摘要
目的 根据翻译起始调控的定量理论及利用翻译增强子序列 ,提高人干扰素α1型变种IFN α1C基因的原核表达。方法 采用“步移式”聚合酶链反应 (PCR) ,对IFN α1C基因 5′端核苷酸序列进行三种不同程度的碱基突变 ,使IFN α1C在pBV2 2 0载体中形成的翻译起始区域 (TIR)的二级结构自由能 (△G)逐渐降低 ,同时 ,采用PCR技术 ,将翻译增强子cDNA序列引入pBV2 2 0中的SD序列上游 ,构建一种新型载体pBVE。结果 三种IFN α1C改造基因的表达量均有所提高 ,而且随△G从原有的 - 5 0 2 4 1.6J mol降至 - 2 2 190 .0J mol(绝对值 ,下同 ) ,其表达量有逐渐升高趋势 ,最高可达 2 .4 3×10 8U L ,约为IFN α1C母体的 13倍。选用IFN α1C及其三种改造基因为报导基因 ,结果显示以pBVE为载体的抗病毒活性比在pBV2 2 0中增强了 2～ 5倍。结论 含有翻译增强子的pBVE为一种新型原核高效表达载体。翻译调控的定量关系理论 ,可有效地运用于提高IFN α1C基因在大肠埃希菌中的表达。
Objective To increase prokaryotic expression level of IFN α1C gene through the quantitative theory of translational control and the “translational enhancer” sequence. Methods “Stepwise” polymerase chain reaction (PCR) was used to alter the 5′ terminal cDNA sequence of IFN α1C in three different grades of base mutation. In this way, the free energy (△G) of the secondary structure in translational initiation region(TIR) was decreased gradually. An expression plasmid (pBVE) was constructed to contain the “translational enhancer” cDNA sequence by modifying pBV220 upstream of the SD region. Results The expression levels of three kinds of IFN α1C modified gene were all increased.Furthermore, it presented an increasing trend with decreasing in △G varying from -50 241.6 to -22 190.0 J/mol. The highest expression was 2.43×10 8 U/L, covering twelve times more than its original cDNA. IFN α1C gene and its modified cDNA was inserted into pBVE as reporting genes. E.Coli cells harbouring pBVE/IFN α1Cs cDNA produced two to five times more IFN than cells harbouring pBV220/IFN α1Cs. Conclusion pBVE containing “translational enhancer” is a high level prokaryotic expression vector. The theory of quantitative translational control can effectively be used to enhance the IFN α1C gene expression level in E.coli .
出处
《中华实验和临床病毒学杂志》
CAS
CSCD
北大核心
2002年第4期315-318,共4页
Chinese Journal of Experimental and Clinical Virology
