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  

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

作　　者：陈治中[1,2] 王琳[1] 王晓蓓[1] 毛晓露[1] 陈凤花[1] 刘峰[1] 胡丽华[1] 

机构地区：[1]华中科技大学同济医学院附属协和医院检验科,武汉430022 [2]广西壮族自治区人民医院检验科,南宁530021

出　　处：《中国免疫学杂志》2012年第2期104-108,113,共6页Chinese Journal of Immunology

基　　金：国家自然科学基金资助项目(30672008)

摘　　要：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。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 regulat

关 键 词：T细胞免疫球蛋白粘蛋白-4基因 生物信息学分析 克隆 

分 类 号：R392.11[医药卫生—免疫学]