兔IL-10重组基因表达及其亚细胞定位的研究

Expression and Sub-cellular Localization of Rabbit IL-10 and EGFP Fusion Protein

本研究旨在克隆新西兰白兔白细胞介素10(IL-10)基因的cDNA,并探讨其生物信息学功能,进一步通过增强型绿色荧光蛋白(EGFP)融合蛋白实现该基因亚细胞水平的表达定位。实验采用RT-PCR方法克隆兔IL-10基因的cDNA,初步进行生物信息学分析,构建含有EGFP报告基因的融合表达载体pEGFP-IL-10。脂质体(LTX)介导基因转染NIH-3T3细胞,48 h后在荧光倒置显微镜下观察基因表达产物的分布,RT-PCR检测mRNA在体外水平的表达。结果表明:实验成功克隆出兔IL-10基因537 bp长的完整编码区(CDS),共编码178个氨基酸,GenBank登录号为NM_001082045;IL-10蛋白的分子量为20.15 ku,理论等电点为8.20;信号肽区域预测结果显示,IL-10蛋白含有一小段信号肽序列,属于分泌型蛋白,并且信号肽酶的切割位置在第21和第22个氨基酸之间;荧光倒置显微镜观察结果发现,融合蛋白在细胞质中表达,而转染pEGFP-C1后荧光蛋白均匀分布于整个细胞;RT-PCR检测mRNA表达明显。结果显示,融合表达载体pEGFP-IL-10构建成功,并在细胞中明显表达,为进一步研究IL-10的生物学功能奠定基础。

The aim of this study was to clone the cDNA of Interleukin 10 （IL-10） gene of New Zealand white rabbit, to explore its bioinformatics function and to analyze the sub-cellular localization of its expression products through enhanced green fluorescent protein （EGFP） fusion protein. The cDNA of rabbit IL-IO gene was cloned through RT- PCR method, the bioinformatics was preliminarily analyzed and the fusion expression vector named pEGFP-IL-10 containing EGFP was constructed, pEGFP-IL-10 was transfected into NIH-3T3 cells through Liposomes （LTX） and then observed under fluorescence inverted microscope 48 h later, mRNA expression level in vitro was detected by RT- PCR. As a result, we successfully cloned rabbit IL-10 gene. The complete CDS size was 537 bp, encoding 178 amino acids with GenBank accession number NM_001082045. The molecular weight was 20. 15 KD, and the theoretical isoelectric point of IL-10 protein was 8. 20. Prediction results of signal region showed that, IL-10 protein was a secreted protein which contained a short signal peptide sequence. Besides, the signal peptidase cleavage site was between the 21^st and 22^nd amino acids. Observation results through fluorescence inverted microscope showed that, the fusion protein was expressed in cytoplasm, while the fluorescent protein transfected with pEGFP-C1 was evenly distributed throughout the whole cell. mRNA expression level in vitro was significant. Results showed that, fusion expression vector pEGFP-IL-10 was successfully constructed and significantly expressed in cells, which provides basis for further study on the biological functions of IL-10.