RESUMO
@#Circumsporozoite protein (CSP) is a sporozoite major surface protein of Plasmodium species. The protein showed promising protection level as a vaccine candidate against Plasmodium falciparum infection. There is a lack of studies on P. knowlesi CSP (PkCSP) as a vaccine candidate due to the high polymorphic characteristic of central repeat region. Recent studies showed the protein has a relatively conserved region at the C-terminal, which consists of T- and B-cell epitopes. This could be the target region for vaccine development against the pre-erythrocytic stage of the parasite. In this study, recombinant PkCSP was expressed using Escherichia coli system. Recombinant PkCSP was immunized in animal models and the antiserum was evaluated using immunoblot analysis. Results showed that PkCSP can be successfully expressed using the bacterial system. Endpoint titre of the antiserum were ranged up to 1:819200. Immunoblot analysis showed the antiserum recognized recombinant PkCSP but not total protein extract from P. knowlesi erythrocytic stage. In conclusion, PkCSP could elicit strong immune response in animal models. However, serum antibodies could not recognize protein from the parasite’s erythrocytic stage extract indicating it is not expressed at the erythrocytic stage. Therefore, PkCSP remains as a potential pre-erythrocytic vaccine candidate against P. knowlesi infection.
RESUMO
BACKGROUND: Pemphigus are chronic autoimmune blistering disorcers characterized by acantholysis. In addition to pemphigus vulgaris(PV), the major clinical variarts are pemphigus foliaceus(PF), paraneoplastic pemphigus(PNP) and drug-induced pemphigus(DP). Detection of pemphigus antigen is important for differential diagnosis as well as research work. Most investigators have identified pemphigus antigens by means of immunoprecipitation using metabolically radiolabeled cultured keratinocytes. However, immunorepitation is generally more expensive, hazardous and time-consuming than immunoblotting. Therefore, establishment of the immunoblotting as a standard technique for the detection of the pemphig us antigens is desirable. OBJECTIVE: To characterized pemphigus antigens by an immunobloting analysis of human epidermal extract and by indirect itnmunofluroscence study using human of cultured keratinocytes as a substraie. METHOD: We performed imrnunoblotting analysis af sera from patieiits with PV, PF, PNP and DP with human epidermal extract as a source of antigen. Indirect immunof uorescence study was also performed using human keratinocytes cultured in high or low calcium media for detection of pemphigus antigens. RESULTS: In an immunoblotting analysis, all(9/9) PV sera showed secific reactivities with a 130-KD protein and all(5/5) FF sera showed reactivities with a 150-KK protein, which is most likely desmoglein 1. Furthermore, one of nine PV serum also reacted with a 150-KD protein, which seems to be the identical antigen detected in PF. All PNP(3/3) sera showed reactivities with two protein bands, 210KD and 190KD. In our indirect imrnunofluorescence study using culltured human keratinocytes as a substrate, when keratinocytes were grown in low calcium media, no pimphigus antigens could be detected. However, when grown in high calciurn media, pemphigus vulga ris and paraneoplastic pernphigus antigens were present t the cell-cell contact areas with a puncta;e pattern, whereas pemphigus foliaceus antigen was not, presint in keratinocytes even when cultured in high calcium media. CONCLUSION: Our results suggests (1) immunoblotting analysis is a reliable technique for defining pemphigus antigen and could be a valuable tool for the differentiation of PV, PF and PNP and(2) PF antigen rnay not be expresseden cultured keratinocytes.