【摘要】 目的构建膜结合型M-CSF(mM-CSF)及其胞内区截短30个氨基酸的剪切体(mM-CSF-Δ)重组逆转录病毒表达载体。方法用DNA重组技术构建并鉴定mM-CSF和mM-CSF-Δ的重组逆转录病毒表达载体MSCVPGK-GFP-mM-CSF、MSCV-PGK-GFP-mM-CSF-Δ,与空载体对照MSCV-PGK-GFP分别转染Phoenix细胞包装病毒,并感染HEK293细胞,通过流式分选术获得3种阳性细胞。结果经Phoenix包装的重组及对照逆转录病毒成功感染HEK293细胞,获得了稳定表达细胞株HEK293-M、HEK293-M-Δ和对照细胞株HEK293-V。RT-PCR以及Western blotting法检测发现HEK293细胞中有mM-CSF和mM-CSF-Δ的表达,HEK293-M细胞和HEK293-M-Δ细胞经流式抗体标记后均能检测到膜蛋白的表达。结论成功构建了mM-CSF及其剪切体的重组逆转录病毒表达载体。更多还原

【Abstract】 Objective To construct membrane-bound macrophage colony-stimulating factor( mM-CSF) and recombinant retroviral expression vector of brachytmema mutation of 30 amino acide located in the intracellular region of mM-CSF( mM-CSF-Δ). Methods The retroviral vectors MSCV-PGK-GFP-m M-CSF and MSCV-PGK-GFP-mM-CSF-Δ were constructed and identified by DNA recombinant techniques. Recombinant and empty vectors were used to transfect the packaging Phoenix cells. HEK293 cells were infected by the viral supernatants. After being sorted by flow cytometry,three positive cell lines were obtained. Results HEK293 cells were successfully infected by retroviruses in packaging Phoenix cells and control retrovirus. Stable expressing cell lines,HEK293-M and HEK293-M-Δ as well as control cell line HEK293-V,were established. The expression of m M-SCF and m M-CSF-Δ was detected by RT-PCR and Western blotting in HEK293 cells and its membrane protein expression was also detected by flow cytometry. Conclusion The m M-CSF and recombinant retroviral expression vector of spliceosome were successfully constructed.更多还原