人α-肿瘤坏死因子基因的高效表达

High Level Expression of Human Tumor Necrosis Factor-a

用人工合成的α-肿瘤坏死因子(TNF-α)基因构建了五个不同的表达质粒,它们不同之处是SD序列与起始密码子ATG间距离(D)各异。计算机模拟计算出翻译起始区域(TIR)中二级结构的最小生成自由能,以(D)为6个核苷酸时最小(绝对值)。它的表达效率也最高,产物TNF-α可达菌体总蛋白的60％。密码子的选用对表达效率有很大的影响,故人工合成TNF-α基因(选用大肠杆菌偏爱的密码子)的表达效率高于sc-DNA(对部分密码子改造的半合成cDNA)。

Five expression plasmids for a total chemically synthesized gene of human tumor necrosis factor-α (TNF-α) were constructed. There are two different points;. 1. the distance (D) between the SD and the initiation codon ATG; 2. the energy- AG°?f298for forming the stable secondary structure in the translation initiation region. The plasmid with a (D) of 6bp has the lowest AG°f298 (absolute value) and showed the highest expression level, up to 60% of the total bacterial proteins. Codon usage will impose much influence on expression level. So it is reasonable and understandable that the total chemically synthesized TNF-α gene with the codons preferable to E. coli usage gave much higher expression level than the sc-DNA in which only partial N-tefminal codons were modified.