犬瘟热病毒N蛋白基因融合蛋白原核表达条件的优化与蛋白纯化  

Optimization of Prokaryotic Expression Conditions of N Protein Gene of Canine Distemper Virus(CDV)and Protein Purification

作　　者：简子健[1] 马素贞[1] 申卫红[1] 翟少华[1] 赵森[1] 

机构地区：[1]新疆农业大学动物医学学院,乌鲁木齐830052

出　　处：《新疆农业科学》2012年第2期336-340,共5页Xinjiang Agricultural Sciences

基　　金：新疆维吾尔自治区高技术项目(200611107)

摘　　要：【目的】提高犬瘟热病毒N蛋白基因在大肠杆菌中表达可溶性GST融合蛋白的含量。【方法】研究诱导剂(IPTG)的最佳加入时间、诱导剂(IPTG)浓度、诱导时间以及不同温度条件对GST-CDV N融合蛋白表达的影响,获得在大肠杆菌中的最佳的表达条件,然后再在最佳条件下大量的表达,用GST-Sepharose 4B亲和层析柱对GST-CDV N融合蛋白进行纯化,得到最大量的可溶性融合蛋白,并对蛋白含量进行测定。【结果】大肠杆菌BL21(DE3)转化菌在37℃培养3.5 h(OD600=1.314)时,加入IPTG使终浓度为1 mmol/L,然后在27℃诱导8 h,GST-CDV N融合蛋白可溶性表达量最高,达到0.862 mg/mL。【结论】确定了犬瘟热病毒N蛋白基因的优化表达条件,为犬瘟热病毒分子免疫学诊断和亚单位疫苗的研制奠定了基础。[ Objective and Method] In order to improve the dissoluble expression level of GST -CDV N fusion protein in Escherichia coli, the influence of various expressing conditions including time of adding IPTG , IPTG concentration, induction time and temperature to GST-CDV N fusion protein was studied in this pa- per. After the optimal expressing conditions were obtained, a considerable GST- CDV N fusion protein in Escherichia coli was expressed under this comdition at this conditions. Then, the GST - CDV N fusion protein was purified by GST - Sepharose 4B affinity column to obtain the highest level dissoluble fusion protein and protein level was determined. [ Result] The experimental results showed that the dissoluble expression of GST -CDV N fusion protein reached the highest level （0.862 mg/lnL） in transformed bacterium strain Escherichia coli BL21（DE3） after pre - culture for 3.5 h （OD600 = 1. 314） ,after IPTG was added to make final concentra- tion to 1 mmol/L and continued to culture 8 h at 27%. [ Conclusion]The platform was established by the op- timized expression conditions of CDV N for the future research on diagnosis of molecular immunology in canine distemper and development of subunit vaccine for this disease.

关 键 词：犬瘟热病毒 N蛋白基因 融合蛋白 原核表达条件的优化 纯化 

分 类 号：S852.655[农业科学—基础兽医学]