Pnlf1 n luciferase expression vector

Four C. trachomatis antigens Pgp3, TmeA, InaC, and HSP60 (Supplementary Table S2) were amplified and cloned into pNLF1-N luciferase expression vector (Promega, Madison, WI, United States) downstream of the Nluc luciferase gene as previously described (49 (link)). All materials and reactive used in this study were consistent with those of Wang. All recombinant plasmids were confirmed by DNA sequencing and transfected into HEK-293 T cells (ATCC CRL-3216). The expressed fusion proteins of Nluc-C. trachomatis in cell lysates were harvested for further confirmation using anti-luciferase antibody, and then stored at −80°C until use. Anti-C. trachomatis antibodies in sera were detected in LISA in which they were first captured by protein G-coated microtiter plate and detected by Nluc-C. trachomatis antigen lysate in the presence of luciferase substrate as described previously (49 (link)). Each sample was tested in triplicate and the relative light units (RLU) were calculated by dividing each sample’s average luciferase light units with the average LU of the control wells. The level of anti-C. trachomatis antibody was expressed as Log₂RLU. The cut-off value of C. trachomatis LISA was determined according to the maximum value of Youden’s index obtained through the receiver operating characteristic (ROC) curve.

Luciferase immunosorbent assay (LISA) was used to detect C. trachomatis-specific antigen pgp3, as described previously [19 (link), 20 (link)], which was more sensitive than enzyme-linked immunosorbent assay (ELISA) [20 (link)]. Briefly, the C. trachomatis pgp3 gene was amplified and sub-cloned into the pNLF1-N luciferase expression vector (Promega, Madison, WI, United States) downstream of the Nluc luciferase gene, which was then transfected into HeLa cells. The cell lysates containing the Nluc-pgp3 fusion protein expressed in HeLa cells were harvested and confirmed using the anti-luciferase antibody. The 96-well white microplate were coated with 50 µL/well Protein G (5 mg/ml; Genscript, Nanjing, China) and incubated overnight at 4 °C. After washing and blocking, 50 µL diluted sera (1:100 dilutions in 2% non-fat dry milk) was added to the wells and incubated for 1 h at 37 °C, followed by washing five times. 50 µL Nluc-Pgp lysates were then added as detection antibody to each well and incubated at 37 °C for 30 min. After washing, 50 µL of the Nano-Glo Luciferase assay reagent was added to determine the luciferase light units of the Nano-Glo Luciferase assay by Fluoroskan Microplate Fluorometer (Thermofisher, United States). The cut-off value of anti-Pgp3 IgG LISA was derived from the average value of negative controls plus 3 standard deviations as described previously.