[Cloning, expression and characterization of a gene encoding signal transduction protein Wnt4 of Schistosoma japonicum].
Wnt proteins together with their downstream effectors forms a set of important signal pathways. The Wnt signal pathway is important in a wide variety of development processes including cell growth, cell differentiation, cell polarity and apoptosis. Wnt4 is a key regulator of gonadal differentiation in humans and mice, playing a pivotal role in early embryogenesis. With RACE technique based on a EST identified in our lab, a novel gene including a complete open reading frame was cloned and named Sjwnt4 (GenBank accession No. DQ643829). Sequence analyses showed that SjWnt4 had a typical characteristics of Wnt family proteins, sharing 43% similarity to Dugesia japonica and 37% to human Wnt4. The ORF of Sjwnt4 contains 1311 nucleotides, encoding 436 amino acid with 49.6 kD molecular weight. Real-time PCR analysis from the worms of various stages of S. japonicum revealed that the mRNA level of Sjwnt4 is highest in the 19 days schistosomula, followed by 44 days female worms, 14 days schistosomula, 31 days adult worms and 44 days male worms, suggesting a stage-and-gender differential express. The Sjwnt4 cDNA fragment was subcloned into a modified expression vector pGEX-4T-2 and transformed into E. coli BL21 (DE3) cells, and the production of recombinant Sjwnt4 protein fused to a GST tag was analysed. In the presence of IPTG, the 76kD fusion protein was expressed in included bodies. Western-blotting revealed that the fusion protein could be recognized by the rabbit serum specific to Schistosoma japonicum adult worm antigen preparation. The study provides important basis for investigating the regulation mechanism of the Wnt signaling pathway during the development especially gonadal differentiation processes of Schistosoma japonicum.
Tao
Li-Hong
LH
Shanghai Veterinary Research Institute of CAAS, National Laboratory of Animal Schistosomiasis Control, Key Laboratory of Animal Parasitology Ministry of Agriculture, Shanghai 200232, China.
Yao
Li-Xiao
LX
Fu
Zhi-Qiang
ZQ
Feng
Xin-Gang
XG
Liu
Jin-Ming
JM
Shi
Yao-Jun
YJ
Yuan
Chun-Xiu
CX
Cai
You-Min
YM
Lin
Jiao-Jiao
JJ
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005260:Female; D018507:Gene Expression Regulation, Developmental; D015801:Helminth Proteins / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D007106:Immune Sera / Q000276:immunology; D008297:Male; D008969:Molecular Sequence Data; D011817:Rabbits; D011994:Recombinant Proteins / Q000276:immunology / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012549:Schistosoma japonicum / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D012737:Sex Factors; D015398:Signal Transduction / Q000235:genetics*; D013997:Time Factors; D051153:Wnt Proteins / Q000235:genetics* / Q000276:immunology / Q000378:metabolism
D018076:DNA, Complementary; D015801:Helminth Proteins; D007106:Immune Sera; D011994:Recombinant Proteins; D051153:Wnt Proteins