噬菌体VB_SauM_Lm44穿孔素HolLm44的克隆表达及其抑菌效果研究  

Study on Cloning and Expression of Holin HolLm44 of Bacteriophage VB_SauM_Lm44 and Its Antibacterial Effect

作　　者：张欣然 廖毅婷 熊东炜 李鹏[1] 杨淑华[1] 龙淼[1] ZHANG Xinran;LIAO Yiting;XIONG Dongwei;LI Peng;YANG Shuhua;LONG Miao(Key Laboratory of Ruminant Infectious Disease Prevention and Control(East),Ministry of Agriculture and Rural Affairs,Key Laboratory of Livestock Infectious Diseases,Ministry of Education,College of Animal Science and Veterinary Medicine,Shenyang Agricultural University,Shenyang 110866,China)

机构地区：[1]沈阳农业大学动物科学与医学学院,重要家畜疫病研究教育部重点实验室,农业农村部反刍动物重大疫病防控重点实验室(东部),沈阳110866

出　　处：《动物营养学报》2025年第3期2081-2089,共9页CHINESE JOURNAL OF ANIMAL NUTRITION

基　　金：辽宁省教育厅基本科研项目(JYTYB2024027)。

摘　　要：本试验旨在克隆表达金黄色葡萄球菌噬菌体VB_SauM_Lm44穿孔素并研究其抑菌效果。首先,通过分析VB_SauM_Lm44基因组,获得穿孔素基因序列,并利用PCR扩增穿孔素基因(命名为HolLm44,其表达的穿孔素蛋白命名为HolLm44);然后,将目的基因片段与双酶切处理后的线性pET-28a(+)质粒连接、转化入大肠杆菌DH5α克隆感受态细胞,提取重组质粒并转化入大肠杆菌表达菌株BL21(DE3)及BL21(DE3)plysS,进行目的蛋白诱导表达和纯化;最后,通过平板抑菌法及刃天青染色法验证穿孔素的抑菌效果,探究穿孔素HolLm44发挥其活性的最适作用条件。结果显示:穿孔素HolLm44具有2个跨膜区,其N-端和C-端都分布于细胞质内,属于Ⅱ类穿孔素。穿孔素HolLm44最适作用温度为37℃,最适作用pH为5.2。在作为指示菌的20株金黄色葡萄球菌中,只有1株为对穿孔素HolLm44表现出敏感性。本研究成功克隆表达出了噬菌体VB_SauM_Lm44中具有活性的穿孔素HolLm44,并证实其具有良好的抑制金黄色葡萄球菌的效果。This experiment aimed to clone and express of holin of Staphylococcus aureus phage VB_SauM_Lm44 and to study its antibacterial effect.Firstly,the holin gene sequence was obtained by analyzing the VB_SauM_Lm44 genome,and the holin gene(named HolLm44,the expressed perforin protein was named HolLm44)was amplified by PCR.Then,the target gene fragment was ligated with the linear pET-28a(+)plasmid after double enzyme digestion and transformed into the Escherichia coli DH5α cloned competent cells;the recombinant plasmid was extracted and then transformed into the expression strains BL21(DE3)and BL21(DE3)plysS of Escherichia coli to enable the induced expression and subsequent purification of the target protein.The antibacterial effect of holin HolLm44 was evaluated through both the plate inhibition assay and the resazurin staining technique,thereby establishing the optimal conditions under which holin HolLm44 exerts its activity.The results showed that holin HolLm44 had two transmembrane regions,and its N-terminal and C-terminal were distributed in the cytoplasm,which belonged to classⅡ holin.The optimal temperature and pH of holin HolLm44 were 37℃ and 5.2,respectively.Of the 20 Staphylococcus aureus strains used as indicator bacteria,only one was sensitive to the holin HolLm44.The results demonstrate that this study successfully cloned and expressed the active holin HolLm44 from phage VB_SauM_Lm44,confirming its potent inhibitory effect against Staphylococcus aureus.

关 键 词：噬菌体VB_SauM_Lm44 穿孔素 克隆表达 抑菌 金黄色葡萄球菌 

分 类 号：Q785[生物学—分子生物学]