College of Food Science and Technology,Huazhong Agricultural University
沙门氏菌是引起食源性疾病的一类重要的病原菌,对其进行有效的控制具有重要意义。本文在对鼠伤寒沙门氏菌噬菌体T139基因组进行分析的基础上,获得一个编码裂解酶中内肽酶的基因序列,采用蛋白质二级结构分析和异源表达纯化,成功验证该内肽酶基因orf 40表达产物LysT40对沙门氏菌具有裂解活性,得到一个潜在的抗沙门氏菌的噬菌体裂解酶。研究结果表明：噬菌体T139基因组全长38,854 bp,平均GC含量为49.10%,不包含tRNA基因。进化树分析表明噬菌体T139属于T7噬菌体属,共预测出43个ORFs,有23个被赋予已知功能。多序列比对及保守结构域分析表明,orf 40基因编码的蛋白(LysT40)包含126个氨基酸残基,具有内肽酶保守结构域,为可能的噬菌体裂解酶。LysT40二级结构中存在大量的螺旋结构结构(70.63%),使得该蛋白能够保持结构稳定。将orf 40进行异源表达、纯化,经过SDS-PAGE和Western Blot分析,在10-15 kDa处获得单一目标蛋白条带,纯化的蛋白浓度为280 μg/mL。LysT40在30 min内对氯仿或EDTA预处理的鼠伤寒沙门氏菌具有较好的裂解活性,吸光值OD600相比于对照组分别下降0.18和0.31。本研究可为开发噬菌体裂解酶作为抗菌剂用于沙门氏菌的控制奠定一定的研究基础。
Salmonella is an important group of pathogens causing food-borne diseases, and its effective control is of great importance. In this study, according to the analysis of the genome of Salmonella Typhimurium phage T139, a gene sequence encoding an endolysin was obtained. Protein secondary structure analysis and heterologous expression purification were used to successfully verify the lytic activity of the endopeptidase gene orf 40 expression product LysT40 against Salmonella, and obtained a potential anti-Salmonella phage endolysin. The T139 genome was 38,854 bp, with an average GC content of 49.10%, and contained no tRNA genes. Evolutionary tree analysis indicated that phage T139 belongs to the genus T7 phage, and a total of 43 ORFs were predicted, with 23 being assigned known functions. Multiple sequence alignment and conserved structural domain analysis showed that the orf 40 gene encodes a protein (LysT40) containing 126 amino acid residues with a conserved structural domain of phage endopeptidase, making it a possible phage endolysin. Presence of a large number of helical structural structures (70.63%) in the secondary structure of LysT40 allowed the protein to maintain structural stability. The endopeptidase gene orf 40 was heterologously expressed, purified and analyzed by SDS-PAGE and Western Blot to obtain a single target protein band at 10-15 kDa at a purified protein concentration of 280 μg/mL. LysT40 showed significantly lytic activity against Salmonella Typhimurium pretreated with chloroform or EDTA within 30 min, with absorbance values OD600 decreasing by 0.18 and 0.31 respectively compared to the control. This study may provide a basis for the development of phage endolysin as antimicrobial agents for the control of Salmonella Typhimurium.