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目的:制备抗耻垢分枝杆菌(Smeg)GlmU的多克隆抗体,并对其特异性进行鉴定。方法:用PCR技术扩增SmegglmU,构建可在大肠杆菌中表达的重组质粒pET29b-SmegglmU,用IPTG诱导表达,经Ni-NTA-Agarose柱层析纯化后,以其为免疫原制备抗SmegGlmU的多克隆抗体,并以间接ELISA方法测定抗体效价,以Western blot方法鉴定抗体的特异性。结果:在大肠杆菌中得到了可溶性表达的SmegGlmU;以其免疫小鼠获得抗血清,Western blot鉴定显示获得了能特异性地作用于耻垢分枝杆菌GlmU的多克隆抗体,ELISA测定表明其效价高于1∶6400。结论:获得了能特异性地作用于耻垢分枝杆菌GlmU的高效价多克隆抗体,为应用反义RNA技术研究glmU基因的功能奠定了基础。
Objective: To prepare polyclonal antibodies against GlmU of Smeg and to identify their specificity. Methods: SmegglmU was amplified by PCR. The recombinant plasmid pET29b-SmegglmU was constructed and expressed in E.coli. The recombinant plasmid pET29b-SmegglmU was induced by IPTG. After purified by Ni-NTA-Agarose column chromatography, Antibody titers were determined by indirect ELISA. The specificity of the antibody was identified by Western blot. Results: SmegGlmU was successfully expressed in Escherichia coli. The antiserum was obtained from the mice immunized with it. The results of Western blot showed that polyclonal antibody specific to GlmU of M. smegmatis was obtained. Price is above 1: 6400. CONCLUSION: A high titer polyclonal antibody that can specifically act on GlmU of M. smegmatis was obtained, which laid the foundation for the study of glmU gene function using antisense RNA technology.