M-CSF胞质内稳定表达细胞系的构建和鉴定

Construction and Identification of M-CSF Stably Expressing Cell Line in Cytoplasm

目的构建真核细胞pCMV/cyto/myc-M-CSF载体,建立M-CSF胞质内稳定表达细胞系,为进一步研究M-CSF的胞内作用奠定基础。方法构建靶向定位载体pCMV/cyto/myc-M-CSF,PCR及双酶切鉴定后转染HeLa细胞,G418筛选阳性克隆,RT-PCR和免疫细胞化学鉴定M-CSF的表达及M-CSF的蛋白定位。结果重组质粒pCMV/cyto/myc-M-CSF用M-CSF特异性的引物进行PCR扩增,结果显示插入片段约为1 400 bp,双酶切后分别得到约5.0 kb和1 400 bp的两条带,M-CSF分子大小与预期一致。RT-PCR、免疫细胞化学结果表明转染M-CSF的HeLa细胞高表达M-CSF(P<0.05),并且转染M-CSF的HeLa细胞表达的M-CSF蛋白定位于细胞质。结论成功构建了稳定高表达胞质M-CSF的HeLa细胞系。

Objective To establish a cell line that stably expresses cytoplasmic M - CSF to explore the further effect of cytoplasmic M - CSF. Methods The constructed pCMV/eyto/mye - M - CSF plasmid was identified by PCR and double digestion. Both pCMV/cyto/mye and pCMV/eyto/mye - M - CSF vectors were trasfected into HeLa cells by using liposome and screening with G418. Then RT - PCR and immunocytochemistry were confirmed to detect the expression and localization of M - CSF in HeLa cells. Results The size of the recombinant vector pCMV/cyto/mye - M - CSF was 1400bp. After transfeeting,high levels of both M - CSF mRNA and M - CSF protein were expressed and localized to the cytosol in M -CSF- transfected HeLa cells（ P 〈0.05 ） ,which suggested that a cell line that highly expressed cytoplasmic M - CSF was successfully established. Conclusion A cell line that highly expressed cytoplasmic M - CSF was established.