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目的扩增日本血吸虫的酪氨酸羟化酶(Schistosoma japonicumTyrosine Hydroxylase,Sj TH)编码基因,构建pcDNA3.1(+)-SjTH真核表达载体,并检测其在COS-7细胞中的表达情况。方法以日本血吸虫成虫cDNA为模板,RACE PCR扩增Sj TH编码基因,并与pGEM-T连接进行亚克隆,双酶切后回收目的基因,并与真核表达载体pcDNA3.1(+)连接,PCR和双酶切初步鉴定后测序,纯化无内毒素重组质粒pcDNA3.1(+)-Sj TH,转染入COS-7细胞,G418筛选阳性克隆,RT-PCR和Western blot鉴定重组Sj TH蛋白的表达。结果 RACE PCR扩增出SjTH编码基因,大小约1392bp,经双酶切鉴定、测序及Blast分析鉴定重组真核质粒构建成功。脂质体介导无内毒重组真核质粒pcDNA3.1(+)-SjTH转染入COS-7细胞,G418筛选出阳性克隆,RT-PCR证实阳性单克隆细胞带有SjTH编码基因,Western blot鉴定单克隆细胞表达重组SjTH蛋白,大小约54kD。结论真核表达载体pcDNA3.1(+)-SjTH构建成功,G418筛选出阳性克隆,真核表达重组SjTH蛋白,为后续研究SjTH蛋白功能奠定基础。
Objective To amplify the gene encoding Schistosoma japonicum Tyrosine Hydroxylase (Sj TH) and construct the eukaryotic expression vector pcDNA3.1 (+) - SjTH and detect its expression in COS-7 cells. Methods The cDNA encoding Schistosoma japonicum was used as a template to amplify Sj TH gene by RACE PCR. The gene was subcloned into pGEM-T and double-digested. The target gene was recovered and ligated with eukaryotic expression vector pcDNA3.1 (+), PCR and restriction endonuclease digestion. The recombinant plasmid pcDNA3.1 (+) - Sj TH was successfully transfected into COS-7 cells and positive clones were screened by G418. The recombinant Sj TH protein was identified by RT-PCR and Western blot expression. Results SjTH gene was amplified by RACE PCR and its size was about 1392bp. After double enzyme digestion, sequencing and Blast analysis, the recombinant eukaryotic plasmid was successfully constructed. The recombinant plasmid pcDNA3.1 (+) - SjTH was transfected into COS-7 cells without lipofection. The positive clones were screened by G418. The positive clones were confirmed by RT-PCR with SjTH encoding gene. Western blot Monoclonal cells were identified as recombinant SjTH proteins with a size of about 54 kD. Conclusion The eukaryotic expression vector pcDNA3.1 (+) - SjTH was successfully constructed. The positive clones were screened by G418 and the recombinant SjTH protein was expressed in eukaryotic cells, which laid the foundation for the subsequent study on the function of SjTH protein.