克隆、测序布鲁氏菌疫苗株M5—90外膜蛋白基因OMP31,原核表达OMP31并对其检测。从羊种布鲁氏菌M5-90中PCR获得OMP31基因,连接到pBS—T克隆质粒并测序;将测序正确的基因片段克隆人大肠杆菌表达载体pET-28a,进行SDS—PAGE,而后Western—blot检测。结果M5-90疫苗株的OMP3I基因序列与羊种布鲁氏菌参考株16M的同源性为99.03%;外膜蛋白基因OMP31在大肠杆菌表达后能够被Western—blot检测到。结论:表达的布鲁氏菌疫苗株M5-90外膜蛋白OMP31可以被布鲁氏菌特异性血清识别,表现出良好的抗原性,为以后实验室诊断和疫苗的研究做好坚实的上游工作。
Brucella melitensis M5-90 vaccine strain OMP31 gene was cloned, sequenced,expressed and detected. OMP31 gene was amplified by PCR,cloned into pBS-T vector and sequenced. The objective gene was recombined into pET-28a vector, expressed and detected by SDS-PAGE and Western-blot. The results showed there was 99.03% identity for OMP31 gene between B. melitensis M5-90 vaccine strain and 16M reference strain and the expressed OMP31 was detected by Western-blot. The conclusion conveyed us that the special serum against bruceUa wild strain could recognize the expressed OMP31 and show it is an immunodominant antigen. This experiment gives us a good upstream work to laboratory diagnosis and study of vaccine.