鼠伤寒沙门菌Ssek3基因的序列特征分析与表达纯化

Sequence analysis, prokaryotic expression and purification of Salmonella typhimurium Ssek3 protein

目的 研究鼠伤寒沙门菌Ssek3基因的序列结构特征及蛋白表达纯化.方法 从鼠伤寒沙门菌SL1344株获得Ssek3基因的序列,利用生物信息学方法进行系统性分析,并进行原核表达及纯化.结果 软件预测分析结果显示,SseK3蛋白由335个氨基酸,72个氨基酸残基组成,蛋白质理论相对分子质量（Mr）为37.89×103,分子式为C1700 H2629 N463O497S12,等电点为6.7,属于稳定存在的亲水蛋白质.该蛋白质无信号肽也无跨膜区,属于膜内蛋白,含有5个N-糖基化位点、3个O-糖基化位点、33个磷酸化位点、22个B细胞线性结合位点和11个T细胞结合位点以及21个二硫键位点.二级结构中,α-螺旋（Hh）占34.03％（114个）,伸展链（Ee）占21.49％（72个）,β-折叠（Tt）占8.96％（30个）,无规则卷曲（Cc）占35.52％（119个）.实验结果显示,Ssek3基因大小为1008 bp,SDS-PAGE分析显示,Ssek3基因在原核表达系统中以分泌蛋白形式存在,表达的融合蛋白大小（Mr）约为40×103.结论 本研究成功克隆了鼠伤寒沙门菌Ssek3基因,对其进行序列分析和原核表达,为深入探究Ssek3蛋白在鼠伤寒沙门菌感染宿主细胞中的作用提供资料.

Objective To study the sequence structure of Salmonella typhimurium Ssek3 gene and to express it at protein level in a prokaryotic expression system .Methods Sequence of Ssek3 gene was ob-tained from Salmonella typhimurium SL1344 strain.Bioinformatics methods were used for systematic analy-sis .A prokaryotic expression system for expressing Sse3k gene was constructed and the expressed protein was purified by Ni-NTA affinity chromatography .Results Sequence analysis showed that the Ssek3 gene of Sal-monella typhimurium was 1008 bp in length, encoding a protein of 335 amino acids and 72 amino acid resi-dues.The molecular weight, molecular formula and isoelectric point of Ssek3 protein was 37.89×103, C1700 H2629 N463 O497 S12 and 6.7, which indicated that it was a stable and hydrophilic protein .Ssek3 protein was a membrane protein without signal peptide or transmembrane region , containing five N-glycosylation sites , three O-glycosylation sites , 33 phosphorylation sites , 22 linear B-cell epitopes , 11 T-cell epitopes and 21 di-sulfide bonds.The secondary structure of Ssek3 protein contained 114 α-helices （Hh） （34.03%）, 72 ex-tended chain （Ee） （21.49%）, 30β-sheets （Tt） （8.96%） and 119 random coils （Cc） （35.52%）.Re-sults of SDS-PAGE showed that the fusion protein Ssek 3 expressed in the prokaryotic expression system was a secretory protein with a molecular weight of about 40×103 .Conclusion The Ssek3 gene of Salmonella typh-imurium is successfully cloned , sequenced and expressed in this study , which will lay a foundation for fur-ther studying the role of Ssek3 protein in host cells during Salmonella typhimurium infection.