ANG-1基因重组腺相关病毒获取及转染猪骨髓干细胞的研究
R654.2; 目的:构建携带血管形成素-1(ANG-1)基因腺相关病毒(AAV)载体,通过病毒包装和纯化及滴定,获得高纯度高感染性的病毒液,并转染到乳猪骨髓干细胞中,获得有效表达,为进行血管再生的基因治疗研究奠定基础.方法:利用PCR技术,获取目的基因ANG-1,通过重组DNA技术得到ANG-1与pUC119的重组质粒,采用SalⅠ和BglⅡ双酶将pUC119中的目的基因ANG-1基因片断切出,再克隆到质粒pSNAV2.0中.用Lipofectamine将pSNAV ANG-1质粒转染293细胞后,用G418选择培养获得293/AAV2ANG-1细胞株,产生了有传染性的病毒颗粒,纯化并进行病...
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| Published in | 浙江大学学报（医学版） Vol. 38; no. 4; pp. 370 - 376 |
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| Main Authors | , , , , |
| Format | Journal Article |
| Language | Chinese |
| Published |
浙江省台州医院心胸外科,浙江,临海,317000%江苏省肿瘤医院胸外科,江苏,南京,210000%阜外医院临床心血管药理中心,北京,100037
2009
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| Subjects | |
| Online Access | Get full text |
| ISSN | 1008-9292 |
| DOI | 10.3785/j.issn.1008-9292.2009.04.008 |
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| Abstract | R654.2; 目的:构建携带血管形成素-1(ANG-1)基因腺相关病毒(AAV)载体,通过病毒包装和纯化及滴定,获得高纯度高感染性的病毒液,并转染到乳猪骨髓干细胞中,获得有效表达,为进行血管再生的基因治疗研究奠定基础.方法:利用PCR技术,获取目的基因ANG-1,通过重组DNA技术得到ANG-1与pUC119的重组质粒,采用SalⅠ和BglⅡ双酶将pUC119中的目的基因ANG-1基因片断切出,再克隆到质粒pSNAV2.0中.用Lipofectamine将pSNAV ANG-1质粒转染293细胞后,用G418选择培养获得293/AAV2ANG-1细胞株,产生了有传染性的病毒颗粒,纯化并进行病毒DNA颗粒滴度测定.分别将绿色荧光蛋白(GFP)基因重组腺相关病毒(rAAV2GFP)及rAAV2ANG-1感染猪骨髓干细胞,并对转染效率及转染后表达情况进行初步研究.结果:测序证实ANG-1与GenBank提供的原始序列完全一致.重组载体经酶切鉴定与PCR筛选证实重组质粒和预期结果完全一致,在新的重组质粒中,ANG-1有一套CMV启动子和PolyA终止子系统.293细胞包装病毒效果良好,携带ANG-1的感染性AAV滴度为9×1011v.g/ml,用Western杂交法检测显示猪骨髓干细胞中表达ANG-1.1×106v.g/ml rAAV2GFP感染CD34阳性细胞48h后55%左右的细胞有明显的荧光.结论:ANG-1基因AAV构建成功,并能在骨髓干细胞中有效表达,为严重缺血性疾病基因治疗的研究奠定了基础. |
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| AbstractList | R654.2; 目的:构建携带血管形成素-1(ANG-1)基因腺相关病毒(AAV)载体,通过病毒包装和纯化及滴定,获得高纯度高感染性的病毒液,并转染到乳猪骨髓干细胞中,获得有效表达,为进行血管再生的基因治疗研究奠定基础.方法:利用PCR技术,获取目的基因ANG-1,通过重组DNA技术得到ANG-1与pUC119的重组质粒,采用SalⅠ和BglⅡ双酶将pUC119中的目的基因ANG-1基因片断切出,再克隆到质粒pSNAV2.0中.用Lipofectamine将pSNAV ANG-1质粒转染293细胞后,用G418选择培养获得293/AAV2ANG-1细胞株,产生了有传染性的病毒颗粒,纯化并进行病毒DNA颗粒滴度测定.分别将绿色荧光蛋白(GFP)基因重组腺相关病毒(rAAV2GFP)及rAAV2ANG-1感染猪骨髓干细胞,并对转染效率及转染后表达情况进行初步研究.结果:测序证实ANG-1与GenBank提供的原始序列完全一致.重组载体经酶切鉴定与PCR筛选证实重组质粒和预期结果完全一致,在新的重组质粒中,ANG-1有一套CMV启动子和PolyA终止子系统.293细胞包装病毒效果良好,携带ANG-1的感染性AAV滴度为9×1011v.g/ml,用Western杂交法检测显示猪骨髓干细胞中表达ANG-1.1×106v.g/ml rAAV2GFP感染CD34阳性细胞48h后55%左右的细胞有明显的荧光.结论:ANG-1基因AAV构建成功,并能在骨髓干细胞中有效表达,为严重缺血性疾病基因治疗的研究奠定了基础. |
| Abstract_FL | Objective: To construct recombinant adeno-associated virus (rAAV) vector containing angiopoietin-1(ANG-1) gene and to express the ANG-1 in targeting cells. Methods: ANG-1 cDNA was obtained from human spleen by RT-PCR and was inserted into AAV vectors to form rAAV ANG-1,the virus stocks in high titer were harvested.The rAAVANG-1 and rAAV GFP were transferred into pig mesenchymal stem cells and the expression of ANG-1 was detected by Western blot. Results: The cloned ANG-1 cDNA was 1515bp in length which was in accordance with that reported previously.Titration of rAAVANG-1 stock was 9×1011v.g/ml.The expression of ANG-1 gene was detected in transfected cells.Forty-eight hours after rAAV GFP was transfected into mesenchymal stem cells,55% cells expressed GFP. Conclusion: The constructed rAAV ANG-1 vector has successfully transfered and expressed in pig mesenchymal stem cells. |
| Author | 包卫光 朱成楚 陈仕林 唐礼江 刘玉清 |
| AuthorAffiliation | 浙江省台州医院心胸外科,浙江,临海,317000%江苏省肿瘤医院胸外科,江苏,南京,210000%阜外医院临床心血管药理中心,北京,100037 |
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| Author_FL | ZHU Cheng-chu CHEN Shi-lin BAO Yu-guang TANG Li-jiang LIU Yu-qing |
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| DocumentTitle_FL | Construction of adeno-associated virus vector containing ANG-1 gene and its expression in pig mesenchymal stem cells |
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| Keywords | 血管生成素1/代谢 Adenoviridae/genet 骨髓干细胞 Angiopoietin-1/metab 腺相关病毒载体 骨髓祖代细胞/代谢 rAAV 腺病毒科/遗传学 Myeloid progenitor cells/metab Transfection 重组,遗传 转染 Recombination,genetic Mesenchymal stem cells |
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| Publisher | 浙江省台州医院心胸外科,浙江,临海,317000%江苏省肿瘤医院胸外科,江苏,南京,210000%阜外医院临床心血管药理中心,北京,100037 |
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| Snippet | R654.2; 目的:构建携带血管形成素-1(ANG-1)基因腺相关病毒(AAV)载体,通过病毒包装和纯化及滴定,获得高纯度高感染性的病毒液,并转染到乳猪骨髓干细胞中,获得有效表达,为进... |
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| Title | ANG-1基因重组腺相关病毒获取及转染猪骨髓干细胞的研究 |
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