羊布鲁氏菌omp25基因原核表达载体的构建与鉴定

目的:利用PCR反应从羊布鲁氏菌基因组DNA中克隆omp25基因,并将其构建到原核表达载体pET-32a中。方法:试剂盒法提取羊布鲁氏菌基因组DNA,设计合适的引物,通过PCR反应从基因组中扩增外膜蛋白OMP25的编码基因(omp25)。将得到的基因片段连接到克隆载体pMD19-T的多克隆位点(MCS)中,将连接体pMD19-T-omp25转化感受态大肠杆菌DH5(并进行筛选和克隆扩增,提取质粒并进行酶切分析与序列测定。利用酶切与连接反应将目的基因(omp25)插入载体pET-32a的多克隆位点中,将连接体pET-32a-omp25转化感受态大肠杆菌DH5(并进行筛选和克隆扩增,提取质粒并进行酶切分析。结果:PCR扩增得到的基因片段与GenBank中已公布的羊布鲁氏菌omp25基因同源性为99%。酶切分析结果表明,omp25基因成功地插入载体pET-32a中。结论:成功地扩增得到了羊布鲁氏菌omp25基因并构建了携带该基因的重组原核表达载体pET-32a-omp25,为后续的研究提供了可靠的基础。

CONSTRUCTION OF OMP25 GENE IN PROKARYOTIC EXPRESSION VECTOR FROM B. MELITENSIS

Objective: To explore the function of outer membrane protein OMP25 in Brucella melitensis M5 strain,a prokaryotic expression vector containing omp25 was constructed.Methods: The genome DNA of B.melitensis was isolated with commercial available kit.The PCR product containing omp25 gene was inserted into vector pMD19-T.The recombinant plasmids pMD19-T-omp25 was transformed into competent E.coli DH5α.Then the omp25 gene fragment was cut out and cloned into the prokaryotic vector pET-32a which was transformed into E.coli DH5α.Results: An expected gene fragment was isolated from the bacterial genome by PCR amplification and confirmed with sequencing.Conclusions: The recombinant expression plasmid pET-32a-omp25 was correctly constructed,which could provide the materials for OMP25 function analysis.