人T细胞免疫球蛋白黏蛋白与凋亡细胞的相互作用研究

发布时间：2018-02-01 02:43

  本文关键词： T细胞免疫球蛋白粘蛋白-1 T细胞免疫球蛋白粘蛋白-3 RT-PCR SYBR GreenⅠ 基因表达 mRNA T细胞免疫球蛋白粘蛋白-1 T细胞免疫球蛋白粘蛋白-3 T细胞免疫球蛋白粘蛋白-4 生物信息学分析 克隆 融合蛋白 T细胞免疫球蛋白粘　出处：《华中科技大学》2011年博士论文　论文类型：学位论文

【摘要】：第一部分人TIM-1和TIM-3 mRNA实时SYBR Green I定量RT-PCR检测方法的建立 目的：建立实时SYBR Green I定量RT-PCR检测人TIM-1和TIM-3 mRNA的方法。 方法：从人外周血单个核细胞提取的总RNA中逆转录扩增人TIM-1和TIM-3的cDNA,将将纯化的人TIM-1和TIM-3的扩增产物分别与pMD18-T Simple载体进行连接,转化宿主菌DH5α,提取重组质粒DNA, PCR鉴定并测序分析。纯化质粒并检测260 nm吸光值,确定重组质粒原液的拷贝浓度并以此制备荧光定量PCR梯度浓度标准品,进行实时荧光定量PCR实验。 结果：建立了TIM-1和TIM-3基因基因mRNA表达实时荧光定量PCR检测方法,检测灵敏度达103拷贝,线性范围为103-107拷贝。阈值循环数(Ct)与PCR体系中起始模板量的对数值之间有着良好的线性关系,线性相关系数分别为1.00和1.00,扩增效率分别为1.070和1.023,批内及批间变异系数5%。熔解曲线分析表明,产物为特异的单峰。 结论：我们成功建立检测人TIM-1和TIM-3的实时荧光定量PCR方法,为进一步研究人TIM-1和TIM-3功能奠定基础。 第二部分人T细胞免疫球蛋白粘蛋白基因家族及其融合蛋白真表达载体的构建和生物信息学分析 目的：分别构建人T细胞免疫球蛋白粘蛋白基因3成员及其融合蛋白真核表达载体,并对TIM基因家族3个成员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基因家族3个成员及其胞外(结构)域基因片段和人IgG1 Fc基因片段。并将它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IgGl Fc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。并应用生物信息学初步分析TIM基因家族的物理化学性质、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基因家族3个成员及其胞外(结构)域基因和人IgG1 Fc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-1蛋白为不稳定亲水性蛋白,有1个跨膜螺旋结构,相对分子量是39.2KD,等电点pI为6.44。该蛋白含约23.08%的α-螺旋,29.67%的延伸链,47.25%的不规则卷曲,1段20个氨基酸组成的信号肽。TIM-1蛋白亚细胞定位于内质网、高尔基体、细胞膜上。功能分析预测TIM-1蛋白具有受体、信号转导、免疫应答功能。 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。 TIM-4蛋白为不稳定亲水性蛋白,有1个跨膜螺旋结构,相对分子量是41.6KD,等电点pI为5.75。该蛋白含约10.32%的α-螺旋,29.89%的延伸链,59.79%的不规则卷曲,1段24个氨基酸组成的信号肽。TIM-4蛋白亚细胞定位于细胞质、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TIM-4蛋白具有受体和信号转导功能。 结论：成功构建TIM基因家族成员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基因家族成员的性质特征,为进一步研究该基因家族奠定基础。 第三部分人T细胞免疫球蛋白黏蛋白与凋亡细胞的相互作用研究 目的：为了研究人T细胞免疫球蛋白粘蛋白基因家族与凋亡细胞之间的相互作用,以进一步探讨TIM基因家族在细胞凋亡中作用。 方法：我们构建含人TIM基因三个成员不同长度片段的9种pEGFP-N1真核表达载体及其Ig融合蛋白表达载体。将这些构建的真核表达载体瞬时转染CHO细胞,并收集上清液。直接采用TIM-EGFP融合蛋白与PI为探针,检测TIM蛋白与凋亡细胞的相互作用。 结果：人TIM基因家族的3个蛋白都能直接识别和结合凋亡细胞,而与活细胞不能结合。并且,sTIM-1-EGFP、sTIM-3-EGFP和sTIM-4-EGFP融合蛋白与凋亡细胞之间的相互作用被相应的TIM-1-Ig、TIM-3-Ig和TIM-4-Ig所阻止。而且,结果表明人TIM基因家族的3个蛋白通过其IgV区直接识别和结合凋亡细胞。 结论：人TIM基因家族的3个蛋白充当凋亡细胞的受体,可能在细胞凋亡调控中起重要作用。人TIM蛋白可以作为新的蛋白用于细胞凋亡的检测。 第四部分人TIM-EGFP融合蛋白的在大肠杆菌中的表达和特征的研究 目的：为了探讨人TIM-EGFP融合蛋白在大肠杆菌中的表达和纯化,并评价它们的生物活性。 方法：采用PCR分别扩增人TIM基因家族3成员和EGFP片段,并且将它们克隆至原核表达载体pET-28a中。构建的重组质粒pET-28a-TIM-EGFP分别转化大肠杆菌BL21(DE3),经IPTG诱导表达TIM-EGFP融合蛋白。表达的融合蛋白经Ni-NTA树脂纯化,并且通过荧光显微镜检测它们与凋亡细胞的结合活性。 结果：我们分别成功构建人TIM-1-EGFP、TIM-3-EGFP和TIM-4-EGFP的融合蛋白表达载体,并在大肠杆菌中表达。我们结果证明TIM-EGFP融合蛋白3个成员都能直接识别和结合凋亡细胞,而与活细胞不能。我们更进一步证实TIM-4-EGFP与凋亡细胞的相互作用可以被相应的TIM-Ig融合蛋白阻止。 结论：我们分别成功构建人TIM-1-EGFP、TIM-3-EGFP和TIM-4-EGFP的融合蛋白表达载体,并在大肠杆菌中表达。据我们所知,这是目前首次在大肠杆菌中表达TIM基因家族的3个成员。我们结果也表明人TIM基因家族介导与凋亡细胞的识别和结合。而且,纯化的融合蛋白可以作为准备好的生物活性的TIM-1、TIM-3和TIM-4来源,这为进一步研究人TIM-1、TIM-3和TIM-4基因和它们的相应受体功能和调节机制奠定基础。
[Abstract]:The first part of human TIM-1 and TIM-3 mRNA real-time SYBR Green I quantitative RT-PCR detection method
Objective: to establish a real-time SYBR Green I quantitative RT-PCR method for the detection of human TIM-1 and TIM-3 mRNA.
Methods: the reverse transcription of total RNA mononuclear cells extracted from human TIM-1 was amplified and TIM-3 cDNA of peripheral blood will be amplified and purified TIM-1 and TIM-3 respectively with pMD18-T Simple vector and transformed into E. coli DH5 alpha, extraction of recombinant plasmid DNA PCR and sequenced. Purification of plasmid and detection the 260 nm absorption value, determine the concentration of recombinant plasmid solution and to prepare fluorescent quantitative PCR concentration gradient standard, real-time fluorescence quantitative PCR experiments.
Results: the mRNA gene TIM-1 and TIM-3 gene expression detection method of real-time fluorescence quantitative PCR detection sensitivity was 103 copies, 103-107 copies. The linear range of threshold cycle number (Ct) and template PCR system volume on the value had good linear relationship between the linear correlation coefficients were 1 and 1, amplification the efficiency were 1.070 and 1.023, the intra batch and inter batch coefficient of variation of 5%. melting curve analysis showed that the product was a single peak.
Conclusion: we have successfully established a real-time fluorescence quantitative PCR method for detecting human TIM-1 and TIM-3, which lays the foundation for further study of the function of human TIM-1 and TIM-3.
Construction and bioinformatics analysis of the second human T cell immunoglobulin gene family and the true expression vector of the fusion protein
Objective: to construct human T cell immunoglobulin 3 gene and its fusion protein eukaryotic expression vector respectively, and bioinformatics analysis of 3 members of TIM gene family.
Methods: Trizol method was used to extract total mRNA from peripheral blood mononuclear cells, using two step RT-PCR amplification of TIM gene family and 3 members of the extracellular domain (structure) gene fragment and IgG1 gene fragment of Fc. And they were cloned into eukaryotic expression vector pcDNA3.1 (+), and were identified by PCR sequencing and sequence analysis. The TIM-3 extracellular domain (structure) gene fragment was subcloned into has cloned the human IgGl gene fragment of Fc eukaryotic expression vector pcDAN3.1 (+); and were identified by PCR and enzyme digestion. And the application of bioinformatics analysis of physical and chemical properties of TIM protein gene family. Domain function.
Results: the successful extraction from PBMC and reverse transcription cDNA amplified TIM gene family and 3 members of the extracellular domain (structure) gene and IgG1 Fc gene; by PCR, enzyme digestion and sequencing analysis showed that the sequence information provided by GenBank and they are exactly the same. The bioinformatics analysis results show that the TIM-1 protein is not stable hydrophilic protein with 1 transmembrane helix structure, relative molecular weight is 39.2KD and isoelectric point pI is the 6.44. protein containing 23.08% alpha helix, extended chain 29.67%, 47.25% random coil, the signal peptide of.TIM-1 protein sub cellular localization of the 1 segment of 20 amino acids in the endoplasmic reticulum, Golgi apparatus, cell membrane. Functional analysis of the predicted TIM-1 protein has receptors, signal transduction, immune response function.
TIM-3 protein is a stable hydrophilic protein with 1 transmembrane helix structure, relative molecular weight is 33.4KD and isoelectric point pI is the 5.54. protein containing 32.56% alpha helix, extended chain 8.27%, 49.17% random coil, the signal peptide of.TIM-3 protein subcellular 1 21 amino acids located in the endoplasmic reticulum net, Golgi, vacuole, cell membrane. Functional analysis of prediction of TIM-3 protein has the function of receptors and signal transduction.
TIM-4 protein is a unstable hydrophilic protein with 1 transmembrane helix structure, relative molecular weight is 41.6KD and isoelectric point pI is the 5.75. protein containing 10.32% alpha helix, extended chain 29.89%, 59.79% random coil, 1 24 amino acid signal peptide.TIM-4 protein sub cellular located in the cytoplasm, nucleus, vesicle secretion system of vesicles, mitochondria, endoplasmic reticulum, Golgi. Function analysis and prediction of TIM-4 protein has the function of receptors and signal transduction.
Conclusion: the TIM gene family members and their fusion protein eukaryotic expression vectors were successfully constructed, and bioinformatics analysis was performed by bioanalysis software. We could understand the characteristics and characteristics of TIM gene family members, and lay the foundation for further study of the gene family.
Study on the interaction of immunoglobulin mucin and apoptotic cells in the third part of human T cells
Objective: To investigate the interaction between human T cell immunoglobulin and mucin gene family and apoptotic cells, so as to further explore the role of TIM gene family in cell apoptosis.
Methods: 9 we construct eukaryotic pEGFP-N1 containing human TIM gene three members of different length fragment expression vector and Ig fusion protein expression vector. The constructed eukaryotic expression vector was transfected into CHO cells, and the supernatants were collected. Using TIM-EGFP fusion protein and PI as probe, the interaction of TIM protein and apoptosis detection cells.
Results: 3 protein TIM gene family can direct the recognition and binding of apoptotic cells, and combined with living cells. And, sTIM-1-EGFP, sTIM-3-EGFP and sTIM-4-EGFP fusion protein and the interaction between cell apoptosis by the corresponding TIM-1-Ig, TIM-3-Ig and TIM-4-Ig stop. Moreover, the results show that the 3 protein TIM gene the family through its IgV direct recognition and binding of apoptotic cells.
Conclusion: the 3 proteins of human TIM gene family play an important role in the regulation of apoptosis, which serve as the receptors of apoptotic cells. Human TIM protein can be used as a new protein for the detection of apoptosis.
Study on the expression and characteristics of the fourth part of human TIM-EGFP fusion protein in Escherichia coli
Objective: To investigate the expression and purification of human TIM-EGFP fusion protein in Escherichia coli and evaluate their bioactivity.
Methods: using PCR amplification of human TIM gene family members and 3 EGFP fragments, and then they will be cloned into the prokaryotic expression vector pET-28a. The recombinant plasmid pET-28a-TIM-EGFP was transformed into Escherichia coli BL21 (DE3), the expression of TIM-EGFP fusion protein was induced by IPTG. The expression of the fusion protein purified by Ni-NTA resin, and by fluorescence microscopy detection of their binding activity and apoptosis of cells.
Results: we successfully constructed TIM-1-EGFP fusion protein expression vector TIM-3-EGFP and TIM-4-EGFP, and its expression in Escherichia coli. Our results demonstrated that TIM-EGFP fusion protein 3 members can direct recognition and binding of apoptotic cells, but not with living cells. We further confirmed the interaction between TIM-4-EGFP cells and apoptosis can be the corresponding TIM-Ig fusion protein prevents.
Conclusion: we successfully constructed TIM-1-EGFP fusion protein expression vector TIM-3-EGFP and TIM-4-EGFP, and its expression in Escherichia coli. To our knowledge, this is the first expression of 3 members of TIM gene family in Escherichia coli. Our results also show that the recognition and binding of human TIM gene family mediated apoptosis and. Moreover, the purified fusion protein can be used as ready for the biological activity of TIM-1, TIM-3 and TIM-4 of the source, for the further study of human TIM-1, TIM-3 foundation and TIM-4 genes and their corresponding receptor function and regulation mechanism.

【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R392

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