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目的研究沙门菌感染宿主细胞的过程中,沙门菌III型分泌系统分泌的效应蛋白SopB对宿主细胞蛋白激酶MAPK的激活作用,同时寻找SopB蛋白与MAPK激活有关的结构域。方法通过基因克隆的方法将沙门菌SopB全长基因以及各种SopB truncation均被克隆至pCS2-EGFP质粒载体中,利用磷酸钙转染法将该SopB重组质粒转染至HeLa细胞,24h后通过westernblot方法利用磷酸化的pERK1/2、p-p38以及pJNK抗体检测HeLa细胞MAPK蛋白激酶受SopB激活的情况。结果在HeLa细胞中过量表达SopB蛋白能够显著诱导蛋白激酶MAPK的磷酸化激活,进一步在HeLa细胞中表达SopB各种truncation片段证实,SopB对HeLa细胞MAPK蛋白激酶的激活与其N末端29个氨基酸直接相关。结论 SopB可以激活宿主细胞MAPK信号途径,且这种活性与其N末端29个氨基酸有关。
Objective To study the activation of SopB, a Salmonella type III secretion system, on the activation of protein kinase MAPK in Salmonella typhimurium infected host cells, and to find out the domains of SopB protein involved in MAPK activation. Methods The full-length gene of Salmonella SopB and various SopB truncation genes were cloned into pCS2-EGFP plasmid vector by gene cloning method. The SopB recombinant plasmid was transfected into HeLa cells by calcium phosphate transfection method. After 24 h, Methods The phosphorylation of pERK1 / 2, p-p38 and pJNK antibodies were used to detect the activation of MAPK protein kinase in HeLa cells by SopB. Results Overexpression of SopB protein in HeLa cells induced phosphorylation of protein kinase MAPK, and further expressed various truncation fragments of SopB in HeLa cells. The results showed that activation of MAPK protein kinase in HeLa cells was directly related to the N-terminal 29 amino acids . Conclusion SopB can activate the MAPK signaling pathway of host cells, and this activity is related to its N-terminal 29 amino acids.