To detect the binding of RdFV CP with sperms in vitro, His-tag-fused CP was expressed in Escherichia coli strain Rosetta, and the proteins were purified using nickel-nitrilotriacetic acid resin (Qiagen). Sperm smears collected from testes of RdFV-free R. dorsalis were successively incubated with the purified CP (0.5 μg/μl) for 1 h, smeared on poly-lysine-treated glass slides, immunolabeled with CP-rhodamine (0.5 μg/μl), stained with DAPI (2.0 μg/ml), and then processed for immunofluorescence microscopy.
In neutralization experiments to test the direct interaction between RdFV CP and HongrES1, mature sperms excised from the testes of RdFV-free R. dorsalis were pre-incubated for 30 min with pre-immune antibody (0.5 μg/μl) or HongrES1 antibody (0.5 μg/μl), and then the in vitro CP-sperm binding experiment was performed as described above.
In neutralization experiments to test the direct interaction between RGDV particles and HongrES1, mature sperms excised from RGDV-free leafhoppers were pre-incubated for 30 min with pre-immune antibody (0.5 μg/μl) or HongrES1 antibody (0.5 μg/μl), incubated with the purified RGDV particles (1.0 μg/μl) for 1 h, smeared on poly-lysine-treated glass slides, immunolabeled with P8-FITC (0.5 μg/μl), stained with DAPI (2.0 μg/ml), and then processed for immunofluorescence microscopy.
In neutralization experiments to test the direct interaction between RdFV CP and HongrES1, mature sperms excised from the testes of RdFV-free R. dorsalis were pre-incubated for 30 min with pre-immune antibody (0.5 μg/μl) or HongrES1 antibody (0.5 μg/μl), and then the in vitro CP-sperm binding experiment was performed as described above.
In neutralization experiments to test the direct interaction between RGDV particles and HongrES1, mature sperms excised from RGDV-free leafhoppers were pre-incubated for 30 min with pre-immune antibody (0.5 μg/μl) or HongrES1 antibody (0.5 μg/μl), incubated with the purified RGDV particles (1.0 μg/μl) for 1 h, smeared on poly-lysine-treated glass slides, immunolabeled with P8-FITC (0.5 μg/μl), stained with DAPI (2.0 μg/ml), and then processed for immunofluorescence microscopy.
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