【摘要】 [目的]构建融合CD3ζ的TCRαζβζ分子的双表达载体,并观察TCRαζβζ分子中的CD3ζ与细胞膜的共定位情况。[方法]设计重叠PCR引物,利用重叠PCR扩增携带荧光蛋白的融合基因TCRβζ-EYFP和TCRαζ,将其克隆到p IRES2-EGFP载体,构建双表达载体p IRES2-TCRβζ-EYFP/TCRαζ,将重组载体分别转染BEL-7402细胞和Jurkat T细胞,转染48 h后,细胞膜经Di D染料染色,共聚焦显微镜扫描并分析该TCRαζβζ分子的表达以及与细胞膜的共定位情况。[结果]重组双表达载体p IRES-TCRβζ-EYFP/TCRαζ经菌落PCR、酶切鉴定及测序分析证明载体构建成功;共聚焦显微镜扫描结果显示重组载体表达了融合蛋白TCRαζβζ分子;共定位程序分析显示TCRαζβζ分子中的CD3ζ与细胞膜存在共定位关系。[结论]成功构建了融合蛋白TCRαζβζ分子的双表达载体,TCRαζβζ分子中的CD3ζ与细胞膜存在共定位。更多还原

【Abstract】 [Objective] To construct TCRαζβζ fusion protein dual-expression vector,and observe the co-localization of CD3ζ in TCRαζβζ and the plasma membrane in cells.[Methods] Overlapping PCR primers were designed.Then the fusion genes TCRβζ-EYFP and TCRαζ were obtained by overlapping PCR,and were cloned into the pIRES2-EGFP vector to construct the recombined dual-expression vector pIRES2-TCRβζ-EYFP/TCRαζ.After that,the recombinant plasmids were transfected into BEL-7402 cells and Jurkat T cells respectively.After 48 hours of transfection,the cell membrane were stained with DiD dye.Finally,the expression of TCRαζβζ and its co-localization with the plasma membrane in cells were detected and analyzed by confocal laser scanning microscope(CLSM).[Results] The recombinant plasmid pIRES-TCRβζ-EYFP/TCRαζ was constructed successfully confirmed by colony PCR,double restriction enzyme digestion and DNA sequencing.Confocal microscopy showed that TCRαζβζ expressed in cells.Co-location program analysis indicated that CD3ζ in TCRαζβζ was co-localized with the plasma membrane in Jurkat T cells.[Conclusion] The recombined dual-expression vector pIRES-TCRβζ-EYFP/TCRαζ was constructed successfully.The fusion protein TCRαζβζ expressed in cells successfully.CD3ζ in TCRαζβζ molecules co-localized with the plasma membrane in Jurkat T cells.更多还原