日本血吸虫TOR真核表达载体的构建及其在293T细胞中的表达  

CONSTRUCTION OF EUKARYOTIC EXPRESSION VECTOR OF SCHISTOSOMA JAPONICUM TOR AND ITS EXPRESSION IN 293T CELLS

作　　者：宰金丽 马帅[1] 王涛[1] 贾秉光 柴淑梅 张倩[1] 林矫矫[1] 傅志强[1] ZAI Jin-li;MA Shuai;WANG Tao;JIA Bing-guang;CHAI Shu-mei;ZHANG Qian;LIN Jiao-jiao;FU Zhi-qiang(Shanghai Veterinary Research Institute, CAAS, Shanghai 200241, China)

机构地区：[1]中国农业科学院上海兽医研究所,上海200241

出　　处：《中国动物传染病学报》2017年第1期66-71,共6页Chinese Journal of Animal Infectious Diseases

基　　金：国家自然科学基金(31472188);国家科技支撑计划(2015BAI09B04);上海市领军人才后备队计划(201442)

摘　　要：本研究扩增到日本血吸虫Sj TOR完整的蛋白编码区并将其克隆到p XJ40-FLAG载体的HindⅢ、XhoⅠ酶切位点,构建真核表达质粒p XJ40-FLAG-TOR。将测序正确的质粒转染到293T细胞中进行表达,然后应用间接免疫荧光、实时荧光定量PCR、Western blot检测其在293T细胞中的表达情况。测序结果表明Sj TOR蛋白编码区为1245 bp,真核表达质粒p XJ40-FLAG-TOR构建成功。转染293T细胞48 h后,应用间接免疫荧光染色可观察到转染重组质粒的细胞有特异性绿色荧光,空质粒对照则未见。实时荧光定量PCR结果显示,在转染质粒p XJ40-FLAG-TOR 6 h后Sj TOR蛋白的基因已有转录,至转染24 h时转录水平最高,随后开始降低。Western blot结果显示Sj TOR蛋白分子量约53 k Da,可被FLAG单抗和抗Sj TOR-ED1抗血清识别。结果表明Sj TOR蛋白可以在293 T细胞中表达,为进一步研究Sj TOR蛋白的生物学功能和DNA疫苗打下了基础。The DNA fragment encoding Schistosoma japonicum TOR protein were amplifi ed from cDNA templates by PCR and cloned into pXJ40-FLAG through Hin dⅢ、XhoⅠto construct the eukaryotic expression plasmid pXJ40-FLAG-TOR.The recombinant plasmid was verifi ed by sequencing and transfected into293T cells.The expression of the target protein in293T cell was detected by indirect immunofl uorescence,Western blot and quantitative Real-time PCR.Successful expression of Sj TOR protein was confi rmed by the specifi c green fl uorescence in293T cells after transfection for48h and no green fl uorescence in the cell transfected with the control plasmid.Quantitative Real-time PCR results showed that the transcription of Sj TOR gene in293T cells could be detected at6h and came to a head at24h after trandfection,since then began to decline.Western blot results demonstrated that Sj TOR with the molecular weight of53kDa was recognized by Anti-FLAG mouse monoclonal antibody and Sj TOR-ED1antiserum.Results showed that the Sj TOR proteins could be expressed in293T cells,and laid a foundation for further research on Sj TOR biological function and its DNA vaccine.

关 键 词：日本血吸虫 TOR蛋白 基因克隆 真核表达 

分 类 号：S852.735[农业科学—基础兽医学]