枯草芽孢杆菌pgsA基因的克隆及序列分析

Cloning and Sequence Analysis of pgsA Gene of Bacillus subtilis

根据GenBank中已发表的枯草芽孢杆菌pgsA的基因序列,设计合成了一对能扩增1 225 bp基因片段的引物。以枯草芽孢杆菌染色体DNA为模板,经PCR扩增得到目的片断,然后将其克隆到pMD18-T载体上,经蓝白斑筛选和酶切鉴定选择阳性克隆进行序列测定。经DNA Star软件将其与Gen-Bank上的pgsA序列进行同源性比较,结果表明,核苷酸同源性均在90%以上,氨基酸同源性为94%以上。核苷酸序列系统进化树分析,发现该试验株与浙江株亲缘关系最近。经ProtScale软件分析表明,该基因所编码蛋白在靠近其N端存在一个跨膜区,为跨膜表达体系的建立和新型口服基因工程疫苗的研制奠定了一定的基础。

Based on the pgsA gene of Bacillus subtilis published in GenBank, a pair of primers were designed and 1 225 bp fragment of Bacillus subtilis pgsA gene was amplified from chromosome DNA of the bacteria by PCR. The amplified partial gene of pgsA was purified and cloned into pMD18-T vector and the positive recombinants were identified by blue-white screening and restriction endonuclease digestion, and then the gene was sequenced and analyzed. The result showed that pgsA gene is about 90% identical to the other similar sequences from GenBank, while 94 % at the amino acid levels. Phylogenetic tree indicated that tested strain is the nearest relative to Zhejiang strain. In addition,the pgsA protein was analyzed with ProtSeale software, there is one transmembrane domain near its N terminus,whieh suggests that it be a good path for further establishing transmembrane expressing systems and development of new oral vaccine.