초록 ▼

1. rhEGF 유전자 탐색 및 발현
보고되어 있는 hEGF 의 아미노산 서열을 이용하여 최종 아미노산 서열에는 변화를 주지 않으면서 E. coli 내에서 rhEGF 의 발현을 높일 수 있는 유전자를 탐색하였다. 탐색된 유전자 서열에는 5개의 신규한 제한효소 부위를 가지도록 설계하여 조작이 간편하도록 고안, 구성, 합성하였으며 합성된 rhEGF 유전자를 pUC18 벡터에 도입, 유전자의 subcloning을 완료하였다.
2. 발현 system의 구축
rhEGF 유전자를 E. coli의 복제 개시 부위와 복제 조절부위

1. rhEGF 유전자 탐색 및 발현
보고되어 있는 hEGF 의 아미노산 서열을 이용하여 최종 아미노산 서열에는 변화를 주지 않으면서 E. coli 내에서 rhEGF 의 발현을 높일 수 있는 유전자를 탐색하였다. 탐색된 유전자 서열에는 5개의 신규한 제한효소 부위를 가지도록 설계하여 조작이 간편하도록 고안, 구성, 합성하였으며 합성된 rhEGF 유전자를 pUC18 벡터에 도입, 유전자의 subcloning을 완료하였다.
2. 발현 system의 구축
rhEGF 유전자를 E. coli의 복제 개시 부위와 복제 조절부위, 합성된 Omp A 리더서열- 보편적 번역 정지서열- Trp A 전사 정지서열 및 par 부위, 그리고 선별을 위한 tetracycline 내성 마커 유전자를 구성 성분으로하여 제조한 발현벡터에 삽입하였고, 숙주인 E. coli JM101에 형질전화시켜 발현 system을 구축하였다.
3. 배양조건의 최적화
제조된 rhEGF 발현 균주 E. coli JM101/pTE105를 소량 배양하는 과정을 통하여 rhEGF 발현을 확인하였고, PTG, tetracycline에 대한 영향을 관찰하였으며 발현율을 높이기 위한 연구를 진행하였다.
4. 대량 발효 system 구축
발현 최적화 실험을 토대로 발효 방법을 fed-batch 법으로 설정하여 rhEGF의 발현량을 200mg/l 이상의 경제성 있는 수준으로 재현성 있게 생산하는 대량 발효 system을 구축하였다.

Abstract ▼

Studies on the Screening and Expression of Growth Factor Genes
In the field of pharmaceuticals, biotechnology is now vigorously applied in new drug development, and subsequently, a number of biotechnology-derived drugs are already produced or under investigated in many developed countries. In fin

Studies on the Screening and Expression of Growth Factor Genes
In the field of pharmaceuticals, biotechnology is now vigorously applied in new drug development, and subsequently, a number of biotechnology-derived drugs are already produced or under investigated in many developed countries. In finding and making a biologically active materials, biotechnology is much more effective than conventional methods of drug development.
Among the numerous functional proteins that has not been developed as medicines, this study was focused on the human epidermal growth factor(hEGF), which promotes growth and proliferation of various cells, and has been performed to construct the hEGF expression system.
This study has been contributed to establish hEGF research for making a new drug, and it is thought that construction of hEGF expression system by using genetic engineering will improve the domestic biotechnology-related drug development techniques.
1. rhEGF gene design
rhEGF gene was designed to conserve amino acid sequence of mature hEGF which already reported, and to yield high amount of hEGF in E. coli. And, for the convenience of cloning procedure, hEGF gene was designed and synthesized to contain 5 new restriction enzyme sites. The synthesized EGF
gene was subcloned into pUC18 vector and confirmed its base sequence by dideoxy chain termination method.
2. Construction of expression systems.
Subcloned hEGF gene was inserted in newly made expression vector which composed mainly of replication start site and control site of E. coli synthesized Omp A leader sequence, universal translation termination codon, Trp A transcription termination sequence, par site and tetracycline resistant marker. And the resulted expression vector, pTE105, was used to transform E. coli JM101.
3. Optimization of culture condition
After hEGF expression was checked in small scale culture of E. coli JM101/pTE105, the effect of IPTG and tetracycline on the amount of rhEGF expression was studied, and other optimization experiments were also carried out exclusively.
4. Establishment of large scale-fermentation system
On the basis of expression optimization experiment, the fed-batch method was set up as a large scale fermentation system. The amount of hEGF expressed in optimized fed-batch system was consistantly over 200mg/l which met econemical needs
The basis of research on pharmaceutical development of hEGF was accomplished with successful results of new hEGF gene cloning, subsequent expression and large scale fermentation system development.
Also, this genuine expression system is expected to be used as a tool of producing other useful proteins in E. coli. It is prospected that the process successfully performed in this study can be continued to the study on a hEGF large scale purification and other analysis in next year. The research and clinical study related on the pharmaceutical development of hEGF will promise
new approaches to the formulation of biologically active materials and pharmaceutic research at that time.

목차 Contents

• 제1장 서론...15
• 제2장 실험재료 및 방법...17
• 제3장 결과 및 고찰...33
• 제4장 참고문헌...70