Construction and application of Staphylococcus aureus gene knockout plasmid

doi:10.3969/j.issn.2095-4344.2015.05.025

Abstract

Abstract:

BACKGROUND: Methicillin-resistant Staphylococcus aureus has been a primary pathogen of nosocomial infections worldwide. To construct a quick and easy knockout method is an important technique of studying virulence and resistance of methicillin-resistant Staphylococcus aureus.
OBJECTIVE: To construct the Staphylococcus aureus gene knockout plasmid for understanding the antibiotic resistance and virulence of Staphylococcus aureus.
METHODS: pUC19 was considered as a basic skeleton of construction. pLE194Ts temperature-sensitive replicon and tetracycline resistance gene fragment pHY300PLK plasmid in pCL52.1 were bound to EcoR I site in pUC19 by high assurance amplification. All multiple clone sites in pUC19 were reserved. The Escherichia coli-Staphylococcus aureus shuttle plasmid was obtained. The N315 dapB gene knockout plasmid was obtained through gene knockout technology. This strain was eventually identified by multiplex-PCR.
RESULTS AND CONCLUSION: The Escherichia coli-Staphylococcus aureus shuttle plasmid, pYZ1 and pYZ8, was successfully constructed, and had been used in Staphylococcus aureus gene knockout. Homologous recombinant plasmid pYZ-ΔdapB was constructed by restriction enzyme digestion and overlap technique. After genetically modification in RN4220, the constructed gene knockout plasmid pYZ-ΔdapB was introduced to N315 to be screened in the low culture temperature. The deletion strain was successfully obtained after being identified by multiplex-PCR. Above data suggested that pYZ1 and pYZ8 can be successfully used for Staphylococcus aureus gene detection, which provides a tool to study resistance and virulence of clinical Staphylococcus aureus strains.

中国组织工程研究杂志出版内容重点：肾移植；肝移植；移植；心脏移植；组织移植；皮肤移植；皮瓣移植；血管移植；器官移植；组织工程

全文链接：

Key words: Methicillin-Resistant Staphylococcus Aureus, Virulence, Gene Knockout Techniques, Plasmids

CLC Number:

R318

Wei Wei, Li Xiao-ling, Yuan Wen-chang. Construction and application of Staphylococcus aureus gene knockout plasmid[J]. Chinese Journal of Tissue Engineering Research, 2015, 19(5): 799-804.

Figures/Tables 1

2.1 大肠杆菌-金黄色葡萄球菌穿梭质粒pYZ1和pYZ8的构建大肠杆菌-金黄色葡萄球菌穿梭质粒pYZ1、pYZ8是以pUC19为构建的基本骨架，通过overlap的技术手段，把pLE194Ts中温敏复制子的部分1 359 bp大小的片段及四环素抗性基因tetL 1 620 bp或者氯霉素抗性基因cat 1 052 bp的片段，连接入pUC19的EcoRⅠ位点，质粒大小约6.0 kb，并且基本上保留了pUC19中所有的多克隆位点，更有利于质粒构建操作，见图3。质粒pYZ1、pYZ8均可以顺利转入大肠杆菌DH5α以及金黄色葡萄球菌RN4220中，能够复制并提取出来。 2.2 同源重组质粒pYZ-ΔdapB的构建以pYZ1为模板，dapB基因同源前臂、同源后臂以及cat基因通过overlap技术连接后，亚克隆入pYZ1的KpnⅠ和SalⅠ位点后，转入大肠杆菌DH5α，提取后经KpnⅠ和SalⅠ双酶切鉴定，见图4，鉴定成功后转入RN4220，提取质粒后再转入待敲除菌株耐甲氧西林金黄色葡萄球菌 N315。获得含有同源重组质粒pYZ-ΔdapB的N315金黄色葡萄球菌后，由于pYZ1含有温度敏感型复制子pLE194Ts，该质粒在30 ℃以下可以正常复制，但是在42 ℃以上无法正常复制，所以可以通过温度筛选的方法获得dapB基因缺失突变株。在实验过程中，发现用25 ℃以及42 ℃两个温度交替培养，获得突变株的概率较高。经过两轮温度交替培养过后，挑取单菌落至BHI(Cat)以及BHI(Tet)固体培养平板上，37 ℃培养，在氯霉素平板中生长，而在四环素平板中不生长的克隆为可疑的敲除株，见图5。挑取可能的敲除株单菌落培养后，提取全基因组作PCR鉴定。在dapB基因缺失突变株中，应该可以扩增出氯霉素耐药基因cat；突变株无法扩增出dapB基因；同源臂外侧引物AU/BU扩增出的片段大小为2 846 bp，而野生株扩增片段大小为2 050 bp；dapB基因上游同源序列L的外侧引物AU和氯霉素编码基因cat的下游引物Cat-2扩增出的片段大小为1 947 bp左右，野生株中扩增不出；氯霉素编码基因cat的上游引物Cat-dapB-1和dapB基因下游同源序列R的外侧引物BU扩增片段为1 951 bp左右，野生株中扩增不出，见图6。同时对以AU/BU扩增出的片段进行测序，测序结果显示cat基因成功的置换了dapB基因，dapB基因敲除成功。"

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