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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。
Objective To investigate the method of microinjection of exogenous gene into nucleus and to observe the expression and stability of HBV-S gene after it was transfected into human hepatocellular carcinoma cell line SMMC-7721. Methods The plasmid pCR3.1-S containing HBV-S fragment was linearized and injected directly into the nucleus of SMMC-7721 cells by microinjection. After screening by G418, the cell culture supernatants and intracellular were detected by EIA and immunofluorescence HBsAg expression. Results One positive clone was screened from 100 to 150 cells per injection. After the three positive clones were expanded, the culture supernatant of one clone was detected by EIA and HBsAg positive. Immunofluorescence showed HBsAg expression in both cell membrane and cytoplasm, And last 6mo above. Conclusion The HBV-S gene can be stably expressed in SMMC-7721 cells after microinjection.