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目的:获取人组氨酸磷酸酶蛋白PHPT1基因,并构建其C端GFP融合的真核表达载体,通过瞬时转染观察融合蛋白在细胞内的表达和定位,并研究其定位与细胞层状伪足形成的关系。方法:以人宫颈癌细胞株HeLa cDNA为模板,PCR扩增PHPT1的全长编码基因,克隆到pEGFPN2载体中,构建pEGFP-N2-PHPT1真核表达载体,利用脂质体将构建的载体转染到HeLa细胞中,用激光共聚焦扫描显微镜观察C端GFP连接的PHPT1的细胞定位,并进一步探讨其定位与细胞层状伪足形成的关系。结果:成功构建了PHPT1的GFP融合表达载体pEGFP-N2-PHPT1,并在He La细胞中检测到了融合蛋白的表达,发现其定位与细胞层状伪足的形成密切相关,并可直接影响细胞的运动能力。结论:GFP融合形式表达的PHPT1蛋白在细胞质和细胞核中均有表达,并且定位于细胞层状伪足的前沿,影响细胞层状伪足的形成,从而影响细胞运动。
OBJECTIVE: To obtain the human histone phosphatase (PHPT1) gene and construct its C-terminal GFP fusion eukaryotic expression vector. The expression and localization of the fusion protein in the cell were observed by transient transfection and its localization and cell- Foot formation relationship. Methods: The full-length coding gene of PHPT1 was amplified by PCR using HeLa cDNA of human cervical cancer cell line as a template and cloned into pEGFPN2 vector to construct eukaryotic expression vector pEGFP-N2-PHPT1. The constructed vector was transfected by liposome To HeLa cells, the localization of C-terminal GFP-linked PHPT1 cells was observed by laser scanning confocal microscopy and the relationship between PTP1 localization and cell layer pseudopod formation was also investigated. Results: The recombinant fusion protein pEGFP-N2-PHPT1 was constructed successfully and the expression of fusion protein was detected in He La cells. It was found that the localization of GFP fusion protein was closely related to the formation of lamellipodia and could directly affect the expression of Athletic ability. CONCLUSION: The PHPT1 protein expressed by GFP fusion is expressed both in the cytoplasm and in the nucleus, and located at the front of the lamellipodia, affecting the formation of lamellipodia and affecting the cell motility.