重组分泌型TRAIL腺病毒载体的构建及鉴定

CONSTRUCTION AND IDENTIFICATION OF RECOMBINANT SECRETING ADENOVIRAL VECTOR TRAIL

目的构建表达分泌型肿瘤坏死因子相关凋亡诱导配体（TRAIL）基因的重组腺病毒载体。方法采用PCR技术从人肝脏cDNA文库中扩增出编码114-281位氨基酸的TRAIL基因片段。将扩增的TRAIL插入含有分泌性信号肽IL-2的腺病毒穿梭质粒pAdTrack-CMV-IL-2中,将其命名为pAdTrack-CMV-IL-2-TRAIL。pAdTrack-CMV-IL-2-TRAIL经PmeⅠ线性化后,电转化入转化了pAdEasy-1的BJ5183细胞。筛选重组腺病毒质粒pAd-sTRAIL。经HEK293细胞包装后得到复制缺陷型重组腺病毒Ad-sTRAIL,氯化铯密度梯度离心纯化Ad-sTRAIL病毒,并测定病毒滴度。采用RT-PCR法和免疫荧光技术分别检测目的基因的表达。结果纯化后的Ad-TRAIL病毒滴度为3.12×10^15pfu/L。经RT-PCR扩增出长104 bp的基因片段。免疫荧光检测到了目的基因在U251细胞的高效表达。结论成功构建了重组分泌型TRAIL腺病毒载体,为下一步应用于脑肿瘤的基因治疗打下了基础。

Objective To construct a recombinant secreting adenovirus vector expressing TRAIL gene.Methods The TRAIL gene fragment encoding 114-281 amino acids from human liver cDNA library was amplified by PCR.The amplifiable TRAIL was inserted into adenovirus shuttle plasmid containing secreting signal peptide IL-2-pAdTrack-CMV-IL-2,which was named pAdTrack-CMV-IL-2-TRAIL.After linearization by Pmel,pAdTrack-CMV-IL-2-TRAIL was transformed into E.coli BJ5183 harboring pAdEasy-1 by electrotransformation.Recombinant plasmid pAd-sTRAIL was screened.Packing in HEK293 cells,a replication-defective adenovirus-Ad-sTRAIL-was obtained,and purified by cesium chloride density gradient centrifugation,its virus titer was assayed.RT-PCR and immunofluorescence technique were employed to measure the expression of target gene.Results The titer of Ad-sTRAIL was 3.12×1015 pfu/mL after purification.The gene fragment of 104 bp was amplified by RT-PCR.The expression of target gene in U251 cells had been detected by immunofluorescence.Conclusion The recombinant secreting adenoviral vector TRAIL has been constructed successfully,which provides a basis for further application in gene therapy for brain tumors.