Abstract: 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 transformed into E. coli host cells. Protein expression was induced by IPTG and analyzed by SDS-PAGE and Western blot. The results showed that the HSF1 cDNA was 2 036 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.

Key words: Cloning, Expression, Hainan Eld's deer, HSF1

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 factor 1,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2 036 bp,含有1 个1 578 bp 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62 kD 的融合蛋白,用抗His 单克隆抗体进行Western blot,得到一条约62 kD 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。

关键词: 海南坡鹿, HSF1, 克隆, 表达