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【目的】法氏囊活性五肽Bursopentin(BP5)是一种多功能生物学活性因子,不仅具有免疫调节和抗氧化功能,还具有抗肿瘤活性。目前,BP5发挥抗肿瘤生物功能的作用机制尚不清晰。【方法】本研究利用T7噬菌体展示技术构建鸡DT40细胞T7噬菌体c DNA文库,以BP5-BSA为靶分子对文库进行亲和淘选,通过序列测定和生物信息学分析,获得BP5的结合蛋白。通过分析BP5诱导的鸡DT40细胞mRNA基因表达谱和检测BP5对HCT116细胞中p53 Luciferase活性和p53信号通路中相关蛋白表达的影响来探讨BP5抗肿瘤的作用机制。【结果】构建的鸡DT40细胞T7噬菌体c DNA文库原始滴度为1.2×104pfu/m L,重组率为91.7%,扩增后滴度为1.9×109pfu/m L;通过生物淘选获得3条与BP5结合的鸡源蛋白血管内皮生长因子A(vascular endothelial growth factor A)、细胞周期蛋白E1(cyclin E1)和运输蛋白颗粒复合体2(trafficking protein particle complex 2)。基因表达谱分析结果显示,BP5参与调节多条与抗肿瘤功能有关的通路,其中包括p53信号通路,且p53信号通路涉及的5个差异表达基因均与细胞周期调控或抗肿瘤相关,其中SIAH1、TP53(p53)、CIP1(p21)基因被上调,CHEK2、CCNE1基因被下调。RT-PCR方法检测10个表达水平有明显变化的差异基因,其结果与基因芯片分析结果相符;BP5对p53 Luciferase活性的影响实验结果显示,BP5在0.2-20μg/m L浓度下能够明显提高HCT116细胞中p53相对Luciferase活性,Western blotting结果证实,BP5能显著促进p53、p21蛋白的表达水平,而CCNE1和CHEK2蛋白的表达水平则明显降低。【结论】本研究结果表明BP5通过p53信号通路途径发挥其抗肿瘤生物学功能,为进一步研究BP5抗肿瘤具体的分子机制提供了参考依据。
【OBJECTIVE】 Bursopentin (BP5), a multifunctional biological active factor, not only has immunomodulatory and anti-oxidative functions, but also has antitumor activity. At present, the mechanism of action of BP5 on antitumor biological function is not clear. 【Method】 T7 phage display library was used to construct T7 phage cDNA library of chicken DT40 cells. Affinity panning was performed using BP5-BSA as a target. The binding protein of BP5 was obtained by sequence analysis and bioinformatics analysis. The anti-tumor mechanism of BP5 was explored by analyzing the mRNA expression profile of BP5-induced chicken DT40 cells and detecting the effect of BP5 on p53 Luciferase activity and p53-related protein expression in HCT116 cells. [Results] The original titer of T7 phage cDNA library of chicken DT40 cells was 1.2 × 10 4 pfu / m L, the recombination rate was 91.7% and the titer was 1.9 × 109 pfu / m L after being amplified. Three biotinylated strains The chicken source proteins, vascular endothelial growth factor A, cyclin E1, and trafficking protein particle complex 2, which bind to BP5. The results of gene expression profiling showed that BP5 is involved in the regulation of several pathways involved in anti-tumor function, including the p53 signaling pathway. The five differentially expressed genes involved in p53 signaling pathway are all associated with cell cycle regulation or anti-tumor activity. SIAH1, TP53 (p53), CIP1 (p21) gene is upregulated, CHEK2, CCNE1 gene is down-regulated. RT-PCR method was used to detect 10 differentially expressed genes, and the results were in good agreement with the results of microarray analysis. The effect of BP5 on the activity of p53 Luciferase showed that BP5 significantly increased HCT116 concentration at 0.2-20μg / mL P53 relative Luciferase activity in cells, Western blotting results confirmed that BP5 can significantly promote the expression of p53, p21 protein, while the expression of CCNE1 and CHEK2 protein was significantly lower. 【Conclusion】 The results of this study indicate that BP5 exerts its anti-tumor biological function through p53 signaling pathway, which provides a reference for further studying the specific molecular mechanism of BP5 antitumor.