摘要
目的构建CD8α真核表达载体,为构建膜型细胞因子提供重要工具。方法采取RT-PCR从磁珠分选的CD8α+T细胞中得到CD8α的cDNA基因,将其克隆入表达载体pVITRO。经测序后转染SP2/0细胞,通过流式细胞仪和激光共聚焦显微镜检测CD8α分子在SP2/0细胞中的表达情况。结果构建的pVITRO/CD8α载体测序后,Genebank比对证实为CD8α的cD-NA分子,流式细胞仪和激光共聚焦显微镜结果均说明CD8α在SP2/0细胞的细胞膜上得到表达。结论 CD8α在真核细胞膜上能成功表达,为进一步构建膜表达型细胞因子奠定了基础。
Objective To provide an experimental tool to construct membrane-bound form of cytokines,the mouse CD8α were inserted into expressing vector pVITRO.Methods Mouse CD8α＋ T cells were enriched by using CD8α-conjugated microbeads.The total RNA from CD8α＋ T cells were isolated at first,Then RT-PCR were used by oligodT primer and CD8α pair primers respectively.cDNA sequences were cloned into expressing vector pVITRO.After sequencing,the recombinant plasmid pVITRO/CD8α was transfected into SP2/0 cells with liposome.The expression of CD8α on SP2/0 cells was detected by flow cytometery and confocal microscopy.Results The results of sequencing and blasting in the genebank show that we have got a cDNA sequence of CD8α.CD8α expression on SP2/0 cell surface was confirmed by flow cytometery and confocal microscopy.Conclusions CD8α was expressed on SP2/0 cell surface,which laid a solid foundation for constructing the membrane-bound form of cytokines by genetic engineering techniques in future.
出处
《实用临床医药杂志》
CAS
2011年第1期1-3,14,共4页
Journal of Clinical Medicine in Practice
基金
国家自然科学基金资助项目(81001308)
江苏省自然科学基金资助项目(BK2010315)
扬州大学高层次人才科研启动基金资助项目
