Studies on purification isolation and immunological characterization of 47kD and 23kD polypeptide from blood stage P.falciparum parasite

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We have continued this research for several years and have reached the final stage of the study in 1995. The eventual aim of this study is the preparation of 47kD polypeptide which is expressed on the membrane of Plasmodium falciparum parasite at schizont stage of the development. The molecule was focussed in the study on immunoblotting using sera from patients manifesting acute stage clinical symptoms. It looks that the molecule works in the immune response evoked to overcome the acute fulminating stage of the disease. Started from this finding, preparation of the 47kD polypeptide was attempted by means of genetic engineering using cDNA or gDNA libraries. However.successful results were not obtained. In the present study, a preparative SDS-PAGE system was applied to the purification of the target molecule. A large amount of cultured P.falciparum parasites was solubilized and the prepared material was subjected to the purification with the use of Prep-cell econo-auomation system(Bio Rad)by continuous-elution electophoresis. The electrophoresed material was devidedP into 40 fractions each with 8.5ml elutate. A 15mu1 sampled specimen from each fraction was taken and the moleular weight was respectively measured using thin layred polyacril amide gel electrophoresis kit (Excel Gel SDS gradiant 8-18, Pharmacia Biotech). Fractions No.28-32 which contained the polypeptide of target molecular weight were pooled. A new type immuno-affinity chromatography (Affi-Gel Hz lmmunoaffinity) was coupled with anti-47kD monolonal antibody and the pooled material was applied to the prepared column. After running with a large amount of washing buffer, the molecule which was bound to the gel by the monoclonal antibody was eluted by a elution buffer. The resulted material is lyophilized and waits for the study of in vitro immune cell response.