摘要
目的以复制缺陷的腺病毒为载体,用细菌内同源重组法构建含有人的去甲肾上腺素转运子(human neurotransmitter transporter,hNET)基因的腺病毒载体。方法利用限制性内切酶KpnⅠ+XbaⅠ从pCMV5质粒中切下目的基因hNET,亚克隆至经同样酶切的腺病毒穿梭质粒pAdtrack-CMV,形成重组穿梭质粒pAdtrack-CMV-hNET,PmeⅠ酶切线性化后,与pAdeasy-1在大肠杆菌BJ5183中进行同源重组,获得含目的基因的重组体质粒Ad-hNET,PacⅠ酶切后用脂质体Lipofectamine2000转染HEK293细胞,包装成重组体腺病毒。采用PCR技术和Western Blotting对重组体腺病毒进行鉴定,利用穿梭质粒pAdTrack-CMV中带有绿色荧光蛋白报告基因检测表达,经过扩增和纯化,测定病毒滴度。结果测序结果证明hNET序列的正确,PCR、PacⅠ酶切电泳和Western Blotting证实腺病毒重组质粒Ad-hNET构建成功。扩增纯化后测定重组病毒Ad-hNET的滴度为1.2×1010pfu/mL。结论成功构建了能表达hNET基因的重组腺病毒载体,为肿瘤的靶向治疗提供前期的研究工作。
Objective To construct and identify the recombinant replication deficient adenovirus vector which codes for human Norepinephrine Transporter（hNET）gene by using the method of homogenous recombination in bacteria.Methods hNET gene was obtained from the recombinant plasmid pCMV5 via KpnⅠ＋XbaⅠ digestion,and subcloned into E1 deleted expression plasmid pAdtrack-CMV shuttle vector,forming transfer vector pAdtrack-CMV-hNET.Then it was linearized with PmeⅠ followed by homologous recombination with bone plasmid pAdEasy-1 in BJ5183 cells to generate recombinant plasmid Ad-hNET.The DNA of identified Ad-hNET was digested with PacⅠ and transfected to HEK293 cells by liposome-mediated method to package recombinant adenovirus.The PCR technique was applied to detect the target gene and Western Blotting to verify the expression of hNET.The titre of the Ad-hNET was measured with the aid of green fluorescence protein（GFP）expression after multiplication and purification.Results By sequencing,it was confirmed that the product was the gene of hNET.PCR test,restriction endonuclease digestion and Western Blotting confirmed the successful construction of the recombinants Ad-hNET.The titre of purified recombinant adenovirus Ad-hNET was 1.2×10^10 pfu/mL.Conclusion The recombinant adenovirus with the hNET gene was constructed successfully.It will be helpful for the further investigation of its potentiality to be applied in the tumors targeted therapeutic strategy.
出处
《四川大学学报（医学版）》
CAS
CSCD
北大核心
2008年第4期523-526,共4页
Journal of Sichuan University(Medical Sciences)
基金
国家自然科学基金(批准号30400113/C03031702)资助
关键词
腺病毒载体
人去甲肾上腺素转运蛋白
同源重组
Adenovirus vector Human norepinephrine transporter Homologous recombinant
