| Project/Area Number |
05660144
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
食品科学・栄養科学
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| Research Institution | KYUSHU UNIVERSITY |
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Principal Investigator |
SHIRAHATA Sanetaka KYUSHU UNIVERSITY GRADUATE SCHOOL OF GENETIC RESOURCES TECHNOLOGY,ASSOCIATE PROFESSOR, 大学院・農学研究科, 助教授 (90154377)
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| Co-Investigator(Kenkyū-buntansha) |
TACHIBANA Hirofumi GRADUATE SCHOOL OF GENETIC RESOURCES TECHNOLOGY,ASSISTANT PROFESSOR, 大学院・農学研究科, 講師 (70236545)
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| Project Period (FY) |
1993 – 1994
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| Project Status |
Completed (Fiscal Year 1994)
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| Budget Amount *help |
¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1994: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 1993: ¥1,600,000 (Direct Cost: ¥1,600,000)
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| Keywords | Enhancement of productivity of animal cells / activation of promoter / Oncogenes / ras oncogene / CMV promoter / recombinant protein / gene amplification / E1A oncogene / ras / ElA / ヒトインターロイキン-6 |
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| Research Abstract |
In order enhance cellular recombinant protein productivity by using the specific promoter activation with oncogenes, the ras oncogene was cotransfected with the dihydrofolate reductase (dhfr) gene into a recombinant BHK-21 cell line, BCI-8, producing human interleukin-6 (hIL-6) under control of the cytomegalovirus immediate early promoter (CMV promoter). A single step gene amplification of ras using methotrexate (MTX) resulted in the rapid amplification of a stable hIL-6 hyper-producing clone exhibiting about 35 times higher productivity compared to BCI-8 cells. Other oncogenes such as myc, myb, fos, jun and adenovirus E1A did not enhance cellular productivity. To rapidly establish hIL-6 hyper-producing cell lines, we created host cell lines from BHK-21 cells which were 'primed' with amplified ras oncogene expression units prior to the introduction of a producing gene. The primed BHK cell lines were domonstrated to produce many stable highly hIL-6 producing cells achieving about 15 times higher productivity as acompared to original BHK cells upon introduction of the producing gene. Furthermore, the E1A gene elevated the hIL-6 production level in the ras-amplified hIL-6 hyper-producing cells by about 10 times, suggesting a synergistic action between the E1A and ras oncogenes. It was suggested that c-Ha-ras activated NF-kappaB via activation of protein kinase C and E1A participated in the c-Ha-ras dependent activation of transcription. The productivities of other recombinant proteins such as human monoclonal antibodies, human erythropoietin and human granulocyte colony stimulating factor were enhanced by the ras oncogene.
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