日本血吸虫信号转导蛋白Sjwnt_4基因的克隆、表达及功能分析

Cloning, Expression and Characterization of a Gene Encoding Signal Transduction Protein Wnt4 of Schistosoma japonicum

摘要:

由Wnt基因家族产物与其它相关基因产物构成的Wnt信号通路，是细胞发育和生长调节的一个关键途径，对动物的发育特别是生殖系统的发育起重要的调节作用。在人类和小鼠中，Wnt4蛋白是性腺分化过程中主要调节因子，在胚胎发育中起着关键作用。利用RACE技术从日本血吸虫19d童虫中首次扩增到一个Wnt家族基因，序列分析表明该基因的完整编码框含1311bp，编码436个氨基酸，理论分子量49.6kD。同源性分析结果表明，该基因的氨基酸序列具有典型Wnt家族蛋白特征，与日本三角涡虫、人Wnt4的氨基酸序列相似性分别达43％、37％，推测为血吸虫的Wnt4基因，命名为Sjwnt4（GenBank登陆号DQ643829）。实时定量PCR分析显示该基因在14d童虫、19d童虫、31d虫体、44d雌虫及44d雄虫中均有表达，其中19d童虫中的表达量明显高于其它发育阶段，44d雌虫中的表达量明显高于雄虫。构建了该基因的原核表达载体pGEX_4T_2_Sjwnt4，应用大肠杆菌系统进行了表达，表达蛋白以包涵体形式存在，Western印迹显示表达产物能被日本血吸虫成虫粗抗原免疫血清所识别。Sjwnt4基因及其表达产物的获得，为探索Wnt信号通路对血吸虫发育、生殖的调节提供了重要基础。

Abstract:

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, 31days 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.