本文關(guān)鍵詞： T細(xì)胞免疫球蛋白粘蛋白-1 T細(xì)胞免疫球蛋白粘蛋白-3 RT-PCR SYBR GreenⅠ 基因表達(dá) mRNA T細(xì)胞免疫球蛋白粘蛋白-1 T細(xì)胞免疫球蛋白粘蛋白-3 T細(xì)胞免疫球蛋白粘蛋白-4 生物信息學(xué)分析 克隆 融合蛋白 T細(xì)胞免疫球蛋白粘　出處：《華中科技大學(xué)》2011年博士論文　論文類型：學(xué)位論文

【摘要】：第一部分人TIM-1和TIM-3 mRNA實時SYBR Green I定量RT-PCR檢測方法的建立 目的：建立實時SYBR Green I定量RT-PCR檢測人TIM-1和TIM-3 mRNA的方法。 方法：從人外周血單個核細(xì)胞提取的總RNA中逆轉(zhuǎn)錄擴(kuò)增人TIM-1和TIM-3的cDNA,將將純化的人TIM-1和TIM-3的擴(kuò)增產(chǎn)物分別與pMD18-T Simple載體進(jìn)行連接,轉(zhuǎn)化宿主菌DH5α,提取重組質(zhì)粒DNA, PCR鑒定并測序分析。純化質(zhì)粒并檢測260 nm吸光值,確定重組質(zhì)粒原液的拷貝濃度并以此制備熒光定量PCR梯度濃度標(biāo)準(zhǔn)品,進(jìn)行實時熒光定量PCR實驗。 結(jié)果：建立了TIM-1和TIM-3基因基因mRNA表達(dá)實時熒光定量PCR檢測方法,檢測靈敏度達(dá)103拷貝,線性范圍為103-107拷貝。閾值循環(huán)數(shù)(Ct)與PCR體系中起始模板量的對數(shù)值之間有著良好的線性關(guān)系,線性相關(guān)系數(shù)分別為1.00和1.00,擴(kuò)增效率分別為1.070和1.023,批內(nèi)及批間變異系數(shù)5%。熔解曲線分析表明,產(chǎn)物為特異的單峰。 結(jié)論：我們成功建立檢測人TIM-1和TIM-3的實時熒光定量PCR方法,為進(jìn)一步研究人TIM-1和TIM-3功能奠定基礎(chǔ)。 第二部分人T細(xì)胞免疫球蛋白粘蛋白基因家族及其融合蛋白真表達(dá)載體的構(gòu)建和生物信息學(xué)分析 目的：分別構(gòu)建人T細(xì)胞免疫球蛋白粘蛋白基因3成員及其融合蛋白真核表達(dá)載體,并對TIM基因家族3個成員進(jìn)行生物信息學(xué)分析。 方法：采用Trizol法從人外周血單個核細(xì)胞提取總mRNA,利用兩步法RT-PCR技術(shù)擴(kuò)增TIM基因家族3個成員及其胞外(結(jié)構(gòu))域基因片段和人IgG1 Fc基因片段。并將它們分別克隆到真核表達(dá)載體pcDNA3.1 (+)中,通過PCR及測序進(jìn)行鑒定。序列分析后將TIM-3胞外(結(jié)構(gòu))域基因片段亞克隆到已經(jīng)克隆了人IgGl Fc基因片段真核表達(dá)載體pcDAN3.1(+)上；并通過PCR及雙酶切進(jìn)行鑒定。并應(yīng)用生物信息學(xué)初步分析TIM基因家族的物理化學(xué)性質(zhì)、蛋白質(zhì)結(jié)構(gòu)域、功能。 結(jié)果：成功從PBMC中提取并逆轉(zhuǎn)錄的cDNA擴(kuò)增出TIM基因家族3個成員及其胞外(結(jié)構(gòu))域基因和人IgG1 Fc基因；經(jīng)PCR、酶切鑒定、測序分析表明它們與GenBank提供的序列信息完全相同。生物信息學(xué)分析結(jié)果表明TIM-1蛋白為不穩(wěn)定親水性蛋白,有1個跨膜螺旋結(jié)構(gòu),相對分子量是39.2KD,等電點pI為6.44。該蛋白含約23.08%的α-螺旋,29.67%的延伸鏈,47.25%的不規(guī)則卷曲,1段20個氨基酸組成的信號肽。TIM-1蛋白亞細(xì)胞定位于內(nèi)質(zhì)網(wǎng)、高爾基體、細(xì)胞膜上。功能分析預(yù)測TIM-1蛋白具有受體、信號轉(zhuǎn)導(dǎo)、免疫應(yīng)答功能。 TIM-3蛋白為穩(wěn)定親水性蛋白,有1個跨膜螺旋結(jié)構(gòu),相對分子量是33.4KD,等電點pI為5.54。該蛋白含約32.56%的α-螺旋,8.27%的延伸鏈,49.17%的不規(guī)則卷曲,1段21個氨基酸組成的信號肽。TIM-3蛋白亞細(xì)胞定位于內(nèi)質(zhì)網(wǎng)、高爾基體、液泡、細(xì)胞膜上。功能分析預(yù)測TIM-3蛋白具有受體和信號轉(zhuǎn)導(dǎo)功能。 TIM-4蛋白為不穩(wěn)定親水性蛋白,有1個跨膜螺旋結(jié)構(gòu),相對分子量是41.6KD,等電點pI為5.75。該蛋白含約10.32%的α-螺旋,29.89%的延伸鏈,59.79%的不規(guī)則卷曲,1段24個氨基酸組成的信號肽。TIM-4蛋白亞細(xì)胞定位于細(xì)胞質(zhì)、細(xì)胞核、分泌系統(tǒng)的小囊泡、線粒體、內(nèi)質(zhì)網(wǎng)、高爾基體上。功能分析預(yù)測TIM-4蛋白具有受體和信號轉(zhuǎn)導(dǎo)功能。 結(jié)論：成功構(gòu)建TIM基因家族成員及其融合蛋白真核表達(dá)載體,并利用生物分析軟件對其進(jìn)行生物信息學(xué)分析。了解TIM基因家族成員的性質(zhì)特征,為進(jìn)一步研究該基因家族奠定基礎(chǔ)。 第三部分人T細(xì)胞免疫球蛋白黏蛋白與凋亡細(xì)胞的相互作用研究 目的：為了研究人T細(xì)胞免疫球蛋白粘蛋白基因家族與凋亡細(xì)胞之間的相互作用,以進(jìn)一步探討TIM基因家族在細(xì)胞凋亡中作用。 方法：我們構(gòu)建含人TIM基因三個成員不同長度片段的9種pEGFP-N1真核表達(dá)載體及其Ig融合蛋白表達(dá)載體。將這些構(gòu)建的真核表達(dá)載體瞬時轉(zhuǎn)染CHO細(xì)胞,并收集上清液。直接采用TIM-EGFP融合蛋白與PI為探針,檢測TIM蛋白與凋亡細(xì)胞的相互作用。 結(jié)果：人TIM基因家族的3個蛋白都能直接識別和結(jié)合凋亡細(xì)胞,而與活細(xì)胞不能結(jié)合。并且,sTIM-1-EGFP、sTIM-3-EGFP和sTIM-4-EGFP融合蛋白與凋亡細(xì)胞之間的相互作用被相應(yīng)的TIM-1-Ig、TIM-3-Ig和TIM-4-Ig所阻止。而且,結(jié)果表明人TIM基因家族的3個蛋白通過其IgV區(qū)直接識別和結(jié)合凋亡細(xì)胞。 結(jié)論：人TIM基因家族的3個蛋白充當(dāng)?shù)蛲黾?xì)胞的受體,可能在細(xì)胞凋亡調(diào)控中起重要作用。人TIM蛋白可以作為新的蛋白用于細(xì)胞凋亡的檢測。 第四部分人TIM-EGFP融合蛋白的在大腸桿菌中的表達(dá)和特征的研究 目的：為了探討人TIM-EGFP融合蛋白在大腸桿菌中的表達(dá)和純化,并評價它們的生物活性。 方法：采用PCR分別擴(kuò)增人TIM基因家族3成員和EGFP片段,并且將它們克隆至原核表達(dá)載體pET-28a中。構(gòu)建的重組質(zhì)粒pET-28a-TIM-EGFP分別轉(zhuǎn)化大腸桿菌BL21(DE3),經(jīng)IPTG誘導(dǎo)表達(dá)TIM-EGFP融合蛋白。表達(dá)的融合蛋白經(jīng)Ni-NTA樹脂純化,并且通過熒光顯微鏡檢測它們與凋亡細(xì)胞的結(jié)合活性。 結(jié)果：我們分別成功構(gòu)建人TIM-1-EGFP、TIM-3-EGFP和TIM-4-EGFP的融合蛋白表達(dá)載體,并在大腸桿菌中表達(dá)。我們結(jié)果證明TIM-EGFP融合蛋白3個成員都能直接識別和結(jié)合凋亡細(xì)胞,而與活細(xì)胞不能。我們更進(jìn)一步證實TIM-4-EGFP與凋亡細(xì)胞的相互作用可以被相應(yīng)的TIM-Ig融合蛋白阻止。 結(jié)論：我們分別成功構(gòu)建人TIM-1-EGFP、TIM-3-EGFP和TIM-4-EGFP的融合蛋白表達(dá)載體,并在大腸桿菌中表達(dá)。據(jù)我們所知,這是目前首次在大腸桿菌中表達(dá)TIM基因家族的3個成員。我們結(jié)果也表明人TIM基因家族介導(dǎo)與凋亡細(xì)胞的識別和結(jié)合。而且,純化的融合蛋白可以作為準(zhǔn)備好的生物活性的TIM-1、TIM-3和TIM-4來源,這為進(jìn)一步研究人TIM-1、TIM-3和TIM-4基因和它們的相應(yīng)受體功能和調(diào)節(jié)機(jī)制奠定基礎(chǔ)。
[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.

【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2011
【分類號】：R392

【共引文獻(xiàn)】

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2 于蕾;野西瓜極性生物堿A10組分誘導(dǎo)SGC-7901細(xì)胞凋亡線粒體通路的研究[D];北京中醫(yī)藥大學(xué);2010年

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4 佟敬山;ER-α 36在子宮內(nèi)膜癌中的功能及Icaritin誘導(dǎo)的細(xì)胞凋亡的研究[D];吉林大學(xué);2011年

5 吳松一;納米酞菁光敏劑光動力治療脈絡(luò)膜新生血管的實驗研究[D];福建醫(yī)科大學(xué);2011年

6 李文先;TIM-1和TIM-3血清水平和基因多態(tài)性與系統(tǒng)性紅斑狼瘡的相關(guān)性研究[D];安徽醫(yī)科大學(xué);2011年

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