Expression Efficiency of Recombinant Human Interferon Alpha 2b in Chinese Hamster Ovary cells

• Main Authors: Wei-Shih Kao, 高偉適
• Other Authors: Tsu-An Hsu
• Format: Others
• Language: en_US
• Published: 2005
• Online Access: http://ndltd.ncl.edu.tw/handle/22641921705988032894

碩士 === 國立清華大學 === 化學工程學系 === 93 === Most recently, IFN-α2b has become an important anti-virus and anti-cancer drug. However, expressed human IFN-α2b (HuIFN-α2b) at low levels in Chinese hamster ovary (CHO) cells were observed. 1 mg of IFN-α2b production will require 1.1 x 1011 cells which are equivalent to cultured cells in 367 of T75 flasks. For the cytotoxicity of CHO cells induced by HuIFN-α2b, the growth inhibition still has not been observed notably after HuIFN-α2b treatment. Hence, the problem that expressed HuIFN-α2b at very low levels in CHO cells results from inefficient transcription or translation is considered. In results, firstly, copies of HuIFN-α2b is 4- fold higher than that of EGFP reporter in comparison of the cell total and nuclear RNA. Secondly, the copy number difference between HuIFN-α2b and enhanced green fluorescence protein (EGFP) was not distinguished markedly in comparison of cytoplasmic mRNA and nuclear mRNA. Finally, the amount of expressed HuIFN-α2b was significantly lower than that of EGFP in CHO cells. On the other hand, effects of splicing on enhancing protein expression have been demonstrated in the published paper. In spite of the increase in the amount of mRNA levels, HuIFN-α2b expression could not be raised markedly as splicing was employed. Therefore, the reason expressed HuIFN-α2b at low levels in CHO cells might be mRNA inefficient translational initiation or the middle process of translation rather than the transcriptional efficiency. In the future, the translational initiation efficiency and the synthesis rate of HuIFN-α2b in CHO cells will be further studied. The bottleneck in this overall HuIFN-α2b expression process in CHO cells will also be analyzed by kinetics to optimize the expression efficiency.