| 中国斗鸡DJ-1基因克隆与原核、真核表达研究 |
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| 引用本文： | 冯宝刚,朱超,王会,武坤,娜日苏,卢晟盛,关伟军.中国斗鸡DJ-1基因克隆与原核、真核表达研究[J].中国畜牧兽医,2010,37(5):70-74. |
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| 作者姓名： | 冯宝刚 朱超 王会 武坤 娜日苏 卢晟盛 关伟军 |
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| 作者单位： | （1.广西大学动物科学技术学院， 南宁 530004； 2.中国农业科学院北京畜牧兽医研究所， 北京 100193） |
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| 摘 要： | 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0.8 mmol/L时DJ-1蛋白表达量最高；时间梯度结果显示，在8 h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48 h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。
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| 关 键 词： | 中国斗鸡 DJ-1 克隆 真核表达 原核表达 |
The Research of Cloning Prokaryotic Expression and Eukaryotic Expression of DJ-1 Gene from Chinese Gamecock |
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| FENG Bao-gang,,ZHU Chao,,WANG Hui,WU Shen,NA Ri-su,LU Sheng-sheng,GUAN Wei-jun.The Research of Cloning Prokaryotic Expression and Eukaryotic Expression of DJ-1 Gene from Chinese Gamecock[J].China Animal Husbandry & Veterinary Medicine,2010,37(5):70-74. |
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| Authors: | FENG Bao-gang ZHU Chao WANG Hui WU Shen NA Ri-su LU Sheng-sheng GUAN Wei-jun |
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| Institution: | （1.Animal Science and Technology College of Guangxi University，Nanning 530004，China；2.Institute of Animal Science，Chinese Academy of Agricultural Sciences，Beijing 100193，China） |
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| 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-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. |
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| Keywords: | Chinese gamecock DJ-1 clone eukaryotic expression prokaryotic expression |
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