携带Pdcd5基因的三调控增殖性腺病毒的构建及鉴定

本研究旨在构建一种表达pdcd5基因的三调控增殖性腺病毒。应用DNA重组技术将pdcd5cDNA插入三调控条件增殖性腺病毒SG600的E3区，SG600病毒系统是运用人端粒酶反转录酶基因启动子（hTERTp）和缺氧反应元件（HRE）分别调控病毒复制必须基因E1a和E1b的表达，去除E1a基因CR2区中24个核苷酸而构建成的靶向肿瘤细胞增殖的病毒系统。采用酶切及PCR鉴定并筛选重组成功的质粒。荧光显微镜下观察带增强型绿色荧光蛋白（EGFP）基因的重组腺病毒SG611—EGFP对白血病细胞系的感染效率。实时定量PCR检测感染重组腺病毒SG611．pdcd5的K562细胞pdcd5的表达水平。结果显示，构建的se611-pdcd5病毒能扩增出hTERTp、HRE、pd—cd5基因以及腺病毒骨架和11型纤突结基因序列。SG611-EGFP对白血病细胞系K562和MEG-01的感染效率均能达到70％以上。感染SG611-pdcd5的K562细胞pdcd5的表达水平较SG611空载体和携带pdcd5的非增殖性腺病毒（Ad-pdcd5）明显增加（P〈0．001），其表达水平随感染复数增加而升高。结论：成功构建了三调控增殖性腺病毒SG611-pdcd5，后者能高效感染白血病细胞系并高效表达pdcd5，为进一步运用pdcd5靶向肿瘤细胞进行基因治疗的研究提供了基础。

Construction and Verification of A Novel Triple-regulated Oncolytic Adenovirus Carrying Gene Pdcd5

The purpose of this study was to construct a recombinant conditionally replicating adenovirus （CRAd） expressing programmed cell death 5 （ pdcd5 ）. Pdcd5 gene was inserted in the E3 region of SG600 - a CRAd in which the key genes for virus replication E1 a and El b were controlled under the human telomerase reverse transcfiptase promoter （hTERTp） and the bypoxia response element（HRE） respectively, and with a deletion of 24 nucleotides within CR2 region of E1 a. The insertion and orientation of all recombined plasmids were confirmed by restriction enzyme digestion and polymerase chain reaction （PCR）. The infection efficiencies of a recombined virus carrying enhanced green fluorescent protein （EGFP） in leukemic cell lines were observed by using fluorescence microscope. The relative pdcd5 expression levels of K562 after being infected with SG611-pdcd5 were detected by real-time quantitative PCR. The results showed that the construction of SG611-pdcd5 was completed and confirmed. Pdcd5, hTERTp, HRE, skeleton and fiberl 1 of recombinant adenovirus SG611-pdcd5 were successfully amplified. The infection efficiencies of SG611-EGFP were all above 70% in both leukemic K562 and MEG-01 cell lines. SG611-pdcd5 expressed pdcd5 with high efficiency in leukemic cells as compared with Ad-pdcd5 or SG611 （p 〈0. 001 ）. The expression level of pdcd5 increased gradually along with the increase of MOI. It is concluded that the triple-regulated adenovirus of SG611-pdcd5 containing the pro-apoptofic gene pdcd5 has been successfully established with high pdcd5 expression level in leukemic cells, indicating that the recombinant adenovims, SG611-pdcd5, promises further development of targeted tumor gene therapy.