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目的验证外源绿色荧光蛋白基因在日本血吸虫(Schistosoma japonicum)体内能否成功表达并产生绿色荧光。方法采用荧光显微镜观察日本血吸虫不同虫期虫体的背景荧光情况。利用含有由巨细胞病毒(CMV)启动子驱动的绿色荧光蛋白zs Green基因的慢病毒载体感染体外培养的14 d童虫,未感染对照组采用不含慢病毒的RPMI 1640培养液(含10%胎牛血清)培养。感染后48 h,提取童虫基因组DNA和总RNA,合成c DNA,PCR检测zs Green基因是否整合入基因组以及绿色荧光蛋白m RNA的转录表达情况。在感染后24和48 h,以及换为不含慢病毒的RPMI 1640培养液后的不同时间点,使用荧光显微镜观察被感染童虫发出的绿色荧光,并判断其是否为绿色荧光蛋白基因在虫体内表达而产生的绿色荧光信号。结果荧光显微镜观察可见,日本血吸虫童虫无明显自发荧光现象,成虫可自发明显荧光。感染组童虫基因组DNA和总c DNA PCR检测结果显示,均扩增出大小为173 bp的阳性条带,与zs Green基因片段一致,且测序结果正确。荧光显微镜观察可见,而感染组童虫在感染后24 h,肠道内出现绿色荧光亮点,但疑似食物残渣。感染后48 h,童虫肠道发出均匀的绿色荧光,其亮度高于慢病毒培养液的背景荧光亮度。更换为不含慢病毒的RPMI 1640培养液3 d后,童虫肠道内绿色荧光变弱,一周后绿色荧光消失。重新更换为含慢病毒的RPMI 1640培养液感染童虫,2 d后在肠道内再次发现均匀绿色荧光。未感染对照组童虫未出现绿色荧光。结论外源绿色荧光蛋白可在日本血吸虫体内表达,但荧光显微镜下观察到的绿色荧光信号还有待进一步确认。
Objective To verify whether exogenous green fluorescent protein (EGFP) gene can be successfully expressed in Schistosoma japonicum and produce green fluorescence. Methods Fluorescence microscopy was used to observe the background fluorescence of Schistosoma japonicum at different stages of infection. The 14-day-old schistosomula were infected with the lentiviral vector containing the green fluorescent protein zs Green gene driven by the cytomegalovirus (CMV) promoter. The uninfected control group was infected with lentivirus-free RPMI 1640 medium containing 10% Fetal bovine serum). 48 h after infection, genomic DNA and total RNA were extracted from squirrels and c DNA was synthesized. PCR was performed to detect whether zs Green was integrated into the genome and transcription of green fluorescent protein (m RNA) was detected. Fluorescent microscopy was used to observe the green fluorescence of the infected squirrels at 24 and 48 h post-infection, and at different time points after transplanting to RPMI 1640 medium without lentivirus, and to determine if they were green fluorescent protein In vivo expression of the resulting green fluorescence signal. Results Fluorescence microscopy showed that there was no obvious autofluorescence in Schistosoma japonicum and the adults could spontaneously and obviously fluoresce. Genomic DNA and total c DNA of the infected group were detected by PCR. Positive bands of 173 bp were amplified, consistent with the zs Green gene fragment, and the sequencing results were correct. Fluorescent microscopy can be seen, while the infected group of worms 24 h after infection, intestinal fluorescent green spot, but suspected food residue. 48 h after infection, the juvenile worm gut emitted a homogeneous green fluorescence, which was higher than the background fluorescence of lentivirus broth. After replacing with lentivirus-free RPMI 1640 medium for 3 days, the green fluorescence in the intestinal tract of the squirrel was weakened, and disappeared after a week. Re-replace the lentivirus-containing RPMI 1640 culture medium to infect the pest, and then find uniform green fluorescence in the intestine again after 2 days. The uninfected control group did not show green fluorescence in the flies. Conclusion Exogenous green fluorescent protein can be expressed in Schistosoma japonicum, but the green fluorescent signal observed under fluorescence microscope remains to be confirmed.