基于pcDNA3.1框架的p38β真核表达质粒的构建及其功能研究

Study on Construction and Functional Characterization of a pcDNA3.1-Based Eukaryotic Expression Plasmid for p38β

摘要: 目的：为探究p38β在巨噬细胞炎症反应中的调控机制，建立鼠源pcDNA3.1-N-3 × Flag-p38β真核表达质粒，转染RAW264.7细胞建立实验模型。通过脂多糖(LPS)刺激，从细胞增殖、凋亡、促炎因子表达三个方面研究该重组质粒的生物学功能。方法：通过PCR扩增p38β基因，构建基于pcDNA3.1框架的p38β真核表达质粒。利用EcoRI/XhoI限制性位点双酶切，将纯化的目的片段进行体外重组，转化细菌感受态细胞。经酶切鉴定、测序与比对分析后，将重组质粒转染至LPS刺激的RAW264.7细胞中，通过Western blot和qRT-PCR技术及EdU法、ELISA法检测其对LPS诱导的RAW264.7细胞增殖和凋亡的影响，以及肿瘤坏死因子-α (TNF-α)、白细胞介素-6 (IL-6)、白细胞介素-1β (IL-1β)等炎症介质的分泌水平。其中，Western blot技术用于检测凋亡相关蛋白表达，qRT-PCR解析基因转录调控，EdU法检测DNA合成活性，ELISA法检测炎症因子分泌量。结果：双酶切鉴定和Western blot结果显示pcDNA3.1-N-3 × Flag-p38β真核表达质粒构建成功并表达。EdU实验显示：LPS刺激RAW264.7细胞24 h后，p38β过表达组的细胞存活率显著低于空载体对照组。Western blot及PCR结果显示：p38β过表达组细胞的促凋亡蛋白Bax表达高于转染空载体的对照组，抗凋亡蛋白Bcl-2及增殖蛋白PCNA表达低于空载体对照组(均P < 0.05)。Western blot和ELISA结果显示：pcDNA3.1-N-3 × Flag-p38β质粒转染使RAW264.7细胞中TNF-α、IL-6、IL-1β的表达较对照组升高，差异均有统计学意义(P < 0.05)。结论：p38β能抑制LPS刺激的RAW264.7细胞增殖，并促进其凋亡，同时能上调TNF-α、IL-6和IL-1β等炎症介质的表达。

Abstract: Objective: To investigate the regulatory mechanism of p38β in macrophage inflammatory responses, a murine pcDNA3.1-N-3 × Flag-p38β eukaryotic expression plasmid was constructed and transfected into RAW264.7 cells to establish an experimental model. Lipopolysaccharide (LPS) stimulation was used to study the biological functions of this recombinant plasmid in terms of cell proliferation, apoptosis, and pro-inflammatory cytokine expression. Methods: The p38β gene was amplified by PCR to construct the p38β eukaryotic expression plasmid based on the pcDNA3.1 framework. Purified target fragments were recombined in vitro using EcoRI/XhoI restriction sites and transformed into bacterial competent cells. After restriction enzyme digestion, sequencing, and comparative analysis, the recombinant plasmid was transfected into LPS-stimulated RAW264.7 cells. Western blot, qRT-PCR, EdU assay, and ELISA were employed to assess its effects on LPS-induced proliferation, apoptosis, and secretion levels of inflammatory mediators such as tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β). Specifically, Western blot analyzed apoptosis-related protein expression, qRT-PCR elucidated transcriptional regulation, the EdU assay measured DNA synthesis activity, and ELISA quantified inflammatory cytokine secretion. Results: Restriction enzyme digestion and Western blot confirmed the successful construction and expression of the pcDNA3.1-N-3 × Flag-p38β plasmid. The EdU assay revealed that 24 h after LPS stimulation, cell viability in the p38β overexpression group was significantly lower than in the empty vector control group. Western blot and qRT-PCR results demonstrated that the p38β overexpression group exhibited higher expression of the pro-apoptotic protein Bax and lower expression of the anti-apoptotic protein Bcl-2 and proliferating cell nuclear antigen (PCNA) compared to the control group (P < 0.05). Additionally, Western blot and ELISA showed that transfection with pcDNA3.1-N-3 × Flag-p38β significantly upregulated the expression of TNF-α, IL-6, and IL-1β in RAW264.7 cells compared to the control group (P < 0.05). Conclusion: p38β inhibits LPS-induced proliferation and promotes apoptosis in RAW264.7 cells while upregulating the expression of inflammatory mediators such as TNF-α, IL-6, and IL-1β.