Cloning, expression and enzymatic characterization of 3-phosphoglycerate kinase from Schistosoma japonicum.

Abstract

In the present study, a full-length cDNA encoding the Schistosoma japonicum 3-phosphoglycerate kinase (SjPGK) with an open reading frame of 1251 bp was isolated from 42-day-old (42-d) schistosome cDNAs. Real-time quantitative reverse transcription PCR analysis revealed that SjPGK was expressed in all investigated developmental stages and at a higher transcript levels in 21- and 42-d worms. Moreover, the SjPGK mRNA level was significantly downregulated in 10-d schistosomula from Wistar rats (non-susceptible host). SjPGK was subcloned into pET28a(+) and expressed as both supernatant and inclusion bodies in Escherichia coli BL21 cells. The enzymatic activity of recombinant SjPGK protein (rSjPGK) was 125 U/mg. Kinetic analyses with respect to 3-phosphoglycerate (3-PGA) as substrate gave a Km of 2.69 mmol/L and a Vmax of 748 μmol/min/mg protein. rSjPGK was highly stable over a range of pH 8.0-9.0 and temperature of 30°C-40 °C under physiological conditions. Immunolocalization analysis showed that SjPGK was mainly distributed in the tegument and parenchyma of schistosomes. Western blotting showed that rSjPGK had good immunogenicity. We vaccinated BALB/c mice with rSjPGK combined with Seppic 206 adjuvant. However, there were no significant reductions in the numbers of worms of eggs in the liver, as compared to adjuvant or blank control groups in two independent vaccination tests. This study provides the basis for further investigations into the biological function of SjPGK, although it might not be suitable as a potential vaccine candidate against schistosomiasis.

• Authors: Yang Hong, Lini Huang, Jianmei Yang, Xiaodan Cao, Qian Han, Min Zhang, Yanhui Han, Zhiqiang Fu, Chuangang Zhu, Ke Lu, Xiangrui Li, Jiaojiao Lin
• Journal: Experimental parasitology (Exp Parasitol) Vol. 159 Pg. 37-45 (Dec 2015) ISSN: 1090-2449 [Electronic] United States
• PMID: 26299245 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
• Copyright: Copyright © 2015 Elsevier Inc. All rights reserved.

Chemical References

• Glyceric Acids
• 3-phosphoglycerate
• Phosphoglycerate Kinase

Topics

• Amino Acid Sequence
• Animals
• Cloning, Molecular
• Gene Expression Regulation, Enzymologic
• Glyceric Acids (metabolism)
• Male
• Mice
• Mice, Inbred BALB C
• Phosphoglycerate Kinase (chemistry, genetics, immunology, metabolism)
• Phylogeny
• Rabbits
• Random Allocation
• Rats
• Rats, Wistar
• Schistosoma japonicum (enzymology, genetics, immunology)
• Sequence Alignment
• Vaccination