重组hTRAIL腺病毒载体的构建及鉴定

Construction and Identification of Recombinant Adenovirus for Expression of hTRAIL

目的构建表达hTRAIL基因的重组腺病毒载体。方法以重组质粒pThioHisA-TRAIL中提取的TRAIL基因为模板,采用PCR法扩增基因片段。将扩增的基因片段插入穿梭质粒pShuttle-CMV,并转化大肠肝菌DH5α。筛选重组质粒pShuttle-CMV-TRAIL,电转化已转化了pAdEasy-1的BJ5183细胞。筛选重组腺病毒质粒pAdEasy-TRAIL,用Lipofectamine转染293细胞,制备携带人全长TRAIL基因的重组复制缺陷型腺病毒(Ad-TRAIL)。氯化铯密度梯度离心法纯化Ad-TRAIL病毒颗粒,并测定病毒滴度。采用RT-PCR法检测TRAIL基因在293细胞中的转录。以Ad-TRAIL转染YTMLC细胞,采用免疫荧光法和流式细胞术检测TRAIL基因的表达。结果纯化后的Ad-TRAIL病毒颗粒数为2.77×1012VP/ml,感染性滴度为109.5(3.2×109)CCID50/ml。经RT-PCR扩增出约843bp的基因片段,与目的片段大小一致。流式细胞术和免疫荧光法均检测到TRAIL在YTMLC细胞中的表达,表达产物主要存在于细胞质中。结论已成功构建了表达hTRAIL重组腺病毒载体,为肿瘤的基因治疗提供了依据。

Objective To construct a recombinant adenovirus for expression of human TNF-related apeptosis inducing ligand （hTRAIL） gene. Methods Amplify gene fragment by PCR using the TRAIL gene extracted from recombinant plasmid pThioHisA- TRAIL as a template,insert into shuttle plasmid pShuttle-CMV and transform to E. coli DH 5α Screen recombinant plasmid pShutfleCMV-TRAIL and transform to competent BJ5183 cells previously transformed with pAdEasy-1. Screen recombinant plasmid pAdEasyTRAIL and transfect to 293 cells in mediation of Lipefectamine to prepare, replication-deficient adenovirus Ad-TRAIL carrying fulllength of hTRAIL gene. Purify the prepared Ad-TRAIL virus particles by density gradient centrifugation with cesium chloride and determine the titer. Determine the transcription of TRAIL gene in 293 cells by RT-PCR. Transfect YTMLC cells with Ad-TRAIL and determine the expression of TRAIL by IFA and flow cytometry. Results The virus particle count and titer of purified Ad-TRAIL were 2. 77 × 10^12 VP/ml and 10^9.5（3.2 × 10^9 ） CCID50/ml respectively. The gene fragment at a length of about 843 bp was amplified by RT- PCR from transfected 293 ceils, which was consistent with that expected. Both flow cytometry and IFA proved the expression of TRAIL protein in YTMLC cells, and the expressed product mainly existed in cytoplasm. Conclusion Recombinant adenovirus Ad-TRAIL for expression of hTRAIL was successfully constructed, which provided a basis for gene therapy of tumors.