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目的克隆和表达日本血吸虫大陆株酪氨酸激酶(TK4)保守区编码基因,并分析其在日本血吸虫雌、雄虫间的差异表达。方法以日本血吸虫大陆株成虫总RNA为模板,经逆转录PCR(RT-PCR)扩增目的基因片段。纯化PCR产物与原核表达载体pET28a质粒连接构建重组质粒pET28a-SjTK4,在异丙基-β-D-硫代半乳糖苷(IPTG)诱导下表达重组蛋白rSjTK4并进行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)、蛋白质印迹(Western blotting)和生物信息学分析。随后,分别提取日本血吸虫雄虫、雌虫及雌雄混合虫体总RNA后,将其逆转录为cDNA,并进行实时荧光定量PCR,确定TK4基因在雌、雄虫虫体的表达水平。结果 RT-PCR扩增出一条582 bp的基因片段,序列分析表明该片段与SmTK4保守区基因序列同源性为91%,推导的氨基酸序列同源性为98%。SDS-PAGE分析显示,rSjTK4重组蛋白的相对分子质量(Mr)约为26 000(含组氨酸标签)。生物信息学分析显示,该蛋白具有多个酶活性位点。雄虫和雌虫中TK4的cDNA相对拷贝数分别为0.61±0.29和0.03±0.02,雄虫TK4的mRNA表达量为雌虫的18倍。结论日本血吸虫TK4保守区编码基因克隆和表达获得成功,该基因的mRNA在日本血吸虫雄虫的表达量显著高于雌虫。
Objective To clone and express the conserved region of tyrosine kinase (TK4) gene of Schistosoma japonicum in Chinese mainland and analyze its differential expression between female and male. Methods The total RNA of adult Schistosoma japonicum was used as a template to amplify the target gene by RT-PCR. The purified PCR product was ligated with the prokaryotic expression vector pET28a to construct the recombinant plasmid pET28a-SjTK4, and the recombinant protein rSjTK4 was induced under the induction of isopropyl-β-D-thiogalactopyranoside (IPTG) and subjected to sodium dodecyl sulfate- Acrylamide gel electrophoresis (SDS-PAGE), Western blotting and bioinformatics analysis. Subsequently, total RNA was extracted from male and female Schistosoma japonicum, mixed with female and male, respectively, then reverse transcribed into cDNA, and real-time PCR was performed to determine the expression level of TK4 gene in female and male worms. Results A 582 bp gene fragment was amplified by RT-PCR. Sequence analysis showed that the fragment shared 91% homology with the SmTK4 conserved region, and the deduced amino acid sequence homology was 98%. SDS-PAGE analysis showed that the relative molecular mass (Mr) of rSjTK4 recombinant protein was about 26,000 (containing histidine tag). Bioinformatics analysis showed that the protein has multiple enzyme active sites. The relative copy numbers of TK4 in male and female were 0.61 ± 0.29 and 0.03 ± 0.02, respectively. The expression level of TK4 mRNA in male was 18 times that of female. Conclusion The gene encoding TK4 conserved region of Schistosoma japonicum has been successfully cloned and expressed. The mRNA expression of this gene in Schistosoma japonicum was significantly higher than that in females.