摘要
目的原核表达新德里金属-β-内酰胺酶(New Delhi metallo-β-lactamase-1,NDM-1),并制备其多克隆抗体。方法以临床分离的产NDM-1的臭鼻克雷伯杆菌为模板,PCR扩增NDM-1基因,克隆入pET-28a载体,构建重组原核表达质粒pET-28a-NDM-1,转化E.coli BL21(DE3)pLyss,IPTG诱导表达重组蛋白。表达的重组蛋白经硫酸铵沉淀、离子交换层析、Ni亲和层析及分子筛层析纯化后,免疫日本大耳白兔,制备NDM-1多克隆抗体。抗体经硫酸铵沉淀和SPA-Sepharose亲和层析纯化后,Western blot鉴定其特异性。结果重组表达质粒pET-28a-NDM-1经PCR及测序鉴定构建正确;表达的重组蛋白相对分子质量约为28 000,诱导表达的重组菌破菌上清存在较强的β-内酰胺酶活性,表明NDM-1蛋白为可溶性形式表达;最终纯化获得的NDM-1蛋白纯度高于95%;制备的NDM-1多克隆抗体能与诱导表达的重组菌胞外蛋白特异性结合。结论成功原核表达了NDM-1,并制备了其多克隆抗体,为NDM-1的快速检测提供了新的思路。
Objective To express New Delhi metallo-β-lactamase 1（NDM-1）in prokaryotic cells and prepare its polyclonal antibody.Methods NDM-1 gene was amplified by PCR using clinically isolated NDM-1-producing Acinetobacter bacilli as a template,and cloned into vector pET-28a.The constructed recombinant plasmid pET28a-NDM-1 was transformed to E.coli BL21（DE3） pLyss and induced with IPTG.The expressed NDM-1 protein was purified by ammonium sulfate precipitation,ion-exchange,nickel ion affinity and molecular sieve chromatography,with which Japanese white rabbits were immunized.The prepared polyclonal antibody against NDM-1 was purified by ammonium sulfate precipitation and SPA-Sepharose affinity chromatography,and identified for specificity by Western blot.Results Recombinant plasmid pET28a-NDM-1 was constructed correctly as proved by PCR and sequencing.Recombinant NDM-1 protein with a relative molecular mass of 28 000 was expressed.High β-Lactamase activity was detected in the supernatant of recombinant E.coli lysate,indicating that NDM-1 was expressed in a soluble form.NDM-1 protein reached a purity of more than 95% after purification.The prepared NDM-1 polyclonal antibody showed specific binding to the extracellular protein of induced recombinant E.coli.Conclusion NDM-1 was successfully expressed in prokaryotic cells,and its polyclonal antibody was prepared,which provided a new idea for rapid detection of NDM-1.
出处
《中国生物制品学杂志》
CAS
CSCD
2012年第12期1663-1666,1670,共5页
Chinese Journal of Biologicals
基金
广东医学院创新实验项目(2011ZZDC001)
广东省高等学校大学生创业训练计划项目(1057112050)
关键词
新德里金属-β-内酰胺酶
原核细胞
基因表达
多克隆抗体
New Delhi metallo-β-lactamase-1（NDM-1）
Prokaryotic cells
Gene expression
Polyclonal antibody
