Sparc基因的克隆及其在HEK293细胞中的表达

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。

Cloning and Expression Analysis of Sparc in HEK293 Cells

Objective： To investigate the role of SPARC（secreted protein acidic and rich in cysteine） in human tumorigenesis and further explore the relationship between the way that SPARC exerts its function and the type of tumor.Methods： First,Total RNA was isolated from MCF-7 cells.A 1501 bp fragment containing the coding region of Sparc was amplified by RT-PCR and the resulting PCR product was subcloned into PMD20-T vector and sequenced.Second,coding region of Sparc was generated with PCR by using the PMD20-T-Sparc as template,the amplified PCR fragment was inserted into the BamHI and HindⅢ sites of the pcDNA3.1myc-his（-）A expression vector,and the sequence was confirmed by colony PCR and DNA sequencing.In the end,pcDNA3.1 myc-his（-）A-Sparc was transiently transfected into HEK293 cells and the expression of new construct pcDNA3.1 myc-his（-）A-Sparc in HEK293 cells was detected by western blot.Results： The full length coding region of Sparc was obtained and confirmed by sequencing,the expression of Sparc was detected successfully in HEK293 cells.Conclusion： The eukaryotic expression vector of Sparc has been successfully constructed,which will provide a useful tool for designing an in-depth investigation of the role of Sparc in tumorigenesis.