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

QIU Lin; WU Qinghua; XIAO Jun; LI Mingyong

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

更新时间：2023-12-18

- Construction and expression of recombinant adenovirus vector carrying transforming growth factor-β3 gene
- Journal of Chongqing Medical University Vol. 37, Issue 8, Pages: 668-672(2012)
- 作者机构：
  
  重庆医科大学附属儿童医院烧伤整形科,重庆,400014
- 作者简介：
- 基金信息：
- DOI：
  CLC：
- Published：2012，
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QIU Lin, WU Qinghua, XIAO Jun, et al. Construction and expression of recombinant adenovirus vector carrying transforming growth factor-β3 gene. [J]. Journal of Chongqing Medical University 37(8):668-672(2012)
DOI：

QIU Lin, WU Qinghua, XIAO Jun, et al. Construction and expression of recombinant adenovirus vector carrying transforming growth factor-β3 gene. [J]. Journal of Chongqing Medical University 37(8):668-672(2012) DOI：

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.

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

Keywords

transforming growth factor-β3（TGF-β3）enhanced green fluorescent protein（EGFP）adenovirus vector

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