从乳酸片球菌中提取质粒DNA作为模板,根据GeneBank公布的细菌素结构基因,设计一对特异性引物,进行PCR扩增片段,将该片段定向克隆到pTA2载体,测序,与发表的基因序列比较,同源性为100%,然后克隆到表达载体Pet-28a,转化到Escherichia coli DH5α,经过卡那霉素平皿筛选,质粒酶切,PCR两步验证,获得阳性重组质粒,并转化表达宿主菌E.coli BL21(DE3)感受态细胞,以异丙基硫代-βD-半乳糖苷(IPTG)诱导表达,Tricine-SDS-PAGE电泳,显示在4 600 Da处出现条带,与已报道的细菌素分子量大小符合.表达产物对单核细胞增多症李氏杆菌有抗菌作用.
Plasmid DNA was extracted from Pediococcus acidilactici as template.The oligonucleotide primers were designed according to the gene sequence reported by GenBank.Amplied the DNA fragment by means of PCR and cloned to pTA2 Vector to sequenced.Compared the inserted DNA fraction and the Ped-A sequence reported by GenBank,and found sequence homology was 100%.Then inserted the segment to expressing vectorPet-28a.The recombinant plasmid transformed to Escherichia coli DH5,screened by kanamycin resistance and identified by PCR,restriction endonucleases analyzing,obtained positive recombinant plasmid transformed which to E. coli BL21(DE3) receptivity cell,induced with IPTG,Tricine-SDS-PAGE showed its molecularmass as 4 600 Dathe same as thereported pediocin PA-1.
[1] Cotter P D,Hill C,Ross R P.Bacteriocins:developing innate immunity for food[J].Food Microbiology,2005,3:777-788.
[2] Reviriego C,Fernández A,Horn N,et al.Production of pediocin PA-1,and coproduction of nisin A and pediocin PA-1,by wild Lactococcus lactis strains of dairy origin[J].International Dairy Journal,2005,15 (1):45-49.
[3] Willem M de Vos.Gene expression systems for lactic acid bacteria[J].Ecology and Industrial Microbiology,1999,2 (3):289-295.
[4] 周志江,韩烨,韩雪,等.从酸白菜中分离出一株产细菌素的乳酸片球菌[J].食品科学,2006,27(4):89-91.
[5] Francisco B E,Wang J K,Dae Y K.Rapid purification,partial,characterization,and antimicrobial spectrum of the bacteriocin,Pediocin AcM,from Pediococcus acidilactici M[J].International Journal of Food Microbiology,1997,37 (17):1-11.
[6] Marugg J D,Gonzalez C F,Kunka B S,et al.Clonging,expression,and nucleotide sequence of genes involved in production of pediocin PA-1,a bacteriocin from Pediococcus acidilactici PAC1.0[J].Applied and Environmental Microbiology,1992,58(8):2360-2367.
[7] Schoeman H,Vivier M A,Du Toit M,et al.The development of bactericidal yeast strains by expressing the Pediococcus acidilactici pediocin gene(pedA) in Saccharomyces cerevisiae[J].Yeast,1999,15(8):647-656.
[8] 王延华,景强,Pierre Dubus.PCR理论与技术[M].北京:科学出版社,2005.
[9] 迪芬巴赫C w,德维克斯勒G S.PCR技术实验指南[M].种康,瞿礼嘉译.北京:科学出版社,1998:234-256.
[10] 韩雪,周志江.乳酸片球菌细菌素的活性及特性的研究[J].食品研究与开发,2006,27(4):19-21.
[11] Nikki H,Mafia I M,Jose M M,et al.Enhanced production of pediocin PA-1 and coproduetion of nisin and pediocin PA1 by Lactococcus lactis[J].Applied and Environmental Microbiology,1999,65 (10):4443-4450.
[12] Moon G S,Pyun Y R,Kim W J.Expression and purification of a fusion-typed pedioein PA-1 in Escheriehia coil and rocovery of biologically active pediocin PA-1[J].International Journal of Food Microbiology,2006,108(1):136-140.
[13] Beaulieu L,Grolean D,Miguez C B,et al.Production of pediocin PA-1 in the methylotrophic yeast Pichia pastoris reveals unexpected inhibition of its biological activity due to the presence of collagen-like material[J].Protein Expression and Purification,2005,43:111-125.
