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

摘要

本研究扩增到日本血吸虫SjTOR完整的蛋白编码区并将其克隆到pXJ40-FLAG载体的HindⅢ、Xho Ⅰ酶切位点,构建真核表达质粒pXJ40-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SjTOR蛋白编码区为1245 bp,真核表达质粒pXJ40-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pXJ40-FLAG-TOR 6 h后SjTOR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SjTOR蛋白分子量约53 kDa,可被FLAG单抗和抗jTOR-ED1抗血清识别.结果表明SjTOR蛋白可以在293 T细胞中表达,为进一步研究SjTOR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 amplified from cDNA templates by PCR and cloned into pXJ40-FLAG through Hind Ⅲ、Xho I to construct the eukaryotic expression plasmid pXJ40-FLAG-TOR.The recombinant plasmid was verified by sequencing and transfected into 293T cells.The expression of the target protein in 293T cell was detected by indirect immunofluorescence,Western blot and quantitative Real-time PCR.Successful expression of SjrOR protein was confirmed by the specific green fluorescence in 293T cells after transfection for 48 h and no green fluorescence in the cell transfected with the control plasmid.Quantitative Real-time PCR results showed that the transcription ofSjTOR gene in 293T cells could be detected at 6 h and came to a head at 24 h after trandfection,since then began to decline.Western blot results demonstrated that SjTOR with the molecular weight of 53 kDa was recognized by Anti-FLAG mouse monoclonal antibody and SjTOR-ED 1 antiserum.Results showed that the SjTOR proteins could be expressed in 293 T cells,and laid a foundation for further research on SjTOR biological function and its DNA vaccine.