Zhongguo ji sheng chong xue yu ji sheng chong bing za zhi = Chinese journal of parasitology & parasitic diseases · 2012
[Identification of glycosylphosphatidylinositol-anchored protein from Schistosoma japonicum].
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Cao Qin-Yan, Xue Yan-Feng, Shen Li
Аннотация
OBJECTIVE: To identify glycosylphosphatidylinositol (GPI) anchored protein of Schistosoma japonicum.
METHODS: Based on the gene sequence of Schistosoma mansoni GPI anchored protein Sm200 (GenBank Assess No: XM_002569560.1), bioinformatics analysis was performed to find out its homologous gene sequence in S. japonicum, then a selected partial coding sequence (SjGPIs, about 933 bp) from the homologous gene sequence were amplified, and cloned into PET-28a(+) vector. The recombinant plasmid pET-28a(+)SjGPIs were transformed into E. coli Top10 cells and induced with IPTG for protein expression. The recombinant protein SjGPIs was purified with Ni-NTA resin, and the purified recombinant SjGPIs protein was used as antigen to prepare antiserum in New Zealand rabbit. The antiserum was used to detect S. japonicum GPI-anchored protein. To identify a GPI-anchored protein, the detected protein were identified by phosphatidylinositol-specific phospholipase C (PI-PLC) digestion. White blood cells from S. japonicum-infected mice was examined whether they endocytosed GPI-anchored proteins by Western blotting.
RESULTS: The homologous gene sequence of S. mansoni GPI Sm200 gene was found in S. japonicum genome. A 3 495 bp coding sequence was obtained, containing the complete C-terminal sequence. The selected gene sequence (SjGPIs) were amplified and the recombinant plasmid pET-28a(+)-SjGPIs was established. According to the analysis of C-terminal sequence, Western blotting and enzyme digestion of PI-PLC, a GPI-anchored protein was present in S. japonicum tegument (about 1M(r)200000), named SjGPI200. The protein was detected in white blood cells of infected mice.
CONCLUSION: SjGPI200 protein exists in S. japonicum, and anchored to parasite tegument via GPI.
METHODS: Based on the gene sequence of Schistosoma mansoni GPI anchored protein Sm200 (GenBank Assess No: XM_002569560.1), bioinformatics analysis was performed to find out its homologous gene sequence in S. japonicum, then a selected partial coding sequence (SjGPIs, about 933 bp) from the homologous gene sequence were amplified, and cloned into PET-28a(+) vector. The recombinant plasmid pET-28a(+)SjGPIs were transformed into E. coli Top10 cells and induced with IPTG for protein expression. The recombinant protein SjGPIs was purified with Ni-NTA resin, and the purified recombinant SjGPIs protein was used as antigen to prepare antiserum in New Zealand rabbit. The antiserum was used to detect S. japonicum GPI-anchored protein. To identify a GPI-anchored protein, the detected protein were identified by phosphatidylinositol-specific phospholipase C (PI-PLC) digestion. White blood cells from S. japonicum-infected mice was examined whether they endocytosed GPI-anchored proteins by Western blotting.
RESULTS: The homologous gene sequence of S. mansoni GPI Sm200 gene was found in S. japonicum genome. A 3 495 bp coding sequence was obtained, containing the complete C-terminal sequence. The selected gene sequence (SjGPIs) were amplified and the recombinant plasmid pET-28a(+)-SjGPIs was established. According to the analysis of C-terminal sequence, Western blotting and enzyme digestion of PI-PLC, a GPI-anchored protein was present in S. japonicum tegument (about 1M(r)200000), named SjGPI200. The protein was detected in white blood cells of infected mice.
CONCLUSION: SjGPI200 protein exists in S. japonicum, and anchored to parasite tegument via GPI.