| 摘要: |
| 【目的】探讨原核表达猪囊尾蚴TsCL-1蛋白的生物学特性，为研究半胱氨酸蛋白酶(Cysteine proteinase,CP)在猪囊尾蚴与宿主相互关系中的作用奠定基础。【方法】将含有猪囊尾蚴半胱氨酸蛋白酶TsCL-1基因的pGEX-4T TsCL-1重组质粒转入大肠杆菌BL21进行表达，诱导后的重组菌裂解上清通过谷胱甘肽（GST）琼脂糖亲和层析法进行纯化，纯化产物利用明胶蛋白电泳法进行活性检测。【结果】经SDS-PAGE分析显示，猪囊尾蚴TsCL-1基因表达的重组蛋白相对分子质量约为61 ku，表达的目的蛋白约占菌体总蛋白的57.4%。表达产物应用GST琼脂糖凝胶纯化后，纯化蛋白的纯度可达90%以上。明胶蛋白电泳结果显示，纯化蛋白对明胶具有水解活性，并且其水解活性能被半胱氨酸蛋白酶特异性抑制剂E-64抑制。【结论】从重组菌中成功表达了目的蛋白，该蛋白具有明显的水解活性，且E-64对其水解活性有抑制作用。 |
| 关键词: 猪囊尾蚴；半胱氨酸蛋白酶；TsCL1基因 原核表达；纯化；水解活性 |
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| 基金项目:国家高科技研究发展计划“863”项目（ 2006AA10A207） |
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| Prokaryotic expression of TsCL-1 gene from Taenia solium metacestode |
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| Abstract: |
| 【Objective】In order to lay a basis for researching the role of cysteine protease (CP) in the relationship between Cysticercus cellulosae and its host,the study explored the characteristics of the recombinant protein expressed prokaryotically.【Method】The recombinant plasmid pGEX-4T TsCL-1 was transformed into and prokaryotically expressed in E.coli BL21.The expressed products were purified by using GST sepharose FF affinity chromatography.The proteolytic activity of CP was assayed by using zymography.【Result】 SDS-PAGE analysis showed that the molecular weight of recombinant protein was about 61 ku,which corresponded well to the predicted size from the primary sequence of the gene.The purity of the purified protein was more than 90%.The analysis of hydrolysis activity and inhibition experiments showed that CP possessed a hydrolytic activity,and the hydrolytic activity can be specifically inhibited by E-64. 【Conclusion】The target protein was successfully expressed,and the protein has obviously hydrolysis activity.The hydrolytic activity of this protein can be inhibited by E-64. |
| Key words: Cysticercus cellulose cysteine protease TsCL1 gene prokaryotic expression purification hydrolytic activity |
