破伤风毒素保护性抗原在毕氏酵母中的分泌表达

High-level Secretion Expression of Protective Fragment of Tetani Toxin in Pichia pastoris and Affecting Factors in Inducing Expression

采用PCR方法,从C.Tetani 64008菌株中扩增大小为1353 bp的破伤风毒素与靶细胞起结合作用的重链C端基因(Tetc),直接连接pGEM-T载体进行测序,并以pPIC9K为表达载体构建重组表达质粒,经线形化的重组质粒电转化毕氏酵母细胞GS115和KM71,甲醇诱导获得了分泌表达,表达产物存在于培养上清中,占分泌蛋白的10％。通过免疫印迹可以检测到重组表达产物。活性测定表明,重组蛋白具特异结合活性。本研究通过实现破伤风毒素保护性抗原在酵母系统中的分泌表达。研究其影响因素,为其它细菌毒素蛋白高效可溶性表达,及进一步抗原片段介导保护性免疫研究及抗毒素制备奠定了基础。

In order to clone and express the protective fragment of Tetani toxin in Pichia pastoris, the C fragment of toxin was amplified with PCR from strain C. Tetani 64008 and cloned into yeast expression vector pPIC9K. Cloned Tetc gene was expressed in Pichia pastoris GS115 or KM71 cells. Expression product was i-dentified by Western Blotting and the specific binding activity to antiserum was identified in ELISA. Results showed about 1 353 bp DNA fragment containing protective antigenic determinants was amplified from strain of C. Tetani 64008. Cloned gene was expressed in soluble form by secreting into culture mixture. Recombinant Tetc was of 10% of total secreted protein and easily identified by SDS-PAGE analysis and Western blotting. The Recombinant Tetc protein can react specially with Tetani neurotoxin antiserum. It could be concluded that the successful cloning and expression of Tetc with specific antibody binding activity are conducive to further study on immunology and making antitoxin.