简并引物法克隆保加利亚乳杆菌β-N-乙酰氨基己糖苷酶基因

Cloning of β-N-Acetylhexosaminidase Gene Fragment from Lactobacillus delbrueckii subsp.bulgaricus LJJ with Degenerate Oligonucleoide Primers

β-N-乙酰氨基己糖苷酶(β-Hexcase)在细菌、植物和动物中广泛存在,具有典型的外切酶活性并可以催化切除β-N-乙酰氨基己糖的非还原性氨基己糖残基,在细菌细胞分裂中具有重要作用.选取与保加利亚乳杆菌LJJ亲缘关系近的菌种的β-N-乙酰氨基己糖苷酶蛋白序列,利用ICO-DEHOP和CODEHOP在线简并引物设计软件设计β-Hexcase简并引物,选取Hex1-f和Hex1-r引物对,以LJJ基因组DNA为模板经PCR扩增得到614 bp产物.PCR产物连接pGEM-T质粒载体后克隆至大肠杆菌DH5α中,筛选阳性克隆提取质粒进行测序.测序结果显示产物长度为614 bp,该DNA序列经blastx比对发现与其他已知β-Hexcase基因具有相似性,表明所克隆的序列即为LJJβ-Hexcase的基因片段.β-N-乙酰氨基己糖苷酶基因的获得为进一步研究β-N-乙酰氨基己糖苷酶与保加利亚乳杆菌自溶的关系奠定了基础.

β-N-Acetylhexosaminidase （β-Hexcase） , produced by a wide variety of bacteria, plants and animals, behaves like typical exo-enzymes, catalyzing the cleavage of terminal non-reducing N-acetyl-β- hexosamine residues with important physiological roles in cell wall recycling. The β-hexcase sequence of relevant strains genetically close to Lactobacillus delbrueckii subsp, bulgaricus LJJ was selected, and degenerate primers were designed using online ICODEHOP and CODEHOP software. A pair of degenerate primers named hexl-f and hexl-r was chosen for PCR with the LJJ genome DNA as a template. A 614 bp PCR product was obtained, and was transformed into E. coli DHSα using pGEM-T vector and sequenced after extraction of plasmids. Similarity alignment showed that the sequence of the cloned DNA was similar to those of known β-hexcase gene from other bacterial strains. Therefore, the cloned sequence was con- firmed to be the putative β-hexcase gene fragment from LJJ strain. The results obtained in this study would provide basis for studying roles of β-Hexcase bulgaricus strains. in cell wall lysis of Lactobacillus delbrueckii subsp.