title: 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: Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Key Laboratory of Animal Parasitology, Ministry of Agriculture of China, Shanghai 200241, PR China.
authors: Yang
authors: Y
authors: Hong
authors: Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Key Laboratory of Animal Parasitology, Ministry of Agriculture of China, Shanghai 200241, PR China.
authors: Lini
authors: L
authors: Huang
authors: Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Key Laboratory of Animal Parasitology, Ministry of Agriculture of China, Shanghai 200241, PR China.
authors: Jianmei
authors: J
authors: Yang
authors: Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Key Laboratory of Animal Parasitology, Ministry of Agriculture of China, Shanghai 200241, PR China.
authors: Xiaodan
authors: X
authors: Cao
authors: Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Key Laboratory of Animal Parasitology, Ministry of Agriculture of China, Shanghai 200241, PR China.
authors: Qian
authors: Q
authors: Han
authors: Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Key Laboratory of Animal Parasitology, Ministry of Agriculture of China, Shanghai 200241, PR China; College of Animal Science and Technology, Henan University of Science and Technology, Luoyang, Henan Province 471023, PR China.
authors: Min
authors: M
authors: Zhang
authors: Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Key Laboratory of Animal Parasitology, Ministry of Agriculture of China, Shanghai 200241, PR China; College of Animal Science, Henan Institute of Science and Technology, Xinxiang, Henan Province 453003, PR China.
authors: Yanhui
authors: Y
authors: Han
authors: Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Key Laboratory of Animal Parasitology, Ministry of Agriculture of China, Shanghai 200241, PR China.
authors: Zhiqiang
authors: Z
authors: Fu
authors: Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Key Laboratory of Animal Parasitology, Ministry of Agriculture of China, Shanghai 200241, PR China.
authors: Chuangang
authors: C
authors: Zhu
authors: Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Key Laboratory of Animal Parasitology, Ministry of Agriculture of China, Shanghai 200241, PR China.
authors: Ke
authors: K
authors: Lu
authors: College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, Jiangsu Province, 210095, PR China.
authors: Xiangrui
authors: X
authors: Li
authors: Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Key Laboratory of Animal Parasitology, Ministry of Agriculture of China, Shanghai 200241, PR China; Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, Jiangsu Province 225009, PR China. Electronic address: jjlin@shvri.ac.cn.
authors: Jiaojiao
authors: J
authors: Lin
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D015971:Gene Expression Regulation, Enzymologic; D005988:Glyceric Acids / Q000378:metabolism*; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D010735:Phosphoglycerate Kinase / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D010802:Phylogeny; D011817:Rabbits; D011897:Random Allocation; D051381:Rats; D017208:Rats, Wistar; D012549:Schistosoma japonicum / Q000201:enzymology* / Q000235:genetics / Q000276:immunology; D016415:Sequence Alignment; D014611:Vaccination
chemical_list: D005988:Glyceric Acids; C005156:3-phosphoglycerate; D010735:Phosphoglycerate Kinase
keywords: 3-Phosphoglycerate kinase (PGK); Enzymatic activity; Schistosoma japonicum (S. japonicum)