水牛cMyc基因与穿膜肽TAT的融合表达

Fusion expression of buffalo cMyc gene with cell-penetrating peptide TAT

为探讨干细胞转录因子与穿膜肽融合表达蛋白对水牛体细胞诱导重编程的可行性,作者对影响水牛cMyc基因穿膜肽融合蛋白表达的因素进行了探索。应用双酶切定向克隆的方法,将水牛cMyc基因CDS(1 350bp)连接到pET-HA2-TAT载体中,构建成pET-cMyc-TAT载体,经酶切、PCR和测序验证,载体构建正确。将pET-cMyc-TAT载体转化大肠杆菌BL21(DE3)感受态细胞,经IPTG诱导目的蛋白表达,同时探索了其表达形式、诱导表达IPTG最优浓度和最优时间以及蛋白纯化咪唑洗脱的最佳浓度。结果表明,融合蛋白cMyc-TAT在37℃以包涵体的形式大量表达,IPTG终浓度0.001mmol/L,诱导5h为最佳诱导条件,纯化蛋白最佳咪唑洗脱浓度为145.76mmol/L,纯化蛋白经过Western-blot验证具有抗原性。研究结果为建立转录因子穿膜肽融合蛋白诱导水牛iPS细胞技术体系奠定了基础。

To investigate the possibility of using stem cell specific transcription factor and cell-penetrating peptide fusion protein to generate buffalo iPSCs,cMyc and TAT fusion protein expression condition were studied.First,the coding sequence of cMyc gene was directly cloned into pET-HA2-TAT vector as pET-cMyc-TAT.The constructed pET-cMyc-TAT was identified to be correct by enzyme digestion,PCR and sequencing.Then it was transformed into Escherichia coli BL21（DE3） and expressed with induction of IPTG.Fusion protein expressing form,concentration of IPTG,optimal induction time and density of the purification imidazole had been investigated.In result,the fusion protein expressed as inclusion bodies at 37 ℃,with the induction of IPTG at 0.001 mmol/L for 5 hours.After being purified with 145.76 mmol/L imidazole,the cMyc-TAT fusion protein possessed antigenicity in Western-blotting test.In conclusion,this study laid the foundations for preparation of the buffalo iPSCs by induction with fusion protein.