摘要
目的:在乳链菌内表达艰难梭菌A毒素受体结合区并检测其活性. 方法:提取艰难梭菌标准株基因组DNA,扩增编码艰难梭菌毒素A毒素受体结合区的羧基未端基因重复序列(14CDTA),分别连接到乳链菌表达载体pNBC1000及pNBC2000,转化乳链菌,蛋白电泳及Western-blot检测蛋白定位表达情况. 结果:成功构建了14CDTA乳链菌表达载体,并在乳链菌内表达了艰难梭菌受体结合区蛋白,分泌表达的艰难梭菌受体结合区蛋白大小约39.2 ku,膜锚定表达的受体结合区蛋白大小约为55.1 ku,所表达的蛋白均可与艰难梭菌A毒素多克隆抗体发生免疫印迹反应. 结论:表达艰难梭菌受体结合区蛋白的重组乳链菌可以识别艰难梭菌A毒索多克隆抗体,为研制防治艰难梭菌疫苗的口服生态活菌亚单位疫苗奠定了一定基础.
Abstract
AIM: To expreess the Clostridium difficile toxin A receptor binding domain and test its bioactivity. METHODS: The gene of fourteen of the 38 C-terminal toxin A repeats (14CDTA) coding for the clostridium difficile toxin A receptor binding domain was amplified and then cloned into the lactococcus lactis expression system pNBC1000 and pNBC2000 to form pNBC1001 and pNBC2001, the confirmed sequence was cut and ligated to pTRKH2 a shuttle vector for E. coli and Lactococcus lactis to generate pNBCL1001 and pNBCL2001. pNBCL1001 and pNBCL2001 were transformed into Lactococcus lactis by electroporation. Protein electrophoresis and Western-Blot were used to detect the expession of 14CDTA and its bioactivity. RESULTS: The 14CDTA expression vectors were constructed successfully. The secretory form of 14CDTA had a weight of 39.2 ku and the cell anchored form of 14CDTA had a weight of 55.1 ku. All the forms of 14CDTA could be deteced with the clostridium difficile toxin A multicloned antibody by Western Blotting. CONCLUSION: The recombinant lactococcus lactis expressing the 14CDTA may lay a foundation for vaccine against Clostridium difficile.
基金项目
国家自然科学基金自由申请面上项目(30570839)
广东省自然科学基金(05004735)
NETL
NSTL
维普
万方数据