【摘要】 【目的】革兰氏阳性菌的表面蛋白在病原菌致病性方面具有重要作用,表面蛋白锚定到细胞壁过程的关键酶—分选酶成为抗感染的新靶点。【方法】本文利用GenBank中的分选酶A基因(srtA)序列设计特异性引物,以金黄色葡萄球菌基因组DNA为模板进行PCR扩增,获得618bp的DNA片段。按照常规分子克隆操作成功构建两种原核表达载体pet22-srtA和pTRX-srtA,转入大肠杆菌感受态BL21(DE3)中,在1mmol/LIPTG诱导下进行表达。利用SDS-PAGE和Western blot进行鉴定和分析,【结果】结果显示:(1)重组载体pet22-srtA和pTRX-srtA分别表达出相对分子量为约45kDa和39kDa的外源蛋白;【结论】(2)分子伴侣硫氧还蛋白(Trx)有利于分选酶A基因的可溶性表达。该实验为后续的分选酶酶学性质研究特别是抑制剂筛选研究奠定良好基础。更多还原

【Abstract】 [Objective]Sortase is a novel anti-infection target enzyme for its critical action of anchoring surface proteins to the cell wall. [Methods] We amplified the srtA gene from Staphylococcus aureus chromosomal DNA by PCR technique, and then constructed two prokaryotic expression vectors pet22-srtA and pTRX-srtA with regular molecular cloning operation. The pet22-srtA and pTRX-srtA were transformed into Eschericheria coli BL21 (DE3) competent cells and overexpressed under 1 mmol/L IPTG (isopropy-β-D-thiogalactoside) induction. [Results] SDS-PAGE and western blot results show that approximately 45 kDa and 39 kDa proteins were expressed by pet22-srtA and pTRX-srtA respectively. [Conclusion] The molecular chaperone thioredoxin was beneficial to the prokaryotic expression of srtA gene. Moreover, the experiment laid solid foundation to study sortase’s enzymatic property and inhibitors screening especially.更多还原