5/35嵌合型腺病毒载体对脑胶质瘤的抑制作用

Inhibition of 5 /35 chimeric adenoviral vector on glioma

目的通过构建一种新型腺病毒载体,验证肿瘤坏死因子相关凋亡诱导配体(TRAIL基因)治疗胶质瘤的疗效,并证实新型载体的优势。方法经美国华盛顿大学实验室授权获得Ad/35△E1-△E3,即E1和E3缺失的5型腺病毒纤维fiber被35型新型载体。利用基因重组,构建出重组腺病毒质粒Ad5/35△E1-△E3-TRAIL。氯化铯密度梯度离心纯化Ad5/35△E1-△E3-TRAIL,并并测定病毒滴度。采用逆转录酶-多聚酶链反应(RT-PCR)、免疫荧光技术、蛋白质印迹法(Western blot)分别检测目的基因的表达。结果纯化后Ad5/35△E1-△E3-TRAIL的病毒滴度为1.22×1015pfu/l。重组质粒经RT-PCR都扩增出104 bp的基因片段。通过免疫荧光和Western blot等方法检测到目的基因的高效表达。结论新型腺病毒载体质粒Ad5/35△E1-△E3-TRAIL能够高效的在脑胶质瘤细胞中表达。

Objective The efficiency of tumor necrosis factor-related apoptosis-inducing ligand （TRAIL gene） in treating glioma by constructing a new type of adenovirus vector and the advantages of this new carrier are investigated. Methods Authorized by the laboratory of University of Washington USA, AdS/ 35 △E1-△E3 that adenovirus type 5 fiber with E1 and E3 deletion replaced by adenovirus type 35 fiber was used. Gene recombination was utilized to construct recombinant adenobims plasmid Ad5/35 △E1-△E3- TRAIL. Virus Ad5/35△E1-△E3-TRAIL was purified by cesium chloride density gradient centrifugation and its virus titer was assayed. Reverse transcriptase-polymerase chain reaction （ RT-PCR）, immunofluorescence technique and Western blotting were employed to measure the expression of target gene. Results The titer of AdS/35ZXE1-AE3-TRAIL was 1.22 × 1015 pfu/1 after purification. The gene fragment of 104 bp was amplified by RT-PCR. The high expression of targeted gene had been detected by iannunofluorescence technique and Western blotting. Conclusion New type of adenovirus vector plasmid Ad5/35 △E1-△E3-TRAIL can be highly expressed in glioma cells.