蟑螂变应原Cr-PI包涵体的变复性研究  

REFOLDING OF THE COCKROACH ALLERGEN Cr PI FROM INCLUSION BODIES

作　　者：高波[1] 刘志刚[1] 苏东明[2] 罗时文[2] 

机构地区：[1]深圳大学生命科学学院,510680 [2]深圳市微生物基因工程重点实验室,518060

出　　处：《寄生虫与医学昆虫学报》2004年第2期96-100,共5页Acta Parasitologica et Medica Entomologica Sinica

基　　金：国家自然科学基金 (No . 3 9660 0 73 )资助项目~~

摘　　要：本研究优化了GST CrPI融合蛋白在大肠杆菌中的诱导表达条件和GST CrPI包涵体的复性条件 ;采用GlutathioneSepharoseTM 4B亲和层析分离纯化融合蛋白 ,以Xa酶切去掉其GST标签后 ,对CrPI进行了电喷雾电离串联质谱分析 (ESI MS MS)。结果表明 ,IPTG浓度对该蛋白的诱导效果影响不大 ,OD6 0 0 值为 0 6时诱导效果最佳 ;复性蛋白浓度和L 精氨酸浓度对稀释复性结果有重要影响。纯化融合蛋白经Xa酶切、分子筛层析去掉GST标签后 ,重组蛋白的纯度达 95 % 。The induction of GST-Cr PI fusion proteins and the refolding of GST-Cr PI inclusion bodies were optimized. The refolded proteins were purified by affinity chromatography with Glutathione Sepharose 4B. After Xa cleavage of the GST tag, the Cr PI allergen was purified by size exclusion chromatography and subjected to ESI-MS/MS. The results showed the concentration of IPTG had little influence on the induction of GST-Cr PI; a OD 600 of 0.6 was the best bacteria density for the induction.The concentration of renaturing protein and L-arginine was very important for the successful renaturation.After removing GST tag through size exclusion chromatography, the Cr PI reached 95% in purity. MS ESI-MS/MS analysis showed that sequence of the recombinant protein has high identity with that of the major cockroach allergen Cr PI.

关 键 词：蟑螂 变应原 Cr-PI 包涵体 变复性 融合蛋白 大肠杆菌 

分 类 号：R392[医药卫生—免疫学]