人精子顶体膜相关蛋白32真核表达载体的构建及其在NIH3T3细胞中的表达

目的:研究人精子顶体膜相关蛋白32(hSAMP32)mRNA在体外细胞NIH3T3中的表达。方法:利用RTPCR及亚克隆技术构建pcDNA3.1( )hSAMP32质粒,实验组以脂质体介导基因转染法将pcDNA3.1( )hSAMP32真核表达载体导入NIH3T3细胞中,同时设置空白和阴性对照组(转染空载体pcDNA3.1( ))。原位杂交检测hSAMP32mRNA在3组细胞中的表达。结果:①RTPCR扩增产物与预期的目的基因hSAMP32长度一致。亚克隆酶切鉴定可见目的片段。②实验组阳性细胞胞浆中可见hSAMP32mRNA的表达,镜下可见蓝紫色阳性颗粒,空白和阴性对照组未见蓝紫色阳性颗粒。结论:脂质体介导基因转染法能够将pcDNA3.1hSAMP32真核表达载体高效导入NIH3T3细胞中,实现了hSAMP32基因的体外表达。

Construction of hSAMP32 eukaryotic expression-vector and its expression in NIH3T3 cells

Aim: To study the expression of hSAMP32 in NIH3T3 cells in vitro. Methods:To construct the pcDNA3.1( )-hSAMP32 plasmid by PT-PCR and subcloning technique. The pcDNA3.1( )-hSAMP32 DNA was transfected into NIH3T3 cells by liposome. The hSAMP32 mRNA expression was detected by in-situ hybridization.Results: RT-PCR product was consistent with that of target gene, hSAMP32. The target segment was detected through enzyme-digestion in subcloning.The bluish-violet colored granules were located in cytoplasm, but nothing was found in the cells transfected with pcDNA3.1 ( ) or cells not transfected.Conclusion: The pcDNA3.1( )-hSAMP32 DNA could be efficiently transfected into NIH3T3 cells by liposome-mediated gene transfection technique. The hSAMP32 gene expression in vitro is realized.