流产型布鲁氏菌Omp22基因酵母双杂交诱饵载体的构建及鉴定

Construction and Identification of Bait Vector Containing Omp22 Gene from Brucella abortus in Yeast Two Hybrid System

通过PCR获得流产型布鲁氏菌Omp22基因的编码区,将其克隆到pSOS载体中,构建酵母双杂交系统的诱饵载体pSOS-Omp22。测序正确后,将重组质粒导入cdc25H检测其表达产物对酵母细胞有无毒性和自激活作用,且在酵母细胞中定位是否正确。结果表明,获得了正确的流产型布鲁氏菌Omp22基因编码区,并成功克隆到pSOS诱饵载体中,且转化有诱饵载体的cdc25H在SD/Glucose(-L)营养缺陷平板上生长良好,说明表达产物对酵母细胞无毒性,对报告基因也无自激活作用且其定位正确。表明pSOS-Omp22可应用在酵母双杂交系统中,用于寻找巨噬细胞cDNA文库中与Omp22相互作用的蛋白质。

The coding region of Omp22 gene from Brucella abortus was amplified by PCR and then fused with pSOS vector.After confirmation with sequence analysis,the plasmid was transformed into the yeast cell cdc25H,the toxicity and transcriptional auto activation of expressed protein were tested.The results indicated that the coding region was successfully amplified and subcloned into pSOS vector,and the cdc25H transformed with bait plasmid grew well on SD/glucose（-L） plate.These data showed that the bait vectors pSOS-Omp22 was expressed correctly without toxicity and could not auto activate.The pSOS-Omp22 could be applied for the screening of proteins interacting with the Omp22 in macrophages cDNA library in the yeast two hybrid systems.