transduction ability of HOXA5-PTD-EGFP in the cultured human endothelial cell. Recombinant HOXA5-PTD-EGFP was designed to be expressed and purified it in E-coli. Recombinant HOXA5-PTD-EGFP (~33kDa) was successfully identified in the western blot. HOXA5-PTD-EGFP (100 nM) efficiently entered the cells within 1 hour, however, recombinant EGFP itself did not entered. Transduction efficiency of HOXA5-PTD-EGFP at 37℃ was greater than that at 4℃. To evaluate the transduction ability in the endothelial cells, purified HOXA5-PTD-EGFP at the range of 3~200nM was added to cultured endothelial cells for 1hr. after the incubation of HOXA5-PTD-GFP, EGFP, and Tat-GFP, cells were harvested and the change of transduced EGFP was analyzed with western blotting with anti-GFP antibody.

Construction, expression, and purification of HOXA5-PTD-Ref-1. Atheosclerosis is an inflammatory disease of the vessel wall characterized by monocyte infilteration in response to pro-atherogenic factors such as oxidized lipids or inflammatory cytokine. Firm adhesion of monocyte on the endothelial cells was mediated by the expression of molecule in the endothelial cells.

To evaluated the possible role of HOXA5-PTD-APE1/Ref-1 on TNF-α-induced monocyte adhesion to endothelial cells. After incubation with TNF-α for 18hour, monocyte adhesion assay was performed as described in Meterial and Methods. Monocyte adhesion was minimal in un-stimulated endothelial cells. Pretreatment with HOXA5-PTD-APE1/Ref-1 inhibited TNF-α induced monocyte adhesion to endothelial cells. However, APE1/ref-1 protein did not inhibit monocyte adhesion, suggesting that HOXA5-PTD-APE1/ref-1 has specific inhibitory action on monocyte adhesion induced by TNF-α endothelial cells. In conclusion, these data show that protein transduction using the putative protein transduction domain of human HOXA5 may be useful in the suppression of vascular activation or vascular inflammatory disorders.

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