关键词: 中国斗鸡; DJ-1; 克隆; 真核表达; 原核表达

Abstract: 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-N3-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-N3 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.

Key words: Chinese gamecock; DJ-1; clone; eukaryotic expression; prokaryotic expression