Fig. 2

Production of recombinant CpMEDLE-1 and polyclonal antibody. a PCR amplification of the cgd5_4580 gene of C. parvum. Lane M: 100 bp molecular makers; Lane 1: cgd5_4580 PCR product. b Expression and purification of recombinant CpMEDLE-1. Recombinant CpMEDLE-1 protein expressed in E. coli BL21 (DE3) was analyzed by SDS-PAGE (left panel) and western blot (middle panel), while purified CpMEDLE-1 was analyzed by SDS-PAGE alone (right panel). Lane M: molecular weight makers; Lane 1: lysate from recombinant bacteria without IPTG induction; Lane 2: lysate from IPTG-induced recombinant bacteria, with the expected product indicated by an arrow; Lane 3: supernatant of IPTG-induced recombinant bacterial culture; Lane 4: sediment of lysate from IPTG-induced recombinant bacteria; Lane 5: CpMEDLE-1 purified using Ni-NAT affinity chromatography. c Expression of native MEDLE-1 protein in C. parvum sporozoites. Western blots were carried out using pre-immune serum (left panel), anti-CpMEDLE-1 antibodies (middle panel) and post-immune serum (right panel). Lane M: molecular weight makers; Lane 1: purified CpMEDLE-1 protein; Lane 2: crude protein extracted from sporozoites. d Cross-reactivity of anti-CpMEDLE-1 antibodies against other MEDLE proteins as revealed by western blot (left panel) and ELISA (right panel). In western blot analysis, 1 μg/lane of MEDLE proteins including CpMEDLE-1 (Lane 1), CpMEDLE-2 (Lane 2), CpMEDLE-3 (Lane 3) and CpMEDLE-5 (Lane 4) were co-incubated with anti-CpMEDLE-1 antibodies. In ELISA analysis, equal amounts of these four MEDLE proteins coated on plates were incubated with different dilutions of anti-CpMEDLE-1 antibodies, with pre-immune serum as the negative control. Any OD₄₅₀ ratio greater than 2.1 was regarded as positive immunoreactivity