摘要
目的：构建表达小鼠抗菌肽蛋白（Bin1b）的真核表达载体pSG.SS.C3d3.YL-Bin1b，对其在HEK293细胞中的表达进行检测分析。方法应用PCR方法获得Bin1b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，利用间接免疫荧光结合激光共聚焦技术、Western blot技术、免疫组织化学染色技术及流式细胞仪检测技术对Bin1b的表达进行检测。结果构建成功后的pSG.SS.C3d3.YL-Bin1b在HEK293细胞中能够表达Bin1b。结论 Bin1b在真核细胞中的成功表达为后续研究Bin1b的高表达对受精过程的影响奠定了基础。
Objective To construct the eukaryotic recombinant plasmid pSG.SS.C3d3.YL-Bin1b and detect its expression in HEK293 cells. Methods The cDNA fragment of gene Bin1b was first produced by PCR, then inserted into the eukaryotic plasmid pSG.SS.C3d3.YL to form recombinant plasmid pSG.SS.C3d3.YL-Bin1b. After the recombinant plasmid was confirmed by restriction enzymes digestion analysis and sequence, it was transfected into HEK293 cells to express the target protein Bin1b. The expression of Bin1b was detected by immunofluorescence, Western blot, immunohistochemistry staining, and flow cytometry assay, respectively. Results The transfected recombinant vector pSG.SS.C3d3.YL-Bin1b expressed Bin1b protein in HEK293 cells. Conclusion The successful expression of Bin1b in eukaryotic cells will establish a solid base for exploring the effect of high expression of Bin1b on the fertilization process.
出处
《中国男科学杂志》
CAS
CSCD
北大核心
2014年第9期3-7,21,共6页
Chinese Journal of Andrology
基金
国家自然科学基金（81100546）
重庆市自然科学基金（CSTC,2010BB5162）资助
关键词
真核细胞
遗传载体
基因表达
荧光抗体技术
间接
eukaryotic cells
genetic vectors
gene expression
fluorescent antibody technique, indirect
