转化生长因子-β3基因重组腺病毒载体的构建及表达
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Construction and expression of recombinant adenovirus vector carrying transforming growth factor-β3 gene
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    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 2×1010 pfu/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。

    Abstract:

    Objective:To construct the recombinant adenovirus vector containing transforming growth factor-β3(TGF-β3) and en-hanced green fluorescent protein(EGFP) in order to transfect rabbit bone marrow mesenchymal stem cells(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 2×1010 pfu/ml. Western blot demonstrated that TGF-β3 was expressed in rabbit bone marrow MSCs. Conclusions:EGFP-TGF-β3 recombinant adenovirus vector is successful constructed and TGF-β3 protein is effectively expressed in rabbit bone marrow MSCs,which provides the basis for research of gene therapy in preventing hypertrophic scar after wound healing.

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邱 林,吴清华,肖 军,李明勇.转化生长因子-β3基因重组腺病毒载体的构建及表达[J].重庆医科大学学报,2012,37(8):668-672

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  • 在线发布日期: 2012-09-17
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