辐射诱导人TRAIL基因表达的腺病毒穿梭质粒的构建及鉴定

Construction and identification of recombinant adenoviral shuttle vector with radiation-inducible TRAIL gene

目的:构建辐射敏感Egr-1启动子诱导人肿瘤坏死因子(TNF)相关凋亡诱导配体(TRAIL)基因表达的腺病毒穿梭质粒pshuttle-Egr1-hTRAIL。方法:利用逆转录-聚合酶链式反应(RT-PCR)方法从pACCMV-hTRAIL质粒中扩增得到TRAIL基因片段,并将其连接到pMD19T载体进行测序,利用基因重组技术构建含有辐射诱导启动子Egr-1的腺病毒穿梭质粒pshuttle-Egr1-hTRAIL。结果:PCR扩增出约820 bp的片段,测序结果证实扩增片段的序列与GenBank公布(NM_003810)的一致。pshuttle-Egr1-hTRAIL用EcoRⅠ、KpnⅠ双酶切重组得到大小为3 540和4 299 bp的2个片段,用SmaⅠ酶切得到1 517、2 282和4 040 bp的3个片段,用BamHⅠ酶切得到3 304和4 535 bp的2个片段,与预期结果完全一致。结论:成功克隆了hTRAIL基因,并构建了辐射诱导表达的腺病毒穿梭质粒pshuttle-Egr1-hTRAIL。

Objective To construct a recombinant adenoviral shuttle vector pshuttle-Egrl-hTRAIL containing radiation-sensitive Egr-1 promoter and TNF-related apoptosis-inducing ligand （TRAIL）. Methods The TRAIL gene fragment was acquired from the plasmid pACCMV-hTRAIL by RT-PCR. Then the TRAIL gene was ligated to pMD19T vector and sequenced. With the gene recombinant technique, the recombinant plasmid pshuttle-Egrl- hTRAIL with radiation-inducible promoter Egr-1 was constructed. Results A fragment about 820 bp was amplified by PCR, and the sequence of acquired bTRAIL gene was totally in concordance with that published in GenBank （NM_003810）. Moreover, the recombinant plasmid pshuttle-Egrl-hTRAIL was digested by EcoR I and Kpn I double-enzyme and BamH I singly both into two fragments, with the length of 3 540 and 4 299 bp, 3 304 and 4 535 bp, respectively. The Sma I enzyme could digest it into three fragments with lengths of 1 517, 2 282 and 4 040 bp. The results of enzyme identification were all in concordance with that expected. Conclusion The hTRAIL gene is cloned and the recombinant plasmid pshuttle-Egrl-hTRAIL is constructed successfully.