基于PML-RARα融合基因的特异性T细胞免疫反应和基因疫苗构建研究
发布时间：2018-07-24 17:02
【摘要】：目的：了解PML-RARα多肽和蛋白诱导脐血T细胞的T细胞受体(TCR)Vβ谱系表达和克隆性情况，，并分析其诱导的T细胞的特异性杀伤效应。并构建具有抗急性早幼粒细胞白血病(APL)作用的PML-RARα基因疫苗。 方法：分别以NB4细胞和不同浓度(16.7μg／ml，33.3μg／ml或50μg／ml)的PML-RARα多肽联合IL-2、抗CD3单抗以及抗CD28单抗诱导脐血T细胞增殖，利用RT-PCR-基因扫描技术分析诱导一定时间(第5、10天或第3、6、9、12天)的脐血TCR Vβ亚家族的利用和克隆性增殖的特点，同时利用LDH法检测其细胞杀伤性。用RT-PCR方法扩增PML-RARα基因序列，分别克隆到真核表达载体pIRES和分泌载体pSecTag中。将经测序鉴定正确的重组质粒转染K562细胞，RT-PCR方法检测重组质粒在真核细胞中的转录情况，点杂交和SDS-PAGE凝胶电泳检测蛋白的表达情况。 结果：限制性表达和克隆性增殖的TCR Vβ亚家族T细胞均可见于PML-RARα多肽和NB4细胞诱导后的脐血T细胞，经LDH法检测其细胞杀伤活性，显示此T细胞具有较强的细胞毒作用。成功构建了PML-RARα／pIRES和PML-RARα／pSecTag重组质粒，经测序鉴定二者序列均正确，将其转染K562细胞后经RT-PCR电泳鉴定表明在转录水平能够表达，经点杂交检测显示重组质粒在真核细胞中可表达PML-RARα蛋白，经SDS-PAGE电泳检测在预期的位置亦可见一目的条带，大小约为17KD。 结论：PML-RARα多肽和NB4细胞可在体外诱导脐血T细胞呈克隆性增殖，此优势增殖的克隆性T细胞具有特异性细胞毒作用。成功构建PML-RARα融合基因疫苗，体外真核细胞中能表达PML-RARα蛋白。
[Abstract]:Aim: to investigate the expression and cloning of T cell receptor (TCR) V 尾 in cord blood T cells induced by PML-RAR 伪 peptide and protein, and to analyze the specific cytotoxicity of T cells induced by PML-RAR 伪 peptide and protein. PML-RAR 伪 gene vaccine with anti-(APL) effect in acute promyelocytic leukemia was constructed. Methods: cord blood T cell proliferation was induced by NB4 cells and different concentrations (16.7 渭 g / ml 33.3 渭 g/ml or 50 渭 g/ml) of PML-RAR 伪 peptide combined with IL-2, anti-CD3 monoclonal antibody and anti-CD28 monoclonal antibody, respectively. RT-PCR- gene scanning technique was used to analyze the utilization and clonal proliferation of TCR V 尾 subfamily in umbilical cord blood induced for a certain period of time (5th day, 10th day or 3rd day, 912 day). Meanwhile, LDH assay was used to detect the cytotoxicity of human umbilical cord blood TCR V 尾 subfamily. PML-RAR 伪 gene was amplified by RT-PCR and cloned into eukaryotic expression vector pIRES and secretory vector pSecTag, respectively. The transcription of recombinant plasmid in eukaryotic cells was detected by RT-PCR, and the expression of protein was detected by dot blot and SDS-PAGE gel electrophoresis. Results: the restricted expression and clonal proliferation of TCR V 尾 subfamily T cells could be found in PML-RAR 伪 peptide and NB4 cells induced T cells in umbilical cord blood. The cytotoxicity of these T cells was detected by LDH assay. The recombinant plasmids PML-RAR 伪 / Pires and PML-RAR 伪 / pSecTag were successfully constructed. The sequence of the two recombinant plasmids was confirmed by sequencing. After transfection into K562 cells, RT-PCR electrophoresis showed that they could be expressed at the transcriptional level. Dot blot analysis showed that the recombinant plasmid could express PML-RAR 伪 protein in eukaryotic cells, and a target band was also found by SDS-PAGE electrophoresis. The size of the recombinant plasmid was about 17kD. Conclusion the cloned T cells of cord blood can be induced to proliferate in vitro by NB4 cells and 1: PML-RAR 伪 polypeptide. The dominant clonal T cells have specific cytotoxic effects. PML-RAR 伪 fusion gene vaccine was successfully constructed and expressed PML-RAR 伪 protein in eukaryotic cells in vitro.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R392
本文编号：2142053
[Abstract]:Aim: to investigate the expression and cloning of T cell receptor (TCR) V 尾 in cord blood T cells induced by PML-RAR 伪 peptide and protein, and to analyze the specific cytotoxicity of T cells induced by PML-RAR 伪 peptide and protein. PML-RAR 伪 gene vaccine with anti-(APL) effect in acute promyelocytic leukemia was constructed. Methods: cord blood T cell proliferation was induced by NB4 cells and different concentrations (16.7 渭 g / ml 33.3 渭 g/ml or 50 渭 g/ml) of PML-RAR 伪 peptide combined with IL-2, anti-CD3 monoclonal antibody and anti-CD28 monoclonal antibody, respectively. RT-PCR- gene scanning technique was used to analyze the utilization and clonal proliferation of TCR V 尾 subfamily in umbilical cord blood induced for a certain period of time (5th day, 10th day or 3rd day, 912 day). Meanwhile, LDH assay was used to detect the cytotoxicity of human umbilical cord blood TCR V 尾 subfamily. PML-RAR 伪 gene was amplified by RT-PCR and cloned into eukaryotic expression vector pIRES and secretory vector pSecTag, respectively. The transcription of recombinant plasmid in eukaryotic cells was detected by RT-PCR, and the expression of protein was detected by dot blot and SDS-PAGE gel electrophoresis. Results: the restricted expression and clonal proliferation of TCR V 尾 subfamily T cells could be found in PML-RAR 伪 peptide and NB4 cells induced T cells in umbilical cord blood. The cytotoxicity of these T cells was detected by LDH assay. The recombinant plasmids PML-RAR 伪 / Pires and PML-RAR 伪 / pSecTag were successfully constructed. The sequence of the two recombinant plasmids was confirmed by sequencing. After transfection into K562 cells, RT-PCR electrophoresis showed that they could be expressed at the transcriptional level. Dot blot analysis showed that the recombinant plasmid could express PML-RAR 伪 protein in eukaryotic cells, and a target band was also found by SDS-PAGE electrophoresis. The size of the recombinant plasmid was about 17kD. Conclusion the cloned T cells of cord blood can be induced to proliferate in vitro by NB4 cells and 1: PML-RAR 伪 polypeptide. The dominant clonal T cells have specific cytotoxic effects. PML-RAR 伪 fusion gene vaccine was successfully constructed and expressed PML-RAR 伪 protein in eukaryotic cells in vitro.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R392
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