提高腺病毒表达载体扩增效率的研究

A study on high efficiency in vitro amplification of adenoviral expression vector

目的探讨在体外扩增重组腺病毒表达载体的有效方法。方法用含10%胎牛血清RPMI1640培养液常规培养人胚肾293细胞,当细胞生长至70%～80%汇合时,加入含有报告基因LacZ的重组5型腺病毒载体(Ad5CMVLacZ)共同培养36～38h。用X-gal组织化学方法检测Ad5CMVLacZ转染和表达的程度。结果293细胞生长至70%～80%汇合时,分裂增殖旺盛,形态为梭形、多角形,有明显分裂相,为指数生长期细胞。加入Ad5CMVLacZ8h后,293细胞开始出现肿胀,细胞间联系疏松,36～48h后细胞变成圆形、球形,对光折射增强,并聚集成葡萄串状。在此时间段作X-gal组化反应显示,约90%的293细胞被转染和表达产物。结论用上述方法可提高体外扩增腺病毒载体的效率,只要把握好病毒转染的时机和收获被转染293细胞的时机,就有可能制备出高滴度的病毒液,从而满足基因治疗神经退行性疾病的需要。

Objective To explore an effective way for increasing amplification efficiency of adenoviral expression vector in vitro. Methods Approximately 70%-80% of confluent human embryonic kidney cells （HEK293） were co-cultured with recombinant adenoviral vector （AdSCMVLacZ） for 36-48 h. X-gal histochemistry was performed to identify the transfection and expression of AdSCMVLacZ. Results Under the normal condition, 70%-80% of confluent HEK 293 cells were logarithmically grown. After 36-48 h of co-culture with AdSCMVLacZ, the shape of HEK 293 cells was gradually changed into spherical, and congregated into grape-like aggregation which is the typical cytopathic effect of Ad virus transfection of host cells. At this stage aboat 90% of transfected HEK 293 cells were staimed blue color by X-gal histochemistry. Conclusion The method is an effective way for amplification of adenoviral expression vector in vitro with paying more attention to ascertaining the time of transfectisn of HEK 293 cells with the viral vector and the time for harversting the transfected ceils so as to obtain enough viral vector for gene therapy.