乙型肝炎病毒S基因经显微注射导入SMMC-7721细胞核内的表达

Expression of HBV-S gene microinjected into SMMC-7721 cell line

目的探讨细胞核显微注射导入外源基因的方法,观察HBV-S基因导入人肝癌细胞株SMMC-7721后的表达及其稳定性。方法将含有HBV-S片段的质粒pCR3.1-S线性化,通过显微注射仪直接注入培养的SMMC-7721细胞核内,G418筛选后,经EIA和免疫荧光法分别检测细胞培养上清和细胞内HBsAg的表达。结果每注射100～150个细胞可筛选出一个阳性克隆,3个阳性克隆经扩大培养后,其中1个克隆的培养上清经EIA法检测HBsAg阳性,免疫荧光显示细胞膜及细胞质均有HBsAg表达,并持续6mo以上。结论 HBV-S基因经显微注射导入SMMC-7721细胞后获得持续稳定的表达。

AIM To study the technology of cell nucleus microinjection and the expression of transfected HBV-S gene in 7721 cell line.METHODS The recombinant plasmid pCR3.1-S, which contains HBV-S, was linearized and microinjected directly into SMMC-7721 cell nucleus under the microinjection apparatus. After G418 selective culture, transformants were examined for the expression of HBsAg by EIA or immunofluroscence assay.RESULTS There was a transformant selected from each 100 150 cells receiving an microinjection. Three transformants were cultured continuously in the common selective pressure. Only 1 transformant was HBsAg positive in the supernatant by EIA, and on the membrane and in the plasma of the transformant by immunoflurescence assay for over 6 months. CONCLUSION HBV-S can permanently express HBsAg after microinjected into the SMMC-7721 cell nucleus.