畜牧与兽医2025，Vol.57Issue(6)：83-91,9.
布氏杆菌分泌蛋白BspD真核表达载体的构建及在小鼠巨噬细胞中的表达
Construction of eukaryotic expression vector of Brucella secretory proteins BspD and expression in murine macrophages
摘要
Abstract
This study was to construct a eukaryotic expressive vector of Brucella bspD and to express this gene in murine macrophage RAW 264.7 cells.Specific primers were designed based on the sequence information of Brucella abortus bspD published in GenBank,and the bspD gene was amplified by PCR.Then,the fragment was cloned into a pcDNA3.1-EGFP vector to obtain a eukaryotic vector pcDNA3.1-EGFP-bspD.The vector was transfected into murine macrophage RAW 264.7 cells by Lipofectamine 3000 immediately after it was confirmed by en-zyme digestion and sequencing analysis.Next,BspD protein expression in RAW 264.7 cells was detected by Western blot.The secretion lev-els of cytokines gamma interferon(IFN-γ)and interleukin-4(IL-4)were detected by an ELISA kit,and the level of cytotoxicity was detected by a water soluble tetrazolium salt-1(WST-1)cytotoxicity detection kit.Finally,cell proliferation was detected using a cell count-ing kit-8(CCK-8).The expression levels of apoptosis genes cysteinyl aspartate specific proteinase-3(Caspase-3)and cysteinyl aspartate specific proteinase-8(Caspase-8)were detected using real-time fluorescence quantitative PCR(RT-qPCR).The results showed that a 936 bp bspD gene fragment was amplified by PCR from the positive cloning vector.The positive cloning vector was verified by restriction en-zyme Kpn Ⅰ and BamH Ⅰ digestion.The sequencing analysis showed that the homology of the gene was 100％with the sequencing informa-tion published in GenBank.These results indicated that a eukaryotic expression vector pcDNA3.1-EGFP-bspD was successfully constructed in this study.The WB detection results showed that the transfection group of pcDNA3.1-EGFP-bspD displayed bands at 36 kDa,which was completely consistent with the expected results,indicating that BspD protein could be expressed in RAW 264.7 cells.The cytokines test re-sults showed that BspD protein could induce RAW 264.7 cells to secrete Th1 cytokine IFN-γ and Th2 cytokine IL-4.BspD protein was non-toxic to RAW 264.7 cells,and it had no significant effect on cell proliferation.The Caspase-3 and Caspase-8.Brucella secreted protein BspD could be expressed in RAW 264.7 cells.This finding provided reference for further research on the function and molecular mechanism of BspD.关键词
布氏杆菌/BspD蛋白/真核表达/巨噬细胞Key words
Brucella/BspD protein/eukaryotic expression/macrophage分类
农业科技引用本文复制引用
李志强,解然,王书利,朱庆妍,蔺秋慧,高丰衣,马亚静,齐德重,祝芳,孙婷..布氏杆菌分泌蛋白BspD真核表达载体的构建及在小鼠巨噬细胞中的表达[J].畜牧与兽医,2025,57(6):83-91,9.基金项目
商丘医学高等专科学校开放课题(KFKT23016) （KFKT23016）
河南省高等学校重点科研项目(24A230014) （24A230014）
