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

Cell Journal

关键词: Toxoplasmosis;    Gene Expression;    Recombinant Protein;    Toxoplasma P35 Protein;   

DOI  :  

来源: DOAJ

【 摘 要 】

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.

【 授权许可】

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