사람 인터루킨-2 (Interleukin : hIL-2)는 항원에 의하여 자극을 받은 T 세포가 분비하는 림포카인 (lymphokine)의 일종으로서, B세포와 T세포를 포함한 여러 면역 세포들의 성장과 활성화를 유도하는 물질로 알려져 있다. 최근에는 암 질환의 치료에 사람 인터루킨-2를 적용하고 있는데 사람 인터루킨-2는 암 질환 뿐 아니라 세균과 바이러스 감염, 면역 결핍증, ...
사람 인터루킨-2 (Interleukin : hIL-2)는 항원에 의하여 자극을 받은 T 세포가 분비하는 림포카인 (lymphokine)의 일종으로서, B세포와 T세포를 포함한 여러 면역 세포들의 성장과 활성화를 유도하는 물질로 알려져 있다. 최근에는 암 질환의 치료에 사람 인터루킨-2를 적용하고 있는데 사람 인터루킨-2는 암 질환 뿐 아니라 세균과 바이러스 감염, 면역 결핍증, 자가 면역 질환 등 광범위한 면역이상 질환에 대하여 적용된다. 동물 발현시스템에서 외래유전자 발현이 외래유전자가 삽입된 염색체상의 위치와 상관없이 일어난다는 점과 전사 종결을 통해 단백질 발현 양을 증가시킨다는 점에 착안하여 이미 개발되어진 pMSG vector를 이용하여 포유 동물세포인 DHFR^(-) CHO (Chinese Hamster Ovary) 세포에서 사람 인터루킨-2 유전자를 발현시켰다. 선별된 DHFR^(+) CHO 세포는 MTX의 적응이 끝난 후, 약 11.4㎍/10^(6)cell/day의 인터루킨-2 단백질을 생산하였다. Southern hybridization과 Northern hybridization을 통하여 증폭된 유전자 copy 수와 transcript 수준에서의 발현을 알아보았다. Two-dimensional electrophoresis를 이용하여 glycosylation 정도를 살펴보았으며, 또한 CTLL(Cytotoxic T-Lymphocyte Line) 세포주에 대한 proliferation test를 통해 재조합 사람 인터루킨-2 단백질이 생화학적 활성을 갖고 있음을 확인하였다. 재조합 사람 인터루킨-2 단백질을 생산하는 세포주를 protein-free 배지에서 적응시켜 suspension 배양하고 배양액으로부터 재조합 사람 인터루킨-2 단백질을 HPLC를 이용한 column chromatography를 통해 순수 분리하였다.
사람 인터루킨-2 (Interleukin : hIL-2)는 항원에 의하여 자극을 받은 T 세포가 분비하는 림포카인 (lymphokine)의 일종으로서, B세포와 T세포를 포함한 여러 면역 세포들의 성장과 활성화를 유도하는 물질로 알려져 있다. 최근에는 암 질환의 치료에 사람 인터루킨-2를 적용하고 있는데 사람 인터루킨-2는 암 질환 뿐 아니라 세균과 바이러스 감염, 면역 결핍증, 자가 면역 질환 등 광범위한 면역이상 질환에 대하여 적용된다. 동물 발현시스템에서 외래유전자 발현이 외래유전자가 삽입된 염색체상의 위치와 상관없이 일어난다는 점과 전사 종결을 통해 단백질 발현 양을 증가시킨다는 점에 착안하여 이미 개발되어진 pMSG vector를 이용하여 포유 동물세포인 DHFR^(-) CHO (Chinese Hamster Ovary) 세포에서 사람 인터루킨-2 유전자를 발현시켰다. 선별된 DHFR^(+) CHO 세포는 MTX의 적응이 끝난 후, 약 11.4㎍/10^(6)cell/day의 인터루킨-2 단백질을 생산하였다. Southern hybridization과 Northern hybridization을 통하여 증폭된 유전자 copy 수와 transcript 수준에서의 발현을 알아보았다. Two-dimensional electrophoresis를 이용하여 glycosylation 정도를 살펴보았으며, 또한 CTLL(Cytotoxic T-Lymphocyte Line) 세포주에 대한 proliferation test를 통해 재조합 사람 인터루킨-2 단백질이 생화학적 활성을 갖고 있음을 확인하였다. 재조합 사람 인터루킨-2 단백질을 생산하는 세포주를 protein-free 배지에서 적응시켜 suspension 배양하고 배양액으로부터 재조합 사람 인터루킨-2 단백질을 HPLC를 이용한 column chromatography를 통해 순수 분리하였다.
Interleukin 2 (IL-2) is a lymphokine produced by T-cells after stimulation by mitogens or antigens. This molecule is a key mediator of the growth and functional activity of B- and T-cells, and cytotoxic cells. Several groups have cloned and expressed the cDNA coding for IL-2 in E.coli, and obtained ...
Interleukin 2 (IL-2) is a lymphokine produced by T-cells after stimulation by mitogens or antigens. This molecule is a key mediator of the growth and functional activity of B- and T-cells, and cytotoxic cells. Several groups have cloned and expressed the cDNA coding for IL-2 in E.coli, and obtained large amounts of unglycosylated IL-2. The glycosylation of rhIL-2 may not be critical for its biological activity, even though it is glycosylated in vivo. However, the glycosylation of rhIL-2 may have a great effect on its stability. Therefore, the production of the glycosylated IL-2 in mammalian cells such as CHO (Chinese Hamster Ovary), can give us a great advantage over the production in E. coli. CHO cell lines producing recombinant human interleukin-2 (rhIL-2) at high level were obtained by using pMSG vector system. It was previously known to confer position-independent (copy number-dependent) expression by insulator and increase the foreign gene expression by the efficient transcription termination. The recombinant expression plasmid carrying rhIL-2 cDNA was transfected into DHFR^(-) CHO cells together with DHFR gene. Most of DHFR^(+) colonies were turned out to be able to express rhIL-2. The level of rhIL-2 expression in CHO cell lines after methotrexate selection was about 11.4㎍/10^(6)cell/day, which is about 6 times higher than those previously reported. Northern and Southern blot analyses show that rhIL-2 mRNAs are highly stable and the amount of rhIL-2 proteins or mRNAs produced in each cell line correlate with the copy number of rhIL-2 cDNA amplified in the chromosomes. The rhIL-2 protein was shown to have the ability to stimulate the proliferation of CTLL-2 cell line, suggesting produced rhIL-2 exists in biologically active form. The rhIL-2 producing cell line was successfully adapted to suspension culture. We have purified the rhIL-2 protein to homogeneity from the conditioned media by column chromatography.
Interleukin 2 (IL-2) is a lymphokine produced by T-cells after stimulation by mitogens or antigens. This molecule is a key mediator of the growth and functional activity of B- and T-cells, and cytotoxic cells. Several groups have cloned and expressed the cDNA coding for IL-2 in E.coli, and obtained large amounts of unglycosylated IL-2. The glycosylation of rhIL-2 may not be critical for its biological activity, even though it is glycosylated in vivo. However, the glycosylation of rhIL-2 may have a great effect on its stability. Therefore, the production of the glycosylated IL-2 in mammalian cells such as CHO (Chinese Hamster Ovary), can give us a great advantage over the production in E. coli. CHO cell lines producing recombinant human interleukin-2 (rhIL-2) at high level were obtained by using pMSG vector system. It was previously known to confer position-independent (copy number-dependent) expression by insulator and increase the foreign gene expression by the efficient transcription termination. The recombinant expression plasmid carrying rhIL-2 cDNA was transfected into DHFR^(-) CHO cells together with DHFR gene. Most of DHFR^(+) colonies were turned out to be able to express rhIL-2. The level of rhIL-2 expression in CHO cell lines after methotrexate selection was about 11.4㎍/10^(6)cell/day, which is about 6 times higher than those previously reported. Northern and Southern blot analyses show that rhIL-2 mRNAs are highly stable and the amount of rhIL-2 proteins or mRNAs produced in each cell line correlate with the copy number of rhIL-2 cDNA amplified in the chromosomes. The rhIL-2 protein was shown to have the ability to stimulate the proliferation of CTLL-2 cell line, suggesting produced rhIL-2 exists in biologically active form. The rhIL-2 producing cell line was successfully adapted to suspension culture. We have purified the rhIL-2 protein to homogeneity from the conditioned media by column chromatography.
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