日本血吸虫酪氨酸激酶TK4保守区编码基因的克隆和分析(英文)  

Cloning and Characterization of Conservative Region of Tyrosine Kinase 4,A Novel Gender-Associated Gene of Schistosoma japonicum

作　　者：臧炜[1] 卢潍媛[1] 徐馀信[1] 曹建平[1] 

机构地区：[1]中国疾病预防控制中心寄生虫病预防控制所、卫生部寄生虫病原与媒介生物学重点实验室、世界卫生组织疟疾、血吸虫病和丝虫病合作中心,上海200025

出　　处：《中国寄生虫学与寄生虫病杂志》2014年第3期167-171,共5页Chinese Journal of Parasitology and Parasitic Diseases

摘　　要：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 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,） 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. Conchlsion 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.

关 键 词：IEI本血吸虫 酪氨酸激酶4 克隆 基因表达差异 

分 类 号：R383.24[医药卫生—医学寄生虫学]