人T细胞免疫球蛋白粘蛋白-4基因真核表达载体的构建和生物信息学分析

目的:构建人T细胞免疫球蛋白粘蛋白-4(TIM-4)基因的真核表达载体,用生物信息学分析了解TIM-4蛋白的特性。方法:采用Trizol法从人外周血单个核细胞提取总mRNA,逆转录合成cDNA,以此为模版,两步法RT-PCR扩增TIM-4,将其克隆至表达载体pcDNA3.1(+)中,构建FTIM-4质粒,通过PCR及测序进行鉴定。同时,我们构建pEGFP-N1-TIM-4真核表达载体,并测序鉴定其准确性;将pEGFP-N1-TIM-4质粒转染CHO细胞,用RT-PCR和荧光显微镜证实其表达。应用生物信息学初步分析TIM-4蛋白的物理化学性质、结构域和功能。结果:从逆转录的cDNA扩增出TIM-4;经PCR、酶切鉴定、测序分析表明扩增产物与GenBank提供的序列完全相同。真核表达载体pEGFP-N1-TIM-4在CHO细胞中稳定表达,表达蛋白主要位于细胞质和细胞膜上。生物信息学分析表明TIM-4蛋白为不稳定亲水性蛋白,有1个跨膜螺旋结构,含约10.32%的α-螺旋,29.89%的延伸链,59.79%的不规则卷曲,有1段由24个氨基酸组成的信号肽。亚细胞主要定位于细胞质、细胞核、分泌系统的小囊泡、线粒体、内质网、高尔基体上。功能分析预测该蛋白具有受体和信号转导功能。结论:成功构建TIM-4基因真核表达载体,利用生物信息学分析洞察了TIM-4蛋白的性质,为进一步研究该基因的免疫调节机制奠定了基础。

Construction of eukaryotic expression vector and bioinformatics analysis of human TIM-4 gene

Objective:To construct eukaryotic expression vector of human TIM-4 gene and explore the biological characteristics of human TIM-4 protein with bioinformatics tools.Methods:Total RNA was extracted from individual PBMC samples using Trizol method according to the manufacturer′s instructions.Two-step RT-PCR was employed to amplify the TIM-4 gene segments.The amplification was cloned into the eukaryotic expression vector pcDNA3.1(+),confirmed by PCR and sequencing.Meanwhile,we constructed pEGFP-N1-TIM-4 eukaryotic expression vector.The integrity of the construct was confirmed by sequencing.CHO cells were stably transfected with the pEGFP-N1-TIM-4 vector.Expression of the fusion construct was verified by RT-PCR and observed by fluorescence microscopy.The physicochemical characteristics,structures and functions of TIM-4 gene were predicted and analyzed with means of bioinformatics.Results:Evidence of DNA sequence analysis,PCR and restriction enzymes digestion showed that the fragments were the same as the sequences provided by GenBank.The human TIM-4 gene was successfully cloned into the eukaryotic expression vectors pcDNA3.1(+) and pEGFP-N1,respectively.The TIM-4-EGFP stable transfectant of CHO cell line was established.The expressed protein was mainly located in the cytomembrane and cytoplasm.The results of bioinformatics analysis suggested TIM-4 protein was a unstable hydrophilic protein,containing one transmembrane domain.The protein included α-helix(10.32%),extended strand(29.89%),random coil(59.79%)and a signal peptide of 24 amino acid residues.Its subcellular localization was mainly in cytoplasm,nucleolus,vesicles of secretory system,mitochondria,endoplasmic reticulum and golgi.Function analysis predicted TIM-4 have functions in receptor and signal transduction.Conclusion:The human TIM-4 gene is successfully constructed and the bioinformatics analysis is made by biological analysis software to investigate its character.It provides foundation for further study the immune regulation mechanism of human TIM-4 gene.