冻干重组人三突变型HIF-1α腺病毒生物学效应考察

Characterization of the biological activities of lyophilized recombinant adenovirus expressing the triple mutant of hypoxia inducible factor-1α

目的研究冻干对重组人三突变型HIF-1α腺病毒(Ad-HIF-1α-564/402/803)生物学效应的影响。方法将前期构建的Ad-HIF-1α-564/402/803在HEK293A细胞中进行扩增,用氯化铯浓度梯度离心法进行腺病毒纯化,X-Gal染色法测定重组腺病毒转染效率,在适宜的条件下制成冻干剂。采用MTS试剂盒观察冻干前后Ad-HIF-1α-564/402/803对hMVECs增殖的影响。分别在冻干前、冻干后1 d、6月、12月四个时间点提取病毒DNA,进行PCR及PCR产物测序鉴定重组人三突变型HIF-1α腺病毒基因;并在4个时间点以Ad-HIF-1α-564/402/803转染hMVECs,提取蛋白进行western blot检测HIF-1α蛋白的表达。结果 X-gal染色显示MOI为100pfu/cell时,转染效率趋于稳定。冻干前后重组人三突变型HIF-1α腺病毒对hMVECs增殖影响无显著差异(P>0.05)。经PCR及基因测序鉴定,在四个时间点,腺病毒所携带的目的基因信息无丢失或突变,HIF-1α蛋白表达无差异(P>0.05)。结论冻干能够长期保持重组人三突变型HIF-1α腺病毒的高生物活性。

Objective To investigate the influence of lyophilization on the biological activity of recombinant adenovirus-mediated triple mutant of hypoxia inducible factor-1α（Ad-HIF-1α-564/402/803）.Methods Ad-HIF-1α-564/402/803 was amplified from HEK293A cells and purified by ultracentrifugation in CsCl gradient solutions.The infection efficiency was observed by X-gal staining.The lyophilized adenovirus was prepared under appropriate conditions.Before and after lyophilization,the effect of Ad-HIF-1α-564/402/803 on hMVEC proliferation was evaluated by MTS assay.The recombinant adenovirus was confirmed by PCR and DNA sequence analysis before and 1 day,6 months and 12 months after lyophilization,and hMVECs infected with Ad-HIF-1α-564/402/803 at these time points were examined for HIF-1α protein expression using Western blotting.Results No significant changes were observed in the effect of lyophilized Ad-HIF-1α-564/402/803 on hMVECs proliferation at the optimal multiplicity of infection of 100 pfu/cell（P0.05）.At the 4 time points,the recombinant adenovirus HIF-1α showed no structural alterations or significant changes in the expression level of HIF-1α protein in the transfected hMVECs（P0.05）.Conclusion Lyophilized Ad-HIF-1α-564/402/803 can maintain its biological activities for a long time.