DMT1第四功能区腺病毒表达载体的构建及其功能的初步研究

Construction and initial study of recombinant adenoviral expressed vector carrying DMT1 domain four

目的:构建携带DMT1第四功能区基因的重组腺病毒表达载体,并对其功能进行初步探讨。方法:采用AdEasy系统构建携带DMT1第四功能区基因的重组腺病毒载体pAd-DMT1-4,脂质体法转染人胚肾293细胞,包装产生重组腺病毒vAd-DMT1-4。vAd-DMT1-4感染C6胶质瘤细胞后,采用RT-PCR检测目的基因表达,用铁孵育感染重组腺病毒的靶细胞后,测定C6胶质瘤细胞内OH.和MDA含量。结果:PCR、序列测定以及限制性酶切证实vAd-DMT1-4基因正确插入穿梭质粒,并与病毒骨架质粒pAdEasy-1重组,重组腺病毒表达载体构建成功。经293细胞包装获得具有稳定感染性的重组腺病毒vAd-DMT1-4,感染重组腺病毒的C6胶质瘤细胞DMT1-4表达增加。用铁孵育感染重组腺病毒的靶细胞后,C6胶质瘤细胞内OH.和MDA含量有增高的趋势。结论:重组腺病毒vAd-DMT1-4可有效感染C6胶质瘤细胞。

Objective：To construct recombinant adenoviral express vector carrying DMT1 domain four gene and to investigate its primary function. Methods：The recombinant adenovirus vector carrying DMT1 domain four gene was constructed with AdEasy system and then transfected 293 cells to produce recombinant adenovirus vAd-DMT1-4. Glioma C6 cells were infected with recombinant adenoviruses . The expression of target gene were tested by RT-PCR. The intracel- lular malondialdehyde（MDA） and Hydroxyl radicals （OH · ） were measured when treated with FeSO4. Results： It was confirmed by PCR, sequencing identification and restrictive analysis that the recombinant adenovirus vector was constructed successfully. The recombinant adenovirus vAd-DMT1-4,which has stable infectivity,was packed in 293 cells. DMT1- 4 expression was increased in glioma C6 cells infected by recombinant adenovirus. Conclusions：The recombinant adenoviruses vAd-DMT1-4 can effectively infect glioma C6 cells.