人白细胞介素-18突变体的虚拟筛选及其活性

Mock Screening and Activity of Human Interleukin-18 Mutant

目的筛选合适的人白细胞介素-18(hIL-18)突变体,并检测其活性。方法运用分子模建工具,模建出IL-18受体及IL-18/IL-18R复合物的分子结构,分别虚拟突变IL-18的4个半胱氨酸为另外19种氨基酸,以野生型IL-18为模板进行同源模建,建立其三维结构模型,计算其IL-18/IL-18R复合物三维结构及分子间作用能变化情况,筛选几种突变方案。运用分子生物学技术构建突变体,表达纯化后进行活性检测。结果根据理论预测情况,筛选了12株突变体。突变的质粒经双酶切及测序鉴定,证明构建正确。突变体蛋白以包涵体形式表达,表达量约占菌体总蛋白的30%,纯化后纯度大于90%。纯化的C38Glu、C127Ser等几株突变体在低浓度时,活性轻微上升,与理论预测结果基本相同。结论已成功构建了多株IL-18的突变体,运用生物信息学方法进行先期筛选有助于加快IL-18的突变研究。

Objective To screen appropriate human interleukin-18 （hIL-18） mutant and determine its activity, Methods Model the molecular structures of human IL-18 receptor （IL-18R） and IL-18/IL-18R complex by using protein homology modeling software, Mock construct the three-dimensional structure of IL-18 mutant in which the cysteine at 4 sites were substituted with the other 19 kinds of amino acids by modeler 8.2 software using wild IL-18 as template. Calculate the intermolecular energy of the complex to screen the program for mutation. The mock screened mutants were constructed by molecular biological technique, then expressed, purified and determined for activity. Results A total of 12 mutants were screened according to the theoretical prediction. Both restriction analysis and sequencing proved that the recombinant plasmids containing the mutant genes were constructed correctly. The mutant proteins in forms of inclusion bodies contained about 30% of total somatic protein and reached a purity of more than 90% after purificatiou. The activities of several purified mutants such as C38Glu and C127Ser, at low concentrations, increased slightly, which were basically consistent with those predicted theoretically. Conclusion Several human IL-18 mutants were successfully constructed, which proved that pre-screening by bioinformatic method was helpful to promoting the study on IL-18 mutation.