关键词: 日本血吸虫, 绿色荧光蛋白, 慢病毒载体

Abstract:

Objective To test if the green fluorescent protein gene can be expressed in Schistosoma japonicum and produce green fluorescence signals there. Methods The spontaneous fluorescence in Schistosoma japonicum at different developmental stages was observed by fluorescence microscopy. The cultured 14-day schistosomula were infected with the lentiviral vector containing cyto megalo virus（CMV）-driven zsGreen gene, or were incubated with RPMI 1640 medium containing 10% fetal bovine serum as a negative control. At 48 h after infection, genomic DNA and total RNA were extracted, and cDNA was synthesized. PCR was performed to detect the genomic integration of zsGreen gene and the expression of zsGreen mRNA. Green fluorescence was observed under a fluorescence microscope at 24 h and 48 h after infection, and at various time points after replacement with fresh culture medium. Results There was no obvious spontaneous fluorescence in schistosomula, but the adult worms showed clear spontaneous fluorescence. PCR results showed a specific band of 173 bp from the schistosomula genomic DNA, which corresponded to the zsGreen gene. Gene sequencing also confirmed this result. At 24 h after infection, green fluorescence was seen in the intestine of schistosomula, which was, however, suspected to be food residues. At 48 h after infection, evenly-distributed green fluorescence emerged across the intestine, and the fluorescence was brighter than the background fluorescence of the lentiviral culture medium. The fluorescence weakened on day 3 after replacement with fresh 1640 medium, and disappeared a week later. The green fluorescence re-emerged 2 weeks after replacement with the lentivirus-containing 1640 medium. No fluorescence was seen in the negative control group. Conclusion The exogenous green fluorescent protein gene can be expressed in Schistosoma japonicum. However, the green fluorescence seen under the fluorescence microscope needs further verification.

Key words: Schistosoma japonicum, Green fluorescent protein, Lentiviral vector