Objective: To analyze the amino acid polymorphism of truncated Staphylococcal enterotoxin-like toxin X (tSElX), and to evaluate its related emetic activities. Methods: Sequence of tselx was compared with both the genome sequence of 145 CC398 strains completed in our research group and the NCBI database. Primers were designed to amplify the target gene of tselx, and the fragment was recombined into pMD18-T vector and sequenced. PCR product was digested with BamHⅠ and EcoRⅠ, and constructed into plasmid pGEX-6P-1 and pET-28a (+). After recombinant plasmid was identified, the protein expression was induced by IPTG. Proteins expressed in the form of inclusion bodies were denatured and renatured, then purified by affinity chromatography and ultrafiltration. Purified tSElX protein was then fed to common marmosets with the dose of 250 μg/kg to observe the vomiting reaction. Results: tselx gene was present in 145 strains of CC398 strains from the different origins (patients, healthy people and animals) in China. Homology of the amino acid sequence of the protein from the Chinese strains appeared 100.0%, while the homology with the four American strains were 97.8%(1) and 98.9%(3), respectively. Through two sets of expression systems and different induction conditions, tSElX was expressed in the form of inclusion bodies. The high purity soluble recombinant tSElX was thus obtained by denaturated and renaturated processes. At the dose of 250 μg/kg, tSElX protein did not cause vomiting in common marmosets. Conclusions: Results of this study showed that the amino acid sequence of tSElX was highly conserved and was universally present in a particular clone group. We obtained soluble recombinant tSElX protein with high purity. We also noticed that tSElX did not have the animal emetic activity at a dose of 250 μg/kg.
目的： 分析缩短的葡萄球菌类肠毒素X（truncated Staphylococcal enterotoxin-like toxin X，tSElX）氨基酸多态性，对其进行克隆表达纯化并对催吐活性进行评价。 方法： 将tselx序列与本课题组前期完成测序的145株CC398菌株基因组序列及NCBI数据库进行比对分析。设计引物扩增tselx目的基因，将该片段重组于pMD18-T载体中并进行测序鉴定。经限制性内切酶BamHⅠ和EcoRⅠ双酶切后将目的片段构建于质粒pGEX-6P-1及pET-28a（+）中，重组质粒鉴定后，IPTG诱导蛋白表达。将以包涵体形式表达的蛋白进行变性和复性，用亲和层析法和超滤法对表达产物进行纯化。将纯化的tSElX蛋白以250 μg/kg喂饲普通狨猴，观察呕吐反应。 结果： tselx基因在我国不同来源（患者、健康人群和动物）的145株CC398菌株均存在。我国菌株编码蛋白的氨基酸序列同源性为100.0%，与4株美国菌株的同源性分别为97.8%（1株）和98.9%（3株）。通过两套表达系统及不同诱导条件，tSElX均以包涵体形式表达。通过变性及复性，获得高纯度的可溶性重组蛋白tSElX。在250 μg/kg剂量下tSElX蛋白不能引起普通狨猴的呕吐反应。 结论： tSElX氨基酸序列在CC398菌株中普遍存在，且具有一定保守性。高纯度的可溶性tSElX重组蛋白在250 μg/kg剂量下不具有普通狨猴催吐活性。.
Keywords: Emetic activity; Enterotoxin; Inclusion body; Recombinant protein; Renaturation.