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

doi:10.11843/j.issn.0366-6964.2023.06.013

摘要/Abstract

摘要： 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。

关键词: PhiC31整合酶, 原核表达, 电穿孔, RMCE, DF-1细胞, 鸡

Abstract: 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.

Key words: PhiC31 integrase, prokaryotic expression, electroporation, recombinase mediated cassette exchange (RMCE), DF-1 cells, chicken

中图分类号:

S831.2

王辉, 许梦缘, 刘灵康, 郑喜邦, 李恭贺, 吴文德. 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[J]. 畜牧兽医学报, 2023, 54(6): 2330-2342.

WANG Hui, XU Mengyuan, LIU Lingkang, ZHENG Xibang, LI Gonghe, WU Wende. PhiC31 Integrase Triggers Site-specific Cassette Exchange in Chicken Fibroblast Cells through Electroporation[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(6): 2330-2342.

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