Cloning, Expression and Characterization of Toxoplasmagondii P35 protein in E. coli

Cloning, Expression and Characterization of Toxoplasma gondii P35 protein in E. coli

Introduction: Toxoplasma gondii is a protozoan parasite which is globallyprevalent in human and animals. Toxoplasma infection is commonlyasymptomatic, but can cause serious medical problems inimmunocompromised individuals and in fetus. Recombinant antigens of theparasite may be helpful in diagnosing the infection more precisely. The goalof this study was to construct and evaluate the functionality of a prokaryoticexpression plasmid pGEX-P35, harboring P35 surface antigen gene of T.gondii and to perform preliminary studies on its ability to detect T. gondiispecific antibodies.Material and Methods: A 450 bp fragment of the P35 gene was amplifiedand inserted into pGEM-T plasmid, sequenced, cut, and then inserted intopGEX-4T-1 plasmid to produce the recombinant plasmid pGEX-P35. Inorder to confirm that the plasmid construct was capable of expressing P35 inbacterial cells, it was transformed into BL21 strain of E. coli and expressed.The resultant recombinant protein was purified and subjected to SDS-PAGEand Western-blot analysis.Results: A 450 band of PCR product was visualized on 1% agarose gel.Comparison of resultant DNA sequence with GenBank databases showed100% identity with AF01275. Restriction enzyme analysis confirmedsubcloning and correction of orientation. SDS-PAGE analysis showed a 42kDa band of purified expressed protein. Western-blot analysis using mouseantibody against RH strain of T. gondii showed that the recombinant P35antigen could be recognized by specific antibodies.Conclusion: Purified and specific recombinant antigens obtained bymolecular biology techniques are attractive alternatives for detection ofserum antibodies. We amplified and cloned a fragment from P35 gene ofToxoplasma gondii encoding P35 tachyzoite-specific surface antigen.Sequenced fragment was accepted by GenBank with an accession numberof DQ092625. This confirms previous results from other countriesestimating just 1% divergence at the level of DNA sequence betweenlineages isolated from different geographical areas. As the recombinant P35(rP35) antigen is recognized by specific antibodies, it is suggested toevaluate the (rP35) for diagnosis of clinical Toxoplasma gondii infections.

Introduction: Toxoplasma gondii is a protozoan parasite which is globally prevalent in human and animals. Toxoplasma infection is commonly asymptomatic, but can cause serious medical problems in immunocompromised individuals and in fetus. Recombinant antigens of the parasite may be helpful in diagnosing the infection more precisely. The goal of this study was to construct and evaluate the functionality of a prokaryotic expression plasmid pGEX-P35, harboring P35 surface antigen gene of T. gondii and to perform preliminary studies on its ability to detect T. gondii specific antibodies. Material and Methods: A 450 bp fragment of the P35 gene was amplified and inserted into pGEM-T plasmid, sequenced, cut, and then inserted into pGEX-4T-1 plasmid to produce the recombinant plasmid pGEX-P35. In order to confirm that the plasmid construct was capable of expressing P35 in bacterial cells, it was transformed into BL21 strain of E. coli and expressed. The resultant recombinant protein was purified and subjected to SDS-PAGE and Western-blot analysis. Results: A 450 band of PCR product was visualized on 1% agarose gel. Comparison of resultant DNA sequence with GenBank databases showed 100% identity with AF01275. Restriction enzyme analysis confirmed subcloning and correction of orientation. SDS-PAGE analysis showed a 42 kDa band of purified expressed protein. Western-blot analysis using mouse antibody against RH strain of T. gondii showed that the recombinant P35 antigen could be recognized by specific antibodies. Conclusion: Purified and specific recombinant antigens obtained by molecular biology techniques are attractive alternatives for detection of serum antibodies. We amplified and cloned a fragment from P35 gene of Toxoplasma gondii encoding P35 tachyzoite-specific surface antigen. Sequenced fragment was accepted by GenBank with an accession number of DQ092625. This confirms previous results from other countries estimating just 1% divergence at the level of DNA sequence between lineages isolated from different geographical areas. As the recombinant P35 (rP35) antigen is recognized by specific antibodies, it is suggested to evaluate the (rP35) for diagnosis of clinical Toxoplasma gondii infections.