This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.1) Plasmid construction and expression of recombinant proteins. To compare the antigenicity of our new p55 protein (p55-v3) with previously described p55 protein (p55-v0), we obtained the p55-v0 plasmid construct from Dr. Smulian, University of Cincinnati. Unexpectedly, we found that this construct did not express recombinant protein. DNA sequence analysis of multiple clones revealed a deletion of a single nucleotide A in the middle of the coding region, resulting in reading frame changes and introduction of multiple stop codons downstream. Therefore, we made new constructs by ourselves, with one construct for the full-length p55-v0 sequence and additional constructs for three overlapping fragments, which are corresponding to the fragments of p55-v3 we had expressed before. 2) Synthesis of peptides and production of antisera: We have obtained synthetic peptides and antisera, one specific for p55-v0 and two specific for p55-v3, from Sigma. The antisera will be used to study the epitope mapping and differential expression patterns of p55-v3 and p55-v0 as proposed in Specific Aim 1. 3) Animal models: We used 40 rats divided into 3 groups. Group A (10 rats) was a control group and housed in SPF conditions and did not receive any medication. Groups B (20 rats) and C (10 rats) were cohoused together in a conventional room (open cages without filter tops). Group B received immunosuppression with Depo-medrol to provoke spontaneous P. carinii pneumoniae (PCP). The dose of Depo-Medrol used was 2 mg/100g body weight subcutaneously twice a week for the first 4 weeks, and once a week for another 6 weeks. Group C didnt receive immunosuppression but were expected to have natural exposure to P. carinii from group B. After 3-4 weeks of immunosuppression all rats in Group B became sick and had apparent weight loss. Five rats died 8-10 weeks after immunosuppression. Examination of the lungs of these 5 rats by staining and PCR found that none of them were positive for P. carinii. In addition we did western blot with whole P. carinii cell lysates and analyzed sera from rats obtained at 8-10 weeks after immunosuppression and sera from control rats. No rats showed positive antibody responses. The reasons why these rats were not infected are unknown. Very recently we obtained live P. carinii organisms from other investigators and did transtracheal inoculation with 5 rats. It has not been determined if any of them are infected.
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