人T细胞免疫球蛋白黏蛋白-3基因及其融合蛋白真核表达载体的构建和生物信息学分析

Bioinformatics analysis and construction of eukaryotic expression vector of human TIM-3 gene and its fusion protein

目的:构建T细胞免疫球蛋白黏蛋白-3基因及其融合蛋白真核表达载体,并对TIM-3基因进行生物信息学分析。方法:采用Trizol法从人外周血单个核细胞提取总mRNA,利用两步法RT-PCR技术扩增TIM-3及其胞外(结构)域基因片段和人IgG1Fc基因片段。并将他们分别克隆到真核表达载体pcDNA3.1(+)中,通过PCR及测序进行鉴定。序列分析后将TIM-3胞外(结构)域基因片段亚克隆到已经克隆了人IgG1Fc基因片段真核表达载体pcDAN3.1(+)上;并通过PCR及双酶切进行鉴定。应用生物信息学初步分析TIM-3基因的物理化学性质、蛋白质结构域、功能。结果:成功从PBMC中提取并逆转录的cDNA扩增出TIM-3及其胞外(结构)域基因和人IgG1Fc基因;经PCR、酶切鉴定、测序分析表明它们与GenBank提供的序列信息完全相同。生物信息学分析结果表明TIM-3蛋白为稳定亲水性蛋白,有1个跨膜螺旋结构,相对分子量是33.4KD,等电点pI为5.54。该蛋白含约32.56%的α-螺旋,8.27%的延伸链,49.17%的不规则卷曲,1段21个氨基酸组成的信号肽。TIM-3蛋白亚细胞定位于内质网、高尔基体、液泡、细胞膜上。功能分析预测TIM-3蛋白具有受体和信号转导功能。结论:成功构建TIM-3基因及其融合蛋白真核表达载体,并利用生物分析软件对其进行生物信息学分析。了解TIM-3基因的性质特征,为进一步研究该基因奠定基础。

Objective：To construct eukaryotic expression vector of human TIM-3 gene and its fusion protein,and conduct bioinformatics analysis of human TIM-3 gene.Method：Total RNA was extracted from human peripheral blood monouclear cells（PMBC）,and human TIM-3 and its ectodomain as well as IgG1 Fc gene fragment were amplified with two-step RT-PCR technique.They were respectively cloned into the eukaryotic expression vector pcDNA3.1（＋）,confirmed by PCR and sequencing.Then the ectodomain of human TIM-3 gene was subcloned into the eukaryote expression vector pcDNA3.1（＋）which contained the fragment of human IgG1 Fc gene after sequence analysis,and identified by PCR combined with double restriction enzyme digestion.The physicochemical characteristics,structures and functions of TIM-3 gene were predicted and analyzed with bioinformatics.Result：Evidence of DNA sequence analysis,PCR and restriction enzyme digestion showed that the fragments were the same as the sequences provided by GenBank.The human TIM-3 gene and its fusion protein constructed were successfully cloned into the eukaryotic expression vector pcDNA3.1（＋）.The results of bioinformatics analysis suggested TIM-3 protein was a stable hydrophilic protein containing one transmembrane domain,with a relative molecular weight of 33.4KD and isoelectric point of 5.54.The protein included α-helix（32.56%）,extended strand（8.27%）,random coil（49.17%）and a signal peptide of 21 amino acid residues.Its subcellular localization was in endoplasmic reticulum,vacuolar,Golgi and plasma membrane.Function analysis predicted TIM-3 would have functions in receptor and signal transduction.Conclusion：The human TIM-3 gene and its fusion protein were constructed successfully and the bioinformatics analysis was made by biological analysis software to investigate its character.It would provide foundation for further study.