麻鸭白细胞介素17的原核表达及鉴定  

Expression of spotted duck interleukin-17 gene in E. coli

作　　者：赵雯[1] 潘志明[1] 耿士忠[1] 康喜龙[1] 方强[1] 丛秋霞[1] 焦新安[1] 

机构地区：[1]扬州大学江苏省人兽共患病学重点实验室,江苏扬州225009

出　　处：《中国预防兽医学报》2011年第5期408-410,共3页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家自然科学基金资助项目(30871860);江苏省自然科学基金资助项目(BK2010039);江苏省高校"青蓝工程"资助项目;扬州大学中青年学术带头人培养人选资助项目

摘　　要：为表达麻鸭白介素17(duIL-17),本研究提取ConA刺激的麻鸭脾脏淋巴细胞总RNA,RT-PCR扩增duIL-17基因片段,将该片段插入克隆载体pCR2.1中构建重组质粒pCR2.1-duIL17。将duIL-17基因片段亚克隆到原核表达载体pGEX-6P-1中,构建重组质粒pGEX-duIL17,将其转化到大肠杆菌BL21中,IPTG诱导表达。测序结果显示,duIL-17与参考序列(EU366165.1)相比较第174位碱基由C突变为T,并且为沉默突变。SDS-PAGE和westernblot分析显示,诱导表达的重组蛋白GST-duIL17主要以包涵体形式存在,分子量大小约为45ku,duIL-17与抗鸡IL-17抗体具有良好反应性。本研究为制备duIL-17单克隆抗体提供了实验依据。The duck interleukin-17 （duIL-17） gene was amplified by RT-PCR from the total RNA of conconavalin A （ConA）-stimulated spotted duck splenic lymphocytes. The gene was cloned into vector pCR2.1 to construct recombinant plasmid pCR2.1-duIL17. Sequencing result showed that duIL-17 had a silent mutation at 174 set from C to T, compared with the reference gene （EU366165.1）. Then duIL-17 gene fragment was subcloned into the expression vector pGEX-6P-1, the resultant recombinant plasmid was transformed into E. coli BL21 and induced by IPTG, SDS-PAGE showed that the expressed fusion protein GST-duIL17 was about 45 ku. Western blot analysis showed that duIL-17 was recognized by chicken IL-17 antibody.

关 键 词：麻鸭 IL-17 原核表达 免疫反应性 

分 类 号：Q786[生物学—分子生物学]