PhiC31整合酶电穿孔鸡成纤维细胞诱发位点特异性基因盒交换

PhiC31 Integrase Triggers Site-specific Cassette Exchange in Chicken Fibroblast Cells through Electroporation

旨在验证直接电穿孔重组蛋白PhiC31能否在鸡DF-1细胞中诱导PhiC31介导的基因盒交换。本研究通过原核表达和经亲和层析纯化获得SUMO-His-PhiC31融合蛋白,利用SUMO酶切除SUMO-His标签,获得天然的PhiC31蛋白。这两种蛋白先与pBCPB+质粒在试管内共孵育进行分子重组反应。之后,先将带有attP TT-DsRed2-attP CT片段的“着陆点”(landing pad,LP)质粒通过电穿孔导入DF-1细胞,然后进行第二次电穿孔,将带有attB TT-EGFP-HiBiT-attB CT的donor质粒连同PhiC31蛋白导入上述DF-1细胞,验证DsRed2-EGFP基因盒交换事件发生。结果显示,成功构建了原核表达载体pET-28a-SUMO-PhiC31,经IPTG诱导,该重组质粒在大肠杆菌中以可溶性方式表达SUMO-His-PhiC31融合蛋白,经亲和层析纯化,SUMO-His-PhiC31蛋白的产量达12 mg·L-1;该融合蛋白和切除SUMO-His标签的天然PhiC31蛋白均成功触发了pBCPB+质粒attP和attB位点之间的重组反应,重组效率基本一致(50%vs.52%);以脂质体转染法将PhiC31真核表达质粒pCMVInt与LP质粒和donor质粒共转染DF-1细胞,荧光显微镜观察证实了DsRed2-EGFP置换现象;以电穿孔方式将LP质粒、donor质粒和PhiC31蛋白先后导入DF-1细胞,获得了相同结果,表明PhiC31蛋白引发了attP TT与attB TT、attP CT与attB CT之间的重组反应。通过原核表达获得的PhiC31蛋白具有生物活性,有望成为一种重要试剂而用于Easi-CRISPR-TARGATT介导的转基因鸡研究。

The present study aimed to evaluate whether the direct protein delivery of PhiC31 integrase through electroporation could induce the PhiC31-mediated recombination in chicken DF-1 cells.Here,prokaryotic expression and affinity chromatography purification was used to produce a small ubiquitin-related modifier(SUMO)-tagged His-PhiC31 fusion protein,and a SUMO proteinase was utilized to generate a natural PhiC31 protein by removing SUMO-His tag.The two types of PhiC31 protein were respectively incubated with pBCPB+plasmid in tubes to induce intermolecular recombination.Subsequently,a landing pad(LP)plasmid harboring the attP TT-DsRed2-attP CT fragment was electroporated into the chicken DF-1 cells,followed by another round of electroporation of the natural PhiC31 protein along with a donor plasmid carrying the attB TT-EGFP-HiBiT-attB CT segment.The results showed that a prokaryotic expression vector(pET-28a-SUMO-PhiC31)was successfully constructed,and SUMO-His-PhiC31 fusion protein was expressed in a resolvable pattern in E.coli with induction of isopropyl-β-D-thiogalactoside(IPTG),and amount of the purified fusion protein reached 12 mg·L-1;both SUMO-His-PhiC31 and natural SUMO-free PhiC31 were capable of triggering the intermolecular recombination between attP and attB sites of pBCPB+plasmid,and the recombinant efficiency of SUMO-His-PhiC31 was comparable to that of PhiC31 proteins(50%vs.52%);co-transfection of DF-1 cells with a PhiC31 expressing vector pCMVInt,LP and donor plasmids resulted in conversion of DsRed2 to EGFP,which was confirmed by fluorescent microscopy;the same results were observed when LP plasmid,the donor plasmid plus PhiC31 protein were successively electroporated to DF-1 cells,implying the occur of PhiC31-mediated recombination between attP TT and attB TT,attP CT and attB CT,respectively.The results indicated that the natural PhiC31 protein is biologically active.The protein is expected to serve as an important reagent in the Easi-CRISPR-TARGATT-mediated genome editing to generate transgenic chickens.