大鼠IgE依赖组胺释放因子重组蛋白纯化效果的优化  

Optimization of the Expression and Purification of Recombinant Rat IgE-Dependent Histamine-Releasing Factor

作　　者：陈晓湘[1,2] 胡旭初[2] 徐劲[2] 余新炳[2] 

机构地区：[1]广州医学院病原生物学教研室,广州510182 [2]中山大学中山医学院寄生虫学教研室,广州510080

出　　处：《热带医学杂志》2008年第12期1203-1205,1209,共4页Journal of Tropical Medicine

基　　金：国家自然科学基金(No.30571634);教育部博士点基金(No.20060558002)

摘　　要：目的提高大鼠IgE依赖组胺释放因子(rHRF)的可溶性表达水平,改善其亲和层析纯化效果,为深入研究该重组蛋白的生物学功能奠定基础。方法选择多克隆酶切位点BamHⅠ和XhoⅠ,将rHRF完整编码区基因从原有的pET-30a-rHRF重组质粒亚克隆至pET-28a载体,构建重组质粒pET-28a-rHRF;转化大肠杆菌BL21(DE3)后,通过改变IPTG浓度(0.1～1.0mmol/L),调节诱导表达温度(25～37℃)和时间(1～5h),筛选能够提高rHRF可溶性表达水平的条件;同时改进亲和层析纯化的条件,提高纯化效果。结果成功构建重组质粒pET-28a-rHRF,不同IPTG浓度、诱导温度和时间对重组蛋白的可溶性表达水平无明显影响。但是,与pET-30a-rHRF转化菌相比,pET-28a-rHRF转化菌所表达的重组蛋白因其N端和C端均带有6×his标签,增强了目的蛋白与树脂的结合力,而使亲和层析纯化效果(重组蛋白的浓度和纯度)显著提高。结论rHRF重组蛋白的可溶性表达水平不受IPTG浓度、诱导表达温度和时间的影响,但是可溶性重组蛋白两端均携带6×his标签可使亲和层析纯化效果显著提高,从而有利于获得足量rHRF用于进一步的生物学功能研究。Objective To improve the expression of the soluble recombinant protein of rat IgE-dependent histamine-releasing factor （rHRF） and to optimize the purification procedures by affinity chromatography. Method The complete coding region of rHRF was subeloned into pET-28a vector from the recombinant plasmid pET-30a-rHRF using the multiple cloning sites BamH I and Xho I . Recombinant pET-28a-rHRF was then transformed into E.coli BL21 （DE3） ceils. Optimal conditions for the expression of the soluble recombinant rHRF were determined by changing the IPTG concentration （0.1 -1.0 mmol/L）, inducing temperature （25-37℃） and inducing time （1 -5 h）. The conditions for affinity chrorhatography were also optimized to improve the purity. Result The recombinant pET- 28a-rHRF was constructed successfully. Expression of the soluble recombinant protein was not affected by the IPTG concentration, inducing temperature and the induction time. Compared with the pET-30a-rHRF transformed cells, recombinant proteins expressed by pET-28a-rHRF transformed cells, due to the presence of 6xhis tags at the N and C terminus, could be easily purified by the affinity chromatography. Conclusion The expression of the soluble recombinant rHRF was not influenced by the concentration of IPTG, inducing temperature and time. The presence of 6 × his tag would greatly facilitate the purification and the subsequence study of the biological functions of rHRF.

关 键 词：大鼠 IgE依赖组胺释放因子 原核表达载体 蛋白质纯化 

分 类 号：R392.8[医药卫生—免疫学]