trachomatis serovar D. Coinci dentally, immunization with CPAF, another secreted chlamydial protein that was dominantly recognized by human antibodies, also induced protective immunity against chlamydial infection. Furthermore, the CPAF induced immunity even reduced pathologies http://www.selleckchem.com/products/Tubacin.html in mouse oviducts induced by chlamydial urogenital challenge infection. It will be interesting to evaluate whether the pgp3 immunization can also decrease the Chlamydia induced pathologies in the mouse oviducts. Given the new knowledge that pgp3 is a highly conformation Inhibitors,Modulators,Libraries dependent antigen, whether immunization with native like pgp3 protein can induce more relevant immunity against chlamydial infection deserves further evaluation. This hypothesis is worth testing since pgp3 also localizes inside the inclusion and may even be a component of the outer membrane complex.
Methods 1. Chlamydial infection The C. trachoamtis serovars D, L2 and MoPn organisms were propagated in HeLa cells, purified, aliquoted and stored as described previously. To infect HeLa cells, cells grown Inhibitors,Modulators,Libraries in either 24 well plates with coverslips or tissue flasks containing DMEM with 10% fetal calf serum at 37 C in an incubator sup Reactivityon Westernantibodies with pgp3 and control fusion plied with 5% CO2 were inoculated with chlamydial organisms at an MOI of 0. 5 as described previously. The infected cultures were processed at different time points after infection for either inmmunofluorescence assays or Western blot analyses as described below. To infect mice, the MoPn organisms were intra vaginally inoculated into mice at a dose of 5 104 IFUs per mouse as described previously.
Inhibitors,Modulators,Libraries The infection was monitored by quantitating the IFUs recovered from the mouse vaginal swabs. Eighty days post infection, mouse blood was col lected for preparing the mouse antisera. The serum sam ples from 5 mice were pooled together at an equal ratio, designated as the pooled mouse antiserum. 2. Fusion protein production and fusion protein ELISA The eight pORFs encoded by the pCHL1 plasmid from C. trachomatis serovar D organisms were cloned into pGEX vectors. The cloned pORFs were expressed as fusion proteins with glutathione Inhibitors,Modulators,Libraries s transferase fused to the N terminus of the chlamydial proteins as previously described. The fusion proteins were purified using immunoprecipitationantibodies with endogenous pgp3 in Reactivity of human antibodies with endogenous pgp3 in immunoprecipitation.
Inhibitors,Modulators,Libraries Various amounts of the pooled positive human antiserum Pacritinib SB1518 was used to precipitate chlamydial endogenous antigens from L2S100 with or with out prior heat treatments either at 56 C for 30 min or boil ing for 10 min. The human antibody precipitates were detected with either the anti pgp3 polyclonal or anti CPAF monoclonal antibodies as shown on the left of the figure. The corresponding protein bands were indicated on the right of the figure.