Gene-cloning, expression and antigenicity analysis of rhoptry protein 17 of Toxoplasma gondii.
OBJECTIVE
To clone and express the rhoptry protein 17 (ROP17) gene of RH strain of Toxoplasma gondii, analyze the antigenicity of recombinant protein.


METHODS
Total RNA was extracted from tachyzoites of RH strain of T. gondii. The open reading frame of TgROP17 gene was amplified with a pair of specific primers which was designed according to the coding sequence of TgROP17 gene (GenBank accession No. AM075203.1), the product of RT-PCR was digested with double restriction enzyme and ligated into a pGEX-6P-1 vector. The recombinant pGEX-6P-1-TgROP17 plasmid was transferred into E. coli DH5alpha and the positive clones were selected through the colony-PCR and confirmed by the double restriction enzyme digestion and sequencing. The constructed pGEX-6P-1-TgROP17 was transformed into E. coli Rosetta (DE3) and induced with IPTG for expression. The expression products were analyzed through SDS-PAGE followed by Coomassie blue staining. Western blotting assay with GST primary antibody and rabbit anti-T. gondii serum was used to confirm the expression of GST-ROP17 and analyze its antigenic properties.


RESULTS
The product of RT-PCR was with 1 850 bp. The recombinant plasmid pGEX-6P-1-TgROP17 was confirmed by colony-PCR, double restriction enzyme digestion and sequencing. A soluble recombinant protein with relative molecular weight of 96,000 was analyzed by SDS-PAGE followed by Coomassie blue staining. The GST tag in GST-ROP17 and the antigenicity of ROP17 were detected efficiently by Western blotting with the GST primary antibody and with the prepared antiserum against T. gondii, respectively.


CONCLUSIONS
The recombinant GST-ROP17 protein has been produced in E. coli and shows specific antigenicity.
Department of Parasitology, Shanxi Medical University, Taiyuan 030001, China.
Hai-long
HL
Wang
Li-tian
LT
Yin
Xiao-li
XL
Meng
Jin-yan
JY
Shen
Hong-li
HL
Liu
Guo-rong
GR
Yin
D000818:Animals; D003001:Cloning, Molecular; D015870:Gene Expression; D008969:Molecular Sequence Data; D010957:Plasmids; D015800:Protozoan Proteins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*; D011817:Rabbits; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D014122:Toxoplasma / Q000235:genetics / Q000276:immunology* / Q000378:metabolism
D015800:Protozoan Proteins; D011994:Recombinant Proteins