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目的构建人SAFB基因真核表达载体,为SAFB基因功能研究提供研究基础。方法设计人SAFB特异性引物,提取人正常结直肠上皮组织总RNA,应用RT-PCR方法提取人SAFBcDNA,通过双酶切及测序鉴定,将SAFB克隆至pcDNA3.1(-),构建人SAFB基因的真核表达载体pcDNA3.1(-)/SAFB,转染SW480细胞,用WesternBlot技术检测目的蛋白的表达。结果成功扩增人SAFB全长cDNA,双酶切鉴定和测序结果证实成功构建pcDNA3.1(-)/SAFB真核表达载体,转染细胞后,可检测出分子量约为140KD的目的蛋白。结论获得人SAFB基因全长cDNA并成功构建了SAFB真核表达载体,证实pcDNA3.1(-)/SAFB转染SW480细胞后可表达SAFB蛋白,为深入研究SAFB基因在肿瘤发生发展中的作用提供了基础。
Objective To construct eukaryotic expression vector of human SAFB gene and provide the basis for the study of SAFB gene function. Methods Human SAFB-specific primers were designed to amplify total RNA of human normal colorectal epithelium. Human SAFB cDNA was extracted by RT-PCR. SAFB cDNA was cloned into pcDNA3.1 (-) by double enzyme digestion and sequencing. The eukaryotic expression vector pcDNA3.1 (-) / SAFB was transfected into SW480 cells and the expression of the target protein was detected by Western Blot. Results The full-length cDNA of human SAFB was successfully amplified. The double digestion and sequencing confirmed that the eukaryotic expression vector pcDNA3.1 (-) / SAFB was constructed successfully. After transfection, the target protein of about 140KD was detected. Conclusions The full-length cDNA of human SAFB gene was obtained and the SAFB eukaryotic expression vector was successfully constructed. The SAFB protein was expressed after transfected with pcDNA3.1 (-) / SAFB. In order to further study the role of SAFB gene in tumorigenesis The foundation.