鲍曼不动杆菌裂解性噬菌体LZ35的分离鉴定和全基因组序列分析

发布时间：2018-05-20 20:35

  本文选题：噬菌体 + 鲍曼不动杆菌　； 参考：《吉林大学》2017年硕士论文

【摘要】：鲍曼不动杆菌(Acinetobacter baumannii)是不动杆菌属重要成员之一,为专性需氧的革兰染色阴性杆菌,广泛存在于自然界的土壤和水中,也可存在健康人的肠道中。流行病学资料证实,鲍曼不动杆菌是引起医院内感染重要的条件致病菌之一,鲍曼不动杆菌引起感染的对象主要为接受化疗的肿瘤患者、器官移植病人、严重外伤和有慢性病的老年患者。此外,鲍曼不动杆菌耐药菌株和多重耐药菌株的不断出现使得抗生素疗法的实际功效明显下降,同时鲍曼不动杆菌可将其携带的耐药基因通过接合、转化和转导的方式传递其他细菌,造成细菌耐药的进一步传播。细菌耐药性的日趋严重和普遍性已成为全球的公共卫生问题。近年来,利用噬菌体预防和控制细菌感染的噬菌体疗法重新引起有关人士的关注。本项研究的目的是通过分离鉴定鲍曼不动杆菌噬菌体并对其生物学特征和遗传信息进行分析,为今后噬菌体用于鲍曼不动杆菌引起的感染提供依据。本研究采用常规方法以鲍曼不动杆菌为宿主菌从环境污水中分离到5株裂解性噬菌体,分别命名为噬菌体LZ12、LZ22、LZ35、LZ57和LZ78。电镜观察发现,噬菌体LZ12、LZ22、LZ35和LZ57的形态学特征符合肌尾病毒科(Myoviridae family)噬菌体,而LZ78则属于足尾病毒科(Podoviridae family)噬菌体。在本研究中,我们对噬菌体LZ35的生物学特性和基因组序列进行了初步的分析。结果显示,噬菌体LZ35的头部呈二十面体立体对称、直径约47 nm,尾部为伸缩性、长约56 nm。噬菌体LZ35的最佳感染复数(MOI)为0.01,一步生长曲线表明,LZ35的潜伏期为10 min,爆发量为149 pfu/cell。LZ35在p H4~10和孵育50℃1h的条件下仍能保持其生物学活性。酶切电泳显示,噬菌体LZ35的基因组中含有Eco RⅠ、BglⅡ、Eco RⅤ、HindⅢ、NdeⅠ、PstⅠ和XbaⅠ的酶切位点。提取和纯化的噬菌体LZ35基因组提交至金维智基因技术服务公司进行测序。结果表明,噬菌体LZ35基因组呈线性双链DNA、大小为44,885 bp,G+C含量是37.95%,未发现t RNA。基因注释显示,LZ35基因组含有83个编码序列(coding sequences,CDS),其中22个编码序列可预测其功能,61个编码序列为未知基因。利用BLASTn软件分析比对表明,噬菌体LZ35的基因组与鲍曼不动杆菌噬菌体IME-AB2(登录号:JX976549.1)和鲍曼不动杆菌噬菌体YMC-13-01-C62(登录号:KJ817802.1)具有很高的同源性(分别为97%和99%)。依据鲍曼不动杆菌噬菌体基因组中的RNA聚合酶核苷酸序列所绘制的进化树发现,噬菌体LZ35与鲍曼不动杆菌噬菌体YMC11/12/R12的进化关系最近,在同一分支上。噬菌体LZ35的全基因组序列已提交至Gen Bank,登录号:KU510289.1。
[Abstract]:Acinetobacter Baumannii is one of the most important members of Acinetobacter Baumannii. Acinetobacter Baumannii is a specific aerobic gram-negative bacilli, widely found in the soil and water of nature, and in the intestinal tract of healthy people. Epidemiological data confirm that Acinetobacter baumannii is one of the most important opportunistic pathogens causing nosocomial infection. Acinetobacter baumannii causes infection mainly among cancer patients receiving chemotherapy and organ transplant patients. Elderly patients with severe trauma and chronic disease. In addition, the continuous emergence of Acinetobacter baumannii resistant strains and multidrug resistant strains significantly reduced the actual efficacy of antibiotic therapy, and Acinetobacter baumannii was able to conjugate the drug-resistant genes it carries. The transformation and transduction of other bacteria causes the further spread of drug resistance. The increasing severity and universality of bacterial resistance has become a global public health problem. In recent years, bacteriophage therapy for the prevention and control of bacterial infection has attracted renewed attention. The purpose of this study was to isolate and identify the bacteriophage of Acinetobacter baumannii and to analyze its biological characteristics and genetic information so as to provide the basis for the use of phage in the future infection caused by Acinetobacter baumannii. In this study, five lytic bacteriophages were isolated from environmental sewage with Acinetobacter baumannii as host bacteria and named as LZ12, LZ22, LZ35, LZ57 and LZ78, respectively. The morphological characteristics of the bacteriophage LZ12, LZ22, LZ35 and LZ57 were similar to that of Myoviridae family, while LZ78 belonged to Podoviridae family. In this study, we analyzed the biological characteristics and genomic sequence of phage LZ35. The results showed that the head of bacteriophage LZ35 was icosahedron stereomorphic, with a diameter of 47 nm, and a retractility of the tail with a length of 56 nm. The one step growth curve showed that the incubation period of LZ35 was 10 min, and the explosion amount of 149 pfu/cell.LZ35 could maintain its biological activity at pH 4N 10 and incubated at 50 鈩，

本文编号：1916137