高效广谱猪链球菌7型前噬菌体裂解酶的挖掘及活性研究

Mining and evaluating the high-effectiveness and broad-spectrum endolysin encoded by prophage harbored in Streptococcus suis serotype 7

摘要:

【目的】噬菌体具有防控耐药性病原菌的抗菌应用潜力，但是有些病原菌噬菌体的获得非常困难，研究发现大多数病原菌存在前噬菌体(prophage)，且由前噬菌体编码的裂解酶(endolysin)具有良好的抗菌应用前景，本研究将挖掘猪链球菌前噬菌体及其编码的裂解酶。【方法】通过对GenBank中登录的数株猪链球菌前噬菌体裂解酶的基因信息分析，挖掘出一株猪链球菌7型菌株前噬菌体编码的裂解酶，研究其生物学活性。以猪链球菌7型菌株7917的基因组为模板，采用PCR技术扩增获得裂解酶基因ly7917，将其克隆至质粒pET28a(+)并转化大肠杆菌DH5α细胞，挑选基因序列正确的阳性克隆、抽提质粒、转化表达菌株大肠杆菌BL21，经IPTG诱导可高效表达裂解酶Ly7917。【结果】平板裂解试验结果显示Ly7917具有高效裂菌活性，能够裂解猪链球菌2型高致病力菌株HA9801；浊度递减试验结果显示该裂解酶能够高效裂解猪链球菌2型、7型、9型和马链球菌兽疫亚种参考株、金黄色葡萄球菌(包括耐甲氧西林金黄色葡萄球菌)等多种革兰氏阳性菌。【结论】基于前噬菌体挖掘的裂解酶Ly7917，具有高效广谱裂菌活性，为临床上利用裂解酶治疗耐药菌的混合感染提供了可能。

Abstract:

[Objective] In recent years, there has been an increasing worldwide prevalence of bacterial resistance to a wide range of antibiotics, resulting in a focus of research interest in bacteriophage. Unfortunately, streptococcus suis (SS) bacteriophage is not easy to isolate, and the endolysin encoded by phage has been examined as a potential agent for SS control. [Methods] In this study, based on the analysis of genomic information of several streptococcus suis deposited in GenBank, we mined and analyzed one endolysin named Ly7917 encoded by a prophage harbored in Streptococcus suis serotype 7 (SS7) genome. The ly7917 gene was amplified by PCR using bacterial genome purified from a SS7 strain 7917 as template and then inserted into the pET28a(+) plasmid. The recombinant plasmid pET28a(+)-ly7917 was transformed into Escherichia coli DH5α, the sequencing of positive colony showed 100% homology with the target gene. The recombinant plasmid was then transformed into E. coli BL21 and induced with IPTG for expression. [Results] The result of plate analysis showed that Ly7917 has highly activity and can lysis Streptococcus suis serotype 2 strain HA9801. In addition, bacterial turbidity test showed that Streptococcus suis serotype 7, serotype 9, most of serotype 2 strains, Streptococcus equi subsp. zooepidemicus strain, Staphylococcus aureus (including Methicillin-resistant Staphylococcus aureus) were highly sensitive to Ly7917. [Conclusion] Effective and broad-spectrum characteristics of Ly7917 mined from prophage brings hope for mixed infection in clinical as an effective therapeutic.