地衣芽孢杆菌2709碱性蛋白酶基因的克隆、序列测定及其表达

以克隆的地衣芽孢杆菌2709碱性蛋白酶编码序列的PCR扩增片段为探针。通过原位杂交从2709基因文库中筛选出两个含有完整的2709碱性蛋白酶基因的阳性克隆：Psci和Psc7。对Psc7中的插入片段构建若干亚克隆后测定了其全部DNA序列，结果显示该插入片段含2709碱性蛋白酶及其信号肽与导肽(Pro—peptide)在内的全部编码序列(1140碱基对)及长度分别为299和832碱基对的上、下游序列，该序列同M．Jacobs等克隆的地衣芽孢杆菌NcIB 6816的subtlisin Carlsberg基因序列显示了极高的同源性。通过枯草杆菌-大肠杆菌穿梭质粒Pbe2将克隆的2709碱性蛋白酶基因转入到蛋白酶缺陷型的枯草芽孢杆菌DB104中，结果表明2709碱性蛋白酶基因在枯草芽孢杆菌中得到了明显的表达。

Cloning, Sequencing and Expression of the Alkaline Protease Gene from Bacillus licheniformis 2709

Using the PCR amplified partial coding sequence of the alkaline protease (subtilisin) from Bacillus licheniformis 2709 as the probe of in situ hybridization,several positive colonies were identified from the 2709 genomic library. Two of them , pSC1 and pSC7, were shown having the intact 2709 gene by physical mapping and partial sequencing. The complete sequence of the insert of pSC7 plasmid has 2271 base pairs including 299bp 5' flanking sequence, 832bp 3'flanking sequence and 1140bp structural sequence. The 2709 subtilisin sequence obtained here is highly homologous to the subtilisin from B. licheniformis NCIB 6816. The cloned gene was introduced into the protease-deficient Bacillus subtilis DB104 and protease activity assay demonstrated that a successful expression of 2709 subtilisin in DB104 was achieved.