论文部分内容阅读
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 a human placental trophoblast cell (HPT-8) that can replicate HBV by transfecting adr subtype HBV-DNA. Methods The recombinant eukaryotic expression plasmid pcDNA3-3HBV was transfected into human placental trophoblast cells by liposome-mediated method. After G418 selection, the transfected cells and the supernatant were harvested by ELISA, immunohistochemistry and PCR Detection. Results After the HPT-8 cells were stably transfected with the pcDNA3-3HBV plasmid, the supernatant of the first and second generation of transfected cells was positive for HBsAg and HBeAg. Immunohistochemistry showed that the positive cells of HBsAg and HBcAg in the transfected cells were positive The HBV-DNA in the culture supernatant was detected by PCR. The titer was 9 × 106 copies / L by fluorescence quantitative PCR, and the presence of HBV-rcDNA and HBV-cccDNA in the transfected trophoblast cells. Conclusion The recombinant plasmid pcDNA3-3HBV can be expressed, transcribed and replicated in human trophoblast cells.