金黄色葡萄球菌糖肽水解酶LytM及其C端的表达、纯化与功能研究

目的利用大肠杆菌重组表达金黄色葡萄球菌（SAU）糖肽水解酶LytM及其C端185～316 aa蛋白（LytM185～316）,检测其生物学活性,并制备多克隆抗体,为检测LytM活性体的天然形式和产生机制以及研究LytM蛋白在SAU感染中的生物学意义奠定基础。方法以SAU 8325-4基因组为模板,PCR扩增lytM及其C端基因,并将其克隆入pET-28a构建重组表达载体,转化入大肠杆菌BL21（DE3）,利用IPTG诱导阳性克隆表达目的蛋白,通过亲和纯化获得目的蛋白LytM及LytM185～316,并制备LytM的多克隆抗体;用薄层层析法测定LytM以及LytM185～316的生物活性,并检测重组蛋白对SAU 8325-4菌体的裂解作用。结果成功构建了表达载体pET-28a-LytM和pET-28a-LytM185～316,获得了纯度较高的重组蛋白LytM及LytM185～316,经测定LytM不能够水解底物而LytM185～316具有较强的糖肽水解酶活性。制备了高效价的抗LytM的多克隆抗体,并证实其可以与LytM及LytM185～316特异性结合。结论只含有C端185～316 aa的重组蛋白能够水解底物,而全长的LytM不具有糖肽水解酶活性。

Expression, purification and function studies of Staphylococcus aureus glycopeptide hydrolase LytM and its C terminal protein

Objective To express and purify Staphylococcus aureus（SAU） glycopeptide hydrolase LytM and its C terminal protein（185-316aa） in Escherichia coli and to study the active form of LytM in SAU.Methods The genes encoding LytM and LytM185-316 were amplified by PCR with SAU 8325-4 genome DNA as the template and cloned into the recombinant expression vectors pET-28a.E.coli BL21（DE3）with the plasmid was induced with IPTG.The protein was expressed and purified by Ni2＋ affinity chromatography before the specific polyclonal antibodies were prepared.The activities of LytM and LytM185-316 were determined by thin-layer chromatography and SAU cell wall lysis assay.Results The purified recombinants LytM and LytM185-316 were expressed and purified.It was demonstrated that LytM185-316 could hydrolyze the substrate（5 Gly） and the cell wall of SAU 8325-4.But the full-length LytM did not have such activities.The prepared polyclonal antibodies of LytM could specifically bind the LytM and LytM185-316.Conclusion The protein containing the C terminal of LytM alone can hydrolyze the substrate.But the full-length LytM doesn′t have such activities.