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目的:为探讨SPARC(secreted protein acidic and rich in cysteine)在人恶性肿瘤发生、发展中的作用及其分子机制,进一步明确SPARC发挥作用的方式及其与肿瘤发生类型的关系。方法:我们首先提取了人乳腺癌细胞系MCF-7的总RNA,在对总RNA进行纯度与定量检测后,利用RT-PCR的方法,以该总RNA为模板,将其反转录为cDNA;再设计引物,以该cDNA为模板,利用PCR扩增出包含Sparc编码区的DNA片段,将该产物纯化后通过T-A克隆连接入pMD20-T载体,利用菌落PCR及DNA测序进行鉴定。以pMD20-T-Sparc为模板,我们设计了特异的针对Sparc全长编码区的引物,并在引物5’端分别加入BamHI、HindIII酶切位点,通过PCR将Sparc编码区扩增出来,经纯化及双酶切后与真核表达载体pcDNA3.1myc-his(-)相连,再经菌落PCR和DNA测序进行鉴定。通过瞬时转染的方法,利用脂质体将所构建的重组SPARC真核表达载体转染HEK293细胞,48h后裂解所培养的细胞,使用western blot检测有无SPARC的表达。结果:测序证实所克隆的Sparc编码区cDNA正确地插入pcDNA3.1myc-his(-)中,western blot检测证实其在HEK293细胞中得到表达,而空载体转染的细胞则无表达,说明所构建的pcDNA3.1myc-his(-)-Sparc能够成功表达。结论:我们成功克隆了人Sparc cDNA,构建了其真核表达载体,并在HEK293细胞中得到有效表达,从而为进一步研究人SPARC的功能及其与肿瘤的关系奠定了基础。
OBJECTIVE: To investigate the role of SPARC (secreted protein acidic and rich in cysteine) in the development and progression of human malignancies, and to elucidate the molecular mechanism of SPARC and its relationship with tumorigenesis. Methods: We first extracted the total RNA of human breast cancer cell line MCF-7. After detecting the purity and quantitation of total RNA, RT-PCR was used to reverse the total RNA as a cDNA . The primers were designed. The DNA fragment containing Sparc coding region was amplified by PCR using the cDNA as a template. The product was purified and ligated into pMD20-T vector by TA cloning. The PCR products were identified by colony PCR and DNA sequencing. Using pMD20-T-Sparc as a template, we designed primers specific to the Sparc full-length coding region. BamHI and HindIII restriction sites were added to the 5 ’end of the primer. The Sparc coding region was amplified by PCR. After purification and double enzyme digestion, it was ligated with the eukaryotic expression vector pcDNA3.1myc-his (-) and identified by colony PCR and DNA sequencing. The constructed recombinant SPARC eukaryotic expression vector was transfected into HEK293 cells by lipofectamine. The cultured cells were lysed 48h later, and the expression of SPARC was detected by western blot. Results: Sequencing confirmed that the cloned Sparc coding region was correctly inserted into pcDNA3.1 myc-his (-). Western blot showed that it was expressed in HEK293 cells but not in empty vector. Of pcDNA3.1myc-his (-) - Sparc was successfully expressed. CONCLUSION: We successfully cloned human Sparc cDNA, constructed its eukaryotic expression vector and expressed it efficiently in HEK293 cells, which laid the foundation for further study on the function of human SPARC and its relationship with tumor.