海南坡鹿HSF1cDNA的克隆与表达  

Cloning and expression of HSF1 cDNA from Hainan Eld's deer

作　　者：成鹰[1] 杜丽[1] 王凤阳[1] 刘涛[1] 李治深[1] 许世英[2] 符运南[2] 林贤梅[2] 吴科榜[1] 林杰材[1] 满初日嘎[1] 

机构地区：[1]海南大学农学院,海口市动物基因工程重点实验室,海口570228 [2]海南大田国家级自然保护区,东方572600

出　　处：《兽类学报》2009年第2期191-197,共7页Acta Theriologica Sinica

基　　金：国家自然科学基金资助项目(30560021);海南省自然科学基金资助项目(30509);海南省教育厅高校科研资助项目(Hj200609)

摘　　要：采用RT-PCR和RACE方法扩增海南坡鹿热休克转录调节因子1(Heat shock transcription factor1,HSF1)cDNA全长,将扩增产物与pMD20-T载体连接,重组质粒经PCR、酶切鉴定后测序并进行生物信息学分析;构建pET28a-hdHSF1表达载体,经IPTG诱导表达后,进行SDS-PAGE和Western blot分析。结果显示,海南坡鹿HSF1cDNA全长为2036bp,含有1个1578bp的开放阅读框,编码525个氨基酸。经生物信息学分析,HSF1是一个等电点为4.93的亲水性蛋白。经IPTG诱导表达后,得到一个带组氨酸标签的约62kD的融合蛋白,用抗His单克隆抗体进行Western blot,得到一条约62kD特异性抗体结合带,表明海南坡鹿HSF1原核表达载体成功构建并表达。Heat shock transcription factor 1 （ HSF1 ）cDNA of Hainan Eld＇ s deer （ Cervus eldi hainanus） was amplified by RT-PCR and RACE, and cloned into pMD20-T vector. The recombinant plasmid was confirmed by PCR, endonuclease digestion, sequencing and bioimformatics analysis. The PCR product was then subcloned into pET28a vector and trans- formed into E. coil host cells. Protein expression was induced by IPTG and analyzed by SDS-PAGE and Western blot. The results showed that the HSF1 eDNA was 2036 bp in length and contained a 1 578 bp ORF encoding 525 amino acids. The encoded protein was a hydrophilic protein which IP was 4. 93 analyzed by bioimformatics. A 62 kD fusion protein with a His tag was induced by IPTG and confirmed by Western blot using anti his tag monoclonal antibody, getting a special antibody binding band, indicated that prokaryotic expression vector of HSF1 from Hainan Eld＇ s deer was constructed and expressed successfully.

关 键 词：海南坡鹿 HSF1 克隆 表达 

分 类 号：Q786[生物学—分子生物学]