小鼠Tim-2基因在L929细胞表达后促进铁蛋白的内吞

发布时间：2018-02-25 21:21

本文关键词： Tim-2基因 BALB/c小鼠 L929细胞真核表达铁蛋白 Tim-2基因昆明小鼠单核苷酸多态性　出处：《华中科技大学》2007年硕士论文　论文类型：学位论文

【摘要】： 第一部分小鼠Tim-2基因在L929细胞表达后促进铁蛋白的内吞目的:获取BALB/c小鼠T细胞免疫球蛋白域粘蛋白域蛋白-(2T cell immunoglobulin and mucin domain containing molecules-2, Tim-2)基因,构建真核表达载体在L929细胞中表达并研究其功能。方法:RT-PCR获取BALB/c小鼠Tim-2基因,T-A克隆后经测序鉴定后酶切,插入pEGFP-N1真核表达载体,脂质体转染L929细胞,荧光显微镜观察外源基因的表达,然后加入铁蛋白与转染细胞作用。结果:外源基因在L929细胞中得到高效表达,荧光显微镜的结果显示,外源基因定位于细胞膜上,并能促进细胞对铁蛋白的内吞。结论:Tim-2基因在真核细胞中表达后定位于细胞膜上,并促进细胞对铁蛋白的内吞。第二部分昆明小鼠Tim-2基因的单个核苷酸变异的研究目的:研究T细胞免疫球蛋白域粘蛋白域蛋白(T cell immunoglobulin-and mucin-domain-containing molecules, Tim)基因家族的成员之一 Tim-2基因在昆明鼠体内的单核苷酸多态性,以提示昆明鼠在肿瘤学和免疫学的相关试验中有可能出现的与国际通用的其它品系小鼠(如BALB/c小鼠)不同的生物学特性。方法:取不同来源的五只昆明小鼠的肝组织,提取总RNA后逆转录得到cDNA,使用特异性引物通过PCR扩增得到Tim-2基因,克隆后测序,然后比较测序图谱。结果:测序结果与Genebank中的Tim-2序列相比,451(TCC→GCC:丝氨酸→丙氨酸),502,504(CCA→TCT:脯氨酸→丝氨酸),586,587(GAT→TCT:天冬氨酸→丝氨酸)。其他变异位点无氨基酸改变。结论:提示昆明小鼠与国际通用的其它品系小鼠(如BALB/c小鼠)Tim-2基因所编码的蛋白质序列有差异,在使用昆明小鼠进行免疫学相关的试验研究的过程中应该注意到由于这种不同所造成的对实验结果的影响。
[Abstract]:The first part of mouse Tim-2 gene expression in L929 cells promotes the endocytosis of ferritin. Aim: to construct the eukaryotic expression vector of BALB/c mouse T cell immunoglobulin domain mucin domain protein (-TX) -2T cell immunoglobulin and mucin domain containing (Tim-2) gene, and to study its function in L929 cells. Methods BALB/c mouse Tim-2 gene was cloned by RT-PCR and sequenced, then inserted into pEGFP-N1 eukaryotic expression vector. L929 cells were transfected with liposome. The expression of exogenous gene was observed by fluorescence microscope, then ferritin was added to the transfected cells. Results: the extraneous gene was highly expressed in L929 cells. Fluorescence microscopy showed that the exogenous gene was located on the cell membrane and could promote the uptake of ferritin in L929 cells. Conclusion the expression of the gene was located on the cell membrane and promoted the endocytosis of ferritin in the eukaryotic cells. Part II study on single nucleotide variation of Tim-2 gene in Kunming mice. Objective: to study the single nucleotide polymorphism of Tim-2 gene of T cell cell immunoglobulin-and mucin-domain-containing (Tims) gene in Kunming mice. These results suggest that Kunming mice may have different biological characteristics from other strains of mice (such as BALB/c mice) in oncology and immunology. Methods: the liver tissues of five Kunming mice from different sources were taken, the total RNA was extracted by reverse transcription, the Tim-2 gene was amplified by PCR with specific primers, cloned and sequenced, and then the sequence map was compared. Results: compared with the Tim-2 sequence in Genebank, the result of sequencing was 451 TCCs. 鈫扜CC: serine. 鈫扐lanine stoma 502CBA 504CCA. 鈫扨roline. 鈫扴erine 586 GAT. 鈫扐spartic acid. 鈫扴erine. No amino acid changes at other mutation sites. Conclusion: the results suggest that there are differences in protein sequences between Kunming mice and other strains of mice (such as BALB/c mice). The effect of this difference on the experimental results should be taken into account in the use of Kunming mice in immunological related experimental studies.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392

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