基于细胞穿透肽Tat的Cre-Loxp重组酶系统的改造

目的：构建基于Tat细胞穿透肽和核定位信号NLS的重组酶Cre蛋白表达载体，引导Cre内化并人核实现细胞水平的基因敲除。方法：在带His标记的细胞穿透肽Tat和增强型绿色荧光蛋白（EGFP）表达载体pET14b—SBP-Tat—EGFP基础上，利用酶切连接的方法将NLS—Cre片段插入带上述表达载体中，构建新的融合蛋白表达载体pET14b—SBP-Tat—NLS-cre-EGFP；经酶切、测序鉴定载体构建正确后，将重组质粒转化BL21（DE3）宿主菌，经异丙基硫代一争I）I半乳糖苷（IPTG）诱导表达后，用Ni^2＋亲和层析纯化得到融合蛋白；将融合蛋白透析、过滤除菌后加入到培养的cdc42基因两端带Loxp位点的C57小鼠腹腔巨噬细胞中，在Zeiss荧光显微镜下观察蛋白转导效率，并用Westernblot方法在蛋白水平检测并验证细胞水平目的基因敲除效果。结果：经酶切、基因测序证实重组载体构建成功，融合蛋白在大肠杆菌中可有效表达；Zeiss荧光显微镜下观察融合蛋白穿透细胞膜并进入细胞，细胞在经融合蛋白内化处理后目的基因蛋白的表达量与对照组没加Tat融合蛋白比较有明显降低。结论：利用Tat细胞穿透肽成功构建了基于细胞穿透肽的带有NLS-Cre的Tat蛋白表达运输载体，建立了可携带Cre进入细胞核进行基因敲除的系统，说明利用该系统可以实现细胞水平的基因敲除。

Transformation of the Cre-Loxp recombination enzyme system based on the cell penetrating peptides Tat

Objective：To construct the fusion expression plasmid based cell penetrating peptides with Cre and nuclear localization signal （NLS） label, and to study gene knockout at cell level. Methods：The fusion protein expression vector pET14b-SBP-Tat-NLFrCre-EGFP incorporating Cre recombinant enzyme and NLS was constructed based on His-tagged pET14b-SBP-Tat-EGFP by insertion method, then it was identified by enzyme digestion and DNA sequence, and the recombinant plasmid was transformed into BL21 （DE3） strain. The H is-SBP-Tat-NLS-Cre -EGFP fusion protein was expressed following IPTG induction and purified with Ni2＋-NTA affinity chromatography. After dialysis and filtration the fusion protein was added into cultured eukaryotic cells, and transduced protein was observed under fluorescence microscope and analyzed by Western blot. Results： Enzyme digestion and DNA sequencing confirmed successful construction of the vector pET14b-SBP-Tat-NLS-Cre-EGFP, and the fusion protein was successfully expressed in E. coll. The fusion protein could rapidly penetrate cell membrane and reach cell nucleus, and it was revealed that in the protein transduction experiments in eukaryotic cells at Loxp site, the targeted gene protein was found to be obviously decreased in the level of protein, indicating that cell penetrating peptides can achieve gene knockout on cell level. Conclusion .. The fusion peptide expression vector based Tat protein and Cre recombinant enzyme for targeted protein delivery to the cell nucleus became an effective system and the system can carry Cre into cells to knock out targeted gene.