Construction and Identification of Transgenic Strain of Toxoplasma gondii High-expressing Virulence Factor ROP18

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Construction and Identification of Transgenic Strain of Toxoplasma gondii High-expressing Virulence Factor ROP18

JIANG Zong-ru1，AN Ran1，YANG Jie2，WAN Li-juan1，DU Jian1，3 *   

  1. 1 Department of Biochemistry and Molecular Biology，Anhui Medical University，Hefei 230032，China；2 The First Clinical Medical School，Anhui Medical University，Hefei 230032，China；3 Anhui Provincial Laboratory of Microbiology and Parasitology，Anhui Medical University，Hefei 230032，China
  • Online:2015-02-28 Published:2015-05-04
  • Supported by:

    Supported by the National Natural Science Foundation of China（No. 81271864，30801329），the Fok Ying-Tong Education Foundation for Young Teachers in the Higher Education Institutions of China（No. 131033），the Fund of Anhui Province for Outstanding Youths（No. 10040606Y19，1308085JGD11）and the National College Students′ Innovative Entrepreneurial Training Plan（No. 201310366008）

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Abstract:

Objective  To construct a transgenic strain of Toxoplasma gondii high-expressing ROP18.  Methods The gene sequence of encoding ROP18 was amplified by RT-PCR with RNA of T. gondii RH strain. The purified PCR product was subcloned into pCR-Blunt Ⅱ-Top vector to construct pROP18. The gene sequence of encoding ROP18 was amplified from pROP18， and subcloned into pTUB8-mycGFPPftail-Ty1. The recombinant plasmid pTUB8-ROP18-Ty1 was electroporated into T. gondii RH strain. Stable transgenic cells were selected in the presence of 25 μg/ml mycophenolic acid and 50 μg/ml xanthine， and parasite clones were isolated by limiting dilution after drug selection. The expression of ROP18 in transgenic parasites was detected by immunofluorescence analysis and Western blotting. Giemsa assay was used to detect the proliferation rate of RH strain and the transgenic strain of T. gondii high-expressing ROP18. Twenty mice were divided into two groups. Each mouse in RH strain group was intraperitoneally injected with 1×103 RH strain, and that of transgenic strain group received 1×103 transgenic high-expressing ROP18 strain.  Results  The full-length sequence of ROP18 gene（1 665 bp） was amplified by RT-PCR. The recombinant plasmid pTUB8-ROP18-Ty1 was identified by restriction enzyme digestion and sequencing methods. Western blotting analysis showed that the transgenic high-expressing ROP18 strain expressed ROP18-Ty1（Mr 56 000）. Immunofluorescence assay showed that ROP18-Ty1 was localized in the rhoptry of T. gondii. Giemsa assay confirmed that ROP18 protein enhanced the proliferation of T. gondii. On the 6th， 12th， and 24th hour after HFF cells infected with T. gondii， the number of tachyzoites in transgenic strain group was 100.0±16.9， 476.0±31.1， and 860.0±52.3， respectively， higher than that of RH strain group （88.0±16.9， 300.0±11.3， 675.0±35.4）（P<0.05）. In RH strain group， all the mice were survival on the 8th day post-infection， the survival rate on the 14th day was 30% （3/10）， and all died on the 16th day. In transgenic strain group， all the mice were survival on the 5th day post-infection， the survival rate on the 8th day was 30%（3/10）， and all died on the 9th day.  Conclusion  The transgenic high-expressing ROP18 strain of T. gondii is constructed.

Key words: Toxoplasma gondii, ROP18 gene, Transgenetic parasite