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cDNA Cloning and Expression of a Human Interferon-$\gamma$ in E. coli
고형곤,현형환,유무영,권병세,주종광,문홍모,김광수,Koh, Hyung-Kon,Hyun, Hyung-Hwan,Yoo, Moo-Young,Kwon, Byung-S.,Jue, Chong-K.,Moon, Hong-Mo,Kim, Kwang-Soo 생화학분자생물학회 1986 한국생화학회지 Vol.19 No.3
인간의 peripheralblood lymphocyte에서 분리된 poly (A)$^+mRNA$를 사용하여 ${\lambda}gt$ 10 cDNA library를 제조하였으며, 화학적으로 합성한 oligonucleotide를 이용하여, 이 cDNA library로부터 IFN-$\gamma$ 유전자를 갖는 DNA을 분리했다. 분리된 clone 가운데 하나인 pBK04는 IFN-$\gamma$의 전체 염기서열을 갖고 있었다. IFN-$\gamma$를 E. coli내에서 발현시키기 위해 lpp promotor를 함유하고 있 는 pINIIIA3 vector를 사용하여 IFN-$\gamma$를 발현시킬 수 있는 재조합 plasmid ($pKS{\gamma}3B$)를 제조하였다. $pKS{\gamma}3B$로써 형질 변환된 bacterial cell을 antiviral activity에 의해 역가 분석을 해본 결과, 높은 정도로 IFN-$\gamma$를 발현시켰으며 이 IFN-$\gamma$는 천연의 IFN-$\gamma$에 대하여 특이성이 있는 monoclonal antibody에 의해서도 인지되었다. High copy number를 갖는 expression vector를 제조 (pHK-3) 하였으며, 이 high copy number vector를 사용하므로써 IFN-$\gamma$의 생성이 증가되는 사실을 확인하였다. A ${\lambda}gt10$ cDNA library constructed with poly $A^+$ mRNA from human peripheral blood lymphocytes was screened by a synthetic oligonucleotide having IFN-$\gamma$ sequence. One of the clones (pBK04) which hybridized to the oligomer was confirmed to have the whole IFN-$\gamma$ coding sequence. The eDNA insert was used to express IFN-$\gamma$ in E. coli under the control of lpp promoter employing pINIIIA3 vector. Cells harboring the recombinant plasmid ($pKS{\gamma}3B$) expressed high level of IFN-$\gamma$ assayed by antiviral activity, The molecule was also recognized by monoclonal antibody specific to a natural IFN-$\gamma$. An expression vector with high copy number (pHK-3) was constructed and was successfully used for the improvement of the expression of IFN-$\gamma$.
대장균에서의 인간 Interferon - γ의 cDNA 크로닝 및 발현
김광수,현형환,유무영,고형곤,권병세,주종광,문홍모 생화학분자생물학회 1991 BMB Reports Vol.19 No.3
A λ gt10 cDNA library constructed with poly A^+ mRNA from human peripheral blood lymphocytes was screened by a synthetic oligonucleotide having IFN-γ sequence. One of the clones (pBK04) which hybridized to the oligomer was confirmed to have the whole IFN-γ coding sequence. The cDNA insert was used to express IFN-γ in E. coli under the control of lpp promoter employing pINTIIIA3 vector. Ce115 harboring the recombinant plasmid (pKS γ 3B) expressed high level of IFN-γ assayed by antiviral activity. The molecule was also recognized by monoclonal antibody specific to a natural IFN-γ. An expression vector with high copy number (pHK-3) was constructed and was successfully used for the improvement of the expression of IFN-γ