人Tim-3启动子的克隆及其逆转录病毒表达载体的构建
发布时间：2018-09-01 07:11
【摘要】： Tim-3(T-cell immunoglobulin-and mucin-domain-containing molecule-3,T细胞免疫球蛋白粘蛋白分子-3)是新近发现的免疫调节分子,特异表达于分化的Th1细胞,参与Th1负调控和免疫耐受形成,在多种免疫性疾病发生中发挥重要作用,阻断其表达可缓解Th2应答引起的过敏性疾病,是多种免疫性疾病的潜在治疗靶标,引起国内外学者广泛关注。但Tim-3研究尚存在诸多问题未能解决,尤其是其表达调控机制研究尚属空白。本研究分三部分内容:初步研究正常人PBMC Tim-3的表达模式;克隆具有特异启动子活性的基因片段,构建人Tim-3启动子报告质粒;构建人Tim-3逆转录病毒表达载体,为进一步研究Tim-3表达调控机制及探索利用Tim-3进行疾病治疗的可行性奠定基础。 第一部分正常人PBMC Tim-3表达模式的初步研究 研究目的 检测经丝裂原刺激的人外周血淋巴细胞Tim-3的表达情况,为进一步研究Tim-3表达调控机制提供实验模型。 研究方法 分离人PBMC,贴壁法去除单核细胞后,分别以PHA、LPS刺激悬浮的淋巴细胞,24h后抽提细胞总RNA,RT-PCR检测Tim-3的表达。未刺激淋巴细胞作对照。 研究结果 RT-PCR检测显示PHA刺激的人外周血淋巴细胞Tim-3表达上调,而LPS刺激的不表达Tim-3,提示处于活化、非极化的T细胞就开始表达该分子。 结论及意义 本部分建立了可供作为研究Tim-3表达调控的细胞模型,为进一步深入研究Tim-3表达调控机制提供实验基础。 第二部分人Tim-3启动子的克隆及其活性的初步研究 研究目的 构建人Tim-3启动子萤火虫荧光素酶报告质粒并分析其活性,为进一步研究Tim-3特异表达于Th1细胞的分子机制奠定基础。 研究方法 1人Tim-3启动子区的预测 利用在线MatInspector软件对人Tim-3基因5′端-2500～+202段进行启动子区预测。 2 Tim-3P1与Tim-3P2 PCR扩增 选定长2325bp的-2083～+242段,并依据该段单一SacⅠ酶切位点,分Tim-3P1(-2083～-838)和Tim-3P2(-948～+242)两部分克隆。设计针对Tim-3P1和Tim-3P2的特异性引物,以人基因组DNA为模板进行PCR扩增。 3含不同长度人Tim-3基因5′端片段报告质粒的构建及鉴定 Tim-3P1 PCR产物,T-A克隆入pGEM-T,测序正确后,亚克隆入pGL3-Basic相应酶切位点构建pGL3-Tim-3P1。Tim-3P2 PCR产物,经SacⅠ、XhoⅠ双酶切、回收后,插入pGL3-Basic多克隆位点(MCS)构建pGL3-Tim-3P2。亦经测序鉴定。为增强所构建报告质粒的启动子活性,设计在上述阳性pGL3-Tim-3P2中引入SV40增强子序列构建Enhancer-Tim-3P2,经双酶切鉴定。 4 RT-PCR检测细胞系内源性Tim-3的表达 收集对数生长期RAW264.7、B16及COS-75×10~5,Trizol法抽提细胞总RNA并行RT-PCR检测。 5报告质粒的转染 将上述构建的报告质粒及pGL3-Basic(阴性对照)分别与pRL-TK(内参照)瞬时共转染RAW264.7、B16和COS-7(详见附图表第二部分),48h收集细胞,对细胞粗提液进行双荧光素酶分析。 6双荧光素酶分析 按双荧光素酶报告基因检测试剂盒说明书将细胞裂解后与底物混合,用荧光计数仪检测所构建报告质粒中萤火虫荧光素酶活性M1与pRL-TK中海肾荧光素酶活性M2,M1/M2的比值即为目的片段的相对活性。每种质粒设2复孔,同一转染实验至少重复3次。 7 Tim-3P1与Tim-3P2转录因子结合位点(TFBSs)分析 同样利用在线MatInspector软件分析Tim-3P1与Tim-3P2中可能存在的TFBSs。 研究结果 1人Tim-3启动子区的预测 分析显示-2500～+202段存在多个TATA box、CAAT box及多个重要TFBSs,可能具有启动子活性。 2成功构建人Tim-3启动子萤火虫荧光素酶报告质粒 测序鉴定证实pGL3-Tim-3P1、pGL3-Tim-3P2构建成功;酶切鉴定证实Enhancer-Tim-3P2构建成功。 3不同细胞系内源性Tim-3的表达 RT-PCR检测显示RAW264.7和B16表达内源性Tim-3,而COS-7不表达。 4报告质粒启动子活性的验证 双荧光素酶分析证实pGL3-Tim-3P1与pGL3-Tim-3P2均具有较弱的启动子活性,其中,pGL3-Tim-3P2相对活性约是pGL3-Basic空载体的2倍,且启动子活性具有细胞特异性;引入增强子的Enhancer-Tim-3P2启动子活性明显升高。 5 TFBSs分析 结果显示Tim-3P1含有下列TFBSs:①1个c-Myb、Ik-1、YY1(Yin and Yang 1,阴阳1)、GATA-3(GATA-binding factor-3)和CHOP;②2个FOXK2、HNF-3(hepatocyte nuclear factor-3,肝细胞核因子-3);③6个TATA box;Tim-3P2含有下列TFBSs:①1个RFX1、p53、GATA-3、NFAT(Nuclear factor of activated T cells,活化T细胞核因子)和CDE/CHR(cell cycle-dependentelement,细胞周期依赖性元件;cell cycle genes homology region,细胞周期基因同源区);②2个MZF1(Myeloid zinc finger protein-1);③3个YY1;④5个TATA box。 结论及意义 本部分成功构建了含人Tim-3启动子的萤火虫荧光素酶报告质粒和带有增强子序列的报告质粒,为进一步研究Tim-3表达调控机制奠定了基础。鉴于Tim-3在免疫应答中的重要作用,深入研究其表达调控将具有重要的理论意义和良好的应用前景。 第三部分人Tim-3逆转录病毒表达载体的构建 研究目的 构建人Tim-3逆转录病毒表达载体,为进一步研究Tim-3表达调控机制、探索利用Tim-3治疗疾病的可行性奠定基础。 研究方法 设计针对人Tim-3 mRNA的特异性引物,以hTim-3-pGEM-T为模板进行PCR扩增。产物克隆入逆转录病毒表达载体pLNCX2的HindⅢ、NotⅠ位点,构建pLNCX2-Tim-3。经PCR、酶切及测序鉴定正确后,转染PT67细胞,48h抽提总RNA并RT-PCR检测Tim-3的表达。PT67空细胞和pLNCX2空载体转染的PT67细胞作对照。 研究结果 PCR、酶切及测序鉴定证实重组载体pLNCX2-Tim-3构建成功;RT-PCR显示pLNCX2-Tim-3在包装细胞PT67中可有效表达Tim-3。 结论及意义 成功构建了有效表达人Tim-3的逆转录病毒表达载体,为进一步研究Tim-3表达调控机制及探索Tim-3过表达治疗疾病的可行性奠定基础。
[Abstract]:Tim-3 (T-cell immunoglobulin-and mucin-domain-containing molecule-3, T-cell immunoglobulin-3) is a newly discovered immunoregulatory molecule, which is specifically expressed in differentiated Th1 cells. It participates in the negative regulation of Th1 and the formation of immune tolerance, and plays an important role in the occurrence of various immune diseases. Blocking its expression can alleviate Th2. Response-induced allergic diseases are potential therapeutic targets for many immune diseases, which have attracted wide attention from scholars at home and abroad. However, there are still many problems unsolved in the study of Tim-3, especially the study of its expression regulation mechanism is still blank. The recombinant human Tim-3 promoter reporter plasmid was constructed from the gene fragment with different promoter activity, and the human Tim-3 retroviral expression vector was constructed to lay a foundation for further study on the regulation mechanism of Tim-3 expression and explore the feasibility of using Tim-3 for disease treatment.
The first part is a preliminary study on the expression pattern of PBMC Tim-3 in normal subjects.
research objective
To detect the expression of Tim-3 in human peripheral blood lymphocytes stimulated by mitogen and provide an experimental model for further study of the regulation mechanism of Tim-3 expression.
research method
After isolation of PBMC and removal of monocytes by adherence method, the suspended lymphocytes were stimulated by PHA and LPS respectively. Total RNA was extracted 24 hours later. The expression of Tim-3 was detected by RT-PCR.
Research results
RT-PCR showed that the expression of Tim-3 was up-regulated in human peripheral blood lymphocytes stimulated by PHA, but not by LPS, suggesting that the non-polarized T cells began to express Tim-3 when they were activated.
Conclusion and significance
This part establishes a cell model which can be used to study the regulation of Tim-3 expression.
The second part of human Tim-3 promoter is cloned and its activity is preliminarily studied.
research objective
To construct human Tim-3 promoter luciferase reporter plasmid and analyze its activity, which will lay a foundation for further study of the molecular mechanism of Tim-3 specific expression in Th1 cells.
research method
Prediction of 1 Tim-3 promoter region
The promoter region of human Tim-3 gene's 5 'end -2500 to +202 segment was predicted by online MatInspector software.
2 Tim-3P1 and Tim-3P2 PCR amplification
A 2325 bp-long segment of -2083~+242 was selected and cloned into Tim-3P1 (-2083~-838) and Tim-3P2 (-948~+242) based on a single Sac I cleavage site. The specific primers for Tim-3P1 and Tim-3P2 were designed and amplified by PCR using human genomic DNA as template.
Construction and identification of 3 '5' end fragment reporter plasmid containing Tim-3 gene with different lengths
Tim-3P1 PCR product, T-A cloned into pGEM-T, sequenced correctly, subcloned into the corresponding digestion site of pGL3-Basic to construct pGL3-Tim-3P1.Tim-3P2 PCR product, digested by Sac I and Xho I, recovered, inserted into pGL3-Basic polyclonal site (MCS) to construct pGL3-Tim-3P2. The promoter activity of the reported plasmid was also sequenced and identified. In the positive pGL3-Tim-3P2, SV40 enhancer sequence was used to construct Enhancer-Tim-3P2, which was identified by double enzyme digestion.
4 RT-PCR was used to detect the expression of endogenous Tim-3 in cell lines.
The logarithmic growth phase RAW264.7, B16 and COS-75 x 10~5 were collected. The total RNA of cells was extracted by Trizol and detected by RT-PCR.
5 transfection of reporter plasmid
The reported plasmids and pGL3-Basic (negative control) were transfected with pRL-TK (internal reference) to RAW264.7, B16 and COS-7 (detailed in the second part of the chart). The cells were collected 48 hours after transfection, and the crude extracts were analyzed by double luciferase assay.
6 double luciferase analysis
The relative activity of the target fragment was determined by fluorescence counting instrument. The ratio of luciferase activity M1 to luciferase activity M2 and M1/M2 in the reported plasmid was determined by fluorescence counting. Repeat 3 times.
7 Tim-3P1 and Tim-3P2 transcription factor binding site (TFBSs) analysis
The online MatInspector software is also used to analyze the possible TFBSs. in Tim-3P1 and Tim-3P2.
Research results
Prediction of 1 Tim-3 promoter region
The analysis showed that there were multiple TATA boxes, CAAT boxes and several important TFBSs in - 2500 to + 202, which might have promoter activity.
2 successfully construct human Tim-3 promoter firefly luciferase reporter plasmid.
Sequencing identification confirmed that pGL3-Tim-3P1 and pGL3-Tim-3P2 were successfully constructed, and enzyme digestion analysis confirmed that Enhancer-Tim-3P2 was successfully constructed.
3 expression of endogenous Tim-3 in different cell lines
RT-PCR detection showed that RAW264.7 and B16 expressed endogenous Tim-3 while COS-7 was not expressed.
4 validation of reporter plasmid promoter activity
Double luciferase assay confirmed that pGL3-Tim-3P1 and pGL3-Tim-3P2 had weak promoter activity, and the relative activity of pGL3-Tim-3P2 was about twice that of pGL3-Basic empty vector, and the promoter activity was cell-specific; Enhancer-Tim-3P2 promoter activity was significantly increased when introducing enhancer.
5 TFBSs analysis
The results showed that Tim-3P1 contained the following TFBSs: 1 c-Myb, Ik-1, YY1 (Yin and Yang 1, Yin and Yang 1), GATA-binding factor-3 (GATA-binding factor-3) and CHOP; 2 FOXK2, HNF-3 (hepatocyte nuclear factor-3, hepatocyte nuclear factor-3); 3 6 TATA boxes; Tim-3P2 contained the following TFBSs: 1 RFX1, p53, GATA-3, NFAT (nuclear factor of activated cells, live). Cell cycle-dependent element; cell cycle genes homology region; 2 MZF1 (Myeloid zinc finger protein-1); 3 YY1; 5 TATA boxes.
Conclusion and significance
In this part, we successfully constructed the glowworm luciferase reporter plasmid containing human Tim-3 promoter and the reporter plasmid with enhancer sequence, which laid a foundation for further study on the regulation mechanism of Tim-3 expression. Prospects.
Construction of the third part of human Tim-3 retroviral expression vector
research objective
Construction of human Tim-3 retroviral expression vector will lay a foundation for further study of the regulation mechanism of Tim-3 expression and explore the feasibility of using Tim-3 to treat diseases.
research method
A specific primer for human Tim-3 mRNA was designed and amplified by PCR using hTim-3-pGEM-T as a template. The product was cloned into the Hind III, Not I site of retroviral expression vector pLNCX2 and constructed pLNCX2-Tim-3. After being identified by PCR, enzyme digestion and sequencing, PT67 cells were transfected and the total RNA was extracted 48 hours and the expression of Tim-3 was detected by RT-PCR. PT67 cells transfected with empty carriers were used as controls.
Research results
PCR, enzyme digestion and sequencing confirmed that the recombinant vector pLNCX2-Tim-3 was constructed successfully, and RT-PCR showed that pLNCX2-Tim-3 could effectively express Tim-3 in PT67 cells.
Conclusion and significance
A retroviral expression vector expressing human Tim-3 was successfully constructed, which laid a foundation for further study on the regulation mechanism of Tim-3 expression and the feasibility of Tim-3 overexpression in the treatment of diseases.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392
本文编号：2216484
[Abstract]:Tim-3 (T-cell immunoglobulin-and mucin-domain-containing molecule-3, T-cell immunoglobulin-3) is a newly discovered immunoregulatory molecule, which is specifically expressed in differentiated Th1 cells. It participates in the negative regulation of Th1 and the formation of immune tolerance, and plays an important role in the occurrence of various immune diseases. Blocking its expression can alleviate Th2. Response-induced allergic diseases are potential therapeutic targets for many immune diseases, which have attracted wide attention from scholars at home and abroad. However, there are still many problems unsolved in the study of Tim-3, especially the study of its expression regulation mechanism is still blank. The recombinant human Tim-3 promoter reporter plasmid was constructed from the gene fragment with different promoter activity, and the human Tim-3 retroviral expression vector was constructed to lay a foundation for further study on the regulation mechanism of Tim-3 expression and explore the feasibility of using Tim-3 for disease treatment.
The first part is a preliminary study on the expression pattern of PBMC Tim-3 in normal subjects.
research objective
To detect the expression of Tim-3 in human peripheral blood lymphocytes stimulated by mitogen and provide an experimental model for further study of the regulation mechanism of Tim-3 expression.
research method
After isolation of PBMC and removal of monocytes by adherence method, the suspended lymphocytes were stimulated by PHA and LPS respectively. Total RNA was extracted 24 hours later. The expression of Tim-3 was detected by RT-PCR.
Research results
RT-PCR showed that the expression of Tim-3 was up-regulated in human peripheral blood lymphocytes stimulated by PHA, but not by LPS, suggesting that the non-polarized T cells began to express Tim-3 when they were activated.
Conclusion and significance
This part establishes a cell model which can be used to study the regulation of Tim-3 expression.
The second part of human Tim-3 promoter is cloned and its activity is preliminarily studied.
research objective
To construct human Tim-3 promoter luciferase reporter plasmid and analyze its activity, which will lay a foundation for further study of the molecular mechanism of Tim-3 specific expression in Th1 cells.
research method
Prediction of 1 Tim-3 promoter region
The promoter region of human Tim-3 gene's 5 'end -2500 to +202 segment was predicted by online MatInspector software.
2 Tim-3P1 and Tim-3P2 PCR amplification
A 2325 bp-long segment of -2083~+242 was selected and cloned into Tim-3P1 (-2083~-838) and Tim-3P2 (-948~+242) based on a single Sac I cleavage site. The specific primers for Tim-3P1 and Tim-3P2 were designed and amplified by PCR using human genomic DNA as template.
Construction and identification of 3 '5' end fragment reporter plasmid containing Tim-3 gene with different lengths
Tim-3P1 PCR product, T-A cloned into pGEM-T, sequenced correctly, subcloned into the corresponding digestion site of pGL3-Basic to construct pGL3-Tim-3P1.Tim-3P2 PCR product, digested by Sac I and Xho I, recovered, inserted into pGL3-Basic polyclonal site (MCS) to construct pGL3-Tim-3P2. The promoter activity of the reported plasmid was also sequenced and identified. In the positive pGL3-Tim-3P2, SV40 enhancer sequence was used to construct Enhancer-Tim-3P2, which was identified by double enzyme digestion.
4 RT-PCR was used to detect the expression of endogenous Tim-3 in cell lines.
The logarithmic growth phase RAW264.7, B16 and COS-75 x 10~5 were collected. The total RNA of cells was extracted by Trizol and detected by RT-PCR.
5 transfection of reporter plasmid
The reported plasmids and pGL3-Basic (negative control) were transfected with pRL-TK (internal reference) to RAW264.7, B16 and COS-7 (detailed in the second part of the chart). The cells were collected 48 hours after transfection, and the crude extracts were analyzed by double luciferase assay.
6 double luciferase analysis
The relative activity of the target fragment was determined by fluorescence counting instrument. The ratio of luciferase activity M1 to luciferase activity M2 and M1/M2 in the reported plasmid was determined by fluorescence counting. Repeat 3 times.
7 Tim-3P1 and Tim-3P2 transcription factor binding site (TFBSs) analysis
The online MatInspector software is also used to analyze the possible TFBSs. in Tim-3P1 and Tim-3P2.
Research results
Prediction of 1 Tim-3 promoter region
The analysis showed that there were multiple TATA boxes, CAAT boxes and several important TFBSs in - 2500 to + 202, which might have promoter activity.
2 successfully construct human Tim-3 promoter firefly luciferase reporter plasmid.
Sequencing identification confirmed that pGL3-Tim-3P1 and pGL3-Tim-3P2 were successfully constructed, and enzyme digestion analysis confirmed that Enhancer-Tim-3P2 was successfully constructed.
3 expression of endogenous Tim-3 in different cell lines
RT-PCR detection showed that RAW264.7 and B16 expressed endogenous Tim-3 while COS-7 was not expressed.
4 validation of reporter plasmid promoter activity
Double luciferase assay confirmed that pGL3-Tim-3P1 and pGL3-Tim-3P2 had weak promoter activity, and the relative activity of pGL3-Tim-3P2 was about twice that of pGL3-Basic empty vector, and the promoter activity was cell-specific; Enhancer-Tim-3P2 promoter activity was significantly increased when introducing enhancer.
5 TFBSs analysis
The results showed that Tim-3P1 contained the following TFBSs: 1 c-Myb, Ik-1, YY1 (Yin and Yang 1, Yin and Yang 1), GATA-binding factor-3 (GATA-binding factor-3) and CHOP; 2 FOXK2, HNF-3 (hepatocyte nuclear factor-3, hepatocyte nuclear factor-3); 3 6 TATA boxes; Tim-3P2 contained the following TFBSs: 1 RFX1, p53, GATA-3, NFAT (nuclear factor of activated cells, live). Cell cycle-dependent element; cell cycle genes homology region; 2 MZF1 (Myeloid zinc finger protein-1); 3 YY1; 5 TATA boxes.
Conclusion and significance
In this part, we successfully constructed the glowworm luciferase reporter plasmid containing human Tim-3 promoter and the reporter plasmid with enhancer sequence, which laid a foundation for further study on the regulation mechanism of Tim-3 expression. Prospects.
Construction of the third part of human Tim-3 retroviral expression vector
research objective
Construction of human Tim-3 retroviral expression vector will lay a foundation for further study of the regulation mechanism of Tim-3 expression and explore the feasibility of using Tim-3 to treat diseases.
research method
A specific primer for human Tim-3 mRNA was designed and amplified by PCR using hTim-3-pGEM-T as a template. The product was cloned into the Hind III, Not I site of retroviral expression vector pLNCX2 and constructed pLNCX2-Tim-3. After being identified by PCR, enzyme digestion and sequencing, PT67 cells were transfected and the total RNA was extracted 48 hours and the expression of Tim-3 was detected by RT-PCR. PT67 cells transfected with empty carriers were used as controls.
Research results
PCR, enzyme digestion and sequencing confirmed that the recombinant vector pLNCX2-Tim-3 was constructed successfully, and RT-PCR showed that pLNCX2-Tim-3 could effectively express Tim-3 in PT67 cells.
Conclusion and significance
A retroviral expression vector expressing human Tim-3 was successfully constructed, which laid a foundation for further study on the regulation mechanism of Tim-3 expression and the feasibility of Tim-3 overexpression in the treatment of diseases.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392
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3 朱汉钢,冯作化,耿辉,张桂梅;肿瘤组织中Tim-3表达的特征及其在肿瘤免疫耐受中的作用[J];细胞与分子免疫学杂志;2005年04期
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