Quinn G B, Reeves I G, Day I N
Department of Biochemistry, College of Medicine, University of South Florida, Tampa 33612-4799.
Clin Chem. 1994 May;40(5):790-5.
Human serum neuron-specific enolase (NSE) is a marker of neurons and of small-cell carcinoma of the lung; improved immunoassays of NSE remain an important goal. Here, we used overlapping complementary DNA (cDNA) clones for reconstruction to express full-length recombinant NSE, and also to express a set of cloned subfragments through the prokaryotic expression vectors pUEX and pUBEX. Subfragments expressed as fusion proteins were used to characterize immunogenic and antigenic regions and epitopes and, expressed as affinity matrices, to derive purified, fractionated polyclonal antibodies. NSE epitope data can be visualized with yeast enolase-1 crystal structure coordinates: The two protein sequences align almost perfectly and are 61% identical. This approach demonstrates the complementarity of cDNA expression with techniques of polyclonal antiserum and monoclonal antibody production and with chemical peptide synthesis in the refinement of immunodiagnostic reagents.
人血清神经元特异性烯醇化酶（NSE）是神经元和肺小细胞癌的标志物；改进NSE的免疫测定法仍然是一个重要目标。在此，我们使用重叠互补DNA（cDNA）克隆进行重组，以表达全长重组NSE，并通过原核表达载体pUEX和pUBEX表达一组克隆的亚片段。表达为融合蛋白的亚片段用于表征免疫原性和抗原性区域及表位，并表达为亲和基质以获得纯化的、分级分离的多克隆抗体。NSE表位数据可以通过酵母烯醇化酶-1晶体结构坐标进行可视化：这两种蛋白质序列几乎完美对齐，且有61%的同一性。这种方法证明了cDNA表达与多克隆抗血清和单克隆抗体制备技术以及化学肽合成在完善免疫诊断试剂方面的互补性。
