天使综合征致病基因UBE3a在大鼠神经元细胞中的克隆和表达检测

目的:调取并构建含有人UBE3a基因全长cDNA的真核表达载体pEGFP-UBE3a,采用磷酸钙转染方法将构建的重组质粒在神经元细胞中表达并观察EGFP-UBE3a蛋白的亚细胞定位。方法:采用PCR技术从人胎脑组织cDNA文库中扩增UBE3a基因的ORF片段并进行酶切构建pEGFP-UBE3a的真核表达重组质粒。用磷酸钙转染方法将重组质粒转染入体外培养的神经元细胞,分别在体外培养8天和14天时通过倒置荧光显微镜观察重组蛋白的表达和定位情况。结果:PCR扩增获得人胎脑组织cDNA文库中UBE3a cDNA全长2559 bp,构建真核表达重组质粒pEGFP-UBE3a成功,经DNA测序与Gen-Bank中的人UBE3a cDNA序列一致。经倒置荧光显微镜观察重组质粒的表达情况,结果显示转染重组质粒的细胞中有高水平的EGFP-UBE3a蛋白表达,且UBE3a在早期神经元的细胞质和细胞核中广泛表达,并弥散在轴突和树突,而在成熟神经元细胞核中的表达明显富集。结论:本实验成功构建了pEGFP-UBE3a真核表达质粒,EGFP-UBE3a蛋白在体外培养的神经元细胞中能够有效表达,且随着神经元的成熟逐步向核内富集,为进一步研究UBE3a基因在天使综合征发生发展中的作用奠定了基础。

The construction and expression of Angelman syndrome related gene UBE3a in rat neuronal cells

Objective:To construct the recombinant eukaryotic plasmids which expressing human UBE3a gene and then detect the expression and location of EGFP-UBE3a protein in cultured neurons in vitro.Methods:The ORF fragment of UBE3a gene was amplified from human embryonic brain cDNA library and then cloned into pEGFP-N1 vector.The recombinant pEGFP-UBE3a plasmids were then transfected into the cultured neurons by calcium phosphate methods.The expression and location of EGFP-UBE3a protein were examined under microscope at DIV(Days in vitro) 8 and 14.Results:The PCR products of UBE3a ORF fragment was 2 559 bp and the sequencing data of pEGFP-UBE3a plasmid was identical to NCBI database.EGFP-UBE3a protein was expressed widely over the cytoplasm in young neurons including axon and dendrites but then focused into nucleus in mature neurons.Conclusion:The pEGFP-UBE3a recombinant plasmids were constructed successfully and expressed well in cultured neurons in vitro.The UBE3a proteins focused into nucleus in mature neurons,which laid the foundation for the functional study of UBE3a gene in human Angelman syndrome.