子痫前期循环系统标志物sFlt-1的重组表达及化学发光检测方法的建立

目的：表达可溶性fms样酪氨酸激酶-1(soluble fms-like tyrosine kinase-1,sFlt-1)重组蛋白，建立针对sFlt-1的化学发光检测方法。方法：构建pcDNA3.4-sFlt-1-His*6表达载体，转染至HEK293细胞进行重组表达。利用链霉亲和素-生物素亲和体系及双抗体夹心法，实现对sFlt-1的定量检测。结果：该方法的最低检测限为2 pg/mL,线性范围为20～40 000 pg/mL,平均回收率为92%,批内及批间精密度变异系数(variable coefficient, CV)均小于10%,与珀金埃尔默sFlt-1检测试剂盒进行比对的相关系数R²为0.925 2。结论：获得了具有天然生物活性的sFlt-1蛋白，建立了具有良好性能的sFlt-1检测方法，后续进一步开发可应用于子痫前期的诊断筛查。

Recombinant Expression of sFlt-1, a Marker of Circulatory System in Preeclampsia,and Establishment of a Chemiluminescence Detection Method

Objective: To express soluble fms-like tyrosine kinase-1 (sFlt-1) recombinant protein and establish a chemiluminescence immunoassay detection method for sFlt-1. Methods: The pcDNA3.4-sFlt-1-His*6 expression vector was constructed and transfected into HEK293 cells for recombinant expression of sFlt-1. Using streptavidin-biotin affinity system and double antibody sandwich method, the quantitative detection of sFlt-1 was realized. Results: The minimum detection limit of the method is 2 pg/mL, the linear range is 20 ~ 40 000 pg/mL, the average recovery is 92%, the intra-assay and inter-assay precision coefficients of variation (CV) are less than 10%, and the correlation coefficient R² compared with the PerkinElmer diagnostic kit is 0.925 2. Conclusion: The sFlt-1 protein with natural biological activity was successfully recombinantly expressed, and a sFlt-1 CLIA detection method with good performance was established, which can be further developed and applied to the diagnosis and screening of preeclampsia.