Biofunctional Human Cystatin-C Expressed in Transgenic Chickens
형질 전환 닭에서 생산하는 생기능성 인간 시스타틴 C

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dc.description학위논문(석사)--서울대학교 대학원 :국제농업기술대학원 국제농업기술학과,2020. 2. 박태섭.-
dc.description.abstract생물 반응기는 생화학 반응을 유용 물질의 생산, 환경오염 물질의 분해, 분석, 의료 등에 응용하는 반응장치를 말한다. 미생물 및 동물 세포를 이용하는 생물 반응기도 있지만, 생기능성 단백질의 효율적인 생산을 위해서는 효율적인 전략이 필요하다. 미생물에서는 치료 단백질 생산 시 글리코실화(포도당화)와 같은 번역 후 변형과정이 제대로 이루어지지 않는다. 또한 동물 세포 시스템은 많은 비용과 시간이 필요하다. 이러한 문제를 해결하기 위해 형질 전환 암탉의 계란을 이용하는 새로운 방법을 고려하였다. 인간 시스타틴-C는 시스타틴 수퍼 패밀리의 구성원이다. 시스타틴-C는 체액과 분비물에서 발견할 수 있고 일정한 속도로 체내에서 생성한다. 또한 이 단백질은 시스테인 프로테아제의 억제제로서 작용하고 미생물 및 기생 침투로부터 몸을 보호한다. 우리는 비바이러스성 piggyBac 트랜스포손(전이인자) 시스템을 사용하여 인간 시스타틴-C 단백질을 발현하는 형질 전환 닭을 생성했다. 우리는 체외 분석을 통해 형질 전환 닭에서의 인간 시스타틴-C의 발현과 생기능성 활동을 확인했다. 따라서, 이 데이터는 형질 전환 닭이 다양한 생물 활성 물질 생산을 위한 생물 반응기로서 효율적으로 사용될 수 있음을 시사한다-
dc.description.abstractBioreactor is the massive production system for bioactive substances as well as therapeutic proteins. Although the various methods such as microorganisms and animal cells, the efficient strategy should be necessary for the efficient production of biofunctional proteins. In the production of therapeutic proteins, post-translational modification is not properly performed in microorganisms. In addition, animal cell systems require more cost and time. To overcome these problems, a new method has been considered with transgenic animals such as the genetically-modified hens eggs. Human cystatin-C is a member of cystatin superfamily. It is produced in the body at a constant rate found in body fluids and secretions. Moreover, this protein acts as an inhibitor of cysteine protease and protects against microbial and parasitic infiltration. We aim to construct transgenic chickens that produce human cystatin-C using a non-viral piggyBac transposon system. We generated transgenic chicken expressing human cystatin-C protein by using non-viral piggyBac transposon system. We analyzed the expression of human cystatin-C in DF1 cells and PGCs, and we produced transgenic chickens using the human cystatin-C expression vector. Expression of human cystatin-C protein in transgenic chickens were verified through Western blot and ELISA. Biofunctional activity of purified human cystatin-C in transgenic chickens was confirmed through in vitro assay. Thus, these data suggests that transgenic chickens could be efficiently used as a bioreactor for production of various bioactive materials.-
dc.description.tableofcontentsIntroduction 1

Review of Literature 4
1. Bioreactor 4
1.1 Definition of bioreactor 4
1.2 Bacterial expression system 5
1.3 Animal cell expression system 7
1.4 Transgenic animal expression system 12
1.4.1 Transgenic chicken expression system 14
2. Cystatin-C 15
2.1 cystatin-C 16
2.2 Kidney function biomarker 18
2.3 function of cystatin-C 21
3. Production of transgenic chicken using non-viral piggyBac transposon system 24
3.1 piggyBac transposon system 24
3.2 PGCs 25

Materials and Methods 28
1. Construction of human cystatin-C expression vector system using piggyBac transposon . 28
2. Transfection of human cystatin-C expression vector to DF1 cell & PGCs 28
3. Microinjection of cultured PGCs into recipient embryos 29
4. Production of transgenic chickens 29
5. Genomic PCR 30
6. Purification of human cystatin-C in egg white and muscle 30
7. ELISA 31
8. Western blotting 31
9. Antimicrobial activity test 32

Results 33
1. Construction of human cystatin-C expression vector 33
2. Identification of vector expression in DF1 cells and PGCs 35
3. Production of germ-line chimera and transgenic offspring 37
4. Identification of human cystatin-C expression in transgenic chickens 39
5. Purification of human cystatin-C in transgenic chickens egg white and muscle 41
6. Antimicrobial activity of human cystatin-C in transgenic chickens egg white 43

Discussion 46

Conclusion 51

References 52
Abstract in Korean 63
dc.publisher서울대학교 대학원-
dc.titleBiofunctional Human Cystatin-C Expressed in Transgenic Chickens-
dc.title.alternative형질 전환 닭에서 생산하는 생기능성 인간 시스타틴 C-
dc.contributor.AlternativeAuthorSeo Woo Kim-
dc.contributor.department국제농업기술대학원 국제농업기술학과-
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Graduate School of International Agricultural Technology (국제농업기술대학원)Dept. of International Agricultural Technology (국제농업기술학과)Theses (Master's Degree_국제농업기술학과)
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