小鼠受精素β亚单位真核表达载体pSG.SS.C3d3.YL-F_β构建及在HEK293细胞中的表达分析

目的:构建表达小鼠受精素β去整合素结构域(Fβ)的真核表达重组载体pSG.SS.C3d3.YL-Fβ,并对其在HEK293细胞中的表达进行分析。方法:应用PCR方法获得Fβ的cDNA片断,将其插入pSG.SS.C3d3.YL中,经酶切鉴定正确后转染HEK293细胞,并利用间接免疫荧光结合激光共聚焦技术、Western印迹技术、免疫组织化学染色技术及流式细胞仪检测技术对Fβ的表达进行检测。结果:成功构建了Fβ的真核表达重组载体pSG.SS.C3d3.YL-Fβ,该重组载体在HEK293细胞中能够表达Fβ。结论:Fβ在真核细胞中的成功表达为后续研究Fβ的高表达对受精过程的影响奠定了基础。

Construction and Expression of Eukaryotic Recombinant of Mouse Fertilin β Subunit in HEK293 Cells

OBJECTIVE: To construct the eukaryotic expression recombinant pSG. SS. C3d3. YL-Fbeta and analyze the expression of mouse fertilin beta subunit in HEK293 cells. METHODS: The cDNA fragment expressing the disintegrin domain of mouse fertilin beta was obtained by PCR, and then inserted into the eukaryotic plasmid pSG. SS. C3d3. YL to get recombinant plasmid pSG. SS. C3d3. YL-Fbeta, which was transfected into the HEK293 cell line to express the target protein Fbeta after identified by restriction enzyme digestion. And then Fbeta was detected by indirect immunofluorescence through confocal laser scanning microscopy, Western blot, immunohistochemistry staining and flow cytometry assay. RESULTS: The recombinant vector pSG.SS.C3d3. YL-Fbeta could express Fbeta in HEK293 cells. CONCLUSION: The expression of Fbeta in eukaryotic cells provides a foundation for further researches on the effect of high F, expression on fertilization process.