携带人TGF-β_1腺病毒载体的构建及鉴定

Reconstruction and Identification of Adenovirus Expression Vector for Human Transforming Growth Factor β_1

目的构建携带人转化生长因子β1(hTGF-β1)的腺病毒载体(Adeno-hTGF-β1),为hTGF-β1基因转染骨髓间质干细胞(BMSCs)的研究奠定基础。方法应用基因重组技术构建hTGF-β1腺病毒表达载体,分别用限制性内切酶酶切和聚合酶链反应(PCR)鉴定,并通过脂质体Fugene 6介导腺病毒DNA转染293细胞。结果重组hTGF-β1腺病毒DNA经限制性内切酶XhoⅠ酶切后,得到14.5、8.1、4.2、4.0、2.5、1.4、0.6 kb片段。与空白腺病毒相比,5.6 kb的片段已被置换成4.2 kb和4.0 kb片段;经PI-SceⅠ/I-CeuⅠ双酶切后,得到32.6 kb和2.6 kb含目的基因片段,片段大小无误,表明重组Adeno-hTGF-β1腺病毒表达系统构建成功。以重组Adeno-hTGF-β1DNA为模板的PCR产物中出现1.35 kb hTGF-β1目的基因片段,表明hTGF-β1基因成功连接至adeno-X腺病毒表达载体中。重组hTGF-β1腺病毒表达载体通过Fugene 6介导转染293包装细胞出现细胞病变效应(CPE)。采用PCR反应鉴定293细胞包装的腺病毒,证实扩增出247 bp的hTGF-β1目的基因片断和312 bp的腺病毒骨架基因片断。结论携带人hTGF-β1的腺病毒载体的构建成功,并能在293细胞包装,为hTGF-β1基因转染的BMSCs在软骨组织工程中的应用奠定了基础。

Objective To construct a recombinant adenovirus expression vector for human transforming growth factor β1 （hTGF-β1） and to lay a foundation for the study of hTGF-β1 gene transfection into bone marrow stromal cells （BMSCs）. Methods An adenovirus expression vector for hTGF-β1 was constructed by recombinant DNA technique. Adeno-hTGF-β1 was confirmed by restriction endonuclease and PCR respectively. Adeno-hTGF-β1 was cotransfected into 293 cells by liposome-mediated （ Fugene 6） method. The generated recombinant adenoviruses were confirmed by PCR. Results The recombinant Adeno-hTGF-β1 was digested with Xho Ⅰ ,and the electrophoresis of the digested products showed 14.5,8.1,4.2,4.0,2.5,1.4,0.6 kb correct fragments. Furthermore, the recombinant Adeno-hTGF-β1 was digested with PI-Sce Ⅰ / I - Ceu Ⅰ , and the digested products showed 32.6 and 2.6 kb correct fragments. The recombinant Adeno-hTGF-β1 was determined by PCR and the product of PCR showed the presence of 1.35 kb hTGF-β1 fragment. The infected 293 cells showed evident cytopathic effect （CPE）. The generated recombinant adenoviruses were confirmed by PCR and the product of PCR showed the presence of 247 bp hTGF-β1 gene fragments and 312 bp gene adenovirus fragments. Conclusion The recombinant Adeno-hTGF-β1 can be constructed correctly and be transfected into 293 cells, which establishes the base for hTGF-β1 gene transfection into BMSCs in the application in cartilage tissue engineering.