日本血吸虫表膜特异结合肽重组质粒ZLW/pEGFP-C2体外转染日本血吸虫童虫的研究  

Recombinant Plasmid ZLW/pEGFP-C2 Transfection into Schistosomula of Schistosoma japonicum

作　　者：刘彦[1,2] 曾庆仁[1] 杨胜辉[1,3] 魏琦[1] 周军[4] 李立新[1] 兰玲梅[1] 

机构地区：[1]中南大学医学细胞与分子生物学实验中心,长沙410013 [2]南华大学医学院寄生虫学教研室,衡阳421001 [3]湖南省中医药大学病原免疫学教研室,长沙410208 [4]中南大学湘雅三医院实验中心,长沙410013

出　　处：《中国寄生虫学与寄生虫病杂志》2011年第1期16-20,共5页Chinese Journal of Parasitology and Parasitic Diseases

基　　金：国家自然科学基金(No.30570952);湖南省卫生厅项目(2009);湖南省科技项目(2010)~~

摘　　要：目的观察日本血吸虫表膜特异结合肽合成基因(ZLW)构建的ZLW/pEGFP-C2重组质粒体外转染日本血吸虫童虫的效率,了解其抗虫作用。方法用0.75%二甲基亚砜(DMSO)和高浓度质粒浸泡法,使用重组质粒ZLW/pEGFP-C2转染机械转化的日本血吸虫脱尾童虫,以瞬时表达的增强型绿色荧光蛋白(EGFP)基因为报告基因,在倒置荧光镜下观察虫体。抽提转染后培养48h虫体的总RNA和全虫蛋白,分别用RT-PCR和蛋白质印迹(Western blotting)检测ZLW基因和EGFP基因在虫体内的表达情况。于转染后培养24、48、72和96h,用美蓝染色法鉴别虫体存活情况,并计数。以上试验均设空质粒组和Tris-HCl缓冲盐溶液(TBS)对照组。结果镜下观察结果显示,转染率约为10%,其EGFP主要分布于虫体皮层,以口、腹吸盘最为明显;空质粒转染组虫体腹吸盘处略带绿色荧光。RT-PCR结果显示,ZLW/pEGFP-C2转染组虫体的扩增产物大小约为259bp,测序结果与ZLW序列一致。Western blotting分析证实EGFP基因在童虫体内表达。抗虫效果显示,转染后24h和48h,ZLW/pEGFP-C2转染组童虫死亡率(14.0%,48.8%)与空质粒组(15.9%,45.7%)和TBS对照组(16.9%,50.3%)间的差异无统计学意义(P>0.05);转染后72h,ZLW/pEGFP-C2转染组死亡率(92.7%)高于空质粒组(73.2%)和TBS对照组(76.8%)(P<0.01);转染后96 h,ZLW/pEGFP-C2转染组童虫死亡率达100%。结论 DMSO作用的高浓度质粒浸泡法可将重组质粒ZLW/pEGFP-C2引入日本血吸虫童虫体内,并获得表达。Objective To study the efficiency of ZLW/pEGFP-C2 plasmid transfection into Schistosoma japonicum schistosomula and observe its in vitro effect of anti-schistosomula.Methods Recombinant plasmid ZLW/pEGFP-C2 was transfected into mechanically transformed schistosomula by immersion in 0.75% DMSO and high concentration of plasmid.Enhanced green fluorescent protein（EGFP） transfected cells were observed under inverted fluorescence microscope.At 48 hours after culture,total RNA and proteins from transfected schistosomula were extracted,and the presence of the transgenes（ZLW and EGFP） in schistosomula were confirmed by RT-PCR and Western blotting.At 24,48,72,and 96 hours after transfection,the schistosomula were counted by light microscope with methylene blue staining.pEGFP-C2 empty plasmid group and TBS group served as controls.Results The transfection rate was about 10%.The fluor-escence of ZLW/EGFP protein was mainly localized in the tegument of the worms,especially abundant around oral sucker and ventral sucker.The expected size of 259 bp fragment was successfully amplified by RT-PCR and confirmed by DNA sequencing.Western blotting analysis showed that ZLW/EGFP was expressed in schistosomula.No statistically significant difference was established for schistosomula mortality among ZLW/pEGFP-C2 group（14.0%,48.8%）,pEGFP-C2 group（15.9%,45.7%） and TBS group（16.9%,50.3%） at 24 and 48 hours after transfection（P0.05）.At 72 hours after trans-fection the mortality rate of ZLW/pEGFP-C2 group（92.7%） was significantly higher than that of pEGFP-C2 group（73.2%）（P0.01）,and after 96 h the mortality in ZLW/pEGFP-C2 group increased to 100%.Conclusion ZLW/pEGFP-C2 plasmid has been introduced into juvenile S.japonicum by immersion in 0.75% DMSO and high concentration of plasmid,and was expressed in the parasite.

关 键 词：日本血吸虫 脱尾童虫 表膜结合肽 转染效率 抗虫效果 

分 类 号：R383.24[医药卫生—医学寄生虫学]