复制缺陷型人TPO重组腺病毒的构建与鉴定

Construction and identification of replication deficient human thrombopoietin recombinant adenoviruses

目的：构建并鉴定携带人ＴＰＯ基因的复制缺陷型重组腺病毒。方法：将含全部编码序列的人ＴＰＯｃＤＮＡ克隆于腺病毒载体穿梭质粒ｐＣＡ３的ＣＭＶ启动子下游，然后与含有腺病毒基因组的质粒ｐＢ－ＨＧ１０共转染２９３细胞，经同源重组产生人ＴＰＯ重组腺病毒（ＡｄＣＭＶＴＰＯ）；体外转染Ａ５４９细胞并检测其表达活性。结果：扩增后获得的病毒滴度达４．５×１０９ｐｆｕ／ｍｌ，经体外转染Ａ５４９细胞检测到ＴＰＯ的表达。结论：所构建的重组腺病毒能有效介导人ＴＰＯ基因的转移，可望用于造血功能重建的基因治疗研究。

Objective: To construct and identify the replication-deficient human thrombopoietin (TPO) recombinant adenoviruses (AdCMVTPO). Methods: The fulllength human TPO encoding TPO cDNA was cloned at the down stream of human CMV promoter of adenoviral shuttle plasmid pCA3. Then the resultant pCA3TPO was cotransfected into 293 cells with the plasmid pBHG10 carrying the adenoviral genome. The AdCMVTPO was successfully generated with homologous recombination. A549 cells were infected with AdCMVTPO and the expression of human TPO was evaluated in vivo. Results: The titer of AdCMVTPO reached 4.5109 pfu/ml after amplification in 293 cells. A549 cells expressed high level of human TPO after infection. Conclusion: The constructed recombinant adenovirus can mediate the human TPO gene transfer efficiently and can be used in the gene therapy of hemopoietic reconstruction.