鼠源pcDNA3.1-3×Flag-c-NUP85真核表达质粒的构建及其部分功能研究

Construction of eukaryotic expression of mouse derived pcDNA3.1-3×Flag-c-NUP85 plasmid and its partial function research

目的构建鼠源pcDNA3.1-3×Flag-c-NUP85真核表达质粒,并观察其对脂多糖(LPS)诱导的RAW264.7细胞中炎症相关因子分泌的影响及对RAW264.7细胞增殖和凋亡的影响。方法通过PCR扩增NUP85基因,构建pcDNA3.1-3×Flag-c-NUP85真核表达质粒。将pcDNA3.1-3×Flag-c载体进行酶切。纯化的PCR产物与载体连接,将连接产物转化细菌感受态细胞。酶切鉴定后,再进行测序和比对分析。然后将其转染至RAW264.7细胞中,通过CCK-8实验和流式细胞术检测其对LPS诱导的RAW264.7细胞增殖和凋亡的影响,并通过Western blot技术和ELISA法检测RAW264.7细胞中肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)的表达情况。结果酶切鉴定和Western blot结果显示pcDNA3.1-3×Flag-c-NUP85真核表达质粒成功构建和表达。CCK-8实验结果显示:24 h后过表达NUP85组细胞的存活率显著低于对照组[(0.55±0.03)vs(0.67±0.05),F=30.98,P<0.05]。流式细胞术结果显示:过表达NUP85组细胞的凋亡率高于对照组[(15.78±1.05)%vs(13.40±0.47)%,F=75.38,P<0.05]。Western blot和ELISA结果显示:转染pcDNA3.1-3×Flag-c-NUP85质粒后,RAW264.7细胞中的TNF-α、IL-6表达较对照组升高,差异均有统计学意义(P<0.05)。结论NUP85能够抑制LPS刺激的RAW264.7细胞的增殖并促进其凋亡,并且NUP85促进LPS刺激的RAW264.7细胞中炎症因子IL-6和TNF-α的表达。

Objective To construct a mouse derived pcDNA3.1-3×Flag-c-NUP85 expression plasmid and observe its effect on expression of inflammation factors in LPS-induced RAW264.7 cells,as well as on the proliferation and apoptosis of RAW264.7 cells.Methods The NUP85 gene was amplified by PCR to construct pcDNA3.1-3×Flag-c-NUP85 eukaryotic expression plasmid.The pcDNA3.1-3×Flag-c vector was divided with enzymes.The purified PCR product was ligated with the vector,and the ligated product was transformed into bacterial competent cells.After identification by enzyme digestion,sequencing and analysis were performed.Then,it was transfected into RAW264.7 cells,and the blank plasmid without NUP85 gene was set as the control group.The effect on cell proliferation and apoptosis were detected by CCK-8 assay and flow cytometry,and the expression of inflammatory cytokines such as tumor necrosis factor-α(TNF-α)and interleukin-6(IL-6)in LPS-induced RAW264.7 cells was detected by Western blot and ELISA.Results Enzyme digestion identification and Western blot results showed that pcDNA3.1-3×Flag-c-NUP85 eukaryotic expression plasmid was successfully constructed and expressed.The results of CCK-8 assay showed that the cell survival rate of NUP85 overexpression group was significantly lower than that of control group after 24 h[(0.55±0.03)vs(0.67±0.05),F=30.98,P<0.05].The results of flow cytometry showed that the cell apoptosis rate of NUP85 overexpression group was higher than that of control group[(15.78±1.05)%vs(13.40±0.47)%,F=75.38,P<0.05].The results of Western blot and ELISA showed that after transfection of pcDNA3.1-3×Flag-c-NUP85,the expression of TNF-αand IL-6 in RAW264.7 cells were higher than those in the control group,with statistical significance(P<0.05).Conclusion NUP85 can inhibit the proliferation and promote apoptosis in LPS-stimulated RAW264.7 cells,and NUP85 can promote the expression of inflammatory cytokines IL-6 and TNF-αin LPS-stimulated RAW264.7 cells.