BMP2和TGFβ3双基因真核表达载体的构建与鉴定
摘要
目的本实验旨在扩增BMP2和TGFβ3基因,并构建含有BMP2和TGF-β3的双基因真核表达载体p工ERS-BMP2-TGFβ3,为研究双基因共转染间充质干细胞及其在体内的表达情况和可能存在的协同效应奠定基础。
方法首先,用PCR方法从质粒pGEMT/BMP2中扩增出BMP2基因全长,并将其连入双基因真核表达载体pIRES,筛选出阳性重组质粒pIRES-BMP2；其次,用RT-PCR的方法从人胚胎组织扩增出TGFβ3基因全长,将TGFβ3基因连入质粒pIRES-BMP2；用酶切的方法筛选出阳性重组质粒pIRES-BMP2-TGFβ3,并进行测序鉴定。
结果成功的克隆出BMP2和TGFβ3基因,并将其连接到目的载体pIRES中,双酶切和质粒测序证实连接正确,且基因的开放阅读框架正确,pIRES-BMP2-TGFβ3双基因真核表达载体构建成功。
结论利用PCR, DNA重组等分子生物学技术,成功构建了双基因真核表达载体pIRES-BMP2-TGFβ3,为进一步研究双基因共转染间充质干细胞,探讨其在体内的表达情况,对间充质干细胞成骨分化的影响及可能存在的协同效应奠定基础。
Objective To construct a bicistronic eukayotic expression vector pIRES-BMP2-TGFB3
Methods Firstly, the BMP2 gene was obtained from pGEMT/BMP2 plasmid by PCR. Then it was inserted into bicistronic eukaryotic expression plasmid vector pIRES. The recombinant plasmid pIRES-BMP2 was obtained; Second, the TGFβ3 was extracted from human embryonal tissue by RT-PCR, then the gene was inserted into the plasmid pIRES-BMP2.The inserted target genes in the plasmid were verified by restriction enzyme digestion and nucleotide sequencing.
Results The direction and sequences of the new bicistronic eukaryotic expression vector pIRES-BMP2-TGFβ3 were correct.
Conclusion The bicistronic eukaryotic expression vector was constructed successfully and formed the foundation for transfecting into mesenchymal stem cells in the next experiment.
方法首先,用PCR方法从质粒pGEMT/BMP2中扩增出BMP2基因全长,并将其连入双基因真核表达载体pIRES,筛选出阳性重组质粒pIRES-BMP2；其次,用RT-PCR的方法从人胚胎组织扩增出TGFβ3基因全长,将TGFβ3基因连入质粒pIRES-BMP2；用酶切的方法筛选出阳性重组质粒pIRES-BMP2-TGFβ3,并进行测序鉴定。
结果成功的克隆出BMP2和TGFβ3基因,并将其连接到目的载体pIRES中,双酶切和质粒测序证实连接正确,且基因的开放阅读框架正确,pIRES-BMP2-TGFβ3双基因真核表达载体构建成功。
结论利用PCR, DNA重组等分子生物学技术,成功构建了双基因真核表达载体pIRES-BMP2-TGFβ3,为进一步研究双基因共转染间充质干细胞,探讨其在体内的表达情况,对间充质干细胞成骨分化的影响及可能存在的协同效应奠定基础。
Objective To construct a bicistronic eukayotic expression vector pIRES-BMP2-TGFB3
Methods Firstly, the BMP2 gene was obtained from pGEMT/BMP2 plasmid by PCR. Then it was inserted into bicistronic eukaryotic expression plasmid vector pIRES. The recombinant plasmid pIRES-BMP2 was obtained; Second, the TGFβ3 was extracted from human embryonal tissue by RT-PCR, then the gene was inserted into the plasmid pIRES-BMP2.The inserted target genes in the plasmid were verified by restriction enzyme digestion and nucleotide sequencing.
Results The direction and sequences of the new bicistronic eukaryotic expression vector pIRES-BMP2-TGFβ3 were correct.
Conclusion The bicistronic eukaryotic expression vector was constructed successfully and formed the foundation for transfecting into mesenchymal stem cells in the next experiment.
引文
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5.Howard, D., Buttery, L.D., Shakesheff, K.M., Roberts, S.J., Tissue engineering:strategies, stem cells and scaffolds. J. Anat.,2008;213(1):66-72.
6.Matsumoto T, Cooper GM, Gharaibeh B, et al. Blocking VEGF as a potential approach to improve cartilage healing after osteoarthritis. J Musculoskelet Neuronal Interact.2008;8(4):316-317.
7.Chen WJ, Li GT, Luo DX, et al. Application of mesenchymal stem cell combining with bone morphogenic protein to improve spine interbody fusion. Zhongguo Jiaoxing Waike Zazhi. 2007;15(3):1012-1014.
8.Jancar J, Slovikova A, Amler E, et al. Mechanical response of porous scaffolds for cartilage engineering. Physiol Res.2007;56 Suppl 1:S17-25.
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