Characterization of immunoreactive proteins of Setaria cervi isolated by preparative polyacrylamide gel electrophoresis.

Filarial parasites are complex mixtures of antigenic proteins and characterization of these antigenic molecules is essential to identify the diagnostically important filaria-specific antigens. In the present study, we have fractionated the somatic extracts from adults of Setaria cervi (bovine filarial parasite) on preparative SDS-polyacrylamide gel electrophoresis and tested the immunoreactivity of the separated gel fractions with polyclonal antibodies against filarial excretory-secretory antigens as well as filarial patients sera. The SDS-PAGE analysis of gel eluted fractions revealed 1 protein band in F-1 fraction, 2 protein bands in F-2 fraction and 2-3 protein bands in all other fractions (F3- F11). Seven gel eluted fractions (F1, F2, F3, F4, F5, F6 and F11) showed high ELISA reactivity with the polyclonal antibody (against excretory-secretory antigen) and four of these fractions (F-1, F-2, F3 and F6) exhibited high ELISA reactivity with antibodies present in filarial patient sera. The reactivities of the gel fractions (F1 and F2), recognized by filarial patients sera, were also tested with the monoclonal antibody (detecting the filarial circulating antigen). The F1 and F2 gel eluted fractions were found to have the target antigen of monoclonal antibody as evident by high reactivity with the monoclonal antibody in ELISA and immunoblotting. The S. cervi gel eluted F1 fraction (containing single antigen) could detect antibodies in filarial patients sera and not in non-filarial sera thereby suggesting its usefulness for specific serodiagnosis of human filariasis.

Immunogenicity of a plasmid DNA vaccine encoding 42kDa fragment of Plasmodium vivax merozoite surface protein-1.

Plasmodium vivax is the second major human malaria parasite that inflicts debilitating morbidity and consequent economic impact in South-East Asian countries. The relapsing nature of P. vivax along with the emergence of drug-resistant P. vivax strains has emphasized the urgent need for a vaccine. However, the development of an effective vivax vaccine is seriously hampered due to the diversity and variation in parasite antigens and non-availability of suitable animal models. DNA based vaccines represent an alternative approach in inducing immunity to multiple targets from different stages of malaria parasite. DNA prime-boosting strategies induce both antibody mediated and cell-mediated immune responses that are the major mechanisms of protection against malaria parasites. We have earlier studied the immunogenicity and protective efficacy of the soluble and refolded forms of recombinant 42kDa fragment of Plasmodium vivax merozoite surface protein-1 (PvMSP-142) using P. cynomolgi rhesus monkey model. In the present study, we have constructed a recombinant DNA vaccine encoding 42kDa fragment of P. vivax MSP-1 and studied the immunogenicity of PvMSP-142 DNA vaccine construct in mice. The 42kDa gene fragment of PvMSP-1 was PCR amplified using gene specific primers and subcloned into pcDNA 3.1 (+) eukaryotic expression vector. In vitro expression of PvMSP-142 plasmid construct was checked by transfection in COS-1 cell line. Indirect immunofluorescence of transfected COS-1 cells probed with monoclonal antibodies against PvMSP-142 exhibited positive fluorescence. Immunization of BALB/c mice with PvMSP-142-pcDNA vaccine construct revealed the immunogenicity of recombinant vaccine plasmid that can be enhanced by prime boosting with recombinant protein corresponding to the DNA vaccine as evidenced by significant elevation of antibody and the cytokines responses.

Cloning, overexpression and characterization of soluble 42kDa fragment of merozoite surface protein-1 of Plasmodium vivax.

Plasmodium vivax represents the second most prevalent malaria species of major public health importance and the global eradication of malaria requires the development of vaccines to prevent infection. The lack of in vitro culture and a suitable animal model for P. vivax malaria are the major problems for the delay in developing a functional vivax vaccine. A number of antigens have been identified for P. vivax as potential malaria vaccine candidates and among these 42kDa fragment of merozoite surface protein-1 (MSP-142) is one of most promising antigen of asexual blood stage. In most of the earlier studies, the MSP-142 of malaria parasites was expressed as insoluble protein in inclusion bodies and it is difficult to get purified protein in conformation form. In the present study, we have cloned, overexpressed and characterized the 42kDa fragment of P. vivax MSP-1 as soluble protein in Escherichiacoli. The 42kDa gene fragment of P. vivax MSP-1 was PCR amplified using specific primers, sequenced and subcloned into pTriEx-4 expression vector. The optimum expression of recombinant P. vivax protein was obtained in SOC growth medium by inducing with 0.2mM IPTG at 37°C for 4h. The SDS-PAGE analysis showed a fusion protein of 55kDa and about 80% was present in soluble form. The purified P. vivax MSP-142 was characterized and found to be correctly folded and in conformation form as evident by CD spectroscopy, presence of 1 free -SH group and the reactivity with reduction sensitive conformational monoclonals against P. vivax MSP-142.