猪白介素家族重要基因的克隆、表达及其结构与功能预测分析

The Molecular Clone, Expression and Prediction of Construction to Key Genes of Interleukin Family of Porcine

【目的】克隆、表达和预测分析猪白介素家族重要成员的结构与功能。【方法】以猪白介素基因家族为对象,通过对猪PBMC刺激诱导,提取RNA,采用RT-PCR技术克隆IL-4、IL-6、IL-18等重要功能基因,并进行原核表达和结构与功能分析。【结果】成功地克隆和表达了IL-4、IL-6和IL-18基因,序列分析发现,克隆的基因与NCBI/GenBank上登载的参考序列的同源性分别为99.25%、99.21%和100%,与其它各物种的基因及其编码蛋白间具有较大的种属差异性;在大肠杆菌中表达的IL-4和IL-6重组蛋白具有较高的生物活性;结构预测表明,这些蛋白分子均含有较多的磷酸化修饰、糖基化修饰(除IL-6)、蛋白激酶以及信号传导结合位点,其中IL-4、IL-6蛋白分子的螺旋化程度较高,均在60%左右,水溶性较低,但这3种蛋白的立体空间结构均为致密的球状,说明该结构特征是其多种生物学功能发挥的基础。【结论】本研究成功地克隆、表达和分析了猪白介素家族的IL-4、IL-6和IL-18等重要分子的结构与功能,为其进一步的生物学功能研究及其应用奠定了基础。

[Objective] Aim This research is to clone, express and analyses the structure and function of major molecular of porcine interleukin family. [Method] Genes of porcine interleukin family was cloned with RT-PCR through stimulated porcine PBMC by LPS and PHA firstly, then expressed in E.coli and predicted the structure and function of these molecular by ExPASY. [ Result ] It was successfully cloned and express the genes of IL-4, IL-6 and IL-18. Products of recombinant expression of IL-4 and IL-6 both have more biological activity, In analysis of these genes with NCBI/GenBank data, the homologies of the nucleotide acid sequence are 99.25%, 99.21% and 100% respectively and have large difference among other animal species. Results of prediction show that these molecular all contain many phosphorylation, glycosylation, protein kinase and signal transduction bonding sites in secondary structure, and all are a compact globularity protein in space configuration. This character of structure is the basis for its more biological functions. [Conclusion] We have successfully cloned, expressed and analysed the structure and function of key molecular of porcine interleukin family.