论文部分内容阅读
目的 观察颗粒酶Ｂ（ｇｒａｎｚｙｍｅ Ｂ）融合蛋白对转染的ＨｅＬａ细胞的作用。 方法用ＲＴ－ＰＣＲ法获取人ｇｒａｎｚｙｍｅ Ｂ全长ｃＤＮＡ，经重组ＰＣＲ将活性型序列的５′端与一段 ＰＥ转膜肽序列融合。将所获重组基因克隆入绿色荧光蛋白（ＧＦＰ）共表达载体ｐＩＲＥＳ２－ＥＧＦＰ，转染ＨｅＬａ细胞。用免疫组化染色法检测目的蛋白的表达，荧光显微镜和电镜观察转染细胞的形态和结构。 结果成功地获得了野生型ｇｒａｎｚｙｍｅ Ｂ ｃＤＮＡ及其融合蛋白基因，并构建了融合蛋白基因与ＧＦＰ基因共表达的真核载体。转染ＨｅＬａ细胞后，检测到目的蛋白的表达。荧光显微镜观察转染细胞有两种变化：一种表现为体积增大并常伴随多核现象；另一种出现细胞质和细胞核的固缩。电镜观察显示，多核巨细胞生长状态良好，固缩的小细胞则呈现凋亡的典型特征。结论ｇｒａｎｚｙｍｅ Ｂ融合蛋白在ＨｅＬａ细胞中异位表达，可使转染细胞的形态发生变化，出现多核的大细胞和核固缩的小细胞。
Objective To observe the effect of granzyme B fusion protein on transfected HeLa cells. Methods The full length cDNA of human granzyme B was obtained by RT - PCR. The 5 ’end of the active sequence was fused with a PE transmembrane peptide sequence by recombinant PCR. The recombinant plasmid was cloned into green fluorescence protein (GFP) co-expression vector pIRES2-EGFP and transfected into HeLa cells. The expression of the target protein was detected by immunohistochemical staining. The morphology and structure of transfected cells were observed by fluorescence microscope and electron microscope. Results The wild-type granzyme B cDNA and its fusion protein gene were successfully obtained and a eukaryotic vector co-expressing the fusion protein gene and GFP gene was constructed. After transfected HeLa cells, the expression of the target protein was detected. Fluorescence microscopy of transfected cells have two changes: one is manifested as the volume increases and often accompanied by multi-nuclear phenomenon; the other appears cytoplasm and nucleus pyknosis. Electron microscopy showed that the multinucleated giant cells grew well and the contracted small cells showed the typical characteristics of apoptosis. Conclusion The expression of granzyme B fusion protein in HeLa cells can change the morphology of the transfected cells, and the appearance of multinucleated macrophages and nuclear pyknotic minicells.