携带角质细胞生长因子和低氧诱导因子-1α双基因减毒沙门菌的遗传稳定性

Genetic stability of attenuated Salmonella TPKH carrying keratinocyte growth factor and hypoxia inducible factor-1α double genes

目的探讨携带角质细胞生长因子(keratinocyte growth factor,KGF)和低氧诱导因子-1α(hypoxia inducible factor-1α,HIF-1α)双基因减毒沙门菌(Ty21a-pIRES-HIF-KGF,TPKH)的遗传稳定性。方法将TPKH连续传代40代,每隔10代检测菌落和菌体的形态、质粒纯度及浓度;每隔5代进行菌落PCR鉴定。取初始代TPKH,接种至大鼠小肠上皮细胞(rat intestinal epithelial cells,IEC-6),于培养12、24、48、72 h时,检测KGF和HIF-1α蛋白的表达水平;取初始代、10、20、30、40代TPKH菌落,接种至IEC-6,培养48 h,检测KGF和HIF-1α蛋白的表达水平。结果 TPKH连续传代40代,菌落和菌体形态基本一致,所含质粒纯度和浓度变化较小,且均可扩增出目的基因条带。初始代TPKH转染IEC-6 12 h,KGF和HIF-1α蛋白水平显著上升(P <0.01);各代TPKH转染IEC-6后,KGF和HIF-1α蛋白水平差异无统计学意义(P> 0.05)。结论 TPKH具有良好的遗传稳定性,本实验为TPKH制剂的大规模生产奠定了基础。

Objective To investigate the genetic stability of attenuated Salmonella TPKH(Ty21a-pIRES-HIF-KGF)carrying keratinocyte growth factor(KGF)and hypoxia inducible factor-1α(HIF-1α)double genes. Methods TPKH was continuously subcultured for 40 passages and identified by colony PCR every 5 passages,of which the morphology,plasmid purity and concentration were observed every 10 passages. Primary TPKH was inoculated to rat intestinal epithelial cells(IEC-6),and determined for the expression levels of KGF and HIF-1α 12,24,48 and 72 h after culture. The TPKH of passages 0,10,20,30 and 40 were inoculated to IEC-6,cultured for 48 h and determined for the expression levels of KGF and HIF-1α. Results The morphology of TPKH subcultured continuously for 40 passages were basically in agreement. The plasmid showed little changes in the purity and concentration,from which the target gene bands were amplified. The expression levels of KGF and HIF-1α in IEC-6 cells 12 h after infection with primary TPKH increased significantly(P < 0. 01). However,the expression levels of KGF and HIF-1α in IEC-6 cells infected with TPKH of various passages showed no significant difference(P > 0. 05). Conclusion TPKH showed good genetic stability,which laid a foundation of large-scale production of TPKH preparations.