论文部分内容阅读
目的:乙酰肝素酶在肿瘤转移过程中起关键作用,其信号肽在该酶翻译后处理过程中起重要作用。本研究旨在探索乙酰肝素酶在哺乳动物细胞中的表达特性及其信号肽对此酶功能活性的影响。方法:从人胎盘cDNA文库中扩增乙酰肝素酶全长编码基因,克隆入pGEM-T载体中,测序鉴定序列完全正确后,将此基因的全长和不含信号肽基因序列分别克隆入真核表达载体pcDNA4.1/Myc-His中构建pcDNA4.1/Myc-His full HPA和pcDNA4.1/Myc-His part HPA,转化COS-7细胞进行瞬时表达,分析这两种乙酰肝素酶蛋白的表达特性及功能活性。结果:在转染了乙酰肝素酶全基因的COS-7细胞裂解液中检测到该酶的功能活性及大小约53×103(Mr)的目的蛋白免疫印迹表达带,为切割激活后乙酰肝素酶羧基端大亚基与标签蛋白的融合体。在转染了敲除信号肽的乙酰肝素酶基因的COS-7细胞裂解液中测到未被切割激活的大小约65×103(Mr)的目的蛋白免疫印迹表达带,未能检测到该酶的功能活性。结论:在COS-7细胞中表达的完整的乙酰肝素酶蛋白和不含信号肽的乙酰肝素酶蛋白均位于细胞内,没有或很少被分泌到细胞外,提示该酶在正常状态下主要分布在细胞内。含信号肽的酶蛋白在翻译后处理过程中被细胞内的蛋白酶切割成2个亚基而被激活,具有功能活性。而不含信号肽的酶蛋白没有被切割而成为有活性的酶,提示乙酰肝素酶的信号肽对其翻译后加工、激活过程有重要的影响。
Objective: Heparanase plays a key role in tumor metastasis and its signal peptide plays an important role in post-translational processing of this enzyme. This study aimed to explore the expression characteristics of heparanase in mammalian cells and the effect of its signal peptide on the functional activity of the enzyme. Methods: The full-length coding gene of heparanase was amplified from the human placenta cDNA library and cloned into pGEM-T vector. After the sequencing was completely correct, the full-length and the signal peptide-free gene sequences were cloned into The pcDNA4.1 / Myc-His full HPA and pcDNA4.1 / Myc-His part HPA were constructed in the eukaryotic expression vector pcDNA4.1 / Myc-His and transformed into COS-7 cells for transient expression. The two heparanase Protein expression and functional activity. Results: The functional activity of the enzyme was detected in the lysate of COS-7 cells transfected with heparanase gene and the expression of the target protein was about 53 × 103 (Mr) Fusion enzyme of carboxyl-terminal large subunit and tagged protein. The Western blot expression band of the target protein of about 65 × 103 (Mr), which had not been cleaved and activated, was detected in the COS-7 cell lysate transfected with the heparanase gene knock-out signal peptide, which was not detected Enzyme functional activity. CONCLUSION: The intact heparanase protein and the signal peptide-free heparanase protein expressed in COS-7 cells are intracellularly and little or no extracellularly secreted, suggesting that the enzyme under normal conditions Mainly distributed in the cell. The signal peptide-containing enzyme protein is activated by the protease cleaving into two subunits during the post-translational processing. However, the signal peptide without signal peptide was not cleaved and became an active enzyme, suggesting that the signal peptide of heparanase has an important influence on the post-translational processing and activation process.