Electrotransformation of Haemophilus parasuis with in vitro modified DNA based on a novel shuttle vector
Electrotransformation of Haemophilus parasuis with in vitro modified DNA based on a novel shuttle vector
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基于新型穿梭载体的体外修饰 DNA 电转化副猪嗜血杆菌
DOI：
10.1016/j.vetmic.2011.08.020
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发表时间：
2012-03-23
影响因子：
3.3
通讯作者：
Chen, Huanchun
中科院分区：
文献类型：
--
作者：
Chen, Liping;Wu, Dongfang;Chen, Huanchun
本研究的目的是建立一种利用新型副猪嗜血杆菌-大肠埃希菌穿梭载体转化猪格拉瑟病病原副猪嗜血杆菌的有效方法。从副猪嗜血杆菌分离株中提取了一个4.2 kb的内源质粒pYC93，并对其进行了完全测序。pYC93与杜氏嗜血杆菌原生质粒pLS88具有高度同源性。以pYC93的起源区、大肠杆菌克隆载体pBluescript SK（+）和Tn903衍生卡那霉素盒为基础，采用重叠PCR策略构建穿梭载体pSHK4。用pSHK4对15株副猪嗜血杆菌血清型参考菌株和1株临床分离株SH0165进行电穿孔，其中只有1株菌株产生转化子，转化效率为8.5 x 10(2) CFUhlg DNA。从同源转化子中重新分离的载体DNA显著提高了转化效率（1.3 × 10(5) CFU/ μ g DNA）。这表明限制修饰系统参与了副猪嗜血杆菌转化的屏障。利用宿主副猪嗜血杆菌的无细胞提取物进行体外DNA修饰，16株菌株中有15株可转化。新的穿梭载体pSHK4和建立的电转化方法为副猪螺旋体的遗传操作提供了有用的工具，以更好地了解病原体。(C) 2011 Elsevier B.V.版权所有
The objective of the present study was to establish a valid transformation method of Haemophilus parasuis, the causative agent of Glasser's disease in pigs, using a novel H. parasuis-Escherichia coli shuttle vector. A 4.2 kb endogenous plasmid pYC93 was extracted from an H. parasuis field isolate and completely sequenced. Analysis of pYC93 revealed a region approximately 800 bp showing high homology with the defined replication origin oriV of pLS88, a native plasmid identified in Haemophilus ducreyi. Based on the origin region of pYC93, E. coli cloning vector pBluescript SK(+) and the Tn903 derived kanamycin cassette, a shuttle vector pSHK4 was constructed by overlapping PCR strategy. When electroporation of the 15 H. parasuis serovar reference strains and one clinical isolate SH0165 with pSHK4 was performed, only one of these strains yielded transformants with an efficiency of 8.5 x 10(2) CFUhlg of DNA. Transformation efficiency was notably increased (1.3 x 10(5) CFU/mu g of DNA) with vector DNA reisolated from the homologous transformants. This demonstrated that restriction-modification systems were involved in the barrier to transformation of H. parasuis. By utilizing an in vitro DNA modification method with cell-free extracts of the host H. parasuis strains, 15 out of 16 strains were transformable. The novel shuttle vector pSHK4 and the established electrotransformation method constitute useful tools for the genetic manipulation of H. parasuis to gain a better understanding of the pathogen. (C) 2011 Elsevier B.V. All rights reserved.