烟草天蛾几丁质酶的表达、纯化及多克隆抗体制备  

Expression, purification, and polyclonal antibody preparation of Manduca sexta chitinase

作　　者：郝婵娟[1] 柴宝峰[1] 王伟[1] 孙毅 梁爱华[1] 

机构地区：[1]山西大学生物技术研究所 [2]山西省农业生物技术研究中心,太原030031

出　　处：《昆虫学报》2005年第3期337-341,共5页Acta Entomologica Sinica

基　　金：国家自然科学基金项目(39970416;30270204;30300038);山西省自然科学基金项目(031010)

摘　　要：将烟草天蛾Manducasexta几丁质酶基因克隆入融合表达载体pET-28a,并在大肠杆菌E.coliBL21(DE3)中进行诱导表达。表达菌株经0.5mmolLIPTG诱导6～8h后,几丁质酶表达并形成包涵体。在Ni2+-NTA亲和柱上经变、复性和纯化,得到可溶的几丁质酶。表达产物经Western印迹鉴定。采用切胶回收的方法切割回收包涵体,并将回收产物免疫新西兰大白兔,ELISA检测抗体效价达1∶20000。Western印迹检测证明抗体特异性良好。The chitinase gene of Manduca sexta was cloned into the fusion expression vector pET_28a and expressed in E. coli BL21(DE3) host cells. After the expression strain was induced for 6-8 hours by 0.5 mmol/L IPTG, the fusion protein chitinase was expressed and detected in inclusion bodies. After denature and renature procedure in Ni^(2+) _NTA affinity chromatography column, soluble chitinase was obtained. The authenticity of in vitro renatured protein was confirmed by Western blot. The inclusion body protein band in SDS_PAGE was excised and the protein was extracted. Then the purified protein was injected into New Zealand rabbits to induce immunoreaction. The induction with two injections lasted for 45 days, and then the anti_serum was prepared. ELISA analysis showed that the titer for this polyclonal antibody was 1∶20 000. Western blot analysis showed that the antibody reacted specifically to the expressed chitinase protein.

关 键 词：烟草天蛾 几丁质酶 原核表达 变性和复性 WESTERN印迹 抗体制备 

分 类 号：Q966[生物学—昆虫学]