关键词: 日本血吸虫, 酪氨酸激酶4, 克隆, 基因表达差异

Abstract: Objective  To clone and express the conservative region of gene encoding tyrosine kinase 4 of Schistosoma japonicum and identify the difference in gene expression between genders of S. japonicum.  Methods  The gene fragment was amplified by reverse transcriptase-polymerase chain reaction（RT-PCR）using the total RNA isolated from adult S. japonicum（Chinese strain）with primers designed according to SmTK4 encoding tyrosine kinase 4. The purified PCR product was ligated with pET28a and the recombinant protein was induced to express, and analyzed by SDS-PAGE, Western blotting and tools of bio-informatics. Subsquently, total RNA was respectively isolated from adult males, females and both worms of S. japonicum. The real-time PCR was performed with corresponding primers after reverse transcription to show the expression levels of the gene in both genders.  Results  A 582 bp in size of the DNA fragment was acquired by RT-PCR. Sequence analysis indicated that the fragment showed 91% in homology to that of SmTK4, and the deduced amino acid sequence showed to be 98% identical with that encoded by SmTK4. SDS-PAGE analysis revealed that the relative molecular weight（M_r）of expressed protein rSjTK4 was approximately 26 000. The bio-information analysis demonstrated that the protein had multiple sites of enzymatic activities. The relative number of copies of SjTK4 in male worms was 0.61±0.29, while 0.03±0.02 in female worms, showing that the mRNA level of TK4 in male worms was 18 times higher than that in females.  Conclusion  The conservative region of gene encoding tyrosine kinase 4 of S. japonicum is successfully cloned and expressed. The mRNA level of TK4 in male worms is significantly higher than that in females.

Key words: Schistosoma japonicum, Tyrosine kinase 4, Clone, Gene expression difference