|
|
|
| Construction and Biological Assay of Human Interferon α1b Mutant 31K |
| TAO Shan-shan1,MAO De-cai2,LIU Jin-yi2,CHEN Yi-jun1 |
1.School of Life Science and Biotechnology，China Pharmaceutical University，Nanjing 210009，China
2.Beijing Tri-Prime Genetic Engineering Co. Ltd.，Beijing102600，China
|
|
|
|
Abstract Interferon alpha are used clinically to treat a group of viral diseases and cancer because of its broad anti-viral，anti-tumor and immune regulation activities. Nevertheless, the clinical usage of interferon-α can still be improved in many ways, such as increasing activity and so on．In order to obtain a new type of molecule with higher potency, human interferon α1b mutant IFNα1b/31K was constructed. ATG was substituted by AAG at 31 of IFN α1b through PCR site-directed mutagenesis in vitro. The amplified fragments were inserted into pET-23b expression vector, and the recombinant plasmid was transformed into Escherichia coli BL21(DE3)．After being cultured in complex auto-inducing media and purification process, IFNα1b/31K was expressed as soluble protein with the yield more than 30% of total bacterial protein. The purity was more than 95%, the anti-viral activity was 1.7 times comparing with IFNα1b, anti-tumor activity was lower than IFNα1b, and the p-value of cytotoxicity & acute toxicity was more than 0.05．
|
|
Received: 07 May 2010
Published: 12 June 2010
|
|
|
| ［1］ 赵敏，熊诗松，巴秋菊，等．国产基因工程干扰素α1治疗慢性乙型肝炎观察．河南医科大学学报，1995，30(2)：200202. Zhao M，Xiong S S，Ba Q J，et al ．Journal of Henan Medical University，1995，30(2)：200202.
［2］ 童福易，唐永煌，白敬羽，等．病毒性肝炎患者α干扰素中和抗体发生率及其时间规律的研究．临床肝胆病杂志，1999，15(1)：3840. Tong F Y，Tang Y H，Bai J Y，et al.Journal of Clinical Hepatology，1999，15(1)：3840.
［3］ Kraus M R，Wilms K．Interferon alpha effect，indications，therapy monitoring and sideeffects．Intemist(Berl), 2000，41(12)：13991404.
［4］ 胡荣，马学军，魏开坤，等．新型基因工程干扰素受体结合域的改造及其生物活性测定．中华实验和临床病毒学杂志，2002，16(2)：132135. Hu R，Ma X J，Wei K S，et al． Chinese Journal of Experimental and Clinical Virology，2002，16(2)：132135.
［5］ 马学军，胡荣，吕海，等．人干扰素α1c/86D AB环突变文库的构建及细胞筛选．病毒学报，1998，14(1)：8386. Ma X J，Hu R，Lv H，et al． Chinese Journal of Virology，1998，14(1)：8386.
［6］ 马学军，杜东毅，尹华丽，等．特异结合人IFNα1b中和抗体的两个短肽．病毒学报，1997，13(2)：169172. Ma X J，Du D Y，Yin H L，et al． Chinese Journal of Virology，1997，13(2)：169172.
［7］ Horisberger M A，Marco S D. Interferon alpha hybrids．Pharmac Ther，1995，66：507534.
［8］ Studier F W．Protein production by autoinduction in highdensity shaking cultures．Protein Expression and Purification，2005，41：207234.
［9］ 中国药典委员会．中华人民共和国药典三部．北京：化学工业出版社，2005．附录5657. Chinese Pharmacopoeia Committee．Chinese Pharmacopoeia Volume Ⅲ．Beijing：Chemical Industrial Press， 2005．Appendix 5657.
［10］ Mounira K，ChelbiAlix，Juana W．Interferon， a growing cytokine family：50 years of interferon research．Biochimie，2007，89：713718.
［11］ BrideauAndersen A D，Huang X J，Sun S C，et al．Directed evolution of geneshuffled IFNα molecules with activity profiles tailored for treatment of chronic viral diseases．PNAS，2007，104(20)：82698274. |
|
Viewed |
|
|
|
Full text
|
|
|
|
|
Abstract
|
|
|
|
|
Cited |
|
|
|
|
| |
Shared |
|
|
|
|
| |
Discussed |
|
|
|
|
