3′端非翻译区影响TNFα cDNA在大肠杆菌中的表达

Effect of 3'-untranslated Region on Expression of TNFa cDNA in Escherichia coli

采用PCR方法改变了表达构建物pRL-rhTNF中的结构,即去掉rhTNFαcDNA3＇端非翻译区序列110bp(命名为pRL-rhTNFα2),转入大肠杆菌后,观察数个阳性转化子的表达情况,并与原型pRL-rhTNFα的表达进行比较。结果表明,去掉3＇端非翻译区的pRL-rhTNFα2能稳定表达rhTNFα,其发酵及纯化产品的生物学特征均等同于原型的,且其表达量比原型的有提高,提示3＇端非翻译区序列对表达有影响。3＇端非翻译区内存在一个相似于TA的重复序列——TTTA TTA七聚体。这可能是本研究中影响TNFα表达量的原因之一。

A construct pRL-rhTNFa2, in which the 110bp 3'-untranslated region of rhTNFa c cDNA was deleted, was transformed in E. colt. The expression level of several positive transformants was determined. The results showed that the expression of pRL-TNFa2 was stable and the biological characteristics of the protein product were as same as that of pRL-TrhTNFa. Furthermore, the expression level of pRL-rhTNFa2 was increased. It suggested that 3'-untranslated region may have some negative effects on gene expression. A TA-rich sequence, TTTATTA, contained in 3'-untranslated region of pRL-rhTNFa may be involved in the inhibitory effect on gene expression.