关键词: 刚地弓形虫, 微线体蛋白2, 原核表达, 可溶性蛋白

Abstract:

 Objective To express Toxoplasma gondii microneme protein 2(TgMIC2) in different Escherichia coli strains, and confirm its protein expression by Western blotting. Methods A primer pair was designed according to the TgMIC2 sequence in GenBank. The TgMIC2 gene was amplified by PCR using first strand cDNA as template after reverse transcription of total RNA from tachyzoites of T. gondii RH strain. The PCR products were digested with NdeⅠand Hind Ⅲ and cloned into the expression vector pET-30a(+). The recombinant plasmid was transformed into E. coli TOP10 and positive clones were confirmed by double restriction digestion and sequenced. The correct plasmid was transformed into E. coli strains BL21(DE3), ArcticExpress(DE3) and Shuffle, and then induced by IPTG for protein expression. The expressed proteins were purified with Ni-NTA affinity chromatography and analyzed by SDS-PAGE. The immune activity of the proteins was analyzed by Western blotting using His-tag monoclonal antibody as primary antibody. Results The TgMIC2 PCR product was 2 000 bp in length. SDS-PAGE analysis showed that TgMIC2 protein had a Mr of 80 000, and the expression pattern and amount of the recombinant protein differed among the strains. Soluble forms and inclusion bodies of recombinant TgMIC2 were seen in BL21 (DE3) and ArcticExpress (DE3) strains with the soluble protein consisting about 10% under different induction conditions(15 ℃, 16 h and 37 ℃, 4 h), while only inclusion body of recombinant TgMIC2 was seen in the Shuffle strain. Western blotting showed that both forms of TgMIC2 could be recognized by His-tag monoclonal antibody after purification. Conclusion pET30a-MIC2 plasmid is constructed and TgMIC2 is successfully expressed in different prokaryotic expression systems.

Key words: Toxoplasma gondii, Miconeme protein 2, Prokaryotic expression, Soluble protein