HBeAg真核表达质粒在293T细胞中表达的研究

Study on the HBeAg Expression of Eukaryotic Expression Plasma of HBeAg in 293 T Cells

目的:观察构建的真核表达质粒pJW4303/HBe在人胚肾细胞(293T)中的表达。方法:将构建好的质粒pJW4303/HBe经脂质体导入293T细胞中,分别在转染后3d、6d、9d及12d收集上清,同时对转染的细胞进行传代和冻存,传代的细胞收集上清,冻存的细胞复苏后收集上清。运用ELISA检测表达量的变化。结果:转染后3d、6d、9d及12d细胞上清液1 8稀释后HBeAg检测的OD值分别为1.047±0.093、1.494±0.112、0.998±0.087、0.678±0.124;不同时间转染传代3d后细胞上清液1 8稀释后HBeAg检测的OD值分别为0.943±0.135、1.378±0.127、0.791±0.145、0.505±0.098,不同时间转染冻存复苏3d后细胞上清液HBeAg检测的OD值分别为0.523±0.113、0.742±0.093、0.41±0.106、0.261±0.089。结论:质粒pJW4303/HBe转染293T细胞6d后HBeAg抗原表达量最高,经传代和冻存复苏后的转染细胞仍然能够表达抗原。

Objective To study the HBeAg expression of eukaryotie expression plasmid of HBeAg （ pJW 4303/HBeAg） in human embryonic renal cell lines （293 T cell）. Methods Eukaryotie expression plasmid of HBeAg was constructed in this laboratory and transfected into 293 T cells （with liposome method）. The tranfeeted cells were cultured and supernatants from the cultures were sampled on d3, d6, d9 and d12 for detection of HBeAg expression （with ELISA）. Culture specimens were further passaged to next generation or frozen - stored on d3 - d12 and HBeAg expression of the next generation cell cultures or resuscitated cell cultured were also ,examined. Results The examined OD value of HBeAg with 1 ： 8 dilution on d3, d6, d9 and d12 was 1.047±0.093, 1.494±0. 112, 0. 998 ± 0. 087 and 0.678 ± 0. 124 respectively. OD value with 1 ： 8 dilution in the next generation cell culture d3 supematants from specimens of d3 - d12 was 0. 943±0. 135, 1. 378±0. 127, 0. 791±0. 145 and 0.505±0.098 respectively, that for resuscitated cell eultures was 0. 523 ± 0.113 , 0. 742 ± 0. 093 , 0. 41±0.106 and 0. 261±0. 089 respectively. Conclusion Expression of HBeAg in 293 T cells after tranfection with eukaryotie expression plasma of HBeAg was higher on d6. Next generation cells and resuscitated cells retained their capability of antigen expression.