牛IFN-α基因高效表达及纯化的研究

Expression and purification of IFN-α gene from bovine

基于对RNA二级结构的预测和密码子偏性计算,通过计算机辅助软件进行牛IFN-α基因优化,经人工合成优化后的基因与pWL表达载体连接后诱导表达,并对表达条件进行优化。SDS-PAGE分析表明,其产物分子量约为17ku,最优表达条件下蛋白表达量占菌体总蛋白的33%,表达产物以包涵体形式存在。经过包涵体溶解、初步复性后利用CM Sepharose Fast Flow离子交换层析柱作为蛋白质复性系统,采用梯度法进行牛α-干扰素包涵体蛋白质的纯化复性。结果表明,梯度离子交换层析法能有效地复性牛α-干扰素包涵体,复性后的重组牛α-干扰素的纯度达95%,通过离子交换层析柱纯化和复性后,重组牛α-干扰素在MDBK/VSV细胞系上的抗病毒活性为5.56×106u/mg。

The bovine IFN-α gene was synthesized using optimized codons based on computer analysis and cloned into pWL expression vector for expression in E.coli. SDS-PAGE analysis showed that the expressed protein had a molecular weight of 17 ku and accounted for 33 % of the total cellular protein. The expression product existed mainly in the form of inclusion body and could be purified by gradient ion exchange chromatography following denaturation/renaturation using the CM Sepharose Fast Flow renaturation system. The renatured BoFN-α protein showed strong antiviral activity of 4.0 × 10^5 u/mg on MDBK/VSV test system.