Synonymous Codon Usage Bias and Overexpression of a Synthetic Gene Encoding Interferon α2b in Yeast  

作　　者：Bin FANG　 Bu-feng LIANG　 Guang-yuan HE 

机构地区：[1]China-UK HUST-RRes Genetic Engineering and Genomics Joint Lab, Huazhong University of Science and Technology （HUST）, Wuhan 430074, China [2]Joint R＆D Lab （Suzhong Pharma Co.,Ltd）, IHS, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China

出　　处：《中国病毒学》2007年第3期226-232,共7页Virologica Sinica

基　　金：The National ‘973’ Basic Research Program (2002CB111302); The National Natural Science Foundation of China (30370807)

摘　　要：To achieve higher level expression of Interferon α2b(IFN-α2b)in methylotrophic yeast(Pichia pastoris),a cDNA fragment coding for the mature IFN-α2b was designed and synthesized based on the synonymous codon bias of P.pastoris and optimized G+C content.The synthetic IFN-α2b was inserted into the secreted expression vector pPICZαA,and then integrated into P.pastoris GS115 genome by electroporation.Multi-copy integrants in the Mut+ recombinant P.pastoris strain were screened by high concentrations of Zeocin.120 hours culturing allowed expression of the IFN-α2b transformant up to 810 mg/L as detected by SDS-PAGE and quantitative methods.In addition,Western blot analysis showed that the recombinant proteins had immunogenicity.The significant antiviral activity of the recombinant IFN-α2b protein was verified by WISH/VSV system,which was 3.3×105 IU/mL.To achieve higher level expression of Interferon α2b （IFN-α2b） in methylotrophic yeast （Pichia pastoris）, a cDNA fragment coding for the mature IFN-α2b was designed and synthesized based on the synonymous codon bias of P. pastoris and optimized G＋C content. The synthetic IFN-α2b was inserted into the secreted expression vector pPICZαA, and then integrated into P. pastoris GS115 genome by electroporation. Multi-copy integrants in the Mut^＋ recombinant P. pastoris strain were screened by high concentrations of Zeocin. 120 hours culturing allowed expression of the IFN-α2b transformant up to 810 mg/L as detected by SDS-PAGE and quantitative methods. In addition, Western blot analysis showed that the recombinant proteins had immunogenicity. The significant antiviral activity of the recombinant IFN-α2b protein was verified by WISH/VSV system, which was 3.3×10^5 IU/mL.

关 键 词：酵母菌 干扰素-Α2B 同义密码子 基因表达 过表达 

分 类 号：Q786[生物学—分子生物学] TQ92[轻工技术与工程—发酵工程]