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[Cloning, prokaryotic expression and immunological identification of Toxoplasma surface antigen IMP1].

OBJECTIVE
To subelone, express and identify the immune mapped protein 1 (IMP1) which encodes a surface antigen of Toxoplasma gondii.


METHODS
The cDNA of T. gondii RH strain was synthesized by reverse transcription PCR, the IMP1 open reading frame (ORF) was amplified by PCR using the T. gondii RH strain cDNA as template, the PCR products were identified by TA-cloning and sequencing, then the IMPI ORF was subcloned into the Nde I and Xho I sites of the vector pET28b, and the positive recombinant pET28b-IMP1 was identified by double-digesting and sequencing. The protein of 6 x His tagged IMP1 was inducibly expressed in E. coli strain BL21 (DE3) with isopropyl β-D-1-thiogalactopyranoside (IPTG), and the induction time, concentration of IPTG and temperature gradients to optimize protein expression conditions were determined. After the cells carried IMP1 were induced by the optimized conditions and harvested, the resulting bacteria were suspended in resuspension buffer and lysed by sonication, and the supernatants were loaded onto the Ni2+ Chelating Sepharose Fast Flow column for affinity chromatography of the N-terminal 6 x His tagged IMP1 protein. Finally, the fusion IMP1 proteins were identified by Western blotting.


RESULTS
The ORF sequence of IMP1 was successfully subcloned from the cDNA of Toxoplasma Gondii RH strain, and the amplified product was sequenced and identified, based on which the IMP1 ORF gene was inserted into the prokaryotic expression vector pET28b, and the recombinant pET28b-IMP1 was constructed successfully. The double-digesting and sequencing results indicated the validity of the recombinant vector. And the optimized conditions for the expression of IMP1 was determined, namely 0.3 mmol/L IPTG induction for 9 h at 20 °C. Furthermore, IMP1 protein was expressed solubly and chelated on Ni2 sepharose beads with high affinity, thus this protein could be purified efficiently by affinity chromatography. The pure fusion protein was confirmed with fine immunocompetence by SDS-PAGE and Western blotting.


CONCLUSIONS
IMP1 protein can be high efficiently expressed by the E. coli prokaryotic expression systems, the protein of IMP1 is soluble and has stable characters. The study may lay a useful foundation for the following works including in vivo expression of IMP1, crystal structure study of IMP1 and anti-toxoplasmosis subunit vaccine development.