中文摘要：

目的构建肿瘤坏死因子α诱导蛋白3相互作用蛋白1（TNIP1）真核表达载体（p EGFP-N3-TNIP1）,观察了解外源TNIP1蛋白的细胞内定位及其在He La细胞中的表达.方法应用RT-PCR从人外周血淋巴细胞中扩增TNIP1基因的开放阅读框（ORF）,将目的片段亚克隆入p EGFP-N3中,筛选阳性克隆,提取质粒测序进行鉴定.将表达质粒转染He La细胞,荧光显微镜观察融合蛋白在细胞内的表达与定位,实时荧光定量PCR（q RT-PCR）和蛋白质免疫印迹分别从m RNA水平和蛋白水平来检测TNIP1在He La细胞中的表达情况.MTT实验检测细胞增殖情况、流式细胞仪检测细胞周期.结果重组质粒双酶切鉴定,在1.9 kb左右有明显的条带,DNA测序结果证实重组质粒p EGFP-N3-TNIP1中含有的目的基因片段与Gen Bank上登录号为BC014008.1的TNIP1基因序列完全一致;荧光倒置显微镜观察重组质粒转染He La细胞后有绿色荧光蛋白的表达,转染48 h后通过q RT-PCR和Western blot分别检测到TNIP1m RNA和蛋白质的表达,均较对照明显升高,外源性的TNIP1蛋白主要定位于细胞质中.结论成功构建了p EGFP-N3-TNIP1真核表达载体,外源表达的TNIP1蛋白主要分布于He La细胞的细胞质部分,过表达TNIP1蛋白不影响He La细胞的细胞周期及细胞生长存活.

英文摘要：

Objective To construct eukaryotic expression vector pEGFP-N3-TNIP1 and to observe the expression in HeLa cells. Methods pEGFP-N3-TNIP1 vector was constructed according to the standard molecular cloning strategies. Total RNA and protein were extracted in 48 h after transfeetion to detect the HeLa expression by quantitative real-time polymerase chain reaction （qRT-PCR） and Western blotting, respectively. MTT was used to examine the growth of HeLa cells that were transformed by pEGFP-N3-TNIP1 plasmid DNA. Flow cytometry was used to analyze the cell cycle. Results The recombinant eukaryotic expression vector pEGFP-N3-TNIP1 was successfully constructed, and the gene sequencing detect result suggested the TNIP1 sequence was identical to that published in GenBank （BC014008.1） . Green fluorescence could be seen by fluorescence microscopy in 24-72 h after transfection and a peak appeared within 48 h. qRT-PCR and Western blot can find TNIP1 mRNA and protein were expressed after transfection. Over-expressing TNIP1 didn＇t directly impact HeLa cells＇ growth. Conclusion TNIP1 eukaryotic expression vector was constructed successfully. Only cytoplasmic location of exogenous TNIP1 protein had been observed in the HeLa cells after transformed by the pEGFP-N3-TNIP1. Over-expressed TNIP1 protein did not directly impact the cell growth of HeLa cells in vitro.