呼和浩特市区牛种布鲁菌bp26和omp10的克隆

The Cloning of bp26 and omp10 Genes of Brucella Abortus in Huhhot

为明确试验布鲁菌和参考布鲁菌菌株与基因库中布鲁菌外膜蛋白BP26和OMP10基因间的同源性。利用布鲁菌外膜蛋白bp26和omp10基因,针对试验布鲁菌和参考布鲁菌菌株进行克隆和序列分析。根据GenBank发表的布鲁菌bp26基因和omp10基因,分别设计合成一对特异性引物,以提取的试验布鲁菌和2株参考布鲁菌的总DNA为模板,通过聚合酶链式反应(PCR)技术扩增得到bp26和omp10基因,回收纯化后将这两个基因分别连接到pMD18-T载体上,热激发转化受体菌大肠杆菌DH5α,质粒提取、PCR鉴定、测序。结果表明,bp26全长为995 bp,包含一个由753 bp组成的完整开放阅读框,试验菌株的同源性为100%,与参考菌株M5、S2的同源性分别为100%和99.9%,与参考序列S19、870的同源性分别为99.9%和100%。omp10全长531 bp,包含一个由396 bp组成的完整开放阅读框,试验菌株的同源性为100%,与参考菌株M5、S2的同源性均分别为100%,与参考序列544的同源性分别为99.7%。证实bp26基因和omp10基因在各种型间的同源性都在99%以上,表明这两个基因在布鲁菌属是高度保守的。

To clear genes homology on gene of the Brucella outer membrane protein BP26 and OMP10 genes among Brucella of testing or reference and Genbank strains,clone and Comparison for their DNA sequences.We designed and synthesized a specific primers with Brucella bp26 and omp10 in Genbank.The genomic DNA of bp26 and omp10 of testing or reference Brucella strains was extracted from each sample,PCR amplification was then carried out and thus obtaining bp26 and omp10.The PCR product was inserted into pMD18-T vector and sequenced.The plasmids were transformed into E.coli DH5α.Extracted E.coli plasmid DNA,PCR detected and sequenced.Sequencing showed that bp26 was 995 bp in length,including a 753 bp open reading of the complete framework.Sequence comparison revealed that homology of bp26 among the testing strains was 100%,homology of bp26 of the testing and the reference M5 or S2 strains were 100% or 99.9%,homology of bp26 of the testing strains and the reference S19 or 870 sequences was 99.9% or 100%.omp10 was 531 bp containing a 396 bp open reading of the complete framework,homology of omp10 among the testing strains was 100%.homology of omp10 of the testing and the reference M5 or S2 strains were 100%.homology of omp10 of the testing and the reference 544 sequence were 99.7%.There are bp26 and omp10 gene homology between the various types in more than 99%.They is highly remain stable.