阴道毛滴虫病毒介导的EGFP在阴道毛滴虫细胞内的表达

Trichomonas vaginalis virus-mediated expression of EGFP in the parasitic protozoan Trichomonas vaginalis

目的研究阴道毛滴虫病毒(Trichomonas vaginalis virus,TVV)介导外源基因进入阴道毛滴虫体内表达的能力,探索TVV作为双链RNA病毒转染载体的可能性。方法根据TVV基因组的序列特征,用绿色荧光蛋白(EG-FP)编码基因替换TVV的全部或部分基因编码区,构建TVV与增强型EGFP编码基因的嵌合体pTVV-EGFP,其体外转录体经电穿孔方法转染携病毒阴道毛滴虫株,RT-PCR及SDS-PAGE方法检测EGFP的表达情况。结果电穿孔转染后培养的虫体在荧光显微镜下观察到绿色荧光信号,且续传15代后仍然存在;RT-PCR检测到EGFP的mRNA,SDS-PAGE检测到转染后虫体及培养上清中有分子质量单位为27 ku的蛋白,与已知EGFP的分子质量相符。结论TVV能成功介导外源性EGFP编码基因在阴道毛滴虫体内表达。

Objective To investigate the capacity for foreign gene expression mediated by dsRNA Trichomonas vaginalis virus （TVV） in the parasitic protozoan T. vaginalis and its potential to serve as a dsRNA transfection vector for T. vaginalis. Methods A chimera of TVV cDNA and enhanced green fluorescent protein （EGFP） was constructed by flanking the firefly luciferase gene with the 5＇and 3＇ untranslated regions （UTRs） of the TVV genome, and the transcript of the construct was synthesized in vitro with T7 RihoMAXTM Express Large Scale RNA Production System （Promega）. After transfection to T. vaginalis already infected with TVV by electroporation, the transcription and expression of EGFP were detected by RT-PCR and SDS-PAGE. Results The results indicated that the transcript of the chimera pTVV-EGFP fluoresced under fluorescence microscopy, and fluorescence was still present in the transfected protozoan after 15 generations. EGFP mRNA was detected by RT-PCR in transfected T. vaginalis and the results of SDS-PAGE indicated that EGFP was expressed in T. vaginalis. Conclusion TVV can mediate the expression of foreign gene EGFP in the parasitic protozoan T. vaginalis.