Gene -cloning,prokaryotic expression,purification and antigenicity analysis of Toxoplasma gondii receptor for activated C kinase 1

Objective To explore the cloning,prokaryotic express and purification of the receptor for activated C kinase 1(RACK1) gene of RH strain of Toxoplasma gondii,and to analyze the antigenicity of recombinant protein.Methods Total RNA was extracted from tachyzoites of RH strain of T.gondii.The open reading frame of TgRACK1 gene was amplified with a pair of specific primers which was designed according to the coding sequence of TgRACK1 gene(GenBank accession No: AY547291),the product of RT-PCR was digested with double restrict enzyme and ligated into a pGEX-6p-1 vector.The recombinant pGEX-6p-1-TgRACK1 plasmid was transferred into E.coli DH5α and the positive clones were selected through the colony-PCR and confirmed by the double restrict enzyme digestion and sequencing.The successful pGEX-6p-1-TgRACK1 construct was transformed into E.coli BL21(DE3) and induced with IPTG to express.The products of expression were analyzed through SDS-PAGE followed by Coomassie blue staining and the recombinant TgRACK1 was purified by using glutathione-triethyleneglycocyl-sepharose 6B.Western blotting assay with GST primary antibody and rabbit anti-T.gondii serum were used to confirm the expression of rTgRACK1 and analyze its antigenic properties.Results The product of RTPCR was 970 bp.The recombinant plasmid pGEX-6p-1-TgRACK1 was confirmed successfully by colony-PCR,double restrict enzyme digestion and sequencing.A recombinant protein with a rough molecular weight of 63 kDa was analyzed via SDS-PAGE followed by Coomassie blue staining.The GST tag in rTgRACK1 and the antigenicity of rTgRACK1 were detected efficiently by Western blotting analysis with the GST primary antibody and with the prepared antiserum against TgRACK1 respectively.The isolated protein was water soluble and showed 95% purity based on SDS-PAGE and gel imaging analysis.Conclusion The TgRACK1 gene is cloned,the recombinant protein TgRACK1 is produced in E.coli and purified and maintained the specific antigenicity.