摘要
目的通过转染adr亚型HBV-DNA,建立可复制HBV的人胎盘滋养层细胞(HPT-8)。方法采用脂质体介导的方法将重组真核表达质粒pcDNA3-3HBV转染人胎盘滋养层细胞,经G418筛选,通过ELISA、免疫组化、PCR等方法对转染细胞和培养上清分别进行检测。结果HPT-8细胞稳定转染质粒pcDNA3-3HBV后,第1、2代转染细胞的培养上清ELISA检测显示HBsAg、HBeAg阳性,免疫组化检测显示转染细胞胞浆中HBsAg、HBcAg呈阳性信号,PCR检测培养上清中HBV-DNA呈阳性,荧光定量PCR检测其滴度达9×106拷贝/L,并且转染滋养细胞中有HBV-rcDNA和HBV-cccDNA存在。结论重组质粒pcDNA3-3HBV能在人滋养层细胞中表达、转录、复制。
Objective To establish the human placental trophoblast （HPT -8） cells for replicating HBV by transfection of pcDNA3 -3HBV adr subtype in vitro. Methods HPT -8 cells were transfected with eukaryotic expression plasmid pcDNA3 - 3HBV by using lipofectamine transfection reagent, after being screened with antibiotic G418. HBsAg and HBeAg were identified by enzyme linked immunosorbent assay （ELISA）, and HBV DNA was tested by polymerase chain reaction （PCR） in the supernatant of transfected HPT - 8 cells. HBcAg and HBsAg were identified by immunocytochemical staining in the transfected HPT - 8 cells. Results After HPT -8 cells were stably transfected with pcDNA3 -3HBV, the ELISA showed, in the supernatant of transfected HPT - 8 cells at passagel and passage2, HBsAg and HBeAg were positive ; the immunocytochemistry showed, in the plasma of transfected HPT- 8 cells, HBsAg and HBcAg were of positive staining; the PCR revealed positive HBV DNA gene in the supernatant ; the real - time quantitative PCR showed copies of HBV genome reach 9 ×10^6 copies/L, and RcDNA and cccDNA was found in the HPT - 8 - HBV cells. Conclusion Recombinant plasmid pcDNA3 - 3HBV could be expressed, transcribed and replicated in HPT- 8 cells.
出处
《中国全科医学》
CAS
CSCD
2008年第1期28-30,共3页
Chinese General Practice
基金
国家自然科学基金重点课题(30230320)
陕西省自然科学基金(2006C248)
