hTERT启动子特异驱动IL-24重组腺病毒的包装及鉴定

Package and identification of recombinant adenovirus containing IL-24 gene specifically driven by hTERT promoter

目的包装并鉴定人端粒酶逆转录酶(hTERT)启动子特异驱动白介素-24(IL-24)的重组复制缺陷型腺病毒。方法全基因合成优化后的hTERT启动子,插入质粒pGL3-basic的多克隆位点(pGL3-phTERT);从人外周血单核细胞扩增IL-24,插入pGL3-phTERT质粒的hTERT启动子下游获得pGL3-phTERT-IL24;随后将phTERT-IL24插入pShuttle中间质粒,使用AdEasyTMAdenoviral Vector System包装Ad-phTERT-IL24重组腺病毒;同时包装CMV启动子驱动IL-24的腺病毒载体为阴性对照(Ad-CMV-IL24),以空载腺病毒Ad作为空白对照。用20 MOI重组腺病毒感染SGC-7901、MKN-45、HF细胞,采用PCR及Western blot检测IL-24在RNA及蛋白水平的表达。结果成功包装Ad-hTERT-IL24重组腺病毒。感染Ad-hTERT-IL24后,hTERT阳性肿瘤细胞内IL-24表达显著上调,但hTERT阴性的HF细胞则无IL-24表达;感染阴性对照Ad-CMV-IL24后,hTERT阳性或阴性细胞内均有IL-24表达,感染空白对照Ad后,hTERT阳性或阴性细胞内均无IL-24表达。结论 AdhTERT-IL24腺病毒载体可有效介导IL-24特异表达于hTERT阳性肿瘤细胞内。

Objective To pack and identify the recombinant replication defective adenovirus which contains IL-24 gene specifically driven by human telomerase reverse transcriptase （hTERT） promoter. Methods The hTERT promoter receiving the total gene synthesis and optimization was inserted into the multi-clone site （pGL3-phTERT） of plasmid pGL3-basic. IL-24 gene was amplified from the human peripheral blood monocytes and inserted into the lower reach of pGL3-phTERT promoter in order to obtain pGL3-phTER-IL24. Then phTERT-IL2d was inserted into the intermediate plasmid of pShuttle. AdEasy^TM Adenoviral Vector System was use to pack Ad-phTERT-IL24 recombinant adenovirus. The adenovirus vector containing the IL-24 driven by CMV promoter was packed and set as the negative control. Blank adenovirus was set as the blank control. SGC-7901, MKN45, and HF cells were infected by recombinant adenovirus at 20 MOI. PCR and Western blot method was used to detect the expression levels of IL-24 in RNA and proteins. Results Ad-phTERT-IL24 recombinant adenovirus was successfully packed. After the hTERT（ ＋ ） tumor cells were infected with Ad-phTERT-II24, the expression level of IL-24 in those cells significantly up-regulated. But there was no expression of IL-24 in hTERT（ - ） HF cells. IL-24 was expressed in both hTERT（ ＋ ） and hTERT（ - ） cells,after those ceils were infected with Ad-CMV- IL24. But after those cells were infected with blank control Ad, there was no expression of IL-24. Conclusion Ad-hTERT-IL24 adenovirus vector can effectively mediate the specific expression of IL-24 in the hTERT（ ＋ ） tumor cells.