pEGFP-N1-omp25荧光真核表达载体的构建及鉴定

Construction and Identification of pEGFP-N1-omp25 Fluorescent Eukaryotic Expression Vector

目的克隆布鲁氏杆菌外膜蛋白omp25基因并构建荧光真核表达载体p EGFP-N1-omp25,检测其在大鼠脑内的表达。方法根据Genebank中omp25基因的核苷酸序列,设计并合成1对引物,以布鲁氏杆菌总基因组DNA为模板,进行PCR扩增得到omp25基因片段,并将omp25基因片段及质粒p EGFP-N1分别进行进行酶切、体外连接,使其定向重组,构建其重组质粒。将制备的重组质粒经侧脑室注射至大鼠,检测p EGFP-N1-omp25在大鼠脑内的表达。结果 PCR扩增出642bp大小的片段,挑取的LB固体培养基上的单菌落经菌液PCR、酶切、测序表明成功构建了p EGFP-N1-omp25荧光真核表达载体。质粒经大鼠侧脑室注射后,脑组织冰冻切片可见荧光表达。结论采用体外重组技术,成功构建了布鲁氏杆菌外膜蛋白omp25基因的真核表达载体,且重组质粒在大鼠脑组织内成功表达。

Objective To clone B. melitensis omp25 gene,to construct its fluorescent eukaryotic expression vector,to detect its expression in the rat brain and to explore the role of omp25 protein in Brucella virulence.Methods A pair of primers was designed and synthesized with Brucella coli total genomic DNA as a template,according to omp25 gene nucleotide sequences in Genebank,and omp25 gene fragments were obtained by PCR amplification. Both PCR product omp25 gene and plasmid p EGFP- N1 DNA were constructed to recombinant plasmid by enzyme digestion,be linked in vitro,and directed recombination. The plasmid was injected into the lateral ventricle of rats,and the recombinant plasmid p EGFP- N1- omp25 distribution and expression in rat brain were detected. Results PCR yielded a fragment of 642 bp. Single clones were identified by double digestion with EcoRI and Sal I. Sequence analysis showed that expression vector p EGFP- N1-omp25 had been constructed successfully. After injection of the plasmid into the lateral ventricle of rats,frozen sections of brain tissue showed fluorescence. Conclusion Using in vitro recombination technology,we successfully constructed Brucella outer membrane protein bacillus omp25 gene eukaryotic expression vector,and the recombinant plasmid successfully expressed in rats. This laid a solid foundation for the subsequent research on the role and the molecular mechanism of Brucella outer membrane protein omp25 gene in the central nervous system infection of Brucella.