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目的 构建在E .coli中表达人La多肽的质粒。方法以人脾cDNA为模板 ,通过PCR获得La多肽 (全长 )的DNA片段。将此片段利用双酶切定向克隆到MBP(麦芽糖结合蛋白 )融合系统的载体pMALTM c中 ,表达可溶性融合蛋白 ,并通过亲和层析予以纯化。结果免疫印迹实验 (IBT)证明 ,表达的融合蛋白具有La抗原多肽的特异性 ,而敏感性超过生化制备的ENA制品。结论重组融合蛋白与天然ENA制品存在免疫学同一性
Objective To construct a plasmid expressing human La polypeptide in E.coli. Methods The DNA fragment of La polypeptide (full length) was obtained by PCR using human spleen cDNA as a template. This fragment was double-digested and cloned into the vector pMALTM c of the MBP (maltose binding protein) fusion system to express the soluble fusion protein and purified by affinity chromatography. Results Western blotting (IBT) demonstrated that the expressed fusion protein possessed the specificity of La antigen polypeptide and its sensitivity was higher than that of biochemically prepared ENA preparations. Conclusion There is immunological identity between recombinant fusion protein and natural ENA preparations