中文摘要：

目的：构建增强型绿色荧光蛋白（EGFP）与T细胞衔接活化因子（Linker for activated of T cells,LAT）融合蛋白的真核表达载体,观测LAT-EGFP在Jurkat细胞中的定位表达。方法：利用RT-PCR技术提取并扩增LAT除去终止密码子外的全部序列,克隆到真核表达载体PEGFP-N3,酶切鉴定并测序。瞬时转染到Jurkat细胞中进行表达,荧光共聚焦显微镜观察LAT-EGFP在Jurkat细胞中的表达及细胞定位。提取转染后细胞总RNA,通过RT-PCR的方法检测LAT-EGFP在转录水平的表达。利用Western blot法进一步鉴定融合蛋白的表达。结果：重组载体经酶切鉴定,切出片段长度在750 bp左右与插入序列长度相符,并进一步进行测序鉴定证实连接完全正确。共聚焦显微镜观察表达的LAT-EGFP融合蛋白定位在细胞膜上,并呈点簇状聚集状态。RT-PCR扩增证实了LAT和EGFP的融合蛋白在Jurkat细胞中在转录水平的表达,Western blot分析进一步证明了LAT和EGFP融合蛋白构建成功,并在蛋白水平上有明显的融合表达。结论：成功构建真核表达载体LAT-EGFP,且融合蛋白LAT-EGFP与野生型LAT在Jurkat细胞中的定位一致,具有功能表达效应,这为后续准确研究具有棕榈酰化位点的跨膜接头蛋白的信号转导作用提供了一种良好的研究载体和方法。

英文摘要：

Objective：To construct a eukaryotic expression plasmid for enhanced green fluorescent protein（EGFP） and LAT fusion protein,and study its localization in Jurkat cells by transient transfection.Methods： The primers were designed according to the cDNA encoding sequence of LAT core sequence excerpt the termination codon.The segments of PCR were cloned into PEGFP-N3 vector,and were identified by restricted enzymes analysis and DNA sequencing.The location of the fusion protein in Jurkat cells was monitored by confocal microscopy.The total RNA was extracted and RT-PCR was applied to identify the expression of LAT-EGFP at transcription level.Western blot was used to tested the expression of fusion protein LAT-EGFP.Results： Restricted enzymes analysis and DNA sequencing showed that the sequences of LAT-EGFP transgenic plasmid was correct.The fusion protein of LAT-EGFP was seen localizing in the membrane.The gene of LAT-EGFP was successfully cloned by RT-PCR and the expression of LAT-EGFP was tested by Western blot.Conclusion： The recombinant eukaryotic expression vector LAT-EGFP has been successfully constructed and the localization of LAT-EGFP has been seen expressing in the membrane.