绵羊PLC-γ1基因真核表达载体的构建及鉴定

Construction and identification of eukaryotic expression vector of Sheep PLC-γgene

目的旨在构建携带红色荧光蛋白(RFP)报告基因的绵羊PLC-γ1非融合性真核表达载体。方法在原有PUC57-PLC-γ1克隆载体基础上,对目的片段N端前加入P2A序列和3个连续Flag序列后使用HindⅢ酶和SalⅠ酶双酶切获得P2A-3×Flag-PLC-γ1片段,将其连接至pDsRed2-C1载体获得重组质粒pDsRed2-C1-P2A-3×Flag-PLC-γ1;然后采用Lip 2000转染试剂将该重组质粒转染至HEK-293T细胞,最后利用荧光显微镜、qRT-PCR、Western blot、免疫共沉淀(IP)和免疫荧光(IF)法鉴定重组质粒pDsRed2-C1-P2A-3×Flag-PLC-γ1的表达及亚细胞定位情况。结果双酶切和测序结果显示质粒pDsRed2-C1-P2A-3×Flag-PLC-γ1构建成功,观察到红色荧光;免疫荧光的亚细胞定位检测出PLC-γ1蛋白在细胞质中表达;qRT-PCR和Western blotting法均能检测出PLC-γ1表达,与对照组有显著性差异(P>0.01);免疫共沉淀也纯化分离出含有Flag标签的PLC-γ1蛋白。结论本研究成功构建绵羊PLC-γ1基因的真核表达载体,在P2A肽作用下实现目的蛋白PLC-γ1与DsRed2的表达,构建的载体可用于研究PLC-γ1在早期胚胎发育中的作用。

Objective To construct sheep PLC-γ1 with red fluorescent protein(RFP)reporter gene non-fusion eukaryotic expression vector.Method It is based on the original cloning vector of PUC57-PLC-γ1,P2A-3×Flag-PLC-γ1 was obtained by adding P2A sequence and three consecutive Flag sequences before the N-terminal of the target fragment and using HindⅢenzyme and SalⅠenzyme fragment.And then connect it to the pDsRed2-C1 vector to obtain recombinant plasmid pDsRed2-C1-P2A-3×Flag-PLC-γ1;After that,the recombinant plasmid is transfected into HEK-293T cells with Lip 2000 transfection reagent.Finally,the expression and subcellular localization of the recombinant plasmid pDsRed2-C1-P2A-3×Flag-PLC-γ1 can be identified by fluorescence microscopy,qRT-PCR,Western blot,immunoprecipitation(IP)and immunofluorescence(IF)methods.Results The results of double digestion and sequencing showed that the plasmid pDsRed2-C1-P2A-3×Flag-PLC-γ1 was successfully constructed and the red fluorescence was observed;The expression of PLC-γ1 protein can be detected of by the subcellular localization of immunofluorescence;Both the methods of qRT-PCR and Western blotting can detect PLC-γ1 expression,and there was a significant difference between the control group and the control group(P>0.01);Immunocoprecipitation also purified and isolated the PLC-γ1 protein with Flag tag.Conclusion This study successfully constructed sheep PLC-γ1 gene eukaryotic expression vector,realizing the expression of the target protein PLC-γ1 and DsRed2 under the action of P2A peptide.The constructed vector can be used to study PLC-γ1 in early embryo development.