HCBP6模拟磷酸化重组质粒的构建与亚细胞定位

目的 构建HCBP6及HCBP6不同位点模拟磷酸化的绿色荧光蛋白重组质粒，明确其亚细胞定位。方法 以构建含有570 bp的HCBP6外显子基因的绿色荧光蛋白重组质粒为基础，应用快速定点突变技术构建HCBP6不同位点模拟磷酸化的重组质粒。将重组质粒测序并进行序列比对。利用jetPRIME转染体系将HCBP6及HCBP6不同位点模拟磷酸化的重组质粒转染至细胞中，应用Real-Time PCR技术检测目的基因mRNA的表达，应用Western blot技术检测目的基因蛋白表达，应用荧光显微镜观察HCBP6不同位点磷酸化质粒的亚细胞定位。结果 将测序的重组序列与目的基因进行比对，发现重组序列与目的基因完全一致，说明HCBP6不同位点模拟磷酸化的重组质粒构建成功。RealTime PCR和Western blot表明，与对照组相比，实验组高表达HCBP6及HCBP6不同位点模拟磷酸化的基因。免疫荧光显微镜可见10Ala主要在细胞质表达，WT、10Asp、151Ala、151Asp主要在细胞核表达。结论 构建的HCBP6及HCBP6不同位点模拟磷酸化的重组质粒可成功转染细胞并在细胞中高表达，HCB...

Construction and subcellular location of HCBP6 mimic phosphorylated recombinant plasmid

Objective To construct green fluorescent protein recombinant plasmids of HCBP6 and clarify its subcellular localization.Methods Based on the green fluorescent protein HCBP6 recombinant plasmid which contained 570 bp HCBP6 exon gene,rapid site-directed mutagenesis was used to construct HCBP6 mimics phosphorylation at different sites of recombinant plasmids.The recombinant plasmids were sequenced and aligned.JetPRIME transfection system was used to transfect HCBP6 and HCBP6 mimic phosphorylated recombinant plasmids into cells.Real-Time PCR technology was used to detect the expression of target gene mRNA.Western blot was used to detect the expression of target gene protein,and fluorescence microscopy was used to observe the subcellular localization of HCBP6 mimic phosphorylated plasmids at different sites.Results It was found that the recombination sequence was completely consistent with the target gene,indicating that the HCBP6 mimic phosphorylated recombinant plasmid was successfully constructed.Real-time PCR and Western blot showed that compared with the control group,the experimental group highly expressed HCBP6 and HCBP6 mimic phosphorylation genes at different sites.Immunofluorescence microscopy showed that 10Ala was mainly expressed in the cytoplasm,and WT,10Asp,151Ala,and 151Asp were mainly expressed in the nucleus.Conclusions HCBP6 mimic phosphorylation recombinant plasmids at different sites and HCBP6 can be successfully transfected into cells and highly expressed.The subcellular localization of HCBP6 mimic phosphorylation recombinant plasmids at different sites were different.