本文旨在探讨TRIM22C端SRPY结构域对其转录、翻译及亚细胞定位的影响。以野生型TRIM22真核表达质粒为模板,扩增出C端SPRY结构域缺失的TRIM22基因片段,并将其克隆入真核表达质粒pcDNA3.1。将野生型和突变型TRIM22真核表达载体分别转染高分化人肝癌细胞株HepG2。通过反转录-聚合酶链反应检测其mRNA表达水平,蛋白质免疫印迹法(Western blot)检测其蛋白表达水平,并通过间接免疫荧光法检测其亚细胞内定位。结果成功构建了C端SPRY结构域缺失的TRIM22真核表达质粒。转染HepG2细胞后,TRIM22SPRY结构域缺失突变体在转录和翻译水平上均与野生型TRIM22无明显差异,但TRIM22SPRY结构域缺失突变体完全丧失了核定位能力,这为进一步研究SPRY结构域在TRIM22抗病毒过程中所发挥的作用及机制提供了有益的启示。

In oeder to investigate the effects of C terminal SPRY domain of TRIM22 on its transcription, translation and subcellular localization, SPRY domain was deleted (ΔSPRY) and amplified through polymerase chain reaction (PCR) and was then inserted into the eukaryotic expression plasmid, pcDNA3.1. Plasmids expressing wild type TRIM22 or TRIM22-ΔSPRY were transfected into HepG2 cells. The mRNA expression level was determined by semiquantitative reverse transcriptase PCR (RT-PCR), the protein expression level was determined by Western blot analysis, and the subcellular localization was determined by immunofluorescence staining. The results showed that plasmid expressing TRIM22-ΔSPRY was successfully constructed. After transfection into HepG2 cells, the TRIM22-ΔSPRY showed no difference from the wild type TRIM22 at both mRNA and protein levels. However TRIM22-ΔSPRY was localized exclusively to the cytoplasm of HepG2 cells in contrast to the nuclear localization of wild type TRIM22. These results may be useful for studying the role of SPRY domain in TRIM22-mediated antiviral activities.