초록 ▼

1. 사람 monocytes cDNA library의 제조 가. 사람 monocytes에 LPS를 처리하여 인터루킨-1 생산을 유도한 후 RNA를 분리하고 Oligo(dT)-cellulose column chromatography에 의하여 mRNA를 분리한다. 나. mRNA로부터 Okayama-Berg 클로닝 시스템을 이용하여 cDNA를 제조하고 균주 JM83을 형질전환시켜 cDNA library를 제조한다.2. 인터루킨-1 cDNA의 클로닝 가. colony hybridization에 의하여 인터루킨-1을 코드하는 cDNA의 클로

1. 사람 monocytes cDNA library의 제조 가. 사람 monocytes에 LPS를 처리하여 인터루킨-1 생산을 유도한 후 RNA를 분리하고 Oligo(dT)-cellulose column chromatography에 의하여 mRNA를 분리한다. 나. mRNA로부터 Okayama-Berg 클로닝 시스템을 이용하여 cDNA를 제조하고 균주 JM83을 형질전환시켜 cDNA library를 제조한다.2. 인터루킨-1 cDNA의 클로닝 가. colony hybridization에 의하여 인터루킨-1을 코드하는 cDNA의 클로닝을 시도하여 oligonucleotides를 probe로 사용한다. 나. 제한효소를 이용하여 클론들을 분석하여 인터루킨-1 cDNA를 분리, 확인한다.

Abstract ▼

Interleukin-1 (IL-1 ) is believed to have multiple physiolocal activities and being developed as anti-cancer and antiinflammatory drugs and a drug for rheumatis-arthritis by many companies. In addition, it has been shown that IL-1 may be used as adjuvants for vaccine, immunostimulants for incurable

Interleukin-1 (IL-1 ) is believed to have multiple physiolocal activities and being developed as anti-cancer and antiinflammatory drugs and a drug for rheumatis-arthritis by many companies. In addition, it has been shown that IL-1 may be used as adjuvants for vaccine, immunostimulants for incurable infectious diseases, and immunosuppressives for allergic reactions and allograft rejection episodes. IL-1 may have protectability against the damages caused by radiation therapy, thus have possibilities to be used for radiation therapy. Recently, IL-1 has been shown to activate human immunodeficiency virus enhancer via nuclear factor KB. Thus, a system to control over AIDS virus may be developed by regulation of IL-1 production. As such, IL-1 has multiple functions, therefore, there may be many different regulatory materials present which may be used for regulation of IL-1 activities. In this research, we isolated IL-1 cDNA as a necessary step to produce IL-1, to study the mechanism(s) of IL-1 activities and to find new IL-1 regulatory activities. 1. We have isolated cDNA clones containing IL-1 cDNA. Total RNA was prepared from LPS-stimulated human monocytes and mRNA was isolated by oligo(dT)-cellulose column chromatography. The cDNA was prepared from mRNA and the cDNA library was prepared using Okayama-Berg cloning system. The cDNA library was screened by colony hybridization. A mixture of two oligonucleotides having complementary sequences to IL-1 cDNA was used as a probe. Four positive clones were identified. Three of them contained partial cDNAS of IL-1. A cDNA clone containing full length IL-1 cDNA was identified by restriction enzyme digestion. 2. We analyzed this IL-1 cDNA by several restriction enzyme digestions and constructed a restriction enzyme map that may be utilized for the construction of IL-1 expression vector in E. coli. We constructed a vector for expression of a portion of IL-1 as preliminary step for construction of IL-1 secretion system using pINIII-ompA3 secretion vector. We are planning to express this IL-1 S1 and also planning to construct vectorcs for full length IL-1 expression.

목차 Contents

• 제1장 서 론...15
• 제2장 실험재료 및 방법...21
• 제1절 실험재료...21
• 1. E. coli균주...21
• 2. 효 소...21
• 3. 화학약품 및 기타...22
• 제2절 IL-1 cDNA의 클로닝...22
• 1. mRNA의 추출 및 분리...22
• 2. cDNA의 합성...23
• 3. E.coli의 형질 전환...23
• 4. IL-1 cDNA의 선별...23
• 5. IL-1 cDNA 클론의 확인...24
• 6. 제한효소를 이용한 분석...24
• 제3절 IL-1 S1 발현벡터...25
• 제3장 결과 및 고찰...26
• 제1절 IL-1 cDNA의 클로닝...26
• 1. cDNA 및 cDNA 라이브러리의 제조...26
• 2. 클론의 확인...26
• 3. 클론의 확인...27
• 4. 제한효소지도 작성...29
• 제2절 IL-1 S1의 발현벡터 제조...32
• 제4장 결론 및 건의사항...35
• 참고문헌...36