重组PLA2R变体分泌表达与纯化用于膜性肾病血清抗PLA2R自身抗体检测

Abstract

Most patients with idiopathic membranous nephropathy (IMN) have autoimmune antibodies specifically against the M-type phospholipase A2 receptor (PLA2R). PLA2R acts as a significant biomarker contributing to the clinical diagnosis of IMN. Herein, we performed the secretory expression of the exocellular domain and immune-dominant regions of PLA2R by using mammalian cells. Using ELISA, we confirmed that the purified His-tagged PLA2R variant of CysRC1C2C7, which contained CysRC1C2 (aa 21–510) and CTLD7 (aa 1097–1246), possesses the strongest binding affinity toward serum anti-PLA2R in IMN patients. The signal peptide of interleukin-2 for secretion was selected, and parameters for transient expression were optimized to achieve the highest titer of CysRC1C2C7. Stepwise purification of CysRC1C2C7 using anion-exchange chromatography and gel filtration apparently increased its capability of anti-PLA2R recognition or interaction in ELISA. Under optimal conditions of expression and purification, the yield of CysRC1C2C7 in monomer form was ∼14.1 mg L−1, with a recovery rate of ∼77%. This recombinant PLA2R variant had decent potential for serological analysis of anti-PLA2R in IMN patients.