School of basic medicine, Weifang medical University, 261053 Weifang, People's Republic of China.
World J Microbiol Biotechnol. 2012 Mar;28(3):1281-5. doi: 10.1007/s11274-011-0761-z. Epub 2011 May 3.
In the present study, we constructed plasmid pUC-ZZ-EGFP to express Pro-ZZ-EGFP using ZZ peptide (a synthetic artificial IgG-Fc-fragment-binding protein derived from the B domain of staphylococcal protein A) and enhanced green fluorescent protein (EGFP). Without induction with isopropyl-β-D: -thiogalactopyranoside, the chimeric protein was effectively expressed in Escherichia coli HB101. Its affinity constant binding IgG was 2.6 × 10(8) M(-1) obtained by competitive enzyme-linked immunosorbent assay, indicating that the ZZ peptide retains the native structure in Pro-ZZ-EGFP. The application of immunofluorescence assay for detecting the Mycoplasma pneumoniae IgG antibody, Pro-ZZ-EGFP, exhibited a good signal comparable in brightness and fluorescence pattern with the signal generated using the fluorescein isothiocyanate-labeled anti-human IgG. The result indicates that Pro-ZZ-EGFP possesses great potential for clinical immunofluorescence IgG test as an alternative versatile fluorescent antibody.
在本研究中，我们构建了质粒 pUC-ZZ-EGFP，通过 ZZ 肽（一种源自葡萄球菌蛋白 A 的 B 结构域的合成人工 IgG-Fc 片段结合蛋白）和增强型绿色荧光蛋白（EGFP）表达 Pro-ZZ-EGFP。在不诱导异丙基-β-D：-硫代半乳糖吡喃糖苷的情况下，嵌合蛋白在大肠杆菌 HB101 中有效表达。通过竞争性酶联免疫吸附试验获得其与 IgG 的亲和常数结合为 2.6×10(8)M(-1)，表明 ZZ 肽在 Pro-ZZ-EGFP 中保留了天然结构。免疫荧光法检测肺炎支原体 IgG 抗体的应用表明，Pro-ZZ-EGFP 产生的信号与使用异硫氰酸荧光素标记的抗人 IgG 产生的信号在亮度和荧光模式上具有良好的可比性。结果表明，Pro-ZZ-EGFP 作为一种替代的通用荧光抗体，具有很大的临床免疫荧光 IgG 检测应用潜力。
