中国斗鸡DJ-1基因克隆与原核、真核表达研究

The Research of Cloning Prokaryotic Expression and Eukaryotic Expression of DJ-1 Gene from Chinese Gamecock

本研究以中国斗鸡λ噬菌体cDNA文库为模板,克隆了DJ-1基因,并构建了pGEX-4T-1-DJ-1原核表达载体和pEGFP-N3-DJ-1真核表达载体,前者用来研究DJ-1蛋白在大肠杆菌表达系统中的优化表达;后者用来转染成纤维细胞系,研究DJ-1蛋白的亚细胞定位和建立转DJ-1基因的细胞模型。试验成功克隆了DJ-1基因的CDS区,并将其定向插入到pGEX-4T-1原核表达载体和pEGFP-N3真核表达载体中。IPTG浓度梯度诱导原核表达结果显示,在0.8mmol/L时DJ-1蛋白表达量最高;时间梯度结果显示,在8h时DJ-1蛋白表达量最高;真核表达载体转染脂尾羊成纤维细胞后48h阳性率最高,DJ-1蛋白在细胞核和胞质中均有表达,但胞质中居多。上述结果表明,本试验已经建立了稳定的DJ-1蛋白的真核、原核优化的表达系统,为进一步研究DJ-1基因的功能奠定了基础。

In this study,DJ-1 gene was cloned from λ phage cDNA library of Chinese gamecock and successfully constructed pGEX-4T-1-DJ-1 prokaryotic expression vector and pEGFP-N3-DJ-1 eukaryotic expression vector. The prokaryotic expression was used to study the DJ-1 protein in E. coli expression system for the optimization of expression and eukaryotic expression was used to transfer fibroblast lines to study DJ-1 protein in subcellular localization and the establishment of DJ-1 gene transferred cell model. To further in-depth study of DJ-1 gene function and lay the foundation for the relationship between the disease. A result,full-CDS areas of DJ-1 gene was cloned,and its orientation inserted into pGEX-4T-1 prokaryotic expression vector and pEGFP-N3 eukaryotic expression vector. The prokaryotic expression induced with IPTG concentration gradient showed that the DJ-1 protein expression was highest in 0.8 mmol/L. In the time gradient showed that the DJ-1 protein expression was highest in 8 h. Eukaryotic expression vector was transfected into fat-tailed sheep fibroblasts with the highest positive rate at 48 h,DJ-1 protein in the nucleus and cytoplasm were expressed,but the cytoplasm was majority. The results indicated that this experiment had established a stable DJ-1 protein in eukaryotic and prokaryotic expression system optimized for the DJ-1 gene function in further studies. This protein could also be used to do antibody preparation,immunization identification and diagnosis research,recombinant eukaryotic plasmid could be used for transgenic animal studies.