서지주요정보
Vaccinia 바이러스를 이용한 동물세포에서의 재조합 단백질 생산 = Factors influencing recombinant protein yields in Vaccinia virus expression system
서명 / 저자 Vaccinia 바이러스를 이용한 동물세포에서의 재조합 단백질 생산 = Factors influencing recombinant protein yields in Vaccinia virus expression system / 신정현.
저자명 신정현 ; Shin, Jung-Hyun
발행사항 [대전 : 한국과학기술원, 1994].
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소장정보

등록번호

8004397

소장위치/청구기호

학술문화관(문화관) 보존서고

MBT 94006

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이용가능

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초록정보

The Vaccinia virus (vSC 8) expression system was employed for the synthesis of $\beta$-galactosidase, a model heterologous proteins. Six cell lines derived from Human cervical carcinoma, African green monkey kidney, Human hepatocellular carcinoma, Mouse brain were evaluated for comparing the amount of recombinant $\beta$-galactosidase. The HeLa HH cells had the highest yield among these cell lines and the Vero cells generally used in plaque assay had 4 fold lower than HeLa HH cells. Different pH, temperature, oxygen partial tension, osmolarity, serum concentrations were tested for the investigation the effects on production of a recombinant $\beta$-galactosidase. The optimal pH range of the $\beta$-galactosidase production and cell growth were between pH 7.0 and pH 8.0. Oxygen partial pressure ranged from 10% to 21% could not show any significant difference in $\beta$-galactosidase production but the production of $\beta$-galactosidase was abruptly decreased when oxygen partial pressure was lower than 1%. The increasing of osmolarity had worse effect on $\beta$-galactosidase production and cell growth. The $\beta$-galactosidase production at $32\,^\circ\!C$ was 3 times higher than at $37\,^\circ\!C$, but cell grew better at $37\,^\circ\!C$ than at $32\,^\circ\!C$. A increase in protein synthesis was also observed when serum concentrations were reduced but cell growth was reduced at lower serum concentration. The conditions that favor maximal cell growth could not lead to maximize the production of virus proteins. To determine $\beta$-galactosidase in animal cells, the cells have to been disrupted. In this study, four different cell disruption methods (freezing and thawing, sonication, homogenization and lysis buffer) were compared with regard to their efficiency in the determination of $\beta$-galactosidase activity. Further, the cell lines' susceptibility to cell disruption methods was investigated by employing three different animal cell lines(HeLa HH, Vero, and HeLa S3 cells) infected by recombinant Vaccinia virus(vSC 8) expression $\beta$-galactosidase gene. Regardless of cell lines used, three different cell disruption methods except homogenization did not show any significant difference in the final $\beta$-galactosidase activity recovered from the cells. Homogenization was quite inefficient. The use of lysis buffer was recommendable because of its convenient to keep cell samples collected during the culture frozen at $-20\,^\circ\!C$ until assay. Since thawing of the cells was enough to recover the $\beta$-galactosidase, further treatment on the frozen cells except thawing was unnecessary.

서지기타정보

서지기타정보
청구기호 {MBT 94006
형태사항 ix, 62 p. : 삽도 ; 26 cm
언어 한국어
일반주기 저자명의 영문표기 : Jung-Hyun Shin
지도교수의 한글표기 : 이균민
지도교수의 영문표기 : Gyun-Min Lee
학위논문 학위논문(석사) - 한국과학기술원 : 생물공학과,
서지주기 참고문헌 : p. 57-59
주제 Viruses.
Gene expression.
Cell culture.
바이러스. --과학기술용어시소러스
유전자 발현. --과학기술용어시소러스
세포 배양. --과학기술용어시소러스
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