可诱导表达hBMP-2基因的慢病毒载体的构建及其慢病毒的产生和鉴定

发布时间：2018-05-12 06:15

本文选题：可诱导慢病毒载体 + hBMP-2基因　；参考：《青岛大学》2010年硕士论文

【摘要】： 目的构建可诱导表达人骨形态发生蛋白-2(hBMP-2)基因的慢病毒载体,转染人胚胎肾上皮细胞系293FT细胞获得相应的病毒颗粒,感染并获得在强力霉素诱导条件下高效表达hBMP-2的人脐带间充质干细胞,为下一步可诱导表达hBMP-2的人脐带间充质干细胞移植治疗骨坏死奠定坚实的实验基础。方法以pcDNA-hBMP-2为模板,采用聚合酶链反应法扩增前后端带有attB重组位点的人骨形态发生蛋白-2(hBMP-2)全长序列。利用Gateway载体构建技术,将扩增产物通过BP反应克隆至pDown载体中,测序正确后,采用LR重组酶将pDown-hBMP-2, pUp-TRE和pLV/Des2-Neo进行重组反应,构建慢病毒载体表达质粒pLV/EXPN2-Neo-TRE-hBMP-2.将pLV/EXPN2-Neo-TRE-hBMP-2与包装质粒(pLV/helper-SL3、pLV/helper-SL4、pLV/helper-SL5)混合,利用脂质体共同转染293FT细胞,包装携带hBMP-2基因的慢病毒颗粒,感染人脐带间充质干细胞,在强力霉素(doxcycline)诱导下,用ELISA检测目的细胞hBMP-2的表达情况。结果通过酶切、聚合酶链式反应及测序验证可诱导hBMP-2真核表达慢病毒载体构建成功；pLV/EXPN2-Neo-TRE-hBMP-2与包装质粒共转染包装细胞293FT细胞,成功获得携带hBMP-2基因的慢病毒颗粒,继之感染人脐带间充质干细胞,在强力霉素诱导条件下,使其高效表达hBMP-2。结论成功构建出可诱导表达hBMP-2基因的慢病毒载体,获得浓缩lentiviral-hBMP-2病毒液,在此基础上最终获得可以在强力霉素诱导条件下高效表达hBMP-2的人脐带间充质干细胞,为下一步可诱导表达hBMP-2的人脐带间充质干细胞移植治疗骨坏死奠定坚实的实验基础。
[Abstract]:Objective to construct a lentivirus vector that can induce the expression of human bone morphogenetic protein-2hBMP-2 gene and obtain the corresponding viral particles by transfection into human embryonic renal epithelial cell line 293FT. Human umbilical cord mesenchymal stem cells expressing hBMP-2 efficiently under doxycycline induction condition were infected and obtained, which laid a solid experimental foundation for further transplantation of human umbilical cord mesenchymal stem cells which can induce the expression of hBMP-2 in the treatment of osteonecrosis. Methods the full-length sequence of human bone morphogenetic protein -2hBMP-2 with attB recombinant site was amplified by polymerase chain reaction using pcDNA-hBMP-2 as template. Using Gateway vector construction technique, the amplified product was cloned into pDown vector by BP reaction. After sequencing correctly, pDown-hBMP-2, pUp-TRE and pLV/Des2-Neo were recombined with LR recombinant enzyme to construct lentivirus vector pLV-EXPN2-Neo-TRE-hBMP-2. PLV/EXPN2-Neo-TRE-hBMP-2 was mixed with pLV- helper-SL3 / pLV-helper-SL4 / pLV-helper-SL5), then the 293FT cells were co-transfected with liposome, and the lentivirus particles carrying hBMP-2 gene were packaged and infected with human umbilical cord mesenchymal stem cells. The expression of hBMP-2 in the target cells was detected by ELISA induced by doxycycline. Results by restriction endonuclease digestion, polymerase chain reaction and sequencing, hBMP-2 eukaryotic expression lentivirus vector was successfully constructed and co-transfected with packaging plasmid into 293FT cells. Lentivirus particles carrying hBMP-2 gene were successfully obtained. Then infected with human umbilical cord mesenchymal stem cells, under the condition of doxycycline induction, hBMP-2 was highly expressed in human umbilical cord mesenchymal stem cells. Conclusion the lentivirus vector which can induce the expression of hBMP-2 gene was successfully constructed, and the concentrated lentiviral-hBMP-2 virus solution was obtained. On this basis, the human umbilical cord mesenchymal stem cells which could efficiently express hBMP-2 under doxycycline induction were obtained. It provides a solid experimental basis for the next step to induce the transplantation of human umbilical cord mesenchymal stem cells expressing hBMP-2 in the treatment of osteonecrosis.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R346

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