【摘要】 目的 运用激光共聚焦显微技术对CDK5RAP3与LHBs进行亚细胞定位研究,为进一步研究HBV影响糖、脂类代谢的分子生物学机制提供一定的思路和方向。方法应用酵母双杂交系统筛选人胰腺cDNA文库中的LHBs结合蛋白基因,构建真核表达质粒pDsRed1-N1-CDK5RAP3,应用激光共聚焦显微技术对CDK5RAP3与LHBs进行亚细胞定位研究。结果成功构建真核表达质粒pDsRed1-N1-CDK5RAP3,转染了pDsRed1-N1-CDK5RAP3质粒的细胞,红色荧光信号较集中分布于细胞浆中,与经DAPI染色的细胞核无重叠。结论证实LHBs基因和CDK5RAP3基因在体内可以表达蛋白,并提示LHBs蛋白和CDK5RAP3蛋白均主要定位在细胞浆内。更多还原

【Abstract】 Objective To screen proteins of human pancreas cDNA library interacting with HBV large protein(LHBs) and observe the location by confocal fluorescence microscope,which brought some new clues for studying the molecular biology mechanism of glucose and lipid.Methods We transformed yeast cell expression vector pGBKT7-core into yeast strain AH109 and performed yeast two-hybrid by mating AH109 containing LHBs gene with Y187 containing plasmids of human pancreas cDNA library and screened of proteins binding to LHBs protein from human pancreas cDNA library.Homo sapiens CDK5RAP3 gene was amplified with reverse transcription polymerase chain reaction(RT-PCR).Constructed eukaryotic expression vector pDsRed1-N1-CDK5RAP3 and transfected into Capan cell lines by using lipofectamine,then observe the location by confocal fluorescence microscope.Results We constructed eukaryotic expression vector pDsRed1-N1-CDK5RAP3.To investigate the subcellular localization of LHBs and CDK5RAP3,the plasmids expressing CDK5RAP3 fusion protein with pEGFP-C1 and pDsRed1-N1 were constructed and confirmed by DNA sequencing analysis and restriction enzyme digestion.After 48 hours,the expression was detected by confocal fluorescence microscope.Conclusion LHBs and Homo Sapiens CDK5RAP3 protein can express in cytoplasm.更多还原