转化生长因子-β3基因重组腺病毒载体的构建及表达  

Construction and expression of recombinant adenovirus vector carrying transforming growth factor-β3 gene

作　　者：邱林[1] 吴清华[1] 肖军[1] 李明勇[1] 

机构地区：[1]重庆医科大学附属儿童医院烧伤整形科,重庆400014

出　　处：《重庆医科大学学报》2012年第8期668-672,共5页Journal of Chongqing Medical University

基　　金：重庆市自然科学基金资助项目(编号:CSTC;2008BB5391)

摘　　要：目的:构建含有转化生长因子-β3(Transforming growth factor-β3,TGF-β3)和增强型绿色荧光蛋白(Enhanced green fluorescent protein,EGFP)的腺病毒载体,使其转染兔骨髓间充质干细胞(Mesenchymal stem cells,MSCs),并在细胞内得到表达。方法:将含有TGF-β3基因克隆至含有EGFP的穿梭质粒,通过在感受态菌DH5α内同源重组,构建pAdxsi-EGFP-TGF-β3。PacⅠ酶切线性化pAdxsi-EGFP-TGF-β3转染HEK293细胞,多轮"乒乓感染法"扩增收集pAdxsi-EGFP-TGF-β3病毒液,氯化铯梯度离心纯化,PCR和荧光显微镜观察EGFP的表达并检测重组腺病毒液的滴度。测定重组腺病毒液转染MSCs细胞转染效率,Western blot检测TGF-β3蛋白表达。结果:成功构建携带TGF-β3和EGFP的重组腺病毒载体pAdxsi-EGFP-TGF-β3,PCR和绿色荧光证实病毒包装成功,并具有感染力,病毒滴度为2×1010pfu/ml。Western blot检测表明Adxsi-EGFP-TGF-β3感染的兔骨髓MSCs细胞有效表达TGF-β3。结论:含有TGF-β3和EGFP的腺病毒载体pAdxsi-EGFP-TGF-β3构建成功和TGF-β3蛋白在兔骨髓MSCs中有效表达,为进一步研究转染TGF-β3基因的兔骨髓MSCs减轻创面愈合后瘢痕组织增生提供实验基础。Objective：To construct the recombinant adenovirus vector containing transforming growth factor-β3 （TGF-β3） and en- hanced green fluorescent protein（EGFP） in order to transfeet rabbit bone marrow mesenchymal stem ceUs（MSCs）. Methods：The cD- NA TGF-β3 was integrated into the shuttle vector of pAdxsi-CMV with EGFP,then the product was transfected into package cell HEK 293 and the recombinant adenovirus vector expressing TGF-β3 gene was generated. The expression of EGFP was identified by PCR and fluorescent microscopy. The titer of pAdxsi-EGFP-TGF-β3 was tested with end-paint dilution assay and its tranfection ef- ficacy was examed. The expression of TGF-β3 was detected by Western blot. Results：The recombinant adenovirus vector pAdxsi- EGFP-TGF-β3 containing TGF-β3 and EGFP was constructed successfully, PCR analysis showed that TGF-β3 cDNA was inserted into the recombinant adenoviral plasmid. The recombinant virus vector was collected by packaging HEK293 cells. The titer of pAdxsi- EGFP-TGF-β3 was 2-10 pfu/ml, Western blot demonstrated that TGF-β3 was expressed in rabbit bone marrow MSCs. Conelusgons： EGFP-TGF-β3 recombinant adenovirus vector is successful constructed and TGF-β3 protein is effectively expressed in rabbit bone marrow MSCs, whieh provides the basis for research of gene therapy in preventing hypertrophic sear after wound healing.

关 键 词：转化生长因子-β3基因 增强型绿色荧光蛋白 腺病毒载体 

分 类 号：Q953.5[生物学—动物学]