Jenkins M C, Fetterer R, Schares G, Björkman C, Wapenaar W, McAllister M, Dubey J P
Animal Parasitic Diseases Laboratory, Beltsville Agricultural Research Center, Agricultural Research Service, USDA, Beltsville, MD 20705, USA.
Vet Parasitol. 2005 Aug 10;131(3-4):227-34. doi: 10.1016/j.vetpar.2005.05.005.
The gene for a dense granule protein (NcGRA6) of Neospora caninum was expressed in Escherichia coli as a His-tag fusion protein and purified by NiNTA affinity chromatography. In a preliminary study, high binding of antibodies from N. caninum-negative cows was observed in enzyme-linked immunosorbent assay (ELISA) using NiNTA-purified NcGRA6. Analysis of NiNTA eluates revealed a significant number of E. coli proteins that co-purified with recombinant NcGRA6. In an attempt to improve the relative sensitivity and specificity of the NcGRA6-based ELISA, the rNcGRA6 eluates were subjected to a secondary purification using reverse phase-high performance liquid chromatography (RP-HPLC). Analysis of RP-HPLC eluates by SDS-PAGE/silver staining revealed the purification of recombinant NcGRA6 from contaminating E. coli proteins. ELISAs using the RP-HPLC purified NcGRA6 (dELISA) or singly purified NcGRA6 (sELISA) for identifying seropositive and seronegative cows in a beef herd experiencing an epidemic outbreak of neosporosis were compared to standard assays based on native tachyzoite protein-immunofluorescence antibody test, immunoblot assay, and ISCOM-ELISA. The relative sensitivity, specificity, and kappa value of the NcGRA6d-ELISA were greatly improved over the NcGRA6s-ELISA when compared to the three native antigen immunoassays. These results indicate that removal of contaminating E. coli proteins improves the performance of recombinant NcGRA6 ELISA in diagnosing bovine neosporosis, and may have applicability to the use of recombinant proteins in diagnosing other infectious agents.
犬新孢子虫致密颗粒蛋白（NcGRA6）基因在大肠杆菌中表达为His标签融合蛋白，并通过NiNTA亲和层析进行纯化。在一项初步研究中，使用NiNTA纯化的NcGRA6进行酶联免疫吸附测定（ELISA）时，观察到来自犬新孢子虫阴性奶牛的抗体有高结合率。对NiNTA洗脱液的分析显示，有大量大肠杆菌蛋白与重组NcGRA6共纯化。为了提高基于NcGRA6的ELISA的相对敏感性和特异性，rNcGRA6洗脱液使用反相高效液相色谱（RP-HPLC）进行二次纯化。通过SDS-PAGE/银染对RP-HPLC洗脱液进行分析，结果显示重组NcGRA6已从污染的大肠杆菌蛋白中纯化出来。将使用RP-HPLC纯化的NcGRA6（dELISA）或单独纯化的NcGRA6（sELISA）来鉴定暴发新孢子虫病疫情的肉牛群中血清阳性和血清阴性奶牛的ELISA，与基于天然速殖子蛋白的免疫荧光抗体试验、免疫印迹试验和免疫刺激复合物ELISA的标准检测方法进行了比较。与三种天然抗原免疫测定法相比，NcGRA6d-ELISA的相对敏感性、特异性和kappa值比NcGRA6s-ELISA有了很大提高。这些结果表明，去除污染的大肠杆菌蛋白可提高重组NcGRA6 ELISA在诊断牛新孢子虫病中的性能，并且可能适用于使用重组蛋白诊断其他感染因子。
