표제지
목차
Ⅰ. 서론 9
Ⅱ. 실험 재료 및 방법 15
1. 균주 및 배지 15
1.1. 균주 및 플라스미드 15
2.2. 배양배지 15
2. 유전자 클로닝 15
2.1. 유전자 증폭을 위한 중합효소연쇄반응 15
2.2. LaboPass™PCR purification kit를 이용한 유전자의 순수분리 21
2.3. Crp, SlyD 유전자의 pGEM-T-easy 벡터 클로닝 21
2.4. 형질전환을 위한 수용성세포의 제조 21
2.5. 형질전환 22
2.6. 플라스미드 DNA 순수 분리 22
2.7. 발현벡터에서 클로닝 23
3. 유전자의 발현 23
4. 분석 방법 24
4.1. DNA 전기영동 24
4.2. 유전자 분석 24
4.3. 단백질 추출 24
4.4. SDS-PAGE 분석 24
4.5. Invision His-tag In-Gel Stain을 이용한 SDS-PAGE분석 25
4.6. Ni-NTA를 이용한 정제 25
4.7. 단백질 분석 27
Ⅲ. 결과 및 고찰 28
1. 유전자의 클로닝 28
2. 염기서열 분석 28
3. crp, slyD 유전자의 발현 33
4. Antisense crp, Antisense slyD의 생장곡선 즉정 33
5. crp, slyD 유전자와 Antisense crp, Antisense slyD 유전자와의 형질전환 33
6. Antisense RNA 유전자와 녹색형광단백질(Green Fluorescent Protein, GFP)과의 형질전환 39
7. Antisense RNA 유전자의 발현실험 42
8. Antisense RNA 유전자의 IPTG 농도에 따른 발현 실험 42
9. Antisense RNA 유전자의 단백질질량분석 46
Ⅳ. 결론 47
참고문헌 48
Abstract 55
Table 1. The contamination proteins of E.coli BL21(DE3) after Ni-NTA purification. 13
Table 2. The list of PCR primers. 18
Figure 1. Structure of Ni-NTA resin. 11
Figure 2. Structures of Histidine and Imidazole. 12
Figure 3. pET21c vector. 16
Figure 4. pRSF Duet-1 vector. 17
Figure 5. Nucleotide sequence of the crp gene (GenBank Accession No: AM946981.2). 19
Figure 6. Nucleotide sequence of the slyD gene (reversed) (GenBank Accession No.: CP001509.3). 20
Figure 7. Protein purification with the Ni-NTA protein purification system. 26
Figure 8. The PCR product of crp and slyD gene. M: 1Kb DNA marker, 1 : crp gene PCR product, 2: slyD gene PCR product. 29
Figure 9. The PCR product of As_crp and As_slyD gene. M: 1Kb DNA marker, 1: As_crp gene PCR product, 2: As_slyD gene PCR product. 30
Figure 10. The confirmation of insert DNA(As_crp, As_slyD) and pRSF Duet-1 vector by restriction enzymes (Not I / Ncol, Xho I... 31
Figure 11. The confirmation of insert DNA in pRSF Duet-1 vector by restriction enzymes. M : 1Kb DNA marker,... 32
Figure 12. BLAST search of As_crp gene. 34
Figure 13. BLAST search of As_slyD gene. 35
Figure 14. Identification of the expressed crp and slyD by SDS-PAGE analysis. M : Protein Size marker, 1 : crp protein total by... 36
Figure 15. Cell growth curve of As_crp. As_slyD and pRSF Duet-1. 37
Figure 16. SDS-PAGE analysis of expression test. M : Protein Size marker, 1 : pRSF Duet-1/pET21c, 2 : pRSF Duet-1/pET21c... 38
Figure 17. SDS-PAGE analysis of Antisense RNA gene and HisGFP after Ni-NTA purification. M : Protein Size marker, 1 :... 40
Figure 18. Invision His-tag In-gel Stain analysis of Antisense RNA gene and HisGFP after Ni-NTA purification.M : Protein... 41
Figure 19. SDS-PAGE analysis of Antisense RNA gene expression test. M : Protein Size marker, 1 : pRSF Duet-1 supernatant after... 43
Figure 20. SDS-PAGE analysis of Antisense RNA gene expression test after Ni-NTA purification. M : Protein Size marker, 1 : pRSF... 44
Figure 21. SDS-PAGE analysis of Antisense RNA gene expression test according to the IPTG concentration.... 45