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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。
Objective To detect the biological activity of LytM and LytM185 ~ 316 protein of S. aureus (SAU) by using Escherichia coli, and to prepare polyclonal antibody for the detection of LytM active The natural form and mechanism of production and the biological significance of LytM protein in SAU infection. Methods The lytM and its C-terminal gene were amplified by PCR using the genome of SAU 8325-4 as a template. The recombinant plasmid was cloned into pET-28a to construct a recombinant expression vector. The recombinant plasmid was transformed into E. coli BL21 (DE3). The recombinant protein was induced by IPTG. , And the target proteins LytM and LytM185 ~ 316 were obtained by affinity purification, and the polyclonal antibody of LytM was prepared. The biological activity of LytM and LytM185 ~ 316 was determined by TLC, and the effect of recombinant protein on the activity of SAU 8325-4 Cleavage effect. Results The recombinant plasmids pET-28a-LytM and pET-28a-LytM185 ~ 316 were constructed successfully. LytM and LytM185 ~ 316 with high purity were obtained. LytM185 ~ 316 was not able to hydrolyze the substrate and LytM185 ~ Glycopeptide hydrolase activity. High titer polyclonal antibody to LytM was prepared and confirmed that it could specifically bind to LytM and LytM185 ~ 316. Conclusion The recombinant protein containing only C-terminal 185 ~ 316 aa can hydrolyze the substrate, while the full-length LytM does not possess glycopeptidase activity.