细菌素产生菌的筛选及其编码基因的克隆与序列分析

Screening of the bacteriocin-producing bacteria and the cloning and sequencing analysis of its bacteriocin gene

细菌素是细菌代谢过程中合成的具有抑菌性质的多肽类物质,它可以广泛地抑制革兰氏阳性菌,尤其是抑制单增李斯特菌等腐败菌和病原菌,大部分细菌素对热稳定,与食品一起在加热处理时使用依然能够稳定存在,并可在人体内进行生物降解和消化,对健康无害。利用分子克隆的方法在大规模筛选细菌素中具有快速准确的特点,根据已知的细菌素DNA序列设计保守的PCR引物,在从杏鲍菇子实体中分离得到56株乳酸菌中,有22株克隆出目的条带,其中只有一个样品稳定性好,经鉴定该样品的乳酸菌株为乳酸乳球菌(Lactococcus lactis)。对克隆的细菌素Nisin-like基因测序结果表明该基因序列全长174bp。生物信息学分析显示该基因编码57个氨基酸,分子量大小5924.93 Da,等电点9.23,二级结构主要由α-螺旋和β-折叠股组成,与报道的有所不同,可能是新的细菌素。

Bacteriocin is a kind of lower-molecular-weight polypeptide produced among metabolic process. It can be widely inhibit gram-positive bacterium, especially the inhibition of Listeria monocytogenes and other spoilage organisms and pathogenic bacteria. Most bacteriocin is thermal stable and can be stay stable in food heating process. The Bioinformatics softwares were used to analysis and compare bacteriocin genes and design the optimal primers, the genomic DNA from 56 Lactic acid bacteria which separated from Pleurotus eryngii Fruitbody were used as the template to clone the DNA sequence ofNisin-like bacteriocin by PCR, 22 strains had the same size target band among all 56 genome, however, only one strain characterized as Laetoeoceus lactis has stable signal. The resuR of sequencing shows that the full length ofNisin-like gene is 174bp, which coded for 57 amino acid residues, and molecular weight is 5924.93 Da. Theoretical PI of 9.23, the secondary structure of protein consist of^-strand and a-helix. It was different with the published one and may encode a new kind of bacteriocin.